Neuroprotection: Models, Mechanisms and Therapies

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Neuroprotection Models, Mechanisms and Therapies Edited by Mathias Bhr

Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

Further Titles of Interest O. von Bohlen und Halbach, R. Dermietzel

Neurotransmitters and Neuromodulators 2002 ISBN 3-527-30318-9

R. Webster (Ed.)

Neurotransmitters, Drugs and Brain Function 2002 ISBN 0-471-97819-1

L. D. Miller, R. L. Hayes, J. K. Newcomb

Head Trauma: Basic, Preclinical, and Clinical Directions 2001 ISBN 0-471-36015-1

R. Shulman, D. Rothman (Eds.)

Brain Energetics and Neuronal Activity: Applications to MRI and Medicine 2004 ISBN 0-470-84720-4

P. Tofts (Ed.)

Quantitative MRI of the Brain: Measuring Changes caused by Disease 2003 ISBN 0-470-84721-2

C. U. M. Smith

Elements of Molecular Neurobiology, Third Edition 2002 ISBN 0-470-84353-5

Neuroprotection Models, Mechanisms and Therapies Edited by Mathias Bhr

Editor: Prof. Dr. Mathias Bhr Department of Neurology University of Gttingen Medical School Robert-Koch-Str. 40 37075 Gttingen Germany

This book was carefully produced. Nevertheless, authors, editor and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at http://dnb.ddb.de. c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation in other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printed in the Federal Republic of Germany. Printed on acid-free paper.

Cover Illustration The cover picture shows an MR scan of an Multiple Systems Atrophy patient with cerebellar and brainsystem involvement (Courtesy of the Editor).

Typesetting hagedorn kommunikation, Viernheim Printing betz-druck gmbh, Darmstadt Bookbinding Litges & Dopf Buchbinderei GmbH, Heppenheim ISBN

3-527-30816-4

Preface In the last decades, the scientific community as well as the public has been overwhelmed with a tremendous amount of data on the cellular, molecular and physiological mechanisms of neuronal proliferation, differentiation, maintenance and degeneration. A plethora of animal models of various neurological disorders has been introduced and used to delineate the mechanisms of cell death and highlight potential targets for neuroprotective or neurorestorative intervention strategies. With great enthusiasm, the complete deciphering of the human genome and the genetic information of various other species has been announced and raised the hope of a final victory over many fatal human diseases. Findings about the genetic or molecular causes of human disorders that were reproduced in animals and positive results from experimental therapies were generalized for human patients, and new experimental treatments were quickly transferred to clinics, sometimes speeded up by high expectations of the public as well as managers from pharmaceutical companies. However, by the same token, many clinical studies which were introduced on the basis of positive data with new pharmacological substances or effective procedures in animal models did not show positive results in humans, and several trials had to be stopped, due to adverse effects which led to a significant deterioration in some patients. From a multitude of substances tested in basic research models, only few have reached clinical testing in patients thus far. Among those substances are neurotrophic factors, mitochondria-stabilizing agents, anti-oxidants, anti-toxins, antibiotics, glutamate receptor and calcium channel blockers as well as caspase-inhibitors (Kermer and Bhr, 2002). However, none of them has so far led to a substantial breakthrough in neurological or psychiatric therapy. Thus, one has to ask why so many promising drugs with positive effects in basic research models failed in the clinic. This is not easy to understand, and there is certainly not a single answer. However, some general aspects can be mentioned which are discussed in more detail in the various chapters of this book: First of all, in animal models, the modes of application, the delay between onset of a pathology and therapy and many side effects are completely different to the human situation. Thus, a substance may show a profound effect after intraparenchymal or intraventricular application minutes after onset of ischemia in animals, but the systemic application hours after onset of a stroke in humans does Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

VI

Preface

not result in a timely and quantitatively sufficient concentration in the affected region or cell in the CNS. Secondly, as mentioned earlier, in standardized animal models, mostly young and otherwise healthy rodents are used. In contrast, patients with neurological disorders, e. g. stroke, are often old with a substantial co-morbidity, which may result in many cross-reactions of a new drug with existing medications and adverse effects that can not be anticipated in young and healthy animals. Finally, the time of onset, the course and the outcome of a given insult vary substantially in patients and, depending on the selection criteria for clinical studies, a potentially effective drug may be given to patients where there is nothing left to rescue at all, or the criteria which are used in animals to demonstrate efficacy are not available for patients (e. g. histological measure of cell survival rates or tissue preservations in animal models which can not be applied in human patients). As a consequence, the whole concept of developing or introducing new drugs on the basis of animal (model) data was challenged, and many big pharmaceutical companies stopped or significantly reduced their efforts to develop new “neuroprotective” substances in their basic research departments. Thus, it appears to be timely to present a comprehensive review on our current knowledge in the field to analyse the potential as well as the limitations of the available model systems for many highly relevant neurological disorders such as stroke, trauma, neurodegenerative disorders or inflammatory diseases of the CNS. Furthermore, it seems to be necessary to have a closer look at some general problems that evolve when data from animals, mostly young and healthy rodents, are to be translated into clinical settings in order to learn from the errors for our future planning of neuroprotective or neurorestorative therapies. Finally, new results from basic as well as applied research need to be presented and evaluated which may open completely new avenues for future therapy concepts. To that end, internationally recognized outstanding experts in their respective fields from several countries have contributed to this first edition of Neuroprotection – Models – Mechanisms – Therapies. We hope that this book provides a guideline for non-experts for a better understanding of the complexity of data that are generated in basic research experiments and the pitfalls of translational research on the one hand and a detailed overview and basis for discussions and future developments in the field for academics involved in basic research or applied neurosciences on the other. Gttingen, January 2004

Mathias Bhr

Acknowledements

The launch of a new book is a multifacetted task which can only be accomplished by team work. I would like to thank all the people who contributed to this book, especially my secretaries Mrs. Heidi Gottfried and Mrs. Barbara Nordmann as well as Dr. Andreas Sendtko from Wiley-VCH.

I

Neurological Disorders – Epidemiology, Clinical Overview, and Model Systems

1. 2. 3. 4. 5. 6. 7.

Stroke Parkinson’s Disease Amyotrophic Lateral Sclerosis Alzheimer’s Disease and Other Neurodegenerative Diseases CNS Inflammation Neurotrauma Spinal Cord Injury

II 8. 9. 10. 11. 12. 13. 14.

Apoptosis and Necrosis Inflammation Metabolic Dysfunctions Protein Misfolding Axonal Growth Inhibition Neurogenesis Excitotoxicity

III 15. 16. 17.

Cellular and Molecular Mechanisms

Therapies

Spinal Cord Trauma Neurodegeneration Perspectives for Neuroprotective Therapies in Basic Research and Clinical Application


Contents

==erst nach HK formatieren==

Preface V List of Contributors XVII I

Neurological Disorders – Epidemiology, Clinical Overview, and Model Systems

1

Stroke 1 Andreas Meisel, Konstantin Prass, Tilo Wolf, Ulrich Dirnagl Abstract 1 Introduction 1 The Penumbra Concept 4 Excitotoxicity 5 Oxygen Free Radicals 5 Tissue Acidosis 6 Peri-infarct Depolarizations 7 Inflammation 8 Damage to the Blood–Brain-Barrier 10 Programmed Cell Death and Apoptosis 11 Ischemia-induced DNA Damage, DNA Repair, and p53 as Genotoxic Sensor 13 Epigenetics 15 Gene Expression 16 Cell Replacement 17 Endogenous Neuroprotection – Ischemic Tolerance 19 Stroke Induced Immune Depression (SIDS) 20 Conclusion 21 References 22

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 1.13 1.14 1.15 1.16

2

2.1 2.1.1

Parkinson’s Disease 29 Marina Romero-Ramos, Matthew Maingay and Deniz Kirik Epidemiology of Parkinson’s Disease 30 Genetic Contribution 30


X

Contents

Environmental Factors 31 Oxidative Stress In Parkinson’s Disease 32 Role of Alpha-Synuclein in Parkinson’s Disease 33 Alpha-Synuclein in Lewy Bodies 33 Alpha-Synuclein and Oxidative Stress 34 The Involvement of Proteosome in PD 36 Parkin Involvement in PD 37 Other Genes Involved in Familial Parkinson’s Disease 39 Neurotoxic PD Models 39 Oxidative Stress 40 Dopamine Homeostasis/Complex I 40 Proteasome 41 Genetic Models of Parkinson’s Disease 41 Alpha-Synuclein Overexpressing Transgenic Animals 41 Parkin-related Genetic Models 42 Viral Vectors for In Vivo Gene Transfer of Disease-related Proteins to the CNS 43 2.7.3.1 Local Overexpression of alpha-synuclein 43 2.7.3.2 Local Overexpression of Parkin-related Proteins 45 Acknowledgements 45 References 45 2.1.2 2.2 2.3 2.3.1 2.3.2 2.4 2.4.1 2.5 2.6 2.6.1 2.6.2 2.6.3 2.7 2.7.1 2.7.2 2.7.3

3

3.1 3.1.1 3.1.2 3.2 3.2.1 3.2.2 3.2.3 3.3 3.3.1 3.2 3.3.3 3.4 3.4.1 3.4.2 3.5

4

4.1

Amyotrophic Lateral Sclerosis 49 Georg Haase Human Motor Neuron Diseases 49 Familial ALS Linked to SOD1 Mutations 50 Juvenile ALS Linked to Alsin Mutations 51 Cell Culture Models of Motoneuron Degeneration 51 Neuronal Cell Lines 51 Isolated Motoneuron Cultures 52 Organotypic Cultures 53 Animal Models of Motor Neuron Disease 53 Axotomy Models 54 Mutant SOD1 Mice 54 pmn Mice 59 Future Neuroprotective Approaches 60 Axonal Protection 60 Mutant SOD1 Knockdown by RNA Interference 61 Conclusion 61 References 61 Alzheimer’s Disease and Other Neurodegenerative Diseases 69 Philipp J. Kahle and Christian Haass Abstract 69 Introduction 70

Contents

4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.4 4.5

Transgenic Invertebrates 70 Transgenic Mice 71 Tyrosine Hydroxylase (TH) Promoter 71 Platelet-derived Growth Factor-b (PDGFb) Promoter 72 Thy1 Promoter 72 Prion Protein (PrP) Promoter 73 Glial Expression 74 Viral Models 75 Conclusion and Outlook 76 References 76

5

CNS Inflammation 83 Christine Stadelmann and Wolfgang Brck Introduction 83 The Pathologic Characteristics of the MS Plaque 84 Axonal Pathology in MS 85 Neuronal Pathology in Multiple Sclerosis 86 Lessons from Animal Models 87 References 88

5.1 5.2 5.3 5.4 5.5

6

6.1 6.2 6.2.1 6.2.2 6.2.2.1 6.2.2.2 6.2.3 6.3 6.3.1 6.3.1.1 6.3.2 6.3.2.1 6.3.2.2 6.3.2.3 6.3.3 6.3.3.1 6.3.3.2 6.3.3.3 6.4

Neurotrauma 93 Ibolja Cernak, Paul M. Lea IV, Alan I. Faden Introduction 93 In Vivo Models 94 Static Brain Injury Models 95 Dynamic Brain Injury Models 95 Indirect Dynamic Brain Injury 95 Direct Dynamic Brain Injury 96 Combined Neurotrauma Models 104 In Vitro Models 104 Tension Stress-induced Injury 107 Stretch Injury Models 107 Compression Injury Models 108 Weight-drop Model 109 Hydrostatic Pressure Models 109 Fluid-percussion Models 109 Shear Injury Models 110 Acceleration Models 110 Hydrodynamic Model 110 Primary Axotomy/Transection Models 111 Conclusion 112 References 113

XI

XII

Contents

7

7.1 7.2 7.2.1 7.2.1.1 7.2.1.2 7.2.1.3 7.2.1.4 7.2.2 7.2.2.1 7.2.2.2 7.2.2.3 7.3 7.4 7.4.1 7.4.2 7.5 7.6 7.7

Spinal Cord Injury 127 Poonam Verma and James W. Fawcett Acute Neuroprotection (preventing neuronal death) 129 Regeneration of Nerve Fiber Tracts (initiating growth following injury) 130 Extrinsic Mechanisms 130 The Glial Scar 131 Blockage of Glial Scar Formation 131 Modification of Growth-inhibitory Molecules 132 Selective Removal of Astrocytes 134 Intrinsic Neuronal Control of Regeneration 134 Neuronal Age 135 Site of Transection 135 Neuronal Variability 136 Bridging Cysts and Scars and Cellular Replacement 136 Replacing Lost Neurons 137 Neural Transplantation 137 Replacing Lost Neurons from Stem Cells 138 Treating Demyelination 138 Enhancing Plasticity 139 Future Expectations 140 References 141

II

Cellular and Molecular Mechanisms

8

Apoptosis and Necrosis 149 Laura Korhonen and Dan Lindholm Introduction 149 Apoptosis versus Necrosis 150 Genetics of Apoptosis and the Proteins Involved 151 Caenorhabditis Elegans as a Model System 151 Caspases 153 Anti-apoptotic Proteins 155 Cellular Mechanisms of Apoptosis 160 The Role of Mitochondria and the Apoptosome Complex 160 Cell Death in Neurodegenerative Diseases 162 References 163

8.1 8.2 8.3 8.3.1 8.3.2 8.3.3 8.4 8.4.1 8.4.2

9

9.1 9.2 9.3 9.4 9.5

Inflammation 169 Harald Neumann Introduction 169 Innate Immune Responses in the CNS 171 Dual Mechanism of Innate Immunity in the CNS 172 Antigen Presentation for Adaptive Immune Responses in the CNS 173 Regulation of Adaptive Immune Responses in the CNS 174

Contents

9.6 9.7

Immune Surveillance of the CNS by T Lymphocytes 176 Effector Mechanisms of Lymphocytes 177 Acknowledgement 180 References 183

10

Metabolic Dysfunctions 187 Konstantin-A. Hossmann Introduction 187 Disturbances of the Energy Metabolism 188 Energy Production and Turnover 188 Reduction of Oxygen Supply 188 Disturbances of Flow-coupling 190 Mitochondrial Dysfunction 191 Importance of Disturbed Energy Metabolism for Injury Evolution 192 Global Brain Ischemia 192 Focal Brain Ischemia 194 Epileptic Seizures 196 Head Trauma 197 Disturbances of Protein Synthesis 198 Mechanisms of Disturbed Protein Synthesis 198 Importance of Disturbed Protein Synthesis on Injury Evolution 200 Global Brain Ischemia 200 Focal Ischemia 201 Epileptic Seizures 203 Head Trauma 203 Therapeutical Implications 204 References 206

10.1 10.2 10.2.1 10.2.2 10.3 10.4 10.5 10.5.1 10.5.2 10.5.3 10.5.4 10.6 10.6.1 10.7 10.7.1 10.7.2 10.7.3 10.7.4 10.8

11

11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8

12

12.1 12.2

Protein Misfolding 209 Milene Russelakis-Carneiro, Claudio Hetz, Joaquin Castilla and Claudio Soto Abstract 209 Introduction 209 Disease Propagation by Replication of Prion Protein Misfolding 210 Prion Biology 211 Prion Pathogenesis 214 Neuronal Targeting 215 Neuronal Apoptosis in Prion Diseases 215 ER-Stress and Apoptosis in Prion Diseases 216 Concluding Remarks 219 References 220 Axonal Growth Inhibition 225 Anne D. Zurn and Christine E. Bandtlow Introduction 225 Intrinsic Properties of CNS Neurons 225

XIII

XIV

Contents

12.2.1 Survival of Injured Neurons 225 12.2.2 Developmental Loss oOf Regenerative Ability 226 12.2.3 Intrinsic Differences in Growth Potential 227 12.2.4 Transient Expression of Growth-associated Molecules 227 12.2.5 Conditioning Lesion 228 12.3 Extrinsic Factors: The CNS as A Non-conducive Growth Environment 229 12.3.1 Cellular Components of Inhibition 229 12.3.1.1 The Glial Scar 229 12.3.1.2 Oligodendrocytes and Myelin (Wallerian Degeneration) 230 12.3.1.3 Secondary Injury: Inflammation, Cell-mediated Immunity, and Glial Cell Loss 231 12.3.1.4 Genetic Influences on Cellular Reactions to CNS Injury 232 12.3.2 Molecular Components of Inhibition 232 12.3.2.1 Axon growth Inhibitors in the Glial Scar 232 12.3.2.2 Myelin-associated Growth Inhibitors 235 12.3.3 Transduction of Axon Growth-inhibitory Signals 238 12.3.3.1 Receptors for Myelin-associated Inhibitors 238 12.3.3.2 Signaling Pathways of Myelin-associated Inhibitors 239 12.4 Experimental Strategies to Overcome Growth Inhibition 241 12.5 Conclusion 243 References 244 13

13.1 13.2 13.2.1 13.2.2 13.3 13.4 13.4.1 13.4.2 13.4.3 13.5 13.5.1 13.5.2 13.5.3 13.5.4 13.5.5 13.5.6 13.6 13.7

Neurogenesis 257 Sebastian Jessberger and Gerd Kempermann Introduction 257 Neural Stem- and Precursor Cells in the Adult Brain 259 Neural Stem Cells in the Adult Brain: In vitro 259 Neural Stem Cells In The Adult Brain: In vivo 260 Maturation and Migration of Adult-generated Neurons in the Adult Brain 262 Neurogenesis in the Adult Brain – Age Dependency, Persistence and Genetic Determinants 265 Neurogenesis in Aged Animals 265 Long-term Persistence of Adult-generated Neurons in the SVZ and Hippocampus 265 Genetic Influence on Adult Hippocampal Neurogenesis 266 Regulation of Adult Neurogenesis 266 Learning 267 Environmental Enrichment 267 Physical Activity 268 Stress and Depression 269 Epilepsy 270 Ischemia and Cortical Trauma 271 Possible Functional Relevance of Neurogenesis in the Adult Brain 272 Therapeutic Strategies for Neural Cell Replacement 274

Contents

13.7.1 Endogenous Neurogenesis 274 13.7.2 Transplantation of Stem- or Precursor Cells into the Damaged Brain 274 Acknowledgements 276 References 276 14

Excitotoxicity 287 Stuart A. Lipton, MD, PhD Abstract 287 14.1 Introduction 288 14.2 The Search for Clinically-Tolerated NMDA Receptor Antagonists 289 14.3 Excitotoxicity 290 14.3.1 Definition and Clinical Relevance 290 14.3.2 Links between Stroke and Vascular Dementia to Excitotoxic Damage 291 14.3.2.1 Possible Links between Excitotoxic Damage and Alzheimer’s Disease 292 14.3.3 Pathophysiology of Excitotoxicity: Role of the NMDA Receptor 292 14.3.3.1 The Importance of Off-Rate from Channel Block 295 14.4 Memantine 296 14.4.1 Background and Pharmacology: Uncompetitive Open-channel Block 296 14.4.2 Voltage Dependence, Partial Trapping, and Other Possible Effects of Memantine 299 14.4.3 Neuroprotective Efficacy 300 14.5 NitroMemantines 302 14.6 Summary 303 Acknowledgments 304 Appendix 304 References 305 III

Therapies

15

Spinal Cord Trauma 311 V. Dietz Abstract 311 Epidemiology 312 Clinical Aspects 312 Acute Stage 312 Dysfunction of the Autonomic Nervous System 313 The Evolution of Signs and Symptoms 314 Principles of Therapy 315 General Concepts 315 Transition from Acute Care to Rehabilitation 316 Basic Aspects of Motor Rehabilitation: Neuronal Activity of the Isolated Spinal Cord 316 Current Approaches in Motor Rehabilitation 317 Spinal Reflex Plasticity 318 Use-Dependent Plasticity – Rehabilitative Approaches 318

15.1 15.2 15.2.1 15.2.2 15.2.3 15.3 15.3.1 15.3.2 15.3.3 15.4 15.4.1 15.4.2

XV

XVI

Contents

15.4.3 15.4.4 15.5 15.5.1 15.5.2 15.6

Task-specific Plasticity 319 Effects of Locomotor Training in Spinal Cord Injury 319 Search for Reliable Clinical Assessment 321 Physical Signs and Function 321 Appropriate Assessment of Function – Basis for New Interventional Therapies 322 Conclusion 324 References 325

16

Neurodegeneration 329 Jrg B. Schulz, MD What Is Neuroprotection? 329 16.1 16.2 Outcome Measures in Clinical Studies 330 16.3 Specific Diseases 331 16.3.1 Wilson’s Disease 331 16.3.2 Parkinson’s Disease 332 16.3.2.1 DATATOP Study 332 16.3.2.2 Dopamine Agonists 332 16.3.2.3 Anti-Excitotoxic Treatment 333 16.3.2.4 Neurotrophins 333 16.3.2.5 MAP Kinase Inhibitors 334 16.3.2.6 Transplantation 334 16.3.2.7 Deep Brain Stimulation 335 16.3.2.8 Coenzyme Q10 335 16.3.3 Multiple System Atrophy and Progressive Supranuclear Palsy 335 16.3.4 Alzheimer’s Disease 336 16.3.4.1 Symptomatic Treatment 336 16.3.4.2 Antioxidants 337 16.3.4.3 Anti-inflammatory Approaches 337 16.3.4.4 Estrogen Replacement 337 16.3.4.5 NMDA Receptor Antagonists 338 16.3.4.6 Experimental Therapies 338 16.3.4.7 Immunization 338 16.3.4.8 Cholesterol-lowering Therapy 340 16.3.5 Huntington’s Disease 341 16.4 Why Do So Many Clinical Studies Fail? What Do We Need? 342 References 343 17

Perspectives for Neuroprotective Therapies in Basic Research and Clinical Application 351 Mathias Bhr and Pawel Kermer References 354 Index 361

List of Contributors Mathias Bhr University of Gttingen Department of Neurology Robert-Koch-Str. 40 D-37075 Gttingen Germany

Ibolja Cernak Department of Neuroscience Georgetown University Medical Center Research Bld, Room EP04 Washington, D.C. 20007 USA

Christine E. Bandtlow Dept. of Neurobiochemistry Institute of Medical Chemistry and Biochemistry Fritz-Pregal-Str. 3 University of Innsbruck A-6020 Innsbruck Austria

Volkmar Dietz Spinal Cord Injury Center University Hospital Balgrist Forchstr. 340 CH-8008 Zrich Switzerland

Wolfgang Brck Institute for Neuropathology Georg-August-University Gttingen Robert-Koch-Str. 40 D-37075 Gttingen Germany Joaquin Castilla University of Texas Medical Branch 301 University Boulevard Galveston, TX 77555 USA

Ulrich Dirnagl Department of Neurology Humboldt-University Berlin Schumannstr. 20–21 D-10098 Berlin Germany Alan I. Faden Department of Neuroscience Georgetown University Medical Center Research Bld, Room EP04 Washington, D.C. 20007 USA James W. Fawcett Brain Repair Centre Cambridge University Cambridge CB2 2PY United Kingdom


XVIII

List of Contributors

Georg Haase Institut de Neurobiologie de la Mediterrane Campus de Luminy F-13288 Marseille Cedex 09 France Christian Haass Laboratory of Alzheimer’s and Parkinson’s Disease Research Department of Biochemistry Ludwig Maximilians University of Munich Schillerstr. 44 D-80336 Munich Germany Claudio Hetz University of Texas Medical Branch 301 University Boulevard Galveston, TX 77555 USA and Instituto de Ciencias Biomdicas Universidad de Chile Santiago, Chile Konstantin-A. Hossmann Max-Planck-Institut fr neurologische Forschung Gleueler Str. 50 D-50931 Kln Germany Sebastian Jessberger Formerly MDC Berlin-Buch, now The Salk Institute for Biological Studies 10010 North Torrey Pines Rd. La Jolla, CA 92037 USA

Philipp J. Kahle Laboratory of Alzheimer’s and Parkinson’s Disease Research Department of Biochemistry Ludwig Maximilians University of Munich Schillerstr. 44 D-80336 Munich Germany Gerd Kempermann Research Group „Neuronal Stem Cells“ Max Delbrck Centre for Molecular Medicine (MDC) Berlin-Buch Robert-Rssle-Str. 10 D-13125 Berlin Germany Deniz Kirik MD, PhD Assistant Professor of Neurobiology Wallenberg Neuroscience Center Lund University BMC A11 S-22184 Lund Sweden Laura Korhonen McLean Hospital Harvard Medical School 115 Mill Street Belmont Massachusetts 02478 USA Paul M. Lea IV Department of Neuroscience Georgetown University Medical Center Research Bld, Room EP04 Washington, D.C. 20007 USA


Dan Lindholm Department of Neuroscience, Neurobiology Uppsala University Husargatan 3 Biomedical Centre, Box 587 S-75123 Uppsala Sweden

Marina Romero-Ramos Wallenberg Neuroscience Center Lund University BMC A11 S-22184 Lund Sweden

Milene Russelakis-Carneiro Apoxis Inc. 18–20 Avenue de Svelin Stuart A. Lipton CH-1004 Lausanne The Burnham Institute The Salk institute for Biological Studies Switzerland The Scripps Research Institute, Jrg B. Schulz and the University of California Department of General Neurology San Diego, La Jolla, California 92037 Center of Neurology and Hertie Institute USA for Clinical Brain Research University of Tbingen Matthew Maingay Hoppe-Seyler-Str. 3 Wallenberg Neuroscience Center D-72076 Tbingen Lund University Germany BMC A11 S-22184 Lund Claudio Soto Sweden Director Protein Misfolding Disorders Lab Andreas Meisel University of Texas Medical Branch Department of Neurology 301 University Boulevard Humboldt-University Berlin Galveston, TX 77555 Schumannstr 20–21 USA D-10098 Berlin Germany Christine Stadelmann Institute for Neuropathology Harald Neumann Georg-August-University Gttingen Head of the Neuroimmunology Unit Robert-Koch-Str. 40 European Neuroscience Institute D-37075 Gttingen Gttingen Germany Waldweg 33 D-37073 Gttingen Poonam Verma Germany Brain Repair Centre Cambridge University Konstantin Prass Cambridge CB2 2PY Department of Neurology United Kingdom Humboldt-University Berlin Schumannstr. 20–21 D-10098 Berlin Germany

XIX

XX


Tilo Wolf Department of Neurology Humboldt-University Berlin Schumannstr. 20–21 D-10098 Berlin Germany

Anne D. Zurn Department of Experimental Surgery Lausanne University Hospital CH-1011 Lausanne Switzerland

I Neurological Disorders – Epidemiology, Clinical Overview, and Model Systems


1 Stroke Andreas Meisel, Konstantin Prass, Tilo Wolf, Ulrich Dirnagl

Abstract

The current understanding of stroke pathophysiology is basically founded on experimental models. Among these, in vitro models with primary cultures of cerebral cells permit stroke pathophysiology to be examined on a molecular level, while animal models (mainly mouse and rat) are used to evaluate medical intervention. There is a highly complex sequence of events leading to the eventual ischemic cerebral damage that follows a well-defined spatio-temporal pattern. Overwhelming excitotoxicity leads to early necrotic cell death in what is to become the core of the infarction, while the tissue damage in the surrounding zone called penumbra happens on a longer time scale. The excitotoxic or inflammatory mechanisms are milder, bearing the biochemical hallmarks of apoptosis. The brain cells, challenged by such a large-scale assault, activate endogenous protective programs. These have been studied by experimentally inducing ischemic tolerance (i. e. ischemic preconditioning). Importantly, cerebral ischemia not only affects the brain, but also impacts other systems. For example, stroke induces dramatic immunodepression through over-activation of the sympathetic nervous system. As a result, severe bacterial infections such as pneumonia occur. The complex signaling cascades not only decide over cell survival in the brain and the neurological deficit, but also over mortality after stroke from extracerebral complications. Their ability to govern not only the maturation of the eventual infarction but also the immune system make them a promising target for intervention and the development of neuroprotective drugs.

1.1

Introduction

In the US more than 600,000 people per year suffer from strokes, and among acutely hospitalized neurological patients stroke patients make up the largest share – about 50 %. Currently, there are about 4 million stroke survivors [1] in Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

4

1 Stroke

the US. Mortality from stroke is an estimated 25 %, which makes it the third major cause of death in industrialized countries. Due to the high rate of severe permanent disability, stroke is a burden not only for the affected patients and their families, but also for national economies: In the US, it is estimated that the annual costs caused by strokes ranges between 30 and 40 billon dollars [2, 3]. In the UK, the cost of one individual patient amounts to £ 30,000 over 5 years [4]. The term stroke accommodates a variety of different conditions. About 85 % of all strokes are caused by cerebral ischemia due to vessel occlusion. Primary cerebral bleeding is rare in comparison –15 %. Of the ischemic strokes, 75 % are caused by emboli, of either arterial or cardial origin, while microvascular occlusion, i. e. hyalinosis or in-situ thrombosis is responsible for 20 % of cases. Hemodynamic ischemia, caused by stenoses of brain-supplying arteries, account for less than 5 % of ischemic strokes [5, 6]. The prospective risk of suffering an ischemic stroke is partially a function of social and behavioral (nutrition, tobacco use, stress) factors and longstanding disorders like hypertension, diabetes, disorders of cholesterol and lipid metabolism, and obesity. Atherosclerosis is not only the main underlying condition in ischemic stroke, but also in coronary heart disease and peripheral vascular disease. Moreover, the genetic background of affected individuals is coming under increasing scientific scrutiny, as might be expected for familial stroke conditions like CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucencephalopathy), which is characterized by relapsing subcortical ischemia. The condition is caused by a mutation in the notch3-gene on chromosome 19 [7, 8]. MELAS (mitochondrial encephalopathy, lactate acidosis, stroke-like episodes) is another rare familial stroke disorder, characterized by migraine, grand-mal seizures, and recurrent cortical infarctions. The condition is caused by a range of mutations in the mitochondrial genome, which explains its maternal inheritance. The severity of the phenotype depends mainly on the mutation locus and on the proportion of mutated mitochondrial genomes in the cerebral mosaic (for review see [9]). However, family and twin studies suggest that genetic factors are likely to be involved in common types of ischemic stroke, not just in rare and well defined familial stroke disorders [10–13]. A locus on chromosome 5q12 described for the Icelandic population seemed to increase stroke susceptibility significantly [14]. The responsible gene has been identified as phosphodiesterase 4D (PDE4D). The risk-conveying polymorphisms are located in the gene-regulatory part. This hints at a defect in the regulation of the encoded cAMP degrading protein [15]. Phosphodiesterase 4D belongs to a group of proteins that are drug targets in the treatment of asthma, erectile dysfunction and inflammation. Symptoms of focal cerebral ischemia depend on the individual affected vessel with its typical supply territory. Table 1 summarizes the frequency of affection [16] and the typical clinical syndromes for the main brain arteries. Stroke mortality is rated between 20 and 30 %. Unfavorable prognostic factors are old age, initial coma, papillary asymmetry, coronary heart disease and heart failure. Pyrexia and infections (pneumonia in particular) deteriorate the prognosis particularly during

1.1 Introduction

the initial phase (see below). Of the surviving patients, one third improves within a week, 40 % remain unchanged in their disability, and 20 % deteriorate further during the first week [17].

Table 1.1

Territorial stroke syndromes.

Anterior territory MCA

(z60 %)

ACA AchA

(4 %) (8 %)

Posterior territory VA/BA (10 %) Cerebellum (PICA/ AICA) PCA

hemiparesis mainly arm, hemihypaesthesia, hemianopia, dysphasia or neglect hemiparesis mainly leg, urinary incontinence, apraxia hemiparesis, hemihypaesthesia, hemianopia

(7 %)

vertigo, diplopia, bilateral hypaesthesia and paresis, crossed syndromes, amaurosis, ataxia, headache, coma headache, ataxia, vertigo, gaze palsy, facial weakness, deafness

(9 %)

hemianopia, dyslexia, visual agnosia

A great deal of our knowledge about the pathophysiology of stroke comes from experimental research. The experimental animal models follow two main paradigms: One is the model of focal cerebral ischemia as a model of ischemic stroke. The other is the model of global cerebral ischemia as model of circulatory arrest. For obvious reasons, we will focus mainly at studies of focal cerebral ischemia. The bulk of experimental studies has been carried out with rats and mice, although some primates have been used. In rodents, ischemia is mostly induced by intravascular occlusion of the MCA, using a monofilament just exceeding the critical vessel diameter. Depending on the duration of occlusion, we distinguish permanent from transient ischemia models. The latter are also used as models for spontaneous reperfusion or for the state after successful lysis therapy with recombinant tissue plasminogen activator (rtPA) in humans, respectively. In models of cerebral ischemia perfusion to the brain is impeded by some form of manipulation. Since the essence of ischemic stroke is vascular occlusion, it is not surprising that in these models the extent of the eventual infarction can be predicted quite accurately from the degree of reduction in regional cerebral blood (rCBF) flow. Thus, if the rCBF is reduced to less than 25 % of normal the likelihood of infarction in a given volume of brain tissue greater than 95 %. In contrast, the likelihood of infarction is less than 5 % if rCBF does not fall below 50 % of normal. These thresholds have been established in comparative human PET and MRI studies [18] and correspond with experimental data from animal models [19]. Thus, the initial reduced rCBF determines the extent of the anticipated infarction. This, however, holds only with the provision that a spontaneous or therapeutic reperfusion does not take place. We now look at ischemic infarction as the result of a complex and prolonged process of infarct maturation rather than a simple result of reduced regional perfu-

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sion. As brain tissue has a high demand for oxygen and glucose, a disruption of perfusion leads to substrate depletion within few minutes, while toxic metabolites accumulate. The ensuing cellular energy deficit leads to a collapse of the established ion gradients and the membrane potential. Neurons and glial cells depolarize. Depending on the extent and the duration of this energy deficiency, the cells will suffer not only a functional but also a structural breakdown. The highly complex sequence of events within the ischemic area follows a fairly well defined stereotypic spatio-temporal pattern, which we will discuss below in more detail. The concept of the ischemic penumbra is crucial to the understanding of these mechanisms. The cascade of ischemic damage begins with excitotoxicity, the formation of reactive oxygen free radicals, the increasing tissue acidosis and the occurrence of periinfarct-depolarizations. It is followed by the stages of inflammation and programmed cell death (apoptosis). This is associated with DNA damage that, in turn, induces DNA repair programs. Although the process is not yet fully understood, we know that chromatin re-modeling, i. e. epigenetic mechanisms, and the activation of transcription factors induce complex gene programs. These changes initiate the expression of destructive proteins involved in inflammation and apoptosis, as well as a host of protective genes that help repair the ischemic damage. It is the activation of these protective genes that builds up ischemic tolerance. Among the many newly discovered protective mechanisms, endogenous and exogenous cell replacement have met with a lot of interest. Apart from these autochthonous mechanisms of the brain tissue, there are other mechanisms on a systemic scale, which bear important clinical significance. For instance, the phenomenon of stroke-induced immunodepression can help to understand why stroke patients are at such high risk of contracting serious bacterial infections. Neuroprotective treatment must be based on an understanding of these mechanisms.

1.2

The Penumbra Concept

In the ischemic brain, we commonly distinguish two tissue volumes – the core of the infarction and the surrounding zone, known as ischemic penumbra [20] – the underperfused and metabolically compromised margin surrounding the irrevocably damaged core. Core and penumbra are characterized by two different kinds of cell death: necrosis and apoptosis (which is also called programmed cell death or delayed neuronal cell death). The severe perfusion deficit in the core causes a breakdown of metabolic processes, cellular energy supply and ion homeostasis, which causes the cells to lose their integrity within minutes. Thus, acute necrosis of cell and tissue prevails in the core. In the penumbra, some residual perfusion is maintained by collateral vessels, which may be unable to maintain the full functional metabolism, but prevents immediate structural disintegration. However, over time, the alteration of cellular homeostasis causes more and more cells to die, and the volume of the infarction increases. The penumbra has thus to be considered as tissue at risk during the maturation of the infarct. In this region,

1.4 Oxygen Free Radicals

apoptosis and inflammatory signaling cascades play an important role. It may initially constitute 50 % of the volume that will end up as infarction. The mechanisms that lead to delayed cell death within the penumbra are the subject of intense research, as they provide targets for a specific neuroprotective therapy in brain regions challenged by ischemia, but which are still viable (for a review see [19, 21]).

1.3

Excitotoxicity

The depolarization of neurons and glia due to local energy deficit causes the activation of voltage-gated calcium channels and the release of excitatory amino acids into the extracellular space. Glutamate in particular, which, under normal cellular energy conditions, would be immediately taken up pre-synaptically or through astrocytes, now remains in the extracellular space where it accumulates dramatically. Through the activation of glutamate receptors NMDA and AMPA, the intracellular Ca2þ level rises. Furthermore, metabotropic glutamate receptors are activated through the induction of phospholipase C (PLC) and inositol triphosphate (IP3), and calcium is mobilized from intracellular stores. Furthermore, the over-activation of AMPA receptors causes a rise of sodium and chloride concentrations. Altogether, the result is a massive disturbance of ion homeostasis, accompanied by passive water influx and cell edema. Ultimately, these massive changes in cell volume account for osmotic cell lysis. This lytic type of cell death, also referred to as necrosis, is primarily observed in the core of the infarction. Cells that escape this most dramatic form of disintegration, as they can not found in the core but in the penumbra where excitotoxicity may be an initiator of molecular events that lead to apoptosis and inflammation (for an overview see [21, 22]).

1.4

Oxygen Free Radicals

As a consequence of ischemia and particularly of reperfusion, reactive oxygen free radicals such as superoxide, hydrogen peroxide and hydroxyl radicals are generated. Nitric oxide is generated via the activation of the calcium-calmodulin-dependent nitric oxide synthase (NOS); it reacts with superoxide radicals and forms thus the highly reactive peroxy-nitrite radical. Further sources of oxygen free radicals in the damaged brain tissue are the breakdown products of the adenosine phosphates, which contribute to radical production via xanthine oxidase and the iron-catalyzed Haber-Weiss reaction. The many different radical species that are thus formed can react with virtually any cellular components (carbohydrates, amino acids, DNA, phospholipids) and damage them. The peroxidation of membrane lipids releases further radicals – and further glutamate. Oxygen free radicals gain even more

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significance when new oxygen reaches the damaged tissue by virtue of reperfusion, or in the penumbra where oxygen supply has not ceased entirely (overview in [21, 22]). Hypoxia itself as well as the elevated intracellular concentration of calcium ions and free radicals disrupt the function of neuronal mitochondria. Consequently, a so-called mitochondrial permeability transition pore (MPT) in the mitochondrial membrane may form. Besides impeding ATP production through loss of mitochondrial potential, the MPT leads to mitochondrial swelling, a burst of free oxygen radicals, and the release of pro-apoptotic molecules. Thus a vicious cycle of further disintegration is fuelled (see below, for review [21, 23]). This vicious cycle is counterbalanced in part by anti-oxidative enzymes like the manganese-superoxide dismutase (Mn-SOD) and the cytosolic forms of the copper-zinc superoxide dismutase (CuZn-SOD). These may prevent the breakdown of the mitochondrial membrane and, thus, the release of cytochrome C, which would be the trigger of apoptosis (see below and for review [24]).

1.5

Tissue Acidosis

In the context of stroke pathophysiology, the proton balance is intimately linked with the glucose metabolism. With reduced oxygen availability, anaerobic glycolysis as only remaining source of ATP production leads to tissue acidosis. It has long been assumed that this acidosis was one of the main noxious mechanisms in ischemic stroke. This so-called ‘lactate-acidosis-hypothesis’ is often quoted as explanation for the “glucose paradox” of cerebral ischemia. This paradox refers to the observation that excessive supply of glucose, the most important source of energy of the brain, during focal cerebral ischemia does not reduce tissue damage as one should think but, instead, augments it [25]. However, by which mechanism this happens and, in fact, whether levels of acidosis reached in brain ischemia can generate brain tissue damage at all is still far from being clear. Possibly, the pH dependent transition of Fe(III) to Fe(II) and the release of iron from molecular storages lead to a facilitation of the Haber-Weiss reaction that forms toxic free oxygen radical species (see above). Besides the production of different species of oxygen free radicals, acidosis also interferes with intracellular protein synthesis. However, the lactate-acidosis-hypothesis is not unchallenged. Particularly the fact that acidosis blocks the NMDA-receptor and thus has an anti-excitotoxic effect indicates the complexity of the role that acidosis plays in cerebral ischemia (for a review [21]). A similarly hotly debated topic are the findings on hyperglycemia during stroke. Experimental data from animal models show a detrimental effect of hyperglycemia during focal cerebral ischemia [26–28]. Clinical data also suggest that hyperglycemia during the acute phase of stroke worsens prognosis [29–31]. Persisting hyperglycemia beyond the acute phase is also an independent prognostic factor for larger infarct volume and poorer functional outcome in stroke patients [32]. It is a matter of debate whether these observations are due to a causal relationship or if hypergly-

1.6 Peri-infarct Depolarizations

cemia is a mere epiphenomenon, possibly due to a stress reaction (i. e. an effect of concomitant catecholamine and glucocorticoid release proportional to initial tissue damage) [34, 34]. The controversial role of an associated lactate-acidosis has already been discussed. An alternative concept is the glucocorticoid hypothesis [35]. It suggests that hyperglycemia enhances the release of glucocorticoids, which are also released during ischemia in the sense of a stress reaction. Glucocorticoids have been shown to have a direct cytotoxic effect [36] and the blockade of glucocorticoid receptors does reduce the damaging effect of hyperglycemia [35]. On the other hand hyperglycemia and acidosis have a protective effect in vitro [37, 38], and some experimental studies demonstrate that hyperglycemia can also have a beneficial effect under special circumstances [39, 40]. However, a recent study on humans seems to shed light on this controversy. Perfusion and diffusion MR imaging and MR spectroscopy shows that stroke patients with hyperglycemia have a higher lactate accumulation in the penumbra. While the perfusion-diffusion-mismatch (as a measure of the size relation of penumbra and core) was similar in the normoglycemic and hyperglycemic groups, the eventual infarct size and disability scores were significantly higher in the hyperglycemic group [41]. While it could still be argued that initially hyperglycemic patients were also likely to be longstanding or latent diabetics with an overall worse microvascular state, we take this study- which links in with much of the experimental animal data – as strong indicator for the vulnerability of the penumbra to hyperglycemia. Indirectly, it also corroborates the lactacidosis theory, which, however, still lacks direct proof.

1.6

Peri-infarct Depolarizations

The anoxic as well as excitotoxic depolarization of neurons and glia increases the concentration of glutamate and potassium in the ischemic core. These diffuse into the penumbral zone where they induce the electrochemical depolarization of more neuronal and glial cells that are “just managing” under the circumstances of oligemia. Depolarization in this context is not to be confused with short-lived action potentials, but means sustained depolarization of the entire cell membrane, a breakdown of the membrane potential that has to be actively rectified. Depolarization also causes a further significant increase in extracellular glutamate and potassium. Thus, waves of sustained electrochemical depolarization spread from the core of infarction into the penumbra, bearing similarity with Leao’s spreading depression. These waves are called peri-infarct depolarizations [42–44]. Peri-infarct depolarizations occur with a frequency of 1–4 per hour and have been detected by functional MRI [45] and near infrared spectroscopy [46]. Since active rectification of the membrane potential requires energy, these peri-infarct depolarizations contribute further to the metabolic compromise and each wave turns a larger area of the penumbra towards irrevocable infarction [43, 44, 47, 48]. In addition, periinfarct depolarizations seem to compromise the impaired microcirculation within

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the penumbra even further [49]. Peri-infarct depolarizations have been detected in patients with head trauma [50].

1.7

Inflammation

The early stage of inflammation, which starts a few hours after the onset of ischemia, is characterized by the expression of adhesion molecules in the vascular endothelium as well as on circulating leukocytes. Thus, leukocytes adhere to the endothelium and transmigrate from the blood into the brain parenchyma, which is of decisive importance for stroke induced brain inflammation (for review see [51–53]). Newly expressed endothelial adhesion molecules like ICAM-1 and VCAM-1 facilitate the interaction between endothelium and leucocytes as first step of transmigration of white blood cells from blood into the tissue. They interact with the b2 -integrins CD11b/CD18 (Mac-1) and CD11a/CD18 (LFA-1) that are, in turn, expressed on leucocytes [54–56]. Neutrophil granulocytes accumulate in the capillaries of the penumbra and interfere with the already impaired microcirculation [57]. Blocking this interaction with CD18, CD11, or ICAM-1 antibodies reduces not only the number of leucocytes but also the size of infarction [58–60]. ICAM-1 knockouts have also smaller infarcts than wild type mice [61]. Next to the b2 -integrins, other integrins that are more specific for lymphocytes like VLA-4(a4 b1) come into action, too. Consequently, blocking the a4 -component reduces also the volume of infarction [62]. A major part in inflammation is ascribed to the population of microglial cells. These cells are the primary immunoeffectors of the CNS and make up about 20 % of the entire glial mass, about the same proportion as neurons. Activation of microglia is a striking feature in the penumbra. The cells change their shape from highly ramified cells to a more plump and amoeba like form, and they proliferate. They also express a number of characteristic surface molecules (for instance MHC I and MHC II) and are capable of antigen presentation [63–66]. Similar to leukocytes, activated microglia cells are able to produce a variety of pro-inflammatory cytokines (but also cytotoxic metabolites (especially oxygen free radicals such as peroxy-nitrite and superoxide) and enzymes (for instance cathepsins). In addition, microglia is phagocytically active. The inhibition of microglial activation turns out to be protective in experimental stroke models [67]. On the other hand, activated microglia have also been shown to release anti-inflammatory cytokines (such as TGF-b1) and growth factors [68]. Because of the Janus-faced nature of microglial products (destructive, e. g. free radicals, vs. protective, e. g. growth factors) the overall role of microglia in cerebral ischemia is not clear at present. It is very likely that microglia play different roles at different times, with protective or regenerative activities occurring days or even weeks after the onset of ischemia. Activated leukocytes (granulocytes, monocytes/macrophages, lymphocytes) as well as neurons and glial cells (astrocytes, microglia) produce cytokines and che-

1.7 Inflammation

mokines [69, 70]. In particular, pro-inflammatory cytokines like TNFa, IL-1 and IL-6 play a major role as mediators of the inflammatory response. They are responsible for the transition from the early, excitotoxic phase to the inflammation phase (for a review: [51–53, 71]. Their regulation depends on transcription factors like NFkB, which, in turn, are activated by oxygen free radicals [72, 73]. CytokinemRNA and also the protein of TNFa and IL-1 can be detected only few hours after induction of an experimental focal ischemia [71]. Expression of IL-6 follows only after about 24 hours [74]. These cytokines are mainly released by microglial cells and macrophages [69, 70]. In stroke patients, it could be shown that the intrathecal concentration of IL-1 and IL-6 correlated with the infarct size [75]. Cytokine receptor antagonists reduce the infarct volume in animal models. For example, blockade of TNFa by TNF-binding proteins reduces brain injury after focal cerebral ischemia [76]. The role of TNFa is not entirely clear, however, as mice lacking the TNFa-receptor (TNFR2) have larger infarctions [77]. In part, these contrasting results may reflect a difference in signal transduction cascades activated by the two TNFa-receptors TNFR1 and TNFR2 [78]. It is also arguable that an increased IL-6 induction may be responsible, since IL-6 knockouts do not have smaller infarctions than their litter mates [79]. Further to the induction of adhesion molecules, the above-mentioned cytokines also render the blood brain barrier more permeable and induce pro-thrombotic functions of the endothelium (for a review [80]). Not only can the pro-inflammatory cytokines be observed, but anti-inflammatory cytokines such as TGF-1b or IL-10 also induced. By down-regulating the inflammation, these cytokines have a protective effect in the context of cerebral ischemia [81–84]. The neuroprotective action of TGF-b1 is well known (for review see [85]). Interestingly, this cytokine seems to play an important part in immunological tolerance. As in ischemic tolerance (see below), it is possible to induce a tolerant state towards ischemia. This protection is induced by immunogenetic proteins (for instance myelin basic protein, MBP) and mediated by a certain lymphocyte population expressing TGF-b1 [86–88]. In contrast to ischemic tolerance, immunological tolerance is longer lasting. Protection lasts for months, whereas in ischemic tolerance it lasts no longer than 7 days. Apart from cytokines, there are two other proteins that dominate the inflammatory phase: the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase 2 (COX-2). Focal cerebral ischemia models have shown that iNOS is induced on the mRNA as well as on the protein level [89]. Protein expression reaches its height after 24 hrs. By inhibiting the expression of iNOS the size of experimental infarcts can be reduced by about 30 %, even if treatment is undertaken only 24 hours after the onset of ischemia [90, 91]. Nitric oxide (NO), which is produced in much greater amounts by iNOS than by the constitutional NO synthases, acts via the formation of peroxynitrite highly cytotoxic [92]. NO triggers apoptosis not only through peroxynitrite but also via the induction of p53, which probably causes direct DNA damage [93]. COX-2 is mainly generated in the penumbra [94]. The enzyme has a destructive effect on the penumbral tissue, mainly through producing oxygen free radicals and toxic prostanoids (for review see [51, 53]). This seems a likely hypothesis, as both genetic and pharmacological inhibition of COX-2 have

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a protective effect [95, 96]. COX-2 as well as iNOS are promising targets for the treatment of stroke patients because blocking them is still effective even 6 to 24 hours after ischemia [91, 96]. Not only the activated residual microglia, but also monocytes from the circulating blood migrate into the affected brain tissue. This transmigration is mediated by a chemokine, the monocyte-attractant protein 1 (MCP-1), which is induced 1 to 2 days after ischemia and causes immigration of monocytes [97]. Moreover, about a third of these transmigrated cells differentiate within 14 days post stroke into microglial cells – virtually indistinguishable from autochthonous microglia [98]. A trans-differentiation into astrocytes has also been observed [99, 100], but our group has not been able to reproduce this finding [101]. Irrespective of that, the important role of astrocytes during inflammation has been recognized; producing both pro-inflammatory as well as neuroprotective factors such as erythropoietin (EPO), TGF-b1, or metallothionein 2 [85, 102, 103]. In general, much further study needs to be conducted before the complex role of inflammation in cerebral ischemia is sufficiently understood to be able to specifically block the damaging proteins and foster protective elements during the inflammation process in stroke.

1.8

Damage to the Blood–Brain-Barrier

The function of the blood-brain-barrier (BBB) depends on the integrity of its cellular matrix which consists of endothelial cells, basal lamina, and astrocytes. Cerebral ischemia causes damage to this matrix and its intercellular signal exchange. Central to these damaging processes are proteases like cathepsins, plasminogen activators (PA), and the matrix metalloproteinases (MMP). The action of the MMPs, a group of more than 20 zinc-endopeptidases, has been extensively studied. Proteolytic destruction of the basal membrane by the MMPs permits the immigration of leukocytes and promotes vasogenic edema. MMP-2 and MMP-9 are expressed within 1 to 3 hours after cerebral ischemia; the level of their expression correlates with the level of BBB-breakdown, the risk of hemorrhagic transformation and the extent of eventual neuronal damage [104–106]. Pharmacological inhibition of the MMPs prevents this breakdown and reduces stroke volumes. Mice with target disruption of MMP9 are partly protected from the effects of ischemia [107, 108]. Of the plasminogen activators, tPA is best known. In the brain, tPA is expressed mainly in neurons, astrocytes and microglia. Studies of mainly primary cortex neurons have shown that tPA acts also as neurotoxin [109], and experiments on tPAknockouts have produced conflicting data [110, 111]. Since recombinant tPA is the sole licensed drug for thrombolytic therapy, these findings reflect also on current therapeutic practice. tPA has furthermore been shown to induce MMP-9 and, thus, – at least indirectly – damaging the BBB [112]. The same holds probably for the recombinant tPA. This may explain why such a high number of secondary hemorrhages have been observed after thrombolytic stroke therapy [106, 113,

1.9 Programmed Cell Death and Apoptosis

114]. Animal experiments showed that delayed rtPA treatment – mimicking reallife delays in treatment – lead to significantly greater damage to the BBB and a major increase in mortality The pharmacological inhibition of MMPs can reverse this effect of rtPA [115]. In addition, tPA enhances NMDA-mediated calcium signaling and subsequent excitotoxic neuronal injury [116]. It is a matter of controversy, whether this detrimental effect is due to a cleavage of the NR1 subunit of the NMDA receptor by tPA [117]. The example of rtPA demonstrates once again that in order to refine treatment of ischemia, a thorough understanding of the complex mechanisms is indispensable. The development of effective and low-risk thrombolytic treatment is, of course, still one of the main objectives of pharmacological stroke research [113]. One promising approach is the development of novel plasminogen activators, such as recombinant desmodus rotundus salivary plasminogen activator (DSPA-a1; Desmoteplase). Experimental evidence indicates that Desmoteplase has pharmacological and toxicological properties superior to rtPA [118].

1.9

Programmed Cell Death and Apoptosis

Apoptosis follows a similar time schedule as inflammation. It sets in after a few hours and continues over days. It is the commonest form of cell damage in the penumbra and is very distinct from the rapid neuronal and glial death in the adjacent ischemic core, where pan-necrosis is complete within few hours. In necrosis, it is the cellular edema that leads to osmolysis (see above). The released cell contents are a potent inflammatory stimulus as discussed above. Caspases, a group of proteases, has a pivotal role in apoptosis. They facilitate the “orderly” destruction of the cell, which is characterized by a condensation of both nucleus and the cell as a whole. The cell contents are fragmented into so-called apoptotic bodies. Endonucleases fragment the cell’s DNA into typical strands of 180 bp length. Other morphologic criteria of apoptosis are membrane blebbing and shrunk cytoplasm with morphologically intact organelles. While the terms “delayed neuronal death” (DND) and “programmed cell death” (PCD) are often used synonymously with “apoptosis”, the two former may well occur without the typical features of the latter. Although most experimental studies show some biochemical evidence for apoptosis to have taken place, histological features are often missing to substantiate the claim. This has led some researchers to question apoptosis as an actual feature of ischemia. A number of reviews have dealt extensively with the significance of apoptosis in the pathophysiology of ischemia (for example [21, 22, 119–122]). This is why we shall focus here on some of its mechanistic aspects in this context. Essentially, apoptosis can be triggered in two ways, by intrinsic (i. e. basically mitochondrial) or extrinsic activation. Binding to the Fas-receptor, for example by TNF-a, triggers the extrinsic pathway. Intrinsic triggers of apoptosis in the context of ischemia are elevated concentrations of intracellular calcium, reactive oxy-

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gen species, and glutamate as well as increased DNA damage. Through damage to the mitochondrial membranes, both pathways directly or indirectly lead to the activation of caspases. The caspases are a hierarchic group of at least 14 cysteine-dependent and aspartate-specific proteases. Their inactive precursor proteins exist in every cell and can be activated through cleavage. Apoptosis is essentially an active and in itself energy dependent cellular deconstruction process. In cerebral ischemia, the caspases 1, 3, 8, and 9 are involved, of which the caspases 8 and 9 are the initiators of a signal cascade that activates the so-called execution caspase 3. Caspase 1 is predominantly engaged in cytokine activation (for review see [119, 121]). Caspase 3 is of predominant importance not only in the context of cerebral ischemia [122], and its genetic or pharmacological inhibition is not only in this context neuroprotective [124, 125]. The substrates degraded by caspase 3 are DNA repair enzymes like poly-(ADPribose)-polymerase (PARP) and the DNA dependent protein kinase (DNA-PK). The latter is important for the repair of double strand breaks. PARP recognizes single strand disruptions and mediates self-repair of the DNA through swift auto-modification with synthesis of highly negatively charged, long and branched ADP-ribose molecules. This process utilizes ATP and thus taps the cellular energy pool. Although not universally accepted, this ATP consumption by PARP is widely believed to be in itself cytotoxic. PARP is also capable of inducing caspase-independent cell suicide via the so-called apoptosis-inducing factor (AIF; see below; [126]). This helps to explain also the partial failure of caspase inhibitors in some models of cerebral ischemia. The genetic or pharmacological blockade of PARP has, however, been demonstrated to be neuroprotective in experimental stroke [127–129]. Of significance is the cleavage by caspase 3 of the inhibitor of caspase-activated DNase (ICAD). ICAD is thus activated and causes the characteristic feature of “laddering” that has been described in all stroke models (for review see [130]). The term laddering draws on the regular appearance of those DNA-fragments as ladder-like bands on a gel electrophoretic separation. The length of the resulting DNA fragments reflects the complex structure of eukaryotic genetic information in the chromatin. Chromatin is a structure, in which most of the DNA is enclosed by specific proteins, the histones (see epigenetics), leaving only few positions accessible for endonucleases. Complete apoptotic laddering leaves DNA-oligomers of about 140 to 180 base pairs in length, which do no longer contain any useful genetic information. Apart from destroying genetic information, caspases also degrade structure proteins of the nucleus and cell, such as laminin, actin, and gelsolin (for a review [120]). Cytochrome C is not only an essential part of the mitochondrial electron transfer chain and hence for the energy production in the cell, but also a crucial mediator in the caspase activation cascade [131, 132]. On the extrinsic pathway of apoptosis, stimulation of the Fas-receptor leads to the formation of the “death-inducing signaling complex” (DISC). DISC activates caspase 8 from its precursor protein, which, in turn, activates “Bid”, a pro-apoptotic protein of the Bcl-2 family (see below). Bid then induces the liberation of cytochrome C from the mitochondrial membrane. Other members of the pro-apoptotic Bcl-2 family are Bad and Bax, im-

1.10 Ischemia-induced DNA Damage, DNA Repair

portant on the intrinsic pathway. Their activation leads to their translocation into the outer mitochondrial membrane and to the establishment of the mitochondrial permeability transition pore (MPTP), leading to the release of cytochrome C. Another way of its release from mitochondria is obviously direct membrane damage by free radicals (for review see [130, 132]). Cytochrome C, together with the cytosolic protein Apaf-1, forms the caspase precursor protein, and, using ATP, the so-called apoptosome. The apoptosome activates caspase 9 through cleavage from its precursor [133]. Whether cytochrome C is eventually released depends, however, on the balance of pro-apoptotic members of the Bcl-2 family like Bid, Bax, Bad, and Bag and their anti-apoptotic counterparts Bcl-2 and Bcl-xL (for review see [134]). Prevailing expression of Bcl-2 and Bcl-xL results in clearly smaller infarction after focal cerebral ischemia [135–137]. Treatment with a Bcl-xL fusion protein containing the TAT domain of the human immunodeficiency virus (HIV) for cell penetration acts also neuroprotectively in models of ischemia [138, 139]. Bcl-2 and Bcl-xL seem to have a stabilizing effect on the mitochondrial membrane; they prevent the formation of MPTP, the ensuing cytochrome C liberation, and the formation of AIF (for review see [132]). Not only the reinforcement of antiapoptotic factors but also the attenuation of pro-apoptotic proteins may be neuroprotective in cerebral ischemia. For instance, the phosphorylation of Bad leads to its sequestration by the protein 14-3-3 and thereby to its inactivation [140]. The PI3K/Akt-pathway, utilized by many neurotrophic factors such as erythropoietin (see below: endogenous neuroprotection), plays an essential role in this [102, 141]. It has been reported that Akt has been activated during cerebral ischemia [142]. Whether this has a functional impact in stroke remains unclear at the moment. Another important protein group involved in stroke are the members of the IAPgroup (inhibitors of apoptosis proteins). The over-expression of the X-chromosomelinked IAP (XIAP) has been shown to be protective in stroke models [143]. IAPs act either indirectly by inhibiting the apoptosome or directly inhibiting caspase 3 (for review see [132]), but can themselves be blocked by other proteins such as DIABLO (in mice, the human homologue is Smac) that is physiologically localized in mitochondria. DIABLO is released after focal ischemia and binds to XIAP. It is yet unclear what its function is during focal ischemia [144]. The data discussed show, however, that apoptosis underlies a complex regulation process which is controlled at many levels.

1.10

Ischemia-induced DNA Damage, DNA Repair, and p53 as Genotoxic Sensor

The emergence of DNA damage and its repair functions in the context of focal cerebral ischemia are particularly interesting. DNA damage is caused by oxygen free radicals (see above). Particularly the interaction of hydroxyl radicals with DNA results in strand breaks and base alterations. Hydroxyl radicals have a very short half-life – with the exception of peroxynitrite, formed from NO and super-

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oxide – and are unlikely to reach the nuclear DNA from outside the cell. They are more likely to be produced in close proximity to the nucleus as a homolytic cleavage product of peroxynitrite, which has the ability to diffuse over longer distances. Under physiological circumstances, DNA lesions are generated with a frequency of about 10,000 events each day [145]. They are recognized and repaired efficiently, provided the repair enzymes are intact and not overloaded. In the wake of ischemia and reperfusion, the tissue is flooded with oxygen free radicals causing abundant DNA damage. Alterations that are typically caused by free radicals [146] have been found in the mouse model of transient focal ischemia, where a massive increase in DNA damage was observed 10 to 20 minutes after reperfusion. Foremost, 8-hydroxy-deoxy guanosine becomes much more prevalent in both nuclear as well as mitochondrial DNA after ischemia [147–150]. These alterations are repaired partially within 4 to 6 hours, and the increase in repair activity is proportional to the degree of DNA damage [148, 151]. There are three basic DNA repair mechanisms – base excision repair (BER), nucleotide excision repair (NER), and what is known as mismatch repair. BER seems to be of particular significance in ischemia. During BER, DNA glycosylases excise damaged or modified bases. The thus generated purine and pyrimidine-free sites (AP-sites) are eliminated from the damaged DNA strand by AP-endonucleases (apurinic/apyrimidinic endonucleases) and replaced by DNA-polymerase b and DNA-ligase with a short strand of 1 to 4 nucleotides [147] based on the complementary blueprint of the remaining undamaged strand. Apart from oxidative DNA damage, deamination of cytosine may produce uracil. In BER, such alterations are sorted out by uracil-DNA-glycosylase (UDG). We found that knockout mice deficient in UDG have substantially larger infarctions than the wild-type controls (Endres, Meisel, Dirnagl, Jaenisch et al. unpublished). DNA repair is neither inexhaustible nor infallible. Persisting mutations are the result of erroneous DNA repair. In models of transient focal ischemia, a dramatic increase in the frequency of mutations has been observed. Half of them were transitions and transversions. The other half were deletions and occasional insertions, mostly resulting in a frame shift (frame shift mutations) of the open reading frame (ORF). It seems that not only the exhaustion of the repair capacity, but also an impaired proofreading ability in the exonuclease-negative DNA polymerase b expressed in the brain is to blame for these mutations. The major role of DNA repair is to ensure transcription of mRNA from intact genes. An obvious consequence of mutations is instability of the genetic information that may lead to faulty proteins, eventually resulting in either cellular malfunction, disintegration, or spawning of tumors [147, 150]. The maintenance of swift and effective DNA repair is therefore essential in postmitotic cells like neurons. In order to adapt to an increased demand, a cell may try to increase its repair efficiency [151]. Apoptosis is another way of avoiding erroneous protein synthesis and the potential emergence of tumors (see above). Both mechanisms are found in focal cerebral ischemia, and it seems to be the extent of the damage that determines which scenario will prevail [147, 149].

1.11 Epigenetics

We have several reasons to believe that the tumor suppressor protein p53 represents the molecular switch on the crossroads of attempted repair and apoptosis [152]. (1) p53 is mainly induced by DNA damage. (2) p53 can halt the cell cycle, and thus prevent the division of non-neuronal cells before complete repair. (3) p53 can induce apoptosis if the damage is extensive [153]. In most models of ischemia, it could be shown that p53 had been induced. [154, 155]. The fact that p53 knockouts have significantly lower infarct volumes accords with p53’s pro-apoptotic function. However, it seems paradoxical that heterozygous p53þ/– mice show even smaller infarctions [156]. An attenuated expression of p53 seems to give better neuroprotection than a total lack of it, which hints at the dual function of the gene. It would be interesting to find out if longer-term neoplasias could be the downside of this short-term effect.

1.11

Epigenetics

Experimental work of the last years has shown that, apart from DNA repair, epigenetic mechanisms like DNA methylation and histone modification play also an important role in ischemic stroke. The transcriptional gene expression is not only regulated by transcription factors, but depends also on epigenetic mechanisms that mainly modify the chromatin structure. In the mammalian genome, DNA methylation is a covalent, postreplicative modification at the carbon 5 position of the pyrimidine ring of cytosine bases (m5C); it typically occurs in CpG dinucleotides. DNA methylation is catalyzed by DNA methyltransferases (DNMT1, DNMT3a, DNMT3b). The main function of DNA methylation lies in the regulation of gene transcription. Methylated genes are barred from expression in a process called “gene silencing” (for a review [157]. First of all, many transcription activators are unable to bind to methylated loci at all. Furthermore, there is a group of methyl-CpG binding domain proteins (MBD [158]) that form a complex with histone deacetylases (HDAC), which, in turn, by deacetylation of histones, cause the chromatin structure to condense. Through this condensation, the gene regulatory loci become inaccessible for transcription factors. Five proteins of the MBD family are known to date: MBD1 through 4 and MeCP2. HDACs deacetylate predominantly histones H3 and H4. Conversely, acetylated histones, open up the chromatin structure, permitting the gene regulatory regions (promoters, enhancers) to become active again, and, ultimately, enabling gene transcription through binding of transcriptional activator proteins. While the state of deacetylation or acetylation of a CpG hypomethylated promoter or enhancer region is comparatively volatile, DNA hypermethylation of these regulatory regions results in a more stable repression [159, 160]. Initially, DNA methylation was thought to be of biological significance only for fundamental phenomena of cell biology like embryogenesis and differentiation, in X-chromosome inactivation, genomic imprinting, and in several neoplastic diseases. In more recent years, its pathophysiological role in a number of further con-

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ditions has been recognized, such as fragile-X-syndrome, ICF (Immunodeficiency, Centromere instability, Facial anomalies) syndrome, or Rett syndrome (for a review [157, 161]). For the first time, we were able to detect the involvement of DNA methyltransferase 1 and DNA methylation in an acute disease, such as ischemic stroke [162– 164]. In addition, a complex pattern of an alterated expression of MBD-family proteins has been described in a model of transient cerebral ischemia [165]. The inhibition of DNA methylation had a protective effect, as had manipulations to the acetylation state of histones. Pharmacological inhibition of a histone acetyltransferase with trichostatin A (TSA) caused enhanced histone acetylation in vitro as well as in vivo and proved to be neuroprotective ([162, Meisel et al. unpublished results). A similar effect was shown recently in a mouse model of Huntington’s disease [166]. Thus, the epigenetic mechanisms of gene regulation may provide yet another axis of neuroprotective therapeutic intervention.

1.12

Gene Expression

The role of transcription factors such as AP-1, HIF-1 and NF-kB in the context of cerebral ischemia has been recognized for much longer [167–169]. They initiate the transcriptional activation of complex programs of gene expression (for a review [170, 171]). The whole hierarchical system of such programs has been unraveled over more than 15 years. Within few hours the transcription of immediate early genes such as c-fos is activated. Some of these, in turn, form transcription factors such as AP-1. They induce a variety of genes that may have protective or destructive effects. Cells that survive the ensuing biochemical negotiation finally express genes that contribute to a structural re-organization and thus to greater plasticity. A great many of the genes involved have been subject to pathophysiological studies in stroke. Their functional significance has been studied either in descriptive ways, i. e. by describing candidate genes on RNA or protein level or using transgenic technology. Especially knockout mice have been extremely useful for this approach. The genetic elimination of specific proteins in mice used for these experiments has been particularly enlightening for the untangling of the signaling cascades involved in and after stroke. In recent years, the advent of genetic screening methods has allowed for the systematic investigation of complex gene expression programs that are initiated by cerebral ischemia. In contrast to candidate-gene research, they permit a more unbiased approach. They are based on the assumption that focal cerebral ischemia does incite complex changes of gene expression. Not just a single, specifically targeted gene is examined for its role in the scenario of stroke but – ideally – all genes. For this new research paradigm terms have been coined like discovery science, genomics, transcriptomics, proteomics, and cellomics. So-called high throughput technologies as well as a highly evolved bioinformatic methodology are a prerequisite for acquiring and managing the vast bulk of information involved. DNA micro

1.13 Cell Replacement

arrays or SAGE technology facilitate the examination of global changes in gene expression on mRNA level (transcriptomics). Global changes on the level of protein synthesis can be described with a combination of two-dimensional gel electrophoresis and identification techniques (proteomics). The first results of a number of studies using these techniques in the context of cerebral ischemia are now available [103, 172–174]. What do these results imply? First of all, they confirm that the assumption of the existence of complex stroke-induced programs of gene expression is correct. Secondly, they demonstrate that they follow a robust temporal and spatial pattern. Not only do the expression patterns differ between core and penumbra, but also within the penumbra, there are many spatial and temporal differentiations. This has even given rise to a concept of “multiple penumbras” [175]. Thirdly, this approach has not only confirmed many findings of candidate gene research but also underpinned our pathophysiological understanding. Thus, a number of heat shock proteins, anti-oxidative enzymes, growth factors, and pro- as well as anti-inflammatory proteins have been detected. Not just transcription factors, but a variety of proteins have been have been found to play essential roles in the metabolism of RNA and DNA. Finally, proteins that contribute to structural and functional reorganization [103, 176–178] are thought to establish the molecular base of the functional healing potential after strokes. Not surprisingly, a number of hitherto unidentified genes have been brought forth by discovery science. Although the results of these screening methods give a great boost to stroke research and open doors to new insights, a full functional characterization of the differentially expressed genes will take many more years. Our understanding of the emerging network of gene expression increasingly depends on not only biological but also mathematical models. However, there are things we do know already; for instance that protein modifications are of great functional importance. Thus, the functional value of numerous proteins depends on their phosphorylation state. For example, the specific phosphorylation of the usually pro-apoptotic protein Bad prevents apoptosis in the ischemic penumbra. The differential function equals thus not simply differential gene expression, and genetic screening methods will never be sufficient to paint the full picture of stroke-induced molecular pathways. The same holds for proteomics, which, just like transcriptomics, neglects the functional importance of RNAs that do not code for proteins. These molecular pathways, however, potentially lead to new, neuroprotective therapies and open up new perspectives for fundamental issues like neuronal plasticity and stem cell research.

1.13

Cell Replacement

In recent years, it has been shown in different models that ischemic brain injury induces neuron formation from neuronal stem cells. Such cells reside in the subgranular zone of the dentate gyrus, in the subventricular zone below the lateral

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ventricles, and periventricularly in the hippocampus. They can migrate into parts of ischemic lesions within the striatum, the dentate nucleus, and the hippocampal CA1 region [179]. It is still a matter of debate to what extent a similar neuroneogenesis also takes place within the penumbra in cortical regions. Neither is it entirely understood to what extent these migrating cells, that doubtlessly express neuronal markers, become functional. Observations so far indicate that it is not a very effective process and that most of the migrating cells perish. However, if these neuronal cells could be enabled or encouraged to assume the function of damaged neurons, this would imply another new therapeutic paradigm (for a review [180, 181]). Indeed, intrathecal infusion of growth factors like FGF-2 and EGF after global cerebral ischemia did not only stimulate the migration and the survival of these cells but resulted also in improved functional results both on cellular and on neurobehavioral level [182]. Astrocytes are newly formed in a similar fashion [183]. For future therapeutic evaluation, it should be kept in mind not only that growth factors such as erythropoietin, FGF-2, EGF, VEGF, SCF, and BDNF have this beneficial effect [182, 184, 185], but also that their expression depends on the stimulation of the NMDA receptor by glutamate [186]. A pharmacological blockade of this receptor with the intention of inducing neuroprotection could interfere with this effect and thus be counterproductive. This is yet another example of an incomplete understanding of the signaling mechanisms, leading to a paradox in the interpretation of our neuroprotective data. Bearing this in mind, it is needless to say that the ongoing experimental studies of cell replacement are still far from clinical implementation. However, the available experimental observations are very exciting: For instance, neural and also haematopoietic pluripotent cells differentiate into neuronal and glial cells when injected into infarction sites (for a review [187]). On the other hand, this potentially helpful strategy may have detrimental effects too. In a mouse model of stroke, for example, murine embryonic stem cells did not migrate into the damaged tissue, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether or not they had been predifferentiated in vitro into neural progenitor cells [188]. As far as safety is concerned, an endogenous pathway of cell regeneration may be more promising. Bone-marrow-derived cells (BMDC) introduced into the brain possess a differentiation potential capable not only of forming microglia (see above; [98]), but also neurons. This apparent plasticity has been attributed to transdifferentiation [189]. However, more recent data suggest in the case of neurons that BMDCs not differentiate into but rather fuse with Purkinje cells in the brain. These observations suggest that cell fusion contributes to the maintenance of neurons [190]. One potential use for haematopoietic cells migrating from blood into ischemic brain tissue could be the therapeutic transfer of protective gene products such as growth factors [98].

1.14 Endogenous Neuroprotection – Ischemic Tolerance

1.14

Endogenous Neuroprotection – Ischemic Tolerance

In the wake of ischemic stroke, not only cascades of destruction are induced, but also endogenous protective mechanisms are activated. These mechanisms are currently studied intensively. Central to this research are models of ischemic preconditioning. The idea behind ischemic preconditioning is to achieve a protected state of a cell, tissue, or whole organism by subliminal exposure to a noxious stimulus (trigger), which is applied just below the level at which damage would occur. The stimulus induces a protective state against insults that would normally be lethal. Subliminal ischemia (‘ischemic tolerance’), hypoxia, reactive oxygen free radicals, inflammation, etc. can serve as tolerance-inducing stimuli. After induction, there are two time windows within which ischemic tolerance can be observed. Early tolerance occurs after about 5 to 120 minutes following stimulation (classical preconditioning), while delayed preconditioning sets in after a latency of about 24 to 72 hours. Seven days after induction, the state of protection is no longer detectable [191, 192]. Studies of patients who had transient ischemic attacks (TIA) before suffering a persistent stroke indicate that ischemic tolerance may also exist in humans. Subsequent to a TIA, brain infarcts were smaller in comparison with patients without preceding TIA. As a TIA, by definition, does not lead to infarction, it may thus be considered as a stimulus that induces tolerance [193, 194]. Unlike severe ischemia, leading to stroke, a preconditioning stimulus induces exclusively protective, but not destructive signaling cascades. There are basically three stages of protective signaling that can be distinguished in delayed preconditioning. In the induction phase molecular sensors (mostly receptors, channels and regulators) are activated by transcription factors. During the transduction phase certain protein kinases, transcription factors and paracrine and autocrine mediators, such as growth factors, amplify the signal and thereby prepare the effector phase. In this phase, proteins with a direct protective impact (anti-apoptotic, anti-inflammatory, anti-oxidative) are switched on. These mechanisms are the molecular basis for ischemic tolerance and have been reviewed in detail elsewhere (for a review [191–193]). Here, we want to give only a brief outline of the concept. On the basis of animal models in mice and rats as well as cell culture experiments, we were able to describe the following signaling cascade: In astrocytes, a tolerance-inducing hypoxic stimulus induces the transcription factor HIF-1 (hypoxia-induciblefactor-1). HIF-1 is one of the central sensors of hypoxia and induces the expression of erythropoietin (EPO; [102, 168, 196]). Erythropoietin is released by astrocytes and binds to the neuronal EPO-receptor. Via the activation of a protein kinase cascade, in particular phosphoinositol-3 kinase, the pro-apoptotic protein BAD becomes phosphorylated and thus inactivated [102]. This neuroprotective signaling cascade mediates approximately 50 % of the protection observed in our model [197]. The remaining 50 % of protection it does not account for suggest the presence of further mechanisms in ischemic preconditioning. As a matter of fact, beside this paracrine cascade, which is mediated by astrocytes and neurons, a number of

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other mechanisms have been described. A host of anti-excitotoxic, anti-inflammatory and anti-apoptotic mechanisms, including programs for regeneration and repair, have been implicated so far (for a review [191–193]). Understanding these mechanisms may enable us in the future to induce or boost endogenous protection in patients as a novel strategy to safeguard the brain against hypoxic/ischemic damage. For example, EPO is a promising drug in stroke therapy. There is solid evidence that exogenously applied EPO is neuroprotective in vivo and in vitro models of cerebral ischemia [102, 198, 199]. Clinical phase I and II trials suggest that EPO is not only safe, but also beneficial in the therapy for acute stroke [200]. In addition, pharmacological inducers of ischemic tolerance in brain could become a promising tool in circumstances when ischemia can be anticipated, e. g. before cardiac or carotid surgery. For example, desferrioxamine, an approved drug for the treatment of hemosiderosis, induces robust ischemic tolerance by inducing erythropoietin in a HIF-1 dependent manner [197]. Combined, the above-mentioned results demonstrate that brain cells are not only challenged by deleterious mechanisms, but also activate innate programs to protect themselves from ischemia. However, cerebral ischemia does not only affect the brain parenchyma, but also other vital organs, in particular the immune system.

1.15

Stroke Induced Immune Depression (SIDS)

Within three days after stroke, up to 61 % of the stroke patients develop fever [201– 205]. Findings from animal experiments show that pyrexia of more than 1hC does not only lead to a dramatic increase of the cytotoxic neurotransmitter glutamate [206], but also to a significantly larger volume of the infarction [207, 208]. Clinical trials demonstrated that both mortality and morbidity rise with the occurrence of fever in stroke [209, 210]. The most common cause of fever in the acute course of stroke is infection [201, 205, 211–213]. Infectious complications occur in 21 to 65 % of stroke patients [214]. Pneumonia is the most frequent infection in stroke patients [215] and a major mortality risk factor [216, 217]. Stroke patients have a much higher risk of developing severe bacterial infections. It is obvious that factors such as immobilization, reduced bulbar reflexes, drowsiness and, subsequently, a higher risk of aspiration promote pulmonary infections. Although these are risk factors for bacterial infection, they cannot fully explain the high risk of infection observed. A large meta-analysis showed that other factors had to be equally important. In particular, a reduced function of the immune system was postulated [218–220]. Very recently, we were able to prove that immunodepression following stroke causes severe bacterial infections [221]. In a mouse model we demonstrated that, three days after stroke, severe bacterial infections (mostly pneumonia and sepsis) develop spontaneously. Stroke induces an over-activation of the sympathetic nervous system and leads to a rapid, severe, and sustained lymphopenia as well as to a disturbed function of lympho-

1.16 Conclusion

cytes and monocytes. Interestingly, there is also evidence for an over-activation of the sympathetic nervous system within the first two days after stroke in humans [222]. Furthermore, we were able to show that a disturbed secretion of interferon-g from T cells and NK cells is central to these severe infections. Both a cellular reconstitution of the immune system and the application of interferon-g prevented infections. In addition, pharmacological inhibition of the sympathetic nervous system with the b-receptor-blocker propranolol not only prevented infections, but also dramatically reduced the high rate of mortality in this model [221]. Our studies are the first to provide a mechanistic explanation for the clinical phenomenon of increased susceptibility to infections after stroke [223]. Approaches to prevent infection after stroke seem to be desirable. Preventive anti-infective therapy is expected to reduce a number of negative prognostic factors such as fever, infection-induced arterial hypotension, and the systemic release of pro-inflammatory cytokines. In addition, with the prevention of severe infections, an earlier mobilization and rehabilitation becomes feasible. In an experimental stroke model, we were able to demonstrate that preventive anti-infective therapy with an antibiotic after focal cerebral ischemia not only reduces mortality and the infarct sizes, but also decreases the functional deficit [224]. These experimental results lead to the initiation of a Phase IIb trial for preventive antibacterial shortterm therapy in patients with acute ischemic media-infarction (Preventive ANti-infective THERapy In Stroke; PANTHERIS, Berlin). Other groups initiated similar trials (A. Chamorro, Spain; M. Hennerici, Germany; personal communications).

1.16

Conclusion

Due to intensive experimental and clinical research, our pathophysiological understanding of cerebral ischemia has greatly improved over the last 15 years. Complex pathways involving excitotoxicity, oxidative and nitrosative stress, peri-infarct depolarizations, inflammation, and apoptosis-like mechanisms have been described in cell culture as well as animal models of cerebral ischemia. Despite impressive results in animal experiments on neuroprotection, the transfer of preclinical results into human stroke therapy has been so far been unsuccessful. Possible reasons for this dilemma include problems with the models used, the design of the clinical trials as well as the drugs, and they have been discussed intensively [21, 225, 226]. However, we believe that although we have often not been able to transfer our experimental results into human stroke therapy, some cautious optimism is justified. More and more experimental data demonstrate that the division of molecular mechanisms in “good” and “bad guys” is an improper simplification, and we need to gather more data in order to identify more suitable molecular targets. Since in stroke there are multiple pathways that cause cell death, it seems reasonable to develop combination therapies targeting all these mechanisms. In addition, the advances in understanding endogenous neuroprotective mechanisms provide new therapeutic options to improve tissue survival after stroke. Targeting systemic com-

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plications like severe bacterial infections in a preventive manner may also improve outcome. Thus, an ever more refined understanding of complex damaging cascades as well as the development of more complex therapeutic strategies will continue to be the basis for more successful therapies of stroke in the future.

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2 Parkinson’s Disease Marina Romero-Ramos, Matthew Maingay and Deniz Kirik Parkinson’s Disease (PD), first described by James Parkinson in 1817, is characterized by the loss of dopamine (DA) innervation in the striatum as a result of neurodegeneration of the dopaminergic cells located in the ventral midbrain. It is estimated that in healthy humans, there are about 400,000 DA-producing cells in this region. Two main cell groups in the midbrain are known to send ascending DA projections to the forebrain. The medially located cell group, known as ventral tegmental area (VTA) cells, mainly project to nucleus accumbens, the prefrontal cortex, septum, olfactory tubercule, and amygdala, whereas the laterally situated cells, known as the substantia nigra pars compacta (SNc) neurons, predominantly project to the striatum. Although neurodegeneration has been documented in both cell groups, the involvement of the SNc appears to be more significant since cell death in this area is more severe compared to the VTA. In 1912, Lewy described the presence of intracytoplasmic eosinophilic inclusion bodies in the surviving dopaminergic nigral neurons of post-mortem tissue from PD patients. They were termed Lewy bodies (LB) and came to be recognized as the pathological hallmark of the disease. Although a definitive diagnosis of PD can only be confirmed by findings from post-mortem specimen, the clinical diagnosis is made based on presence of specific signs and symptoms in the patients. The nigrostriatal pathway is essential for motor-function, and lack of DA results in the dysfunction of motor activation, leading to the appearance of: bradykinesia, resting tremor, rigidity, postural instability and akinesia. Additional confirmation of the diagnosis comes with the response to L-Dopa treatment (for a review see [1]). A diagnosis of “possible PD” can be established when at least two symptoms are found, one must be tremor or bradykinesia, as well as the response to L-DOPA and the exclusion of atypical symptoms. Atypical symptoms include early postural instability (within 3 years of onset), non-therapy-related hallucinations and atypical pathological involuntary movements [2]. Certain factors should be discarded to rule out other diseases, for example a history of bladder instability is related to multiple system atrophy, vertical eye movement abnormalities with progressive supranuclear pulse and early dementia in Dementia with LB (for review see [1, 3]).


32

2 Parkinson’s Disease

2.1

Epidemiology of Parkinson’s Disease

PD is the second most common neurodegenerative disease, with an overall prevalence of 0.1 % in the whole population. It is a disease of an ageing population, as the prevalence increases to 1–2 % in the population over 65. Until recently, primary Parkinsonism was described as an idiopathic disease, and although the cause of the disease still remains largely unidentified, emerging evidence suggests that multiple factors, both genetic and acquired (exposure to environmental factors) contribute to the neurodegeneration of the dopaminergic cells. 2.1.1

Genetic Contribution

The level of genetic contribution in PD seems to be highly dependent on the age of onset, meaning that with an earlier age of onset there is a higher probability of having an affected relative. In fact, if there is already a sibling under 45 affected in the family, the risk of developing PD is 6 times higher. The genetic contribution in early-onset PD has been suggested by twin-studies showing a higher concordance rate for homozygotic than for dizygotic twins with an age of onset under 50 [4]. Although numerous studies with twins have been performed to investigate the genetic component in late onset PD, no concordance was found (for a review see [5]). It is important to take into account that PD has a long preclinical phase, making it very difficult to diagnose subclinical cases, which could figure as false negatives. This can be resolved by studying dopaminergic function using positron emission tomography, a very sensitive tool that will detect early defects in the system, even when symptoms are not yet present. Using this approach, again the concordance was higher in homozygotic twins even for late onset PD [6]. Although family studies could also hint at an environmental factor, as members share the same environment, studies in families with several PD-affected members propose an autosomal dominant inheritance pattern with male predominance of the disease (for a review see [5]). Genealogical studies in Iceland showed that PD patients were more closely related to each other than to matched controls, further supporting the hypothesis of a genetic component of the disease [7]. Additional studies in the same cohort proposed the existence of a susceptibility gene for late-onset sporadic PD [8]. Two other major studies have also screened the genome in search for genes linked to the late onset PD, revealing the involvement of specific loci. However, these results did not coincide with each other, making it impossible to draw any conclusions about the role of any specific loci in the disease [9, 10]. In addition, many other groups used an approach based on the analyses of polymorphism of putative susceptibility genes (for a review see [5]) finding different positive correlation with PD, for example with genes encoding for proteins involved in the dopaminergic system, related to mitochondria or to the antioxidant system. Certainly, the major breakthrough in the genetic studies in the PD field has been a consequence of the finding of autosomal inherited monogenic PD [11]. During

2.1 Epidemiology of Parkinson’s Disease Table 2-1

Genes related to familial Parkinson’s Disease.

Name

Inheritance

Locus

Gene

LB

Onset

PARK1

AD

4q421

a-syn

Yes

Late

PARK2

AR

6q25

Parkin

No*

Juvenile-Early

PARK3

AR

2p13

?

Yes

Late

PARK4

AD

4p15

?

Yes

Late

PARK5

AD?

4p14

UCH-L1

?

Late

PARK6

AR

1p35-36

?

?

Early

PARK7

AR

1p36

DJ-1

?

Early

PARK8

AD

12p11.2-q13.1

?

No

Late

PARK9

AR

1p36

?

1p32

?

Late

Nurr1

Late

PARK10 NR4A2

AD

Juvenile

* One patient has been reported to have LB. AD: autosomal dominant; AR:autosomal recessive.

the last decade familial PD cases have been shown to be the result of defects in different loci (Table 2.1), four of them identified genes encoding for parkin, a-synuclein (a-syn), UCHL-1 and DJ-1. Although these familial cases constitute a very small percentage of the cases, the research based on the involvement of these proteins in PD is helping to better understand the pathophysiology of the disease (see Chapters 3 and 4). 2.1.2

Environmental Factors

The hypothesis proposing the involvement of environmental factors in the etiology of PD initially arose from the discovery of various toxins that, due to their dopaminergic selectivity, were able to induce the same type of neurodegeneration found in patients. Probably the first indication of an involvement of the environment in PD came when a group of drug addicts developed PD in an extremely short period (1–2 weeks), as the result of the exposure to 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant by-product, obtained during the synthesis of a meperidine analog. Later on, it was found that MPTP induces inhibition of complex I of the respiratory chain, which, in turn, leads to oxidative stress and finally to dopaminergic cell death, suggesting that DA neurons were particularly vulnerable to energy depletion and oxidative stress [12]. Other environmental toxins have later been found to induce PD-like neurodegeneration, for example, two pesticides, rotenone [13] and paraquat [14], are both inhibitors of the complex I and

33

34


lead to PD-like neurodegeneration when administered systemically. In addition, other environmental factors, such as exposure to high doses of iron and manganese lead to dopaminergic degeneration – probably due to their role in catalyzing toxic redox reactions (for review see [15–17]). Many of these toxins are now used to develop neurotoxic models of PD, and they have provided valuable clues about the processes leading to neurodegeneration occurring in patients, such as oxidative stress and mitochondrial dysfunction.

2.2

Oxidative Stress In Parkinson’s Disease

The specific neurodegeneration of the dopaminergic cell population in PD has lead to numerous studies about the susceptibility of these cells to different insults. Dopaminergic cells seem to be especially vulnerable to damage, suffering neurodegeneration in every healthy human at an average of 4.4 % per decade. However, in idiopathic PD patients the rate of cell death increases in their early fifties, leading to the appearance of symptoms in their 6th decade of life (for review see [18]). Regarding the underlying mechanism, probably the most extensive studies are those relating to dopaminergic neurodegeneration and oxidative stress in PD. It is well known that even healthy dopaminergic cells have a high vulnerability to oxidative stress due to (1) low antioxidant enzyme levels, (2) high auto-oxidation rates and (3) high iron content [19–21]. Indeed, there is extensive data supporting the involvement of oxidative stress during the neurodegenerative process in PD, as documented by an increase in oxidative damage markers [22–24]. Free radical formation in dopaminergic cells results from the metabolism of DA, which gives rise to H2O2 via MAO, as well as DA auto-oxidation. This process gives rise to quinones via a non-enzymatic conversion catalyzed by iron resulting in H2O2 [19]. One possible mechanism by which oxidative damage can occur in dopaminergic cells is a failure of the antioxidant systems. Certainly, postmortem studies in SN of PD patients have shown decreased glutathione content [25, 26] and increased superoxide dismutase [27]. Also the level of metallic ions is important, as they can catalyze free radical-generating reactions. Therefore, the increase in iron together with the decrease in the ferritin levels found in PD may contribute greatly to the reported oxidative damage [28–30]. Another major cellular source of free radicals is the respiratory chain in the mitochondria. Any dysfunction of this system could lead to oxidative stress. In fact a decrease in mitochondrial activity at the level of the complex I is found in neurons of the SNc in PD patients [31–33], which could also account for the increase in oxidative damage (for a review see [34]). The first event occurring in PD leading to oxidative damage is still controversial. However mitochondrial dysfunction seems to be a secondary event preceded by the decrease in GSH content [35], which will lead to an unbalanced redox status and by oxidative damage to mitochondrial defect.

2.3 Role of Alpha-Synuclein in Parkinson’s Disease

2.3

Role of Alpha-Synuclein in Parkinson’s Disease

In 1997 genetic studies in a small number of Italian and Greek families with early onset autosomal familial PD revealed the presence of a point mutation in the a-syn gene (leading to a change from alanine to threonine at position 53, termed A53T mutant a-syn) [36]. Further support for the involvement of a-syn in PD was obtained when a second a-syn point mutation from alanine to proline at position 30 (A30P) was discovered in a German family with an early-onset of the disease [37]. Although, these mutations have been found only in these few families, further studies involving a-syn in PD resulted in a major breakthrough in the field when it was shown that LBs not only from familial cases but also in sporadic PD invariably immunostained for a-syn [38]. Moreover, a-syn was also found to be the main component in inclusions from all other neurodegenerative diseases characterized by the presence of LBs such as dense Lewy body disease, Lewy body variant of Alzheimer’s disease and Multiple System Atrophy. This has lead to the classification of all the diseases containing a-syn positive aggregates as ‘synucleinopathies’ highlighting the importance of a-syn in neurodegeneration [39]. Alpha-synuclein belongs to a family of highly conserved, small proteins also including beta and gamma synuclein (for a review see [40]). Alpha-synuclein has 140 amino acids and is natively unfolded with an extended structure primarily comprised of random coils [41]. Although a-syn is seen in various tissue types, it is most predominantly and ubiquitously expressed throughout the CNS, where it is located in synaptic terminals, in close proximity to vesicles [42]. The physiological function of a-syn is yet to be determined, however, it has been suggested to be involved in vesicle formation and membrane plasticity. This possible function is supported by a-syn’s ability to bind to membranes and inhibit the phospholipase D2 enzyme, which is involved in vesicle production [43–45]. Accordingly, a-syn knock-out mice show a reduction in the number of vesicles in the distal pool of the presynaptic terminal, suggesting the involvement of a-syn in the regulation of DA storage, release and uptake [46]. In addition, a-syn knock-out mice exhibit an 18 % decrease in striatal DA, a reduction in amphetamine induced activity and increased DA release following paired stimuli, which supports the hypothesis that a-syn may be involved in DA transmission and/or the regulation of synaptic vesicle mobilization at dopaminergic terminals [47, 48]. Alternatively, a-syn shows 40 % homology to the chaperone 14-3-3, suggesting a chaperon-like activity may be stabilizing the conformation of putative binding partners [49]. 2.3.1

Alpha-Synuclein in Lewy Bodies

Post mortem studies from PD patients have shown that the surviving dopaminergic cells in SNc contain intracytoplasmatic aggregates in cell bodies as well as in dendritic and axonal processes (termed Lewy neurites (LN)). At the structural level, LBs contain an electron-dense granular core and a lighter surrounding

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ring formed by radiating fibrils. Fibrils are formed from insoluble assembled a-syn polymers. Fibrilogenesis in vitro is preceded by the formation of metastable soluble oligomers, called protofibrils [50]. The functional consequence of a-syn-positive inclusions are currently viewed in two alternative ways: while the first hypothesis suggest that LBs are deleterious for the cells, the alternative theory proposes them as protective or at least a safer storage form of excessive or altered a-syn. Supporting this later idea, rat-a-syn has been found to form inclusions in dopaminergic cells without neurodegeneration, suggesting that aggregation is not toxic per se [51]. Moreover, it has been proposed that soluble oligomers of a-syn are more deleterious to the cell than the fibrillar forms. The presence of such soluble oligomers in PD brains has recently been documented [52]. In vitro, these oligomers can form amyloid pores disrupting intracytoplasmic vesicles releasing high concentrations of molecules, which, when free in the cytoplasm, would be exposed to catalysis or alterations and could exert toxicity on the cells [53]. Interestingly, the ability to form oligomers is increased in the two mutated forms of a-syn compared with wild-type [54]. However, an increased rate of fibril formation has been documented only for A53T mutated forms, whereas A30P impairs a-syn ability to bind to vesicular membranes and thus may have implications for normal vesicular function [55, 56]. 2.3.2

Alpha-Synuclein and Oxidative Stress

Alpha-synuclein found in inclusions from various synucleopathies, including PD, were recently documented to have oxidative modification on tyrosine residues (nitration). This modification promotes a-syn oligomerization, which could affect its solubility, and lead to aggregation [57, 58]. In fact, oxidative damage in vitro increases self-oligomerization and leads to fibrillar aggregation [57, 59]. Conversely, nitration could occur subsequent to aggregation, once inclusions are already formed. Whether nitration occurs on the soluble a-syn or the aggregated form in LB, it further confirms that oxidative stress is a factor involved in neurodegeneration in PD. In this context, a-syn may not only be a target for oxidative damage, but it could also be a contributor. Alpha-synuclein promotes free radical formation and mitochondrial deficit-generating oxidative damage in vitro; therefore, a-syn could increase the susceptibility to oxidative stress [60–65]. This effect could have a particularly strong impact in the already vulnerable dopaminergic cells, leading to a specific toxicity of a-syn. In fact, the overexpression of a-syn in cell culture leads to an increase in apoptotic cell death only in dopaminergic neurons. Such neurotoxicity was inhibited by antioxidants and completely abolished when DA synthesis was blocked [66, 67]. Therefore, a-syn contribution to oxidative damage seems to be mediated by DA auto-oxidation (Fig 2.1). Alpha-synuclein could induce DA auto-oxidation as a result of an increase of cytoplasmic free-DA, by promoting DA transporter (DAT) clustering, or if mutated (A53T), by inhibiting DA vesicularization [68, 69]. Alternatively, a-syn has been suggested to reduce tyrosine hydroxylase (TH) activity by binding to dephosphorylated TH. A change in the availability of free soluble a-syn, e. g. by aggregation or oligomerization, may lead to impair-

2.3 Role of Alpha-Synuclein in Parkinson’s Disease

Figure 2.1 Diagram representing a-syn relation with the dopaminergic system and the induction of oxidative stress. Dopamine synthesis, which mainly occurs at the terminal level, is regulated by the rate-limiting enzyme tyrosine hydroxylase (TH). Dopamine is then transported to vesicles by the VMAT (vesicular monoamine transporter). Once DA has been released and interacted with dopaminergic receptors, it is taken up by the DAT (DA transporter) to the cytoplasm to be vesicularized again. Otherwise, free-DA is metabolized by the MAO B (monoamineoxidase), or it may undergo auto-oxidation, giving rise to quinones. Both processes would generate H2O2, which is neutralized by antioxidant system or otherwise would induce oxidative stress. a-Synuclein interacts with the dopaminergic system at different levels: It can decrease TH activity by inhibiting its phosphorylation, it interacts with DAT promoting its clustering and, if mutated (A53T), it can impair DA storage process. Mutations on the a-syn (A30P and A53T) can lead to protofibril formation. In addition, a-syn can interact with DA- quinones, inducing protofibril formation. Oxidative stress can lead to oxidative modification of a-syn and other proteins, which then should be degraded by the ubiquitin proteasome system. If this system did not clear these altered proteins they would aggregate leading to Lewy bodies formation.

ment in the inhibition of the TH enzyme, leading to elevated cytosolic DA levels, which in turn may give rise to H2O2 and DA-quinones by auto-oxidation [70]. A further relationship between a-syn, dopamine and oxidative stress is strengthened by in vitro studies, showing that DA-quinones actually interact with a-syn, stabilizing the protofibrils and thereby interfering with the fibril formation [71].

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2.4

The Involvement of Proteosome in PD

The recent identification of mutations in the genes encoding for parkin and ubiquitin carboxy-terminal hydrolase L1 (UCHL1) that lead to the early-onset of PD, has introduced a new window to PD research. These two proteins are involved in a highly specific, tightly controlled system known as the ubiquitin-proteasome system (UPS), which is responsible for the degradation of unwanted intracellular proteins. Post-mortem analysis of brain tissue from sporadic PD patients have identified the presence of parkin and ubiquitin in LB, suggesting that these proteins may be involved in the formation of inclusions [72, 73]. In fact, proteasome activity is decreased in the SN of sporadic PD [74]. Furthermore, a-syn has been shown to indirectly interact with this system providing clues to the involvement of the UPS in PD [75]. In order to explain how these mutations may lead to the onset of PD, we will give a brief overview of the UPS. However, for a more detailed review of this protein degradation system, the readers are referred to the following articles [75, 76]. The UPS is a complex, multi-membered system that requires certain combinations of proteins and enzymes, in order to degrade a specific protein. First an ubiquitinactivating enzyme (E1) transforms ubiquitin into an active intermediate, which can then be recognized by the ubiquitin carrier enzymes known as E2 enzymes. These E2 enzymes transfer the ubiquitin moiety from the E1 to the substrate by a reaction that is catalyzed by an ubiquitin-protein ligase (E3). In mammals, the specialization of the UPS system is based on specific recognition of the substrate by one of the many E3s present in the cell (approximately 1000 in human). However, there are only few E2 enzymes to carry ubiquitin, which can subsequently be activated by the sole E1 enzyme in the cell. This ubiquitination process repeats, with each subsequent ubiquitin linking to the previously bound one to form a polyubiquitinated chain. In general, a chain of at least four ubiquitin attached to the substrate is sufficient to signal to the 26S proteasome that this substrate is unwanted and ready for degradation. The 26S proteasome comprises three parts; a barrel-shaped 20S proteasome, and two 19S caps, one at each end, which open the gates of the 20S proteasome, and facilitates the feeding of the substrate through the proteolytic channel of the 20S proteasome. This process leads to the breakdown of the substrate into short peptide fragments, which are further degraded by other proteases until they reach their most basic amino-acid form to be recycled. The ubiquitin chain is released just prior to the entry of the proteasome, and is broken into its monomeric ubiquitin units by UCHL1 before being recycled back to the beginning of the degradation process. A mutation found in one allele in the gene encoding for UCH-L1 was identified in two siblings with parkinsonism in 1998 [77]. As mentioned, UCH-L1 is the ligase responsible for separating and recycling ubiquitin. This mutation causes a 50 % reduction in the catalytic activity of UCH-L1, which would lead to an insufficiency of ubiquitin in labeling proteins for degradation, eventually leading to molecular crowding directing the cell through the cascade leading to degeneration.

2.4 The Involvement of Proteosome in PD

Furthermore, it has been shown that UCH-L1 is involved in the degradation of asyn [78] and therefore the degeneration may be due to a poor degradation of a-syn rather than an inability to recycle ubiquitin. In UCH-L1 null mice, a loss in both sensory and motor neurons is evident, but there is no sign of the parkinsonism phenotype [79]. However, identified polymorphisms in UCH-L1 associated with a decrease in susceptibility for PD [80] demonstrate that UCH-L1 does play a role in PD. 2.4.1

Parkin Involvement in PD

The first parkin mutations (one or more exon deletions) were identified in 1998 and described as a rare, autosomal recessive juvenile parkinsonism (AR-JP) [81]. Pathological specimens from these patients generally lacked LB, which are otherwise the characteristic pathological hallmark for PD [82]. Since then, over 50 mutations in the parkin gene have been identified, including point mutations, nonsense, missense and splice variants, as well as other variants with mutations inherited in a dominant manner (for a review see [82]). In addition, mutations in the parkin gene have also resulted in PD with the presence of LBs or with a later onset [83, 84]. Parkin is a 465 amino acid residue protein with an ubiquitin-like region, which has been shown to function as an E3 ligase. Several putative substrates have been proposed for parkin, such as CDCrel-1, synphilin-1, Pael-R and a glycosylated form of a-syn (for a review see [85, 86]). Interestingly, the levels and activity of parkin have been found to be either low or void in patients with AR-JP [87, 88], suggesting that the neurodegeneration may be the result of a loss of function. Accordingly, in vitro, reduced levels of proteasome activity were seen when mutant but not wild type parkin was expressed in cell lines [89]. Despite the fact that patients with a mutation have decreased parkin activity in all brain areas, neurodegeneration appears to be limited to the dopaminergic system. Furthermore, proteasome inhibition in vitro, leads to a selective dopaminergic toxicity, whereas other neuronal cells survive [90]. Defects in parkin may lead to a reduced efficiency in the labeling of substrates for degradation, and therefore the build up of excess amounts of its substrate. In fact, an accumulation of Pael-R – a putative G-protein-coupled receptor, which is first ubiquitinated by parkin in order to be degraded – has been found in AR-JP patient brains and may contribute to neurodegeneration [91] (Fig. 2.2). The overexpression of Pael-R in vitro, led to cell death due to endoplasmic reticulum stress [92, 93]. Furthermore, the overexpression of Pael-R in Drosophila has been shown to lead to specific dopaminergic neurodegeneration, which was abolished by concomitant parkin overexpression [94]. Moreover, Pael-R-induced neurotoxicity was DA-dependent since no degeneration was observed when TH activity was inhibited [94]. Parkin-deficient mice showed altered DA handling (see Section 7.2) suggesting the involvement of the protein in dopaminergic system function and neurotransmission [95, 96]. In addition, parkin null Drosophila suffered mitochondria defect,

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Figure 2.2 Diagram representing the degradation of a-syn via the UPS and possible links between UPS defects and PD-like neurodegeneration. a-Synuclein ready to be degraded is ubiquitinated through a cascade of reactions involving at least three distinct enzymes (termed E1, E2 and E3). Polyubiquitinated a-syn is then presented to the proteasome for degradation. End products, short peptides fragments, are dissociated from the polyubiquitin tail, which is broken by the UCHL enzyme into monomeric ubiquitin to be recycled. Mutations or oxidation can modify a-syn affecting its degradation, leading to protofibril and LB formation, which ultimately will induce dopaminergic neurodegeneration. Mutations or oxidation can also induce defects in parkin, decreasing its E3 activity, which will result in the accumulation of its substrates (like Pael-R and glycosylated-a-syn), which are linked to LB formation and/or dopaminergic neurodegeneration (see text). In addition, parkin abnormalities may also induce mitochondrial defects, which have been also connected to oxidative stress, LB formation and dopaminergic neurodegeneration. Mutations on UCHL1 will also lead to dopaminergic cell death; however, the mechanism remains to be determined.

revealing a possible role of parkin in mitochondrial function [97]. Moreover, nonmutated parkin is present in LB of sporadic PD, suggesting its involvement in the disease process. Therefore, dysfunction or decrease in the availability of parkin, for example by aggregation, could affect the dopaminergic system and/or lead to mitochondrial dysfunction which occurs in PD (Fig 2.2). Parkin aggregation may occur by oxidative stress, which leads to the misfolding of the protein [98]. These parkin-accumulations would be cleared by the proteasome, however if this system fails (as it seems to happen in PD) it will lead to inclusion formation [98, 99]. Further involvement of parkin in sporadic PD arises from the finding that a glycosylated form of a-syn is ubiquitinated by parkin, an ability lost in the mutant parkin [100]. It is therefore possible that a decrease in parkin function could result in accumulation of a-syn leading to cell death (Fig 2). Further support for this hypothesis is that a-syn-induced dopaminergic neurodegeneration can be abolished by overexpressing wild type parkin [90, 94].

2.6 Neurotoxic PD Models

The involvement of the UPS in sporadic PD is highly likely. The activity of the UPS declines with age, which would leave the aging brain to be more susceptible to PD [101]. It may be the case that in sporadic PD, which mainly occurs in people over the age of 60, one of the factors involved in the disease is the inability of the UPS to keep up with the requirements of the cell, leading to an overcrowding of toxic substrates, maybe as a result of oxidative stress, and eventually the formation of LB inclusions.

2.5

Other Genes Involved in Familial Parkinson’s Disease

Bonifati and co-workers have recently identified the DJ-1 gene encoding for an ubiquitous protein, mutation which leads to the appearance of monogenic early-onset recessive PD [102]. One of the PD-related mutations of DJ-1 (L166P) induces instability in the protein promoting its degradation through UPS [103, 104]; other patients showed gene deletion. This suggests that a loss of function, by a direct lack of protein or an increase in the protein degradation, results in dopaminergic neurodegeneration. Little is known about this protein; however it seems to be related to the cellular response to oxidative stress [105, 106]. Due to the novelty of this gene discovery, more research is required before determining the link between DJ-1 and PD [107]. Mutations in the gene encoding for the nuclear receptor Nurr1 (NR4A2) has been related to late-onset familial PD [108]. Nurr1 is related to dopaminergic differentiation, maturation and maintenance [109]. Mutations in this gene led to a decrease of Nurr1 mRNA and of TH mRNA, which would result in a disruption of the dopaminergic system.

2.6

Neurotoxic PD Models

During the last few decades, researchers have extensively used models of PD based in the use of neurotoxins. These models are based on local or systemic injections of substances leading to specific dopaminergic neurodegeneration. This selective neurodegeneration leads to the appearance of a Parkinson-like syndrome, which results from the lack of DA in the striatum. The behavioral deficits can be quantified in these animals, constituting a very useful experimental tool for the assessment of therapeutic strategies. The action of the neurotoxins involves at least some aspects of cellular processes related to the course of the disease such as oxidative stress, mitochondrial dysfunction, DA homeostasis or proteasome system deficiency.

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2.6.1

Oxidative Stress

Injection of 6-OHDA in SNc, striatum or the medial forebrain bundle of rats leads to efficient uptake into catecholaminergic neurons, followed by selective and robust degeneration by oxidative damage (for a review see [110, 111]). This neurodegeneration occurs quickly in a very narrow time frame when the drug is injected in SN or medial forebrain bundle, whereas retrograde nigral cell death is a slower process if the drug is released in the striatum [110]. In all these cases, 6-OHDA lesions in rats induce motor impairment, which is usually correlated to the bias in full body turns away from the site of lesion upon apomorphine injection. This effect is believed to be due to the supersensitivity of the DA receptors as a consequence of the lack of the neurotransmitter in the striatum. In addition quantification of rotations after amphetamine injection is a valuable tool to assess the releasable DA pool in the striatum. In this case, lesioned animals turn ipsilateral to the site of the lesion, due to an imbalance in DA content between intact and lesioned site. Alternatively, the lesion can be assessed by performing various drug-free tasks such as the cylinder test, the stepping test and/or the paw reaching test, both based on the ability of the rat to use the paw contralateral to the injection side versus the ipsilateral paw [112, 113]. 2.6.2

Dopamine Homeostasis/Complex I

Upon systemic administration, MPTP is converted to 1-methyl-4-phenylpyridnium (MPPþ) via monoamine oxidase B, is taken up by the DA transporter and leads to cell death by releasing DA from vesicles to the cytoplasm and by inhibiting complex I. This inevitably leads to energy failure, calcium imbalance and free-radical production [114–116]. The primate MPTP model is very useful given that it presents many of the characteristics of PD, such as increased vulnerability of the SNc, response to L-dopa and DA receptor agonists, and behavioral hallmarks such as rigidity, tremor and postural instability [117]. Certain mouse strains (C57Bl/6) are sensitive to this drug, but higher doses may be needed to cause DA depletion in other strains. MPTP lesions in mice show a high individual variability within groups, and spontaneous recovery can occur, making it difficult to assess the results of a treatment in long-term experiments. In addition, the fact that the lesion is bilateral also limits the ability to use a variety of behavioral tests. Some of the tests that are used in rats, such as cylinder tests or paw reaching tests are most valuable in unilaterally lesioned animals. Therefore, more general activity tests, such as open field, are used giving rather limited information about impairment in specific tasks involving limb use. In rats, local intracerebral injections of MPPþ in SNc lead to less robust dopaminergic neurodegeneration, compared to 6-OHDA-induced lesions, with a variable loss of fibers in the striatum.

2.7 Genetic Models of Parkinson’s Disease

More recently rotenone, a non-selective complex I inhibitor, has been used to generate dopaminergic lesion. When infused intravenously via an osmotic mini pump, rotenone can produce a hypokinetic syndrome characterized by bradykinesia, postural instability, unsteady gait and tremor and presents Lewy-body-like inclusions [118]. However, detailed histopathological data suggests that this syndrome may be the result of a degeneration of multiple brain nuclei, including, in addition to SN, the striatum, pedunculopontine tegmental nucleus and locus oeruleus, arguing against a selective action on dopaminergic neurons [119]. Nevertheless, further development of this model with an alternative dosing regime may produce a more representative model of PD [120]. 2.6.3

Proteasome

Due to the involvement of the proteasome in the degradation of damaged proteins, and the fact that the proteasome activity in the SN of PD patients was found to be reduced, a new model was recently described, based on the nigral administration of lactacystin, an inhibitor of 26/20S proteasome system in the rats. Such an approach induced degeneration in the nigral cells, which seemed to be restricted only to neurons with glial cells spared after the injection, except at the highest dose. In this model, a-syn accumulation and aggregation and intracytoplasmic perinuclear inclusions were observed. These changes are accompanied by spontaneous motor dysfunction evident as soon as 2 days post-surgery [121]. This motor behavior remained for 3 weeks and at this point in time animals exhibit contralateral turning upon apomorphine administration. Although this model sounds promising, the specificity of the lesion remains to be assessed. In fact, the accumulation of a-syn in the rats was not restricted to THþ cells only. In addition, as is the case of the other neurotoxic models, the toxicity over the nigral cells is exerted acutely. While this model could be useful to understand the involvement of proteasome system in PD, further studies and a complete characterization of the model need to be done.

2.7

Genetic Models of Parkinson’s Disease 2.7.1

Alpha-Synuclein Overexpressing Transgenic Animals

Transgenic models of PD based on the overexpression of a-syn have been developed not only in mice but also in Drosophila and C. elegans. Overexpression of a-syn in the Drosophila lead to an age-dependent loss of DA neurons, a-syn positive filamentous inclusions and locomotor dysfunction, as assessed by the climbing test [122, 123]. Similarly, dopaminergic neurodegeneration has been documented in C. elegans [124]. It is worth noting that both these species do not normally express

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a-syn. These transgenic animal models are very useful for studying molecular aspects of the disease based on genetic manipulations, but may not be sufficient to provide the required information on novel therapeutic strategies to be used in humans. For this purpose the majority of the experimental data is based on rodent models, while primates are used on selective cases for specific questions. Although a-syn overexpression in the rather simple organisms (worm and fly), consistently leads to a PD like neurodegeneration, this has not been the case in transgenic mouse lines. The first line developed by Masliah and co-workers, where human wt a-syn was expressed under the hPDGFb promoter, showed a reduction in striatal fiber terminal and tissue DA levels, which could be related to the motor impairment found in the mice. However, although a-syn and ubiquitin-immunoreactive inclusions were observed in SN, cell loss was not detectable [125]. Several other lines expressing a-syn under various promoters have been produced, and although they have been reported to generate widespread pathology in the CNS, none of them were able to replicate the selective dopaminergic cell death in SN. In an attempt to target a-syn expression to catecholaminergic neurons, the transgene was driven by the TH promoter in three different lines, however, none of these mice showed specific Parkinsonian pathology in the SN [126–128]. In one of the lines, elevated DAT levels in striatum were observed which could explain the fact that they observed an increased MPTP-induced neurotoxicity [128]. In addition, mice expressing a double mutated a-syn, A30P and A53T, under the regulation of the TH promoter showed an age-dependent dopaminergic dysfunction and motor impairment [128]. Other lines expressing A53T mutated a-syn under the mouse prion protein promoter developed intracytoplasmic neuronal accumulation of a-syn in various areas of CNS, including striatum or dorsal midbrain, but neither cell dysfunction nor death of dopaminergic cells [129, 130]. These mice showed age-dependent motor impairment which seems to be related to the observed motor neuron degeneration [129, 130]. Similarly, transgenic mice where a-syn was expressed under the Thy1 promoter showed degeneration in the spinal cord, where ubiquitin positive a-syn aggregations were observed. However, this promoter did not yield high levels of expression in the SN, nor did it result in dopaminergic cell death [131]. On the other hand, Kahle and collaborators have developed an independent line of mice where the Thy1 promoter drove the expression of a-syn and showed abnormal accumulations in SN and striatum, accompanied with the appearance of a detergent-insoluble fraction of a-syn [132, 133]. 2.7.2

Parkin-related Genetic Models

Since the mutations and genetic defects in the parkin gene related to AR-JP seem to result in a decrease or loss of function, genetic models, based on the deletion of the parkin protein, have been developed. The first line of parkin-deficient mice developed by Goldberg and co-workers has shown behavioral symptoms that may be related to nigrostriatal pathway dysfunction, though they showed no sign of LBs, neuronal loss or increase on parkin-interacting proteins. These mice showed

2.7 Genetic Models of Parkinson’s Disease

some alterations on the dopaminergic system, such as an increase in levels of extracellular DA and a decrease in synaptic excitability of striatal neurons suggesting a role of parkin in the regulation of DA neurotransmission [95]. These findings are consistent with the decrease in striatal levels of DAT and VMAT, and the age-dependent decrease of spontaneous and amphetamine-induced locomotor behavior reported in a second line of parkin-deficient mice [96]. In addition, the second mouse line also showed an increase in DA in limbic areas, and alterations of the dopaminergic metabolism in the striatum [96]. Conversely, parkin-null Drosophila did not show major dopaminergic alterations, although morphologically dopaminergic neurons were shrunken and their TH immunostaining seemed to be reduced [97]. Parkin-deficient animals can be very useful to study the involvement of parkin in the dopaminergic system and motor coordination. However, as these animals do not suffer from a PD-like neurodegeneration, they do not provide good PD models for studies aiming at the identification of new drugs with neuroprotective ability. Another way to trigger pathology secondary to parkin deficiency is to overexpress the substrates of parkin that are known to accumulate in the parkin mutant cases. One such example is based on the overexpression of Pael-R, a presumed G-protein coupled receptor, which is ubiquitinated by parkin (see Section 2.6.3). Pael-R overexpressing Drosophila showed a selective dopaminergic neurodegeneration although again no LB like inclusions were observed [94]. These flies could therefore constitute a better model of PD, or more specifically of AR-JP. 2.7.3

Viral Vectors for In Vivo Gene Transfer of Disease-related Proteins to the CNS Local Overexpression of alpha-synuclein Although transgenic mouse models overexpressing a-syn have failed to generate specific dopaminergic cell loss, introduction of disease-causing genes can also be achieved by somatic gene transfer, using recombinant viral vectors. This approach not only helps to efficiently express the gene of interest in non-dividing cells in the brain, but also provides us with a powerful and flexible tool that can be applied to any adult rodent or primate species. Three studies have been conducted using stereotaxic microinjections of recombinant adeno-associated virus (rAAV2) vectors to transduce nigral DA cells by various forms of a-syn [134–136]. The high affinity of the rAAV vectors to neurons in the SNc makes it possible to express the transgene in a majority of the nigral DA neurons in rats with a single injection of the purified high titer vector stock. rAAV vectors, used to overexpress a-syn in the rat or monkey SN, showed consistent transduction of i95 % of TH-positive cells [134, 135]. In the rat, human a-syn protein was detectable in the nigral neuronal cell bodies at 1-week, and had filled up the processes to the striatum by 3 weeks after the injection of the virus. In this early phase of a-syn overexpression – although no dopaminergic cell death was observed – there was a dopaminergic dysfunction in the nigra (DA and TH enzyme activity reduced by 50 % compared to contralat2.7.3.1

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Figure 2.3 Loss of TH-positive innervation in the striatum following lesions of the nigral dopamine neurons in two different models of Parkinson’s Disease. (A) Representative example of THþ fiber immunostaining of an intact striatum in a rat. Rarely small THþ varicosities were seen (arrow in A). (B) TH-immunostained section showing the extent of TH-positive striatal denervation 8 weeks after intranigral rAAV-a-synuclein injection, as compared with the intact striatum (A). Note in B that not only the density of the fibers is less as compared with the intact (A), but also that the remaining fibers contain large numbers of swollen, dystrophic axons terminals (arrows in B). (C) Loss of TH-positive fibers can be observed in the striatum of a rat after intrastriatal injection of 6-hydroxydopamine: however, the surviving fibers display no dystrophic axons or swollen fibers, but appear relatively normal. Scale bars: 10 mm.

eral untreated side). At 8 to 10 weeks after the transduction, typical PD like neurodegeneration was found and large a-syn-positive inclusions were seen throughout the remaining dopaminergic cells, with some showing shrunken cells bodies, and dystrophic projections (Fig 2.3). When THþ cells were quantified at 8 weeks in rAAV-a-syn injected animals (both in human wt and A53T), the magnitude of cell loss was found to average 50 %, compared to the contralateral side. Individual animals however, varied between 30 % and 80 % in the extent of neurodegeneration [134]. In primates, a similar degree of cell loss (40–75 %) was observed at 16 weeks post-transduction. Comparable neurodegeneration was found when rAAV vector was used to overexpress the A30P mutated a-syn [136]. Cell death seems to be completed by 8 weeks since in animals that were allowed to survive 6 months after transduction, no further decline in nigral TH-positive cells were observed. At 8 weeks-post-transduction, when cell loss in the SN had largely taken place, the reduction in cell numbers paralleled that of fiber innervation, TH enzyme activity and tissue DA levels [134]. This was accompanied by significant motor impairment in those animals whose cell death exceeded 50 %; a requirement previously reported in neurotoxic models of PD. Interestingly, in animals that were allowed to survive for 6 months TH-positive fiber terminal density had partially recovered, although this was not reflected in the biochemical analysis of the DA content or TH activity which remained at levels seen at 3 weeks. Consistent with the reduced levels of tissue DA, the percentage of animals that showed prominent impairments in tests of motor behavioral function, assessed by apomorphine-induced rotation and the skilled paw-use, remained unchanged [134]. In a parallel approach, rLV vector was used to locally overexpress a-syn in SNc [51]. This induced a similar a-syn aggregation in SN and striatum, and also caused selective dopaminergic neurodegeneration. Although the cell death was of a smaller magnitude (24-35 %), the alterations developed in a similar time frame of 3–6 weeks [51]. Interestingly, despite the fact that GABAergic neurons were also

References

transduced by the virus, no neurodegeneration was observed in this neuronal population, pointing out a selectivity in a-syn induced neurodegeneration restricted to THþ cells.

Local Overexpression of Parkin-related Proteins A similar approach of local transduction by viral vectors has been used to locally overexpress one of the proposed parkin substrates, CDCrel-1 [137]. The working hypothesis is again based in the suggested failure of parkin function as one of the factors contributing to PD, which will lead to accumulation of the parkin substrates. rAAV-2 was used to overexpress CDCrel-1 locally in SNc by stereotactically placed injection, which resulted in the transduction of the majority of the nigral neurons where the protein was detected as early as 1 week post-injection. Parkin also filled up the projections reaching striatum in a time-dependent manner and was highly expressed after 2 weeks. This overexpression induced a selective dopaminergic neurodegeneration, starting at 2 weeks and reaching an average loss of 78 % after 6 weeks, which was accompanied by a similar decrease of DA in the striatum. Interestingly, inhibition of the DA synthesis between day 9 and day 15 after virus injection, when the animals were killed, completely abolished the dopaminergic cell death induced by CDCrel-1, suggesting a DA-dependent toxicity. This model seems very promising to study involvement of parkin in PD, however, as in AR-JP, LB were not observed in the animals and no characterization of the behavior of these animals was performed. 2.7.3.2

Acknowledgements

We thank Bengt Mattsson for production of the figures.

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3 Amyotrophic Lateral Sclerosis Georg Haase

3.1

Human Motor Neuron Diseases

Motor neuron diseases are devastating, rapidly progressive and most often fatal disorders in which neurons controlling voluntary movement degenerate while other types of neurons are relatively unaffected. Human motor neuron diseases represent a spectrum of closely related disorders [1, 2] that are traditionally classified according to clinical and pathological criteria. Amyotrophic lateral sclerosis (ALS, Charcot disease or Lou Gehrig’s disease) is characterized by the degeneration of lower motoneurons in spinal cord and brainstem and also upper motoneurons in cerebral cortex [3]. Progressive bulbar palsy is an ALS form that predominantly affects brainstem motoneurons. ALS can be distinguished from progressive muscular atrophy, a condition in which only lower motoneurons are affected [4, 5] and from primary lateral sclerosis, a disease with pure involvement of upper motoneurons. Degenerating motoneurons exhibit various microscopic abnormalities such as accumulation of inclusion bodies, cytoskeletal abnormalities and loss of dendrites [1, 6]. A quantitative reduction of a-motoneurons in the spinal cord has been documented in the cervical [7] and lumbar [8] spinal cord. In ventral roots, myelinated fibers and large caliber fibers were found to be reduced in number [8, 9] whereas myelinated fibers of small and intermediate caliber corresponding, respectively, to autonomous neurons and g-motoneurons, were preserved [10]. As a consequence of denervation, skeletal muscle fibers become atrophic and angulated [11]. Reinnervation of muscle fibers from remaining intact motoneurons results in fiber type grouping as evidenced on immunohistochemical stainings for esterases, ATPases or myosin isoforms. While most ALS cases appear spontaneously, about 10 % are familial (FALS). These can show autosomal dominant, recessive or X-linked inheritance. Genetic studies in FALS are rendered difficult by the late onset of disease, its incomplete penetrance and the short survival of affected family members. Nevertheless, linkage studies and positional cloning have identified several familial ALS loci and Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

52

3 Amyotrophic Lateral Sclerosis Table 3.1 Gene mutations responsible for motor neuron diseases.

Gene

Disease

Locus

Inheritance

Mutation type

References

Sod1

ALS1

21q22.1

dominant*

missense

[13]

Alsin

juvenile ALS (ALS2) progressive lateral sclerosis

2q33

recessive

frameshift

[21, 22]

Tau

ALS/Dementia/ Parkinson complex

17q21

dominant

missense or intronic

[149]

2p13

dominant

missense

[150]

Dynactin (p150 subunit) Androgen receptor

Spinal and bulbar muscular atrophy (SBMA)

Xq12

recessive

CAG triplet expansion

[151, 152]

Survival motor Neuron (Smn)

SMA

5q13

recessive

deletion, missense

[153]

* with the exception of SOD1 D90A

genes [12] (Table 3.1). Only familial ALS forms linked to mutations in the SOD1 and alsin genes will be presented here. Other inherited motor neuron diseases, including spinal and bulbar muscular atrophy, spinal muscular atrophy and hereditary motor neuropathies, have been described in excellent reviews. 3.1.1

Familial ALS Linked to SOD1 Mutations

About 10-20 % of familial ALS forms (1–2 % of total) are linked to mutations in the superoxide dismutase 1 (SOD1) gene. SOD1 is ubiquitously expressed and encodes a dimeric protein. Each subunit contains 153 amino acids and one Cu and Zn ion in the active site. Over 100 different point mutations located at scattered sites throughout the protein have now been shown to produce disease. The vast majority of them show autosomal dominant transmission [13]. It has been proposed that mutant SOD1 might cause disease by catalyzing aberrant free radical chemistry, by perturbing axonal transport or function, by initiating protein aggregation or indirectly by causing excessive glutamate levels toxic to motoneurons. The arguments for and against these hypotheses have been reviewed in detail [14–17].

3.2 Cell Culture Models of Motoneuron Degeneration

3.1.2

Juvenile ALS Linked to Alsin Mutations

Familial ALS cases with disease onset in the first decade of life have been reported by [18] and [19]. Hentati et al studied a large consanguineous Tunisian family with slowly progressive autosomal recessive ALS and established linkage to chromosome 2q33-35 [20]. The responsible gene mutations were identified as small deletions in a new gene termed ALS2 or alsin [21, 22]. It encodes two transcripts of 2.6 kb and 6.5 kb length which can be detected in most tissue types [22]. Computer algorithms have predicted that the alsin protein contains guanine nucleotide exchange factor (GEF) domains for small GTPases. Small GTPases are molecular switches that cycle between an inactive GDP-bound and an active GTP-bound state; they are activated by GEFs which stimulate the exchange of GDP for GTP. Ikeda and colleagues showed that the 183 kD alsin protein acts as guanine-nucleotide exchange factor for the small GTPase Rab5, localizes to early endosomes and enhances their fusion [23]. All eight reported alsin mutations are predicted to result in a failure of Rab5 activation suggesting that they cause neurodegeneration by perturbing endosome dynamics.

3.2

Cell Culture Models of Motoneuron Degeneration

A perfect culture model of motoneuron degeneration should reflect the selective vulnerability of motoneurons, reproduce disease mechanisms and be suitable for high-throughput drug screening. Various culture systems using neuronal cell lines, primary motoneurons and organotypic slices are now available and fulfill these criteria to variable degrees. 3.2.1

Neuronal Cell Lines

Established neuronal cell lines are most often derived from tumors or in vitro transformed cells and only few resemble motoneurons. One of these exceptions is the NSC34 cell line that has been obtained by fusion of mouse neuroblastoma cells with embryonic spinal motoneurons. NSC34 cells display a multi-polar phenotype, express choline acetyltransferase (ChAT), different ion channels and neurofilaments, can generate action potentials and induce acetylcholine receptor clusters on co-cultured myotubes [24, 25]. To unravel the molecular consequences of mutant SOD1 expression, Shaw and colleagues examined stably transfected NSC34 cells by immunoblotting, gene expression profiling [26] and proteome analysis [27]. In cells expressing the SOD1 mutants G37R or G93A, the neurofilaments L and M and several glutathione S-transferases were found to be down-regulated [27, 28] and proteins involved in NO metabolism (argininosuccinate synthase, argininosuccinate lyase and neuronal NO synthase) were up-regulated [27].

53

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3 Amyotrophic Lateral Sclerosis

Some of these changes could be confirmed in mutant SOD1 mice after laser-microdissection of individual motoneurons [27]. Mutant SOD1 expression in NSC34 cells led to functional abnormalities such as decreased cell survival following oxidative stress [29], mitochondrial dysfunction and increased cytochrome c release [30]. 3.2.2

Isolated Motoneuron Cultures

Motoneurons are notoriously difficult to purify and to maintain in primary culture. Current methods rely on three properties of these cells: their characteristic size, their expression of specific surface markers and their dependence on trophic factors for survival and neurite outgrowth. Since motoneurons are larger in size and lower in density than most other neurons and glial cells, they can be enriched by differential centrifugation. Based on this observation, Schnaar and Schaffner collected relatively pure spinal cord motoneurons on the top of a metrizamide gradient [31]. As a further purification step, Henderson and colleagues developed immuno-panning techniques for chicken and rat spinal motoneurons using monoclonal antibodies that recognize specific cell surface antigens such as SC1, BEN or the p75 neurotrophin receptor [32]. More recently, mouse spinal motoneurons have been isolated using antibody-coated microbeads and subsequent magnetic cell sorting [33]. Once purified, motoneurons are seeded in culture dishes coated with appropriate substrates such as polyornithine and laminin. While many motoneurons die during the first hours of culture, the remaining cells develop typical morphologies and are able to survive for several weeks [34] when appropriate trophic factors are added. These procedures are currently limited to embryonic motoneurons (mouse E12, rat E14) from spinal cord and brainstem and not yet available for isolating cortical motoneurons. One of the first cellular motoneuron culture systems relevant to ALS was developed by Durham and colleagues [35]. When plasmid expression vectors for mutant and wildtype SOD1 were microinjected into isolated cultured motoneurons, abnormal protein aggregates appeared specifically in mutant SOD1-expressing cells [35]. Mutant SOD1 expression caused increased apoptotic cell death which could be reduced by co-expression of hsp-70 chaperones [36]. Using liposomes, Estevez and colleagues proposed that SOD1 toxicity was due to loss of zinc binding [37]. Pettmann and colleagues described that activation of the cell surface receptor Fas triggers death of embryonic motoneurons via two parallel pathways, the classical FADD/caspase-8 cascade [38] and a second cascade involving Daxx, ASK1, p38 and transcriptional up-regulation of neuronal NO synthase [39], see Fig. 3.1. They also found that motoneurons from transgenic mice for the SOD1 mutations G37R, G85R or G93A were much more sensitive to Fas- or NO-triggered cell death than motoneurons expressing wildtype human SOD1. No such sensitivity was observed in cerebellar neurons, DRG neurons or astrocytes, reflecting the selective vulnerability of motoneurons in living mutant SOD1 mice [39]. Despite their inherent technical difficulties, motoneuron cultures thus emerge as a promising

3.3 Animal Models of Motor Neuron Disease

tool to screen pharmacological compounds for their effects on disease-related molecular targets and cellular phenotypes. 3.2.3

Organotypic Cultures

In an attempt to study motoneurons in their normal cellular environment, several groups have prepared slice cultures from rat and mouse spinal cord [40–44]. These cultures could be maintained for up to two weeks (adult mouse) or even several months (perinatal rat) [42, 43]. Individual motoneurons in living slice cultures were identified by their large size, the presence of action potentials [41, 45] or retrograde DiI labeling prior to culturing [46]. In fixed slices, motoneuron cell bodies were identified and counted after Nissl staining [44] or immunocytochemical labeling for ChAT [47], Islet 1/2 [46] or Ret [44]. Neuronal outgrowth was visualized and quantified by labeling non-phosphorylated neurofilaments using the SMI-32 antibody [46]. Slice culture systems have also been developed for the study of cortical motoneurons [48, 49]. Slice cultures offer considerable advantages in comparison to isolated motoneuron cultures, but also have limitations: glial overgrowth is often observed and motoneurons have suffered an axotomy which disconnects them from their target muscles and may alter their cell death response. In the context of ALS research, organotypic culture systems have been pioneered by Rothstein and collaborators [40]. They showed that chronic inhibition of glutamate uptake caused slow degeneration of motoneurons. In this model, anti-excitotoxic agents such as CNQX [40], riluzole [50] and topiramate [51], nitric oxide synthase inhibitors [50] and neurotrophic factors such as IGF-1 and GDNF [52] protected against motoneuron degeneration. When organotypic slice cultures were derived from transgenic mutant SOD1 mice and exposed to excitotoxic stimuli, mutant motoneuron death could be blocked with a cyclo-oxygenase-2 inhibitor [53]. Br and colleagues used chronic administration of malonate, an inhibitor of mitochondrial electron transport, to induce motoneuron death in spinal cord slices. In this model, a number of anti-excitotoxic agents, anti-oxidants and caspase-inhibitors showed neuroprotective effects [47].

3.3

Animal Models of Motor Neuron Disease

This section will describe some popular animal models of acute and chronic motor neuron degeneration and discuss how experimental therapies can delay or slow down the degenerative process or improve the function of unaffected motor neurons.

55

56


3.3.1

Axotomy Models

In neonate animals, motor neuron death can be experimentally induced by a peripheral nerve lesion since developing motoneurons depend for their survival on contact with their target muscle and axonal environment. In the most widely used models, the sciatic, facial or hypoglossal nerves are crushed or sectioned and the survival of the lesioned motoneurons is assessed on sections, eventually after prior retrograde labeling. The extent of motor neuron death depends on the age of the lesioned animal and the duration of target deprivation. When the sciatic nerve lesion is performed in 1-day old rats, 90 to 100 % of the corresponding motoneurons are lost [54]. When the same type of lesion is performed at 4 or 5 days of age, 80 to 100 % of the motoneurons survive [55, 56]. Lesioning the peripheral nerve by section or close to the cell body both result in slower target re-innervation and reduced motoneuron survival as compared to lesioning the nerve by section or distally (see references in [57]). Axotomy-induced motoneuron death has many apoptotic features: First, morphological studies on lesioned motoneurons have provided evidence for DNA fragmentation [58], nuclear condensation and mitochondrial disruption [59]. Second, pharmacological inhibition of caspases [60, 61] or mitochondrial pore proteins [62] or over-expression of apoptosis-inhibitory proteins (IAPs) [63] reduced the extent of axotomy-induced motoneuron death. Finally, genetic studies in transgenic mice showed that over-expression of the anti-apoptotic gene Bcl-2 [64] or deletion of the pro-apoptotic gene Bax [65] protected against axotomy-induced cell death. The molecular events in apoptotic motoneuron death seem to involve activation of receptors to TNF [66, 67] and Fas [68] and intracellular production of nitric oxide [69, 70]. Axotomy models have been used to validate the role of various neurotrophic factors including CNTF [71], BDNF [72, 73], NT-4/5 [74], GDNF [75] and CT-1 [34] on motoneuron survival. More recently, several pharmacological compounds such as deprenyl, a monoamine oxidase B inhibitor [76], MK-801, a glutamate receptor antagonist [77], CGP3466B, a GAPDH antagonist [78] and riluzole [79] were shown to protect against axotomy-induced motor neuron death. Axotomy models have thus become popular “in vivo test tubes” that bridge the gap between in vitro cultures and motor neuron disease models.

3.2

Mutant SOD1 Mice

One major breakthrough in ALS research has been the generation of transgenic mutant SOD1 mice. Transgenic mice expressing the human ALS-linked SOD1 mutations G37R, G85R or G93A all showed typical symptoms of motor neuron disease such as progressive muscle atrophy and paresis, gait instability and premature death [80–83]. Disease onset and rate of progression were dependent on the num-

3.2 Mutant SOD1 Mice

ber of transgenes [80, 82, 84] and also the genetic background [85]. Affected mice displayed a reduction in the number of spinal motoneurons [80] and their axons [82] and also signs of astrogliosis [82]. Numerous other histopathological abnormalities found in these mice such as axonal spheroids [86], Lewy-body-like and ubiquitin-positive inclusions [82, 83, 87], fragmentation of the Golgi apparatus [88] and enlarged mitochondria [82, 89] also resembled those previously reported in human ALS [90–92]. Furthermore, electrophysiological studies in mutant SOD mice confirmed the presence of fasciculations and fibrillations, a decrease in the amplitude of nerve-evoked compound muscle actions potential, together with normal sensory conduction velocities [93]. Similar clinical, histological and electrophysiological signs were observed in transgenic rats expressing the SOD1 mutants G93A or H46R [94, 95]. To date, studies in transgenic mice have not unraveled the mechanism(s) whereby mutant SOD1 causes toxicity. The following observations suggest that motoneuron disease is not due to altered enzymatic SOD activity: 1. Some diseasecausing SOD1 mutations such as G93A [80] or G37R [82]) retain enzymatic activity while others such as G85R [83] are inactive. 2. Sod1 knockout mice [96] or transgenic mice overexpressing wildtype SOD1 do not develop ALS-like symptoms [97]. 3. The disease course is not modified in transgenic mutant SOD1 mice in which endogenous SOD activity is lowered (by crossing with Sod1 knockout mice) or raised (by crossing with mice transgenic for wildtype SOD1), see [97]. 4. Knocking out the copper chaperone CCS in mutant SOD1 mice leads to reduced SOD1 enzymatic activity in spinal cord but has no impact on disease onset or progression [98]. While the precise nature of mutant SOD1 toxicity remains controversial, its molecular consequences are beginning to be unraveled: these include sequential activation of caspases [99–101], up-regulation of pro-apoptotic proteins such as Bad and Bax [102], release of cytochrome c from mitochondria [101] and deregulation of the cyclin-dependent kinase cdk5 [103]. The cellular target(s) of mutant SOD1 toxicity also remain enigmatic. Disease in transgenic mice was observed only when the SOD1 transgenes were expressed ubiquitously, under the control of the human SOD1 promoter, but not in transgenic mice in which SOD1 expression was restricted to astrocytes [104] or to neurons [105, 106] (see Fig. 3.2 A-D). Even more puzzling was the observation that elevating mutant SOD1 G93A levels in neurons above ubiquitous mutant SOD1 expression (Fig. 3.2 E) did not exacerbate disease [106]. To begin to settle this issue, Cleveland, Julien and colleagues generated chimeric mice carrying mixtures of wildtype and mutant SOD1 expressing cells in the spinal cord [107], (Fig. 3.2 F). In these chimeras, they observed degenerative signs such as ubiquitin inclusions not only in mutant SOD1-expressing motoneurons, but also in wildtype motoneurons. Furthermore, survival of mutant motoneurons seemed to be enhanced by the presence of non-neuronal cells in their neighborhood. These results suggest that motoneuron degeneration depends on the presence of mutant SOD1 in non-neuronal cells such as interneurons, astrocytes, microglia or others. In the past decade, more than 70 different therapeutic approaches have been tested in mutant SOD1 mice. Only a few of them have resulted in robust positive

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58


Figure 3.1 Isolated motoneurons can be used to study mutant SOD1-related motoneuron death. A. Human superoxide SOD1 is expressed in cultured embryonic motoneurons from transgenic SOD1 G93A mice as revealed by immunolabeling. B. Treatment of SOD1 G85R motoneuron cultures with agonistic antibodies to the cell surface receptor Fas leads to increased apoptosis as detected by combined staining with DAPI (in blue) and immunolabeling for activated caspase 3. An apoptotic motoneuron with strong caspase-3 activation and nuclear condensation c (red arrow) can be distinguished from healthy motoneurons displaying only weak caspase-3 activation and normal chromatin structure (white arrows). Scale bars : 25 mm. C. Motoneurons from transgenic mutant SOD1 G93A, G85R and G37R mice show higher susceptibility to cell death triggered by agonistic anti-Fas antibodies than motoneurons from mice expressing wildtype SOD1. D. Model of Fas-triggered motoneuron death. Cell death involves the classical FADD/ caspase-8 pathway and a parallel pathway leading from Daxx, ASK1 and p38 activation to transcriptional up-regulation of neuronal NO synthase (nNOS) and NO production. The presence of mutant SOD1 sensitizes motoneurons to Fas agonists and NO. Potential sources for these cell death triggers are astrocytes and microglia. Figure 3.2 Motoneuron degeneration in transgenic ALS mice is influenced by non-neuronal cells. n The consequences of mutant SOD1 expression in different cell types of transgenic mice are schematically illustrated on spinal cord cross sections. Mouse genotypes are indicated. Motoneurons are depicted as multipolar cells and glial cells as round cells. Mutant SOD1 expression (mSOD) in these cell types is indicated in gray or black, normal (endogenous) SOD1 expression


is shown inwhite. A. Normal situation. B. In transgenic mice, ubiquitous expression of mutant SOD1 causes typical motor neuron disease. C, D. No disease is observed in transgenic mice expressing mutant SOD specifically in glial cells (C) or in neurons (D). E. Crossing of Thy1: SOD1 mice with transgenic SOD1 G93A mice elevates mutant SOD levels (in black) in motoneurons above the ubiquitous expression levels but does not exacerbate disease. F. Chimeric mice that carry a mixture of wildtype and mutant SOD expressing cells in their spinal cord show later disease onset and longer lifespan than their mutant littermates. These improvements correlate with the proportion of wildtype cells within individual spinal cords. Ubiquitin inclusions are found not only in mutant but also in wildtype motoneurons. Interestingly, mutant SOD motoneurons in the chimeras seem to be protected by wildtype non-neuronal cells in their environment. Taken together, these data suggest that motoneuron degeneration is the result of a complex interplay between motoneurons and surrounding non-neuronal cells. References are given in the text section.

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effects (see Table 3.2). Following treatment with riluzole, currently the only FDAapproved drug for ALS, the mean survival of SOD G93A mice was improved by 11 %, but histological or functional effects could not be documented [108]. Beal and colleagues showed that feeding the animals with creatine increased survival and provided some motoneuron protection [109]. Numerous ALS patients have since then taken creatine, but a recent placebo-controlled clinical trial disclosed any beneficial effect of oral creatine at 10 g per day [110]. Encouraging results were obtained by Friedlander and colleagues by intrathecal infusion of the broad caspase inhibitor zVAD-fmk [111]. Recently, several groups have also reported positive effects of minocycline which might be related to the inhibitory actions of this tetracycline analogue on caspase activation, mitochondrial cytochrome c-release [112], cdk5 deregulation or microglial cell activation [113]. When minocycline was administered in combination with riluzole and the calcium channel blocker nimodipine, the therapeutic benefit was further enhanced [114]. Neurotrophic factors have been regarded with some skepticism despite their robust survival effects in neuronal cultures and axotomy models. This certainly reflects their failure in clinical trials where they were delivered by subcutaneous injection or via minipumps. To deliver these factors in a more constant and targeted manner, several groups have developed gene therapy methods. Adenovirus or AAV vectors coding

Table 3.2 Selected therapeutic trials in mouse motor neuron disease models.

Treatment

Delivery Begin* Mode

Outcome Lifespan Neuroincrease muscular function

SOD G93A

early

oral

11 %

not reported

not reported

[108]

pmn

early

oral

I10 %

yes

not reported

[154]

Creatine

SOD G93A

early

oral

18 %

yes

yes

[109]

Minocyclin

SOD G37R

early

oral

z 6%

yes

yes

[113]

Minocyclinþ Riluzoleþ Nimodipin

SOD G37R

early

oral

13 %

yes

yes

[114]

ZVAD-fmk

SOD G93A

early

intrathecal 21 %

yes

yes

[111]

NT-3 Adeno

pmn

early

i. m.

50 %

yes

yes

[125]

GDNF AAV

SOD G93A

early

i. m.

14 %

yes

yes

[116]

CT-1 Adeno

SODG93A pmn

early early

i.m i. m.

8% 18 %

yes yes

yes yes

[115] [127]

IGF AAV

SOD G93A

late

i. m.

18 %

yes

yes

[117]

Riluzole

Model

Motoneuron protection

Reference

* Begin of treatment is classified with respect to disease onset: “early” treatments are initiated in the presymptomatic period, “late” treatments are started at or after clinical disease onset.


for cardiotrophin-1 [115], GDNF [116] and more recently IGF-1 [117] were injected into skeletal muscle of mutant SOD1 mice. These treatments resulted in significantly improved neuromuscular function, reduced motoneuron degeneration and increased survival of the treated animals. These studies are encouraging, but leave open important questions. Are the observed effects due to release of the factors into the bloodstream or to retrograde vector transport from muscles to spinal motoneurons? What are the best combinations of factors? Can neurotrophic factors be targeted to motoneurons in order to reduce their unwanted side effects on nonneuronal cell types [118]? 3.3.3

pmn Mice

Mice with progressive motor neuronopathy (pmn) [119] suffer from an even more severe form of motor neuron disease than mutant SOD1 mice. Mice homozygous for the mutation develop first symptoms at two weeks of age when hindlimb and pelvic girdle muscles become atrophic. The disease rapidly extends to the forelimbs and other muscle groups and leads to death around 40 days of age, most probably by respiratory failure. Electromyography showed fibrillations indicating neurogenic atrophy [120]. Histopathological analyses have revealed a massive loss of motor axons in peripheral nerves and ventral roots [119] and also a reduction in the number of motoneurons in brainstem nuclei [121, 122]. Therapeutic studies in this model have been facilitated by the introduction of the semidominant phenotypic marker Extratoe (Xt) close to pmn [123]. In this system, homozygous Xt pmnþ/Xt pmnþ mice die in utero, double heterozygous Xtþ pmn/ Xt pmnþ mice (“Xt mice”) can be recognized by the presence of a clearly visible extra digit on their hindfeet, and homozygous Xtþ pmn/Xtþ pmn mice (“pmn mice”), identified by a normal number of digits, can be treated as early as in the neonate. The pmn mice have paved the way to the development of new therapeutic strategies including gene therapy. Sendtner and colleagues [121] first described that implantation of genetically-engineered tumor cells secreting CNTF in pmn mice reduced degeneration of phrenic motor axons and facial motor neuron cell bodies. Similar results were obtained by Aebischer and colleagues who subcutaneously implanted encapsulated fibroblasts [124]. Using intramuscular injection of adenovirus vectors, our group showed that the neurotrophic factors NT-3 [125], CNTF [126] and CT-1 [127] were able to significantly improve neuromuscular function, reduce motoneuron degeneration and increase lifespan of treated pmn mice. More recently, we showed that a non-viral gene transfer method, intramuscular injection and electroporation of plasmid DNA [128], was as efficient as the adenovirusbased strategy to attenuate disease symptoms. These studies provided a proof of concept, but the relationship of pmn to human motor neuron disease remained uncertain. Recently, the pmn mutation was identified as a point mutation in the Tbce gene [129, 130] which encodes one of five chaperone proteins that assist in the folding of tubulins, the constituents of microtubules [131]. In collaboration with Gunet and colleagues we showed that Tbce is

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expressed in various tissues and cells, including spinal motoneurons. We also demonstrated that the pmn mutation at the last residue of TBCE results in decreased protein stability, diminished concentrations of tubulins in peripheral nerves and progressive loss of microtubules [129]. In line with these findings, Sendtner and colleagues reported that isolated cultured motoneurons from embryonic pmn mice exhibit shorter and more irregularly shaped axons than wildtype motoneurons [130]. Human TBCE gene mutations have been described in Eastern Arabian subjects suffering from inherited Sanjad Sakati syndrome with hypoparathyroidism, mental retardation, facial dysmorphism and eventually osteosclerosis and fulgurant infections [132]. It is thus conceivable that the TBCE mutations in humans and mice impair different functions of the TBCE chaperone or that motor neuron disease symptoms in the human patients are obscured by the other even more devastating symptoms.

3.4

Future Neuroprotective Approaches 3.4.1

Axonal Protection

In the past, numerous studies have focused on the apoptotic death of motor neurons in ALS. Recent studies however suggest that the axon degenerates weeks to months before the cell body: Electrophysiological and histopathological studies for instance demonstrated that the axon is affected early in patients with ALS [133] and spinal muscular atrophies (SMA) [134] and also mouse models of ALS [93, 135] and SMA [136]. Moreover, the molecular mechanisms of axonal degeneration seem to differ from those involved in cell body apoptosis. This is nicely illustrated by experiments in mice with the semi-dominant mutation Wallerian degeneration slow (WldS, Ola). In normal mice, a peripheral nerve lesion leads to axonal disintegration and reactive proliferation of Schwann cells in the distal nerve stump, a process called Wallerian degeneration. In WldS mice however, the distal nerve stump remains intact following a lesion and continues to conduct action potentials over several weeks. Coleman, Perry and colleagues have identified the WldS mutation as a tandem triplication on mouse chromosome 4 [137, 138] that results in production of a chimeric protein [139] that contains the N-terminus of the ubiquitination factor Ube4b fused in frame to the enzyme nicotinamide mononucleotide adenylyltransferase (Nmnat). The WldS (Ube4b/Nmnat) fusion protein protects axons not only against lesion injury, but also against several other forms of injury. Taxol neurotoxicity for instance was attenuated in WldS mice [140] and Vincristin neurotoxicity was inhibited in cultured sensory neurons transduced with an adenoviral WldS vector [141]. Crossing WldS mice with pmn mice reduced loss of neuromuscular synapses and motor axons and increased the animals lifespan by 50 % [142]. Beneficial effects of WldS have also been observed in P0 knockout mice, a model of peripheral neuropathy [143], and, according to a preliminary report, in

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mutant SOD G93A mice [144]. The mode of action of WldS seems to be indirect since the WldS protein is localized to the nucleus, but not the axon [145]. 3.4.2

Mutant SOD1 Knockdown by RNA Interference

A new therapeutic approach in mutant SOD1-related ALS is based on the RNA interference technique (RNAi). RNA interference has been discovered as an endogenous gene silencing mechanism in which double-stranded RNA triggers the degradation of homologous messenger RNAs with extraordinary sequence specificity [146]. The mediators of mRNA degradation are small, 21 to 23 nucleotide long, RNAs (siRNAs). Since almost all toxic SOD1 mutations represent single nucleotide substitutions, it is in principle possible to design RNA sequences that specifically silence mutant SOD1 alleles [147] without affecting the expression of the wildtype allele. These RNAs are introduced into the target cells by transfection of doublestranded oligonucleotides or expressed as small hairpin RNAs from plasmid DNA vectors [147] or lentiviral vectors [148]. Further studies will be required to determine the appropriate target cell and the level of gene silencing necessary to achieve therapeutic effects remain to be determined.

3.5

Conclusion

In the last 15 years, experimental research in motor neuron diseases has greatly benefited from the discovery of human SOD1 gene mutations and the development of new cell culture and animal models. In cellular models, motoneuron degeneration can be caused by diffusible factors such as excess glutamate, reactive oxygen and nitrogen species, etc. Intracellular events comprise activation of caspases, damage to mitochondria and aggregation or deregulation of cellular proteins. Some of these mechanisms have also been shown to contribute to motor neuron disease in vivo, in transgenic mutant SOD1 mice. More recently, axon degeneration has been identified as an early disease phenomenon and the molecular mechanisms of motor axon maintenance begin to be unraveled. Translating these findings into new clinically relevant therapies will be the major challenge of the future.

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4 Alzheimer’s Disease and Other Neurodegenerative Diseases Philipp J. Kahle and Christian Haass 0) Abbreviations

Ab, amyloid b-peptide; AD, Alzheimer’s disease; LB, Lewy body; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine; MSA, multiple system atrophy; PD, Parkinson’s disease; PDGF, platelet-derived growth factor; PrP, prion protein; SN, substantia nigra; SYN, synuclein; TH, tyrosine hydroxylase; wt, wild type.

Abstract

Although a-synuclein (aSYN) is commonly associated with Parkinson’s disease, it is of wider relevance for neurodegenerative dementias. aSYN comprises the major building block of Lewy bodies, which occur not only in Parkinson’s disease, but also in a variety of neurodegenerative dementias, including Alzheimer’s disease. Several invertebrate and vertebrate animal models have been generated, based on transgenic expression of human aSYN. Ectopic aSYN expressed in Drosophila melanogaster was deposited in Lewy body-like structures, and caused loss of dopaminergic neurons. As the flies aged, they displayed movement dysfunction. The phenotype appeared to depend on aSYN misfolding because it was ameliorated by stimulation of the molecular chaperone Hsp70. A number of transgenic mouse models have been generated using a variety of promoters to express human wild-type and mutant aSYN. Somatodendritic accumulation of the transgenic aSYN with neuritic distortions was a common finding. In some mouse models aSYN aggregated into typical fibrillar amyloid. Phenotypic mice showed hyperphosphorylation and oxidation of aSYN, whereas ubiquitynation was a less consistent post-translational modification in transgenic mice. Concomitant with the formation of fibrillar aSYN deposits, the animals developed locomotor dysfunction. It will be interesting to investigate the cognitive function in mouse models of cortical a-synucleinopathy. Transgenic synuclein animal models will contribute to the molecular understanding of neurodegenerative diseases, and provide a test bed for anti-aggregation and neuroprotection therapies.


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4.1

Introduction

a-synuclein (aSYN) was originally identified in the electric organ of Torpedo californica [41] and its canary ortholog “synelfin” was found to be involved in songbird learning [13]. aSYN first became suspicious when its central fragment was isolated as “non-amyloid component” from Alzheimer’s disease (AD) plaques [67]. aSYNimmunoreactive dystrophic neurites surround plaques in AD brain [7, 42] and transgenic mouse models [64, 65, 73]. Moreover, aSYN inclusions (Lewy bodies; LBs) are found in the limbic regions of AD patients, especially those with familial AD [22, 39, 51]. Missense mutations [32, 54, 74] and triplications [10, 58] of the aSYN gene are associated with autosomal-dominant hereditary Parkinson’s disease (PD). aSYN is the major fibrillar component of LBs and Lewy neurites [59], the hallmark lesions in the brains of patients with PD and other LB diseases (dementia with LBs, LB variant of AD, and neurodegeneration with brain iron accumulation type 1, formerly known as Hallervorden-Spatz syndrome) [18]. Interestingly, the synaptic protein aSYN is not only deposited in neuronal inclusions, but also constitutes the main fibrillar component of glial cytoplasmic inclusions that characterize multiple system atrophy (MSA) [18]. The synucleins comprise a family of three homologs: aSYN, bSYN, and gSYN [14]. aSYN and bSYN are widely expressed in brain neuropil [38]. A striking difference in the primary sequences of aSYN and bSYN is the absence of 11 amino acids in the central domain of bSYN, which has dramatic consequences for the pathological aggregation properties. Whereas aSYN has a strong tendency to self-aggregate, bYN does not [15, 27]. In fact, bSYN even inhibits the pathological aSYN aggregation [23, 50, 68]. The physiological roles of the synucleins remain to be elucidated in detail; current data suggests that aSYN regulates neurotransmission [1, 6].

4.2

Transgenic Invertebrates

Ectopic expression of wild-type (wt) aSYN and (A53T)aSYN in Caenorhabditis elegans caused motor deficits when pan-neuronal (aex-3) or motoneuron-specific (acr-2, unc-30) promoters were used [33]. Specific expression in the four pairs of dopaminergic neurons was achieved with a dat-1 promoter; these worms showed some 25 % reduction of dopaminergic markers without obvious inclusion body formation [33]. The first animal system that comprised the most wanted features of a PD model, namely fibrillar aSYN aggregates and degeneration of dopaminergic neurons, was the transgenic Drosophila melanogaster. The molecular pathology in this invertebrate model shared with human Lewy pathology the presence of electron-dense aSYN filaments [11] as well as age-dependent, disease-specific post-translational

4.3 Transgenic Mice

modifications of aSYN, namely ubiquitination and phosphorylation of serine-129 [4, 62]. aSYN transgenic flies developed an age-dependent locomotor phenotype that read out as failure to climb up a straw. Pharmacological evidence linking the locomotor phenotype with the dopaminergic neuron loss came from the finding that L-DOPA and dopaminergic agonists rescued this phenotype, whereas dopaminergic antagonists exacerbated the locomotor deficiency in the aSYN flies [52]. Thus, aSYN transgenic D. melanogaster is a highly promising model organism for the rapid screening of novel anti-PD drugs. The neurotoxic effect of aSYN misfolding in the D. melanogaster model was demonstrated by the suppression of dopaminergic neuron loss upon genetic and pharmacological up-regulation of the molecular chaperone Hsp70 [3, 4]. Interestingly, Hsp70 gene transfer prevented dopaminergic neuron loss despite the continued buildup of aSYN inclusions. Thus, it may not be the mature LBs that mediate neurotoxicity, but rather some toxic folding intermediate [19, 70].

4.3

Transgenic Mice

The fact that synucleins are only encoded in vertebrate genomes might explain the relative ease to elicit aSYN fibrillization and dopaminergic neuron loss in D. melanogaster, which contrasts with the remarkable capability of transgenic mice to withstand adverse effects of transgenic aSYN expression for a rather long period of time. Nevertheless, a number of transgenic mouse models have been recently developed that recapitulate aSYN pathology. 4.3.1

Tyrosine Hydroxylase (TH) Promoter

Expression of wt or mutant aSYN in transgenic mouse catecholaminergic neurons led to specific accumulation of transgenic aSYN within dopaminergic cell bodies [45, 55]. A significant fraction of the transgenic aSYN was also detected in the striatum, indicating that aSYN mutations do not cause a loss of synaptic transport. Moreover, (TH)-(A30P)aSYN mice were not sensitized to 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) toxicity, judging from the normal THþ neuron count in the substantia nigra (SN) and unaffected levels of striatal dopamine [55]. Richfield et al. engineered transgenic mice to express an aSYN molecule harboring both the A30P and A53T mutation in one molecule under control of the TH promoter [56]. The cellular and subcellular distribution of double-mutant aSYN was similar to the single-mutant mouse models. However, these transgenic mice showed a marked loss of locomotor activity in the acute phase of MPTP intoxication, and amphetamine-stimulated ambulance was reduced in the double-mutant transgenic mice [56]. In addition, the (TH)-(A30P/A53T) aSYN mice became significantly less active upon aging, concomitant with an age-dependent reduction of dopamine and its metabolites in the striatum [56]. Thus, expression of a potentially

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very aggressive double-mutant aSYN leads to adverse effects in the nigrostriatal dopamine system, causing behavioral deficits in transgenic mice. 4.3.2

Platelet-derived Growth Factor-b (PDGFb) Promoter

Masliah et al. employed the pan-neuronal PDGFb promoter to drive expression of human wt aSYN in transgenic mice. The highest-expressing mouse line showed granular inclusions of transgenic aSYN in neuronal cell bodies and neurites [43]. There was a slight reduction in the density of THþ terminals in the striatum, and aged transgenic mice displayed some impaired locomotor performance on the rotarod [43]. This relatively mild phenotype aggravated after crossbreeding with a mouse line in which transgenic expression of human amyloid precursor protein, bearing both the Swedish and the London familial AD mutations, causes a very high plaque load [44]. The rotarod deficits develop at younger ages in the bigenic mice, accompanied with a greater number of aSYN inclusions. Thus, the highly fibrillogenic amyloid b-peptide (Ab42) may stimulate aSYN aggregation and hence, LB formation. Indeed, unlike the classical course of a-synucleinopathy that emerges from the brain stem [5], in familial AD aSYN aggregation appears to be locally induced in limbic areas such as the amygdala [39]. Although Ab42 induces aSYN aggregation in vitro [44], it is not known how the predominantly extracellular Ab42 impinges upon the intracellular protein aSYN. Nevertheless, a similar pro-aggregative effect of Ab42 has been reported for the aggregation of tau into neurofibrillary tangles [21, 37]. A potential drug target might arise from the finding that bSYN is not only incapable of forming pathological fibrils, but actually inhibits aSYN aggregation [23, 50, 68]. When (PDGFb)-h(wt)aSYN mice were crossbred with transgenic mice expressing bSYN throughout the brain, a significant reduction of the number of aSYN inclusions was detected [23]. Likewise, the reduction of THþ synaptic terminals was partially ameliorated upon crossbreeding with bSYN mice, and there was a slight improvement of locomotor function. Because a molar excess of bSYN is required to prevent fibrillization of the very abundant aSYN protein [23, 68], the therapeutic potential of full-length bSYN is probably very limited. It will be interesting to identify the molecular determinants of the anti-amyloidogenic properties of bSYN and to develop small molecule aSYN aggregation inhibitors based on this principle. 4.3.3

Thy1 Promoter

Van der Putten et al. described a transgenic mouse model in which human mutant (A53T)aSYN was expressed under control of the CNS neuron-specific promoter, Thy1. Somatodendritic accumulation of granular aSYN was prominent throughout the brain and spinal cord, and ubiquitin was occasionally detected in these pathological profiles [69]. (Thy1]-(A53T)aSYN mice displayed a rapid deterioration of

4.3 Transgenic Mice

rotarod performance and died prematurely. The severe locomotor phenotype was ascribed to degeneration of the neuromuscular junctions [69]. Kahle et al. employed the Thy1 promoter to drive human (A30P)aSYN and (wt)aSYN in CNS neurons [26, 27]. The subcellular distribution of the majority of transgenic aSYN was normal. Even the mutant (A30P)aSYN with its apparently reduced affinity for lipid membranes was effectively transported to the synapses where it loosely interacted with small vesicles [26]. Thus, aSYN mutations appear not to cause PD by loss of synaptic vesicle binding. However, a significant fraction of transgenic aSYN, wt as well as mutant, accumulated in a detergent-insoluble form within neuronal cell bodies and swollen neurites [27]. At this early stage, there was no sensitization of (Thy1]-(A30P)aSYN mice to MPTP [55], in accord with the results from (TH)-(A30P)aSYN mice (see above). This early phenotype was subsequently found to aggravate in an age- and gene dose-dependent manner. The detergent-insoluble, non-fibrillar transgenic aSYN within neuronal cell bodies and neuritic swellings appears to be tolerated by (Thy1]-(A30P)aSYN mice for several months. However, upon aging, some of the transgenic aSYN protein acquires a fibrillar “amyloid” conformation, as evidenced by electron microscopy, thioflavin S staining, and proteinase K resistance [47]. Moreover, the pathological phosphorylation at serine-129 [12] became apparent at this stage [47]. Additional pathological modifications included oxidation and ubiquitination [47], both established markers of human a-synucleinopathy. Thus, Lewy pathology was recapitulated in this mouse model, and closely correlated with the onset of a lethal locomotor phenotype. The mice became hypokinetic, and within a few weeks, gait and posture disturbances aggravated until the mice showed spastic paralysis of the limbs. The animals eventually lost their capabilities to rear and move, so that they were incapable of nourishing themselves and required euthanasia. Loss of locomotor control is likely to be due to neurodegeneration, which was evident from massive astrogliosis in brain stem and spinal cord. However, the SN was remarkably spared from pathology, and the levels of dopamine and its metabolites were normal even in end-stage (Thy1]-(A30PaSYN mice [47]. Thus, fibrillization of aSYN leads to neurodegeneration in vivo. As in early-stage human patients, the brain stem is a predilection site of a-synucleinopathy in transgenic mice. In midbrain, however, the reticular formation and deeper layers of the superior colliculus rather than the SN are affected in the transgenic mice [48]. 4.3.4

Prion Protein (PrP) Promoter

Neurotoxic fibrillization of transgenic aSYN in the brain stem and spinal cord with preserved SN is not a particularity of (A30PaSYN driven by the Thy1 promoter, but was also described for mice that expressed high levels of (A53TaSYN under control of a PrP promoter [16, 36]. Transgenic aSYN fibrils could be isolated and characterized by immuno-electron microscopy [16] as well as with biochemical methods [16, 36]. The predilection sites of transgenic a-synucleinopathy in these animal models included spinal cord, brainstem, deep cerebellar nuclei, and parts of the

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midbrain. Thus, these brain regions contain neuronal subpopulations that are particularly vulnerable to a-synucleinopathy upon strong overexpression of transgenic aSYN. Short-term electrophysiological consequences of aSYN overexpression were reported. In hippocampal slice cultures of (PrP)-(A30P)aSYN mice, paired-pulse depression was enhanced [61]. The converse was reported for aSYN knockout mice [1], suggesting a role for aSYN in controlling excessive neurotransmitter release. Adverse effects of aSYN overexpression only became apparent upon aging. With increasing age of the animals, neurodegeneration became evident through GFAP-positive astrogliosis [16, 20, 36], ultimately leading to a specific locomotor phenotype. After several months of viable and fertile life, the mice develop hypokinesia and dystonia (hunchback). Tremulous movements are occasionally noted. The limbs eventually become paralyzed, so that the mice are incapable of feeding themselves and die of dehydration. This lethal phenotype typically progresses over a time course of few weeks. The nigrostriatal dopamine system was not impaired, even in end-stage animals. Thus, late-stage symptoms of the recently developed transgenic aSYN mouse models were remarkably similar, regardless whether PrP or Thy1 promoters were used, or whether the A53T or A30P mutant aSYN was expressed [16, 20, 36, 47]. Transgenic overexpression of aSYN in neurons therefore recapitulates human a-synuclein pathology and demonstrates the neurotoxicity of aSYN fibrillization. 4.3.5

Glial Expression

In addition to the classical Lewy pathology within neurons, aSYN inclusions have been observed in glial cells [24, 72]. In fact, (oligodendro)glial cytoplasmatic inclusions of aSYN are diagnostic for MSA [2, 60, 71]. Biochemical studies revealed the pathological nature of aSYN deposited in white matter extracted from MSA patients [8, 9, 66]. In order to study the consequences of ectopic aSYN expression in oligodendrocytes in vivo, transgenic mouse models were generated in which human aSYN was driven by oligodendrocyte-specific promoters. Using a myelin protolipid protein promoter, Kahle et al. achieved sustained expression of transgenic aSYN in white matter throughout the brain [28]. Unlike the disease-irrelevant control green fluorescent protein, expressed under control of the same promoter, which evenly distributed throughout oligodendrocytes, aSYN was asymmetrically clustered in the cytosol of oligodendrocytes. The transgenic aSYN-positive profiles resembled the typical triangular and half moon-shaped glial cytoplasmic inclusions of MSA patients [34] at the light microscopic level. Moreover, the pathological phosphorylation at serine-129 was recapitulated in (PLP)-aSYN mice. Biochemical analysis confirmed the pathological insolubility of hyperphosphorylated, transgenic aSYN in this mouse model. However, in contrast to transgenic mice expressing high levels of aSYN in neurons, oligodendroglial transgenic aSYN did not fibrillize upon aging. Possibly oligodendrocyte-specific promoters do not

4.4 Viral Models

lead to very high cytosolic aSYN concentrations, or oligodendrocytes have a greater capacity to suppress aSYN misfolding, either because of an abundance of small heat shock proteins (such as aB-crystallin) [46] or because oligodendrocytes have a lower susceptibility to pro-aggregative risk factors compared to aged neurons. One such risk factor might be concomitant tauopathy. Giasson et al. employed a 2’,3’-cyclic nucleotide 3’-phosphodiesterase promoter to express human aSYN in transgenic mouse oligodendrocytes [17]. No particular pathology was noted in these animals unless they were cross-bred with mice that express [P301L]tau in oligodendrocytes [17]. A subset of bigenic oligodendrocytes was thioflavin S-positive, and it is tempting to speculate that in oligodendrocyte cytosol, aSYN and tau synergistically form individual fibrils, as was shown in vitro [17]. Tau pathology has been noted in some MSA individuals [17, 25, 53], but more systematic studies are warranted to distinguish the potentially synergistic co-fibrillization of tau and aSYN from, e. g., co-morbid MSA and progressive supranuclear palsy [63]. An alternative approach leading to a mouse model with aSYN mismetabolism in white matter (oligodendrocytes) was to overexpress the a1B-adrenergic receptor in transgenic mice [49]. Systemic overexpression of a constitutively active mutant a1B-adrenergic receptor caused multiple organ failures, including locomotor dysfunction that could be ameliorated with L-DOPA [75]. Administration of the a1-adrenergic receptor antagonist terazosin prolonged survival and reduced the pathology in this animal model [49]. It will be interesting to determine whether genuine aSYN fibrils form in this model, and how a-synucleinopathy contributes to the generalized neurological phenotype in this mouse model [57].

4.4

Viral Models

In striking contrast to the apparent resistance of the nigrostriatal dopamine system of aSYN transgenic mice, acute viral aSYN gene delivery into the SN of adult rats caused loss of dopaminergic cells. When packed into an adeno-associated virus vector, both (A30P)aSYN [31] and (A53T)aSYN [29] administered to rat midbrain caused a selective loss of roughly half the THþ neurons in the SN. Lentiviral delivery of wt and both mutant forms of aSYN into rat SN caused a similar selective reduction of THþ neurons [40]. Loss of nigral dopaminergic neurons was accompanied by a reduction of striatal dopaminergic innervation [29, 40]. As expected for such partial lesions, only mild, drug-induced locomotor deficiencies were observed [29]. Neurodegeneration in viral models was demonstrated by fluoro-jade staining [29, 35, 40]. Similar to the early a-synucleinopathy that characterizes most transgenic mouse models, virally delivered aSYN forms granular aggregates within neuronal cell bodies as well as in swollen, dystrophic neurites. Essentially the same results (nigrostriatal dopamine depletion and somatodendritic accumulation of adeno-associated virally delivered aSYN) were achieved for marmosets [30], validating this experimental system for non-human primates.

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4.5

Conclusion and Outlook

Unlike in D. melanogaster whose genome does not encode synucleins and therefore may not have evolved cellular anti-aggregation (chaperone) systems against a-synucleinopathy, the formation of aSYN fibrils in mammals may require a long incubation time of high transgenic human aSYN concentrations. These conditions are fulfilled in some transgenic mouse models. Molecular defense mechanisms may protect transgenic mice from a-synucleinopathy for many months, but might exhaust upon aging. Then aSYN fibril formation becomes neurotoxic, ultimately leading to the lethal locomotor phenotype of some aSYN transgenic mice. It will be interesting to study cognitive deficits in transgenic a-synucleinopathy models, in order to provide a model for LB dementias.

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Abeliovich A, Schmitz Y, Farias I, Choi-Lundberg D, Ho W-H, Castillo PE, Shinsky N, Garcia Verdugo JM, Armanini M, Ryan A, Hynes M, Phillips H, Sulzer D, Rosenthal A (2000) Mice lacking a-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25: 239–252. Arima K, Uda K, Sunohara N, Arakawa K, Hirai S, Nakamura M, Tonozuka-Uehara H, Kawai M (1998) NACP/a-synuclein immunoreactivity in fibrillary components of neuronal and oligodendroglial cytoplasmic inclusions in the pontine nuclei in multiple system atrophy. Acta Neuropathol 96: 439–444. Auluck PK, Bonini NM (2002) Pharmacological prevention of Parkinson disease in Drosophila. Nat Med 8: 1185–1186. Auluck PK, Chan HYE, Trojanowski JQ, Lee VM-Y, Bonini NM (2002) Chaperone suppression of a-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science 295: 865–868. Braak H, Del Tredici K, Rb U, de Vos RAI, Jansen Steur ENH, Braak E (2003) Staging of brain pathology

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5 CNS Inflammation Christine Stadelmann, and Wolfgang Brck

5.1

Introduction

Multiple Sclerosis is a chronic neurological disease affecting approximately one million young adults worldwide with major personal and socio-economic consequences. Approximately 50 % of MS patients will become dependent on a walking aid, and a significant percentage of these patients will need a wheelchair after 15 years of disease duration. Natural history studies have shown unequivocally that a progressive disease course is the most important determinant of a poor outcome in MS. Pathologically, multiple sclerosis (MS) is regarded as a chronic inflammatory disease leading to multifocal demyelinated plaques in the central nervous system (CNS). The pathological hallmarks of the lesions are (1) an inflammatory infiltrate consisting of T cells, few B cells and macrophages/microglia, (2) demyelination in the white and gray matter with loss of oligodendrocytes in the chronic disease stage, (3) damage to the axons with significant axonal loss and (4) gliosis with astrocyte proliferation and intensive glial fiber production [1; 2]. The majority of MS patients starts with a relapsing-remitting disease course in which the inflammatory-demyelinating component of the disease is predominant. In the progressive disease stage, which may be either primary or secondary, an additional neurodegenerative component appears to be involved [3]. This neurodegeneration leads to extensive neuroaxonal damage in the MS brain [4]. Axon loss seems to be the major determinant of the persistent neurological deficit in progressive MS [5]. However, there is still no therapy available to protect axons and neurons in MS – in contrast to the early inflammatory demyelinating phase in which anti-inflammatory and immunomodulatory therapy strategies were established [6–9]. The lack of neuroprotective therapies in MS may be due to the fact that the exact mechanisms and effector molecules of axonal and neuronal degeneration are still not defined, and any axon- or neuroprotective therapy is yet to be established.


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5.2

The Pathologic Characteristics of the MS Plaque

The traditional description of an MS lesion is the demyelinated plaque in the white matter of the CNS with relative axonal sparing and glial scar formation [1]. In addition, remyelination may occur in MS lesions, particularly at early stages of the disease [10]. Studies in recent years added significant knowledge to the disease concept and changed the view of MS pathology. Traditionally, MS has been considered a T cell-mediated autoimmune disorder which leads to myelin and oligodendrocyte destruction. Recent studies on a large collection of MS lesions, however, have indicated that the structural and immunopathological features of MS plaques are extremely variable and the events in MS immunopathogenesis may be more complicated [11–15]. This is not surprising, given the heterogeneity observed with respect to the clinical, radiographic, genetic and morphological features of the disease. Oligodendrocytes and the myelin sheath are the major targets of the autoimmune response in MS. Both necrosis and apoptosis of oligodendrocytes have been described in MS. Oligodendrocytes are susceptible to damage by a broad panel of immunological or toxic effectors, including cytokines such as TNFalpha [16], reactive oxygen or nitrogen species, excitatory amino acids such as glutamate [17], complement components, proteolytic and lipolytic enzymes, T cell-mediated injury via T-cell products (perforin/lymphotoxin) [18], the interaction of Fas antigen with Fas-ligand [19], interaction of TRAIL with its receptor [20], CD8þ class I MHC-mediated cytotoxicity [21] or persistent viral infection [22]. Remyelination during the early stages of MS lesions can be extensive [23, 24], but clearly depends on the presence of oligodendrocytes within the lesion [25–27]. Systematic studies on larger samples of MS tissue suggested that the fate of the oligodendrocyte as well as evidence for oligodendrocyte apoptosis is uniform among all the active lesions in a single patient, but is extremely variable when lesions are compared between patients [15, 28]. These data suggest that the mechanisms of tissue injury in MS differ between individual MS patients. Recent analyses of MS lesions confirmed this hypothesis and showed differences in the plaques concerning the nature of the inflammatory infiltrate, oligodendrocyte preservation or loss, damage to the axon and degree of demyelination or remyelination. These observations suggest that the pathogenic events involved in MS may be more complicated than in purely T cell-mediated autoimmune disease [13, 15]. This concept of a heterogenous MS lesion development was supported by the detection of different immunopathological mechanisms of myelin destruction in actively demyelinating MS lesions. The majority of active MS plaques were characterized by T cell/macrophage-mediated myelin damage or by the deposition of activated complement components in the areas of active demyelination. Other cases showed phenomena similar to those observed during a primary oligodendrocyte dystrophic process with either a selective loss of myelin-associated glycoprotein (MAG) and oligodendrocyte apoptosis, or degeneration of oligodendrocytes in a small rim of periplaque white matter adjacent

5.3 Axonal Pathology in MS

to sites of active demyelination [13]. These observations suggest that different pathogenic mechanisms of demyelination may operate in different subgroups of MS patients.

5.3

Axonal Pathology in MS

Although MS is generally considered an inflammatory demyelinating disease with relative axonal sparing. Pathology of axons including loss, transections or disturbed axonal transport, however, have long been recognized in MS pathology [29]. The timing in relation to the disease stage and plaque evolution as well as the effector mechanisms of axon damage, however, have not yet been identified. Recent studies suggested that axon damage occurs early in the disease and during lesion development. Two different phases of axon degeneration were characterized, the first occurring during active myelin breakdown and the second in chronic demyelinated plaques in which the naked axon seems more susceptible to further damage. The major impact of axon degeneration for the MS patient is its massive effect on the permanent neurological deficit, for which it appears to be the major determinant [5]. Loss of axons as a permanent consequence of axon damage in chronic lesions on the one hand, and acute axonal damage on the other, have been described in detail in several recent studies. In chronic MS lesions, a significant reduction in axon density of about 60 % has been observed [30, 31]. Axonal loss has also been detected in the otherwise unaffected MS white matter [32, 33]. The loss of axons is likely to be the effect of accumulation of extensive axon damage that occurs from the early disease stages on. Markers for identifying acute damage to axons in MS tissue sections exist and include (1) dephosphorylation of neurofilaments, (2) disturbances of axonal transport, (3) expression of specific calcium channels or even (4) axonal transections [34–36]. Disturbances of axonal transport or axonal transections are best detected by proteins which are transported along the axons. These proteins accumulate at sites of transport disturbances or transections. A commonly used marker is amyloid precursor protein (APP) which is produced in neurons and transported in the periphery along the axonal cytoskeleton. Subtle changes in axonal transport or axon transections lead to APP deposits in the axon that are easily detectable by immunocytochemistry. In an autopsy study of multiple sclerosis patients, transected axons inside the lesions were more frequent in areas with a higher number of inflammatory cells than in hypocellular areas [34]. Further systematic analyses showed that axonal damage occurs within both active and chronic MS plaques and is also present in the apparently normal white matter (NAWM) of MS brains [37, 38]. The timing and pathogenesis of axon destruction, however, are not yet exactly defined. Axon damage may occur either in parallel with myelin destruction or in a second phase when the axon is demyelinated and more susceptible to any damaging event. Recent studies confirmed both of these two phases of axon degen-

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eration [38, 39]. A high amount of axon damage occurs when the myelin sheath is being attacked, suggesting that axonotoxic components are present among the inflammatory mediators damaging the myelin/oligodendrocyte unit. The second phase of axon degeneration concerns the naked, demyelinated axon which may be more susceptible to soluble or cellular immune mediators present in the chronic plaque. It is also possible that axons lose trophic support from the oligodendrocyte – a hypothesis which is supported by the fact that remyelinated axons are protected from further damage [39]. However, the exact mediators of axon damage have not yet been clarified. The mechanisms mediating axonal damage are not yet clear. However, inflammation may be involved in axonal degeneration as the number of APP expressing axons correlated with the number of certain inflammatory cell types [37, 39]. This finding is in agreement with other reports [35]. Macrophages/microglia and CD8positive cells are the candidate cell types which were identified to correlate with acute axonal injury. There is some experimental data which support this hypothesis on the engagement of macropghages/microglia and CD8-positive cells in axonal injury [40]. There is evidence from experimental studies that many cellular, soluble or trophic factors may be involved in mediating axonal damage. In MS lesions, as described above, there is a correlation between the extent of axon damage and the severity of inflammation during active demyelination. An association was particularly found between the numbers of CD8þ T cells and the extent of axon damage [39]. In vitro, CD8þ T cells were shown to transsect MHC class I positive neurites [41]. Additionally, in an animal model of multiple sclerosis, axon damage was absent in MHC class I-deficient mice [40]. Macrophage-derived toxic molecules may also lead to axonal damage. Nitric oxide or glutamate have been shown to cause axonal degeneration in vitro and in vivo [42, 43]. Axonal integrity clearly depends on the interaction between the axon and the myelin-forming cell, the oligodendrocyte which delivers trophic survival signals to the axon. If this interaction is interrupted, as is the case in myelin-deficient animals, axonal degeneration occurs spontaneously [44]. The final execution phase in the destruction of the axonal cytoskeleton is the influx of Ca2þ ions into the axon which then activates a broad range of proteinases such as calpain [45, 46]. This activation finally leads to the disintegration of the axon. The latter process may be prevented by blocking Naþ or Ca2þ entry into the axon [47, 48].

5.4

Neuronal Pathology in Multiple Sclerosis

Demyelination in the cortex with formation of so-called cortical plaques has long been described in the MS literature. A systematic description of these lesions identified seven plaque types, depending on the topography within the cortex [49]. The main groups of cortical plaques are located in the subpial superficial cortical layers, at the cortical-subcortical junction or completely intracortically. Recent studies con-

5.5 Lessons from Animal Models

firmed these findings and focussed on the extensive subpial demyelination in MS cases [50]. These cortical plaques are generally not associated with an increased lymphocyte infiltration [51], and microglial activation predominates in cortical MS plaques [52]. Apoptotic neurons have been detected in the demyelinated cortex and this may impact on neuronal and cognitive disability in MS. Additionally, in an animal model of MS, experimental autoimmune encephalomyelitis (EAE), apoptotic neurons were detected in the retinal ganglion cell layer [53] and reduced synaptic and dendritic density was found in the spinal cord [54]. These data indicate that MS is not restricted to the white matter, but also involves the gray matter matter with damage of neurons. Since the main therapeutic approaches at the moment focus on modulating the immune system, new concepts have to be developed to protect the axon and the neurons in MS.

5.5

Lessons from Animal Models

Stimulated by findings in imaging and pathology of the human disease, mechanisms of axonal and neuronal damage in inflammatory CNS disease have been the focus of recent studies in autoimmune, viral, and transgenic non-inflammatory animal models. Axonal and neuronal damage may be caused by either a direct attack of cellular or humoral immune mediators on neuronal structures or by indirect damage through immune reactions directed at the myelin/oligodendrocyte unit. Furthermore, demyelination alone may suffice to cause axonal degeneration and in any case render axons much more vulnerable to an inflammatory environment. The neuronal cell body may be damaged directly by inflammatory mediators or suffer indirectly after axonal transection close to the cell body; atrophy (retrograde degeneration) or cell death may ensue. An important role of T cells, especially CD8-positive T cells, in mediating axonal damage has been shown in a viral encephalomyelitis model, thus supporting data from the human disease [40, 55]; a direct attack of T cells on neurons in EAE is suggested by Smith et al. [56]. However, it is not clear thus far whether specific cellular immunity directed against axonal/neuronal antigens is operating in autoimmune as opposed to viral models of CNS inflammation. Various soluble mediators have been implicated in axonal damage, such as an excess of glutamate [56, 57], of nitric oxide [42], and an increase in intraaxonal Ca2þ levels [36]. A combination of nitric oxide exposure and electrical activity may cause permanent axonal damage [42]. Furthermore, demyelination per se may not only render the axon more vulnerable to pro-inflammatory mediators in the lesion: a disturbed myelin/oligodendrocyte unit such as is seen in transgenic models lacking myelin proteins (and with no obvious disturbance in myelination) leads to axonal degeneration independent of demyelination and inflammation, and suggests an important role of myelin/oligodendrocytes in the functional integrity of axons [44, 58]. Mechanisms of neuronal cell death are less well examined. In an animal model of optic neuritis, death of retinal ganglion cells was found to follow a caspase-3-dependent apoptotic pathway

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[53, 59]. Recently, interest has been stimulated not only in neuronal cell death, but also in more subtle changes in neuronal functioning in CNS inflammation. Consequently, a reduction of synaptic markers was found in an autoimmune EAE model [54]. In summary, T cell-mediated cytotoxicity, cytokines such as nitric oxide, ionic imbalances and energy failure caused by mitochondrial damage have been suggested to be mediators and mechanisms of axonal and neuronal damage in CNS inflammation. However, further research particularly into more functional neuronal changes is necessary, given the recent finding of extensive cortical involvement in MS patients.

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Prineas JW, Barnard RO, Kwon EE, Sharer LR, Cho ES (1993) Multiple Sclerosis: remyelination of nascent lesions. Ann Neurol 33:137–151. Raine CS, Scheinberg L, Waltz JM (1981) Multiple sclerosis. Oligodendrocyte survival and proliferation in an active established lesion. Lab Invest 45:534–546. Lucchinetti CF, Brck W, Rodriguez M, Lassmann H (1996) Distinct patterns of Multiple Sclerosis pathology indicates heterogeneity in pathogenesis. Brain Pathol 6:259–274. Kornek B, Lassmann H (1999) Axonal pathology in multiple sclerosis. A historical note. Brain Pathol 9:651–656. Mews I, Bergmann M, Bunkowski S, Gullotta F, Brck W (1998) Oligodendrocyte and axon pathology in clinically silent multiple sclerosis lesions. Mult Scler 4:55–62. Lovas G, Szilgyi N, Komoly S, Palkovits M, Komoly S (2000) Axonal changes in chronic demyelinated cervical spinal cord plaques. Brain 123:308–317. Ganter P, Prince C, Esiri MM (1999) Spinal cord axonal loss in multiple sclerosis: a post-mortem study. Neuropathol Appl Neurobiol 25:459–467. Evangelou N, Esiri MM, Smith S, Palace J, Matthews PM (2000) Quantitative pathological evidence for axonal loss in normal appearing white matter in multiple sclerosis. Ann Neurol 47:391–395. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L (1998) Axonal transection in the lesions of multiple sclerosis. New Engl J Med 338:278– 285. Ferguson B, Matyszak MK, Esiri MM, Perry VH (1997) Axonal damage in acute multiple sclerosis lesions. Brain 120:393–399. Kornek B, Storch MK, Bauer J, Djamshidian A, Weissert R, Wallstroem E, Stefferl A, Zimprich F, Olsson T, Linington C, Schmidbauer M, Lassmann H (2001) Distribution of a calcium channel subunit in dystrophic axons in multiple sclerosis

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and experimental autoimmune encephalomyelitis. Brain 124:1114– 1124. Bitsch A, Schuchardt J, Bunkowski S, Kuhlmann T, Brck W (2000) Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation. Brain 123:1174– 1183. Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, Linington C, Schmidbauer M, Lassmann H (2000) Multiple sclerosis and chronic autoimmune encephalomyelitis. A comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am J Pathol 157:267–276. Kuhlmann T, Lingfeld G, Bitsch A, Schuchardt J, Brck W (2002) Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time. Brain 125:2202–2212. Rivera-Quinones C, McGavern D, Schmelzer JD, Hunter SF, Low PA, Rodriguez M (1998) Absence of neurological deficits following extensive demyelination in a class I-deficient murine model of multiple sclerosis. Nature Med 4:187–193. Medana I, Martinic MA, Wekerle H, Neumann H (2001) Transection of major histocompatibility complex class I-induced neurites by cytotoxic T lymphocytes. Am J Pathol 159:809– 815. Smith KJ, Kapoor R, Hall SM, Davies M (2001) Electrically active axons degenerate when exposed to nitric oxide. Ann Neurol 49:470–476. Werner P, Pitt D, Raine CS (2001) Multiple sclerosis: altered glutamate homeostasis in lesions correlates with oligodendrocyte and axonal damage. Ann Neurol 50:169–180. Griffiths I, Klugmann M, Anderson T, Yool D, Thompson C, Schwab HM, Schneider A, Zimmermann F, McCulloch M, Nadon N, Nave K-A (1998) Axonal swellings and degeneration in mice lacking the major proteolipidprotein of myelin. Science 280:1610–1613.

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References erative CNS diseases. Trends Neurosci 25:313–319. 56 Smith T, Groom A, Zhu B, Turski L (2000) Autoimmune encephalomyelitis ameliorated by AMPA antagonists. Nature Med 6:62–66. 57 Pitt D, Werner P, Raine CS (2000) Glutamate excitotoxicity in a model of multiple sclerosis. Nature Med 6:67–70. 58 Garbern JY, Yool DA, Moore GJ, Wilds IB, Faulk MW, Klugmann M, Nave K-A, Sistermans EA, van der Knaap MS, Bird TD, Shy ME, Kam-

holz JA, Griffiths IR (2002) Patients lacking the major CNS myelin protein, proteolipid protein 1, develop lenght-dependent axonal degeneration in the absence of demyelination and inflammation. Brain 125:551–561. 59 Diem R, Hobom M, Maier K, Weissert R, Storch MK, Meyer R, Bahr M (2003) Methylprednisolone increases neuronal apoptosis during autoimmune CNS inflammation by inhibition of an endogenous neuroprotective pathway. J Neurosci 23:6993– 7000.

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6 Neurotrauma Ibolja Cernak, Paul M. Lea IV, Alan I. Faden

6.1

Introduction

The goal of experimental models of traumatic brain injury (TBI) is to reproduce selected pathological components of clinical injury in experimental animals in order to address pathology and/or treatment [1]. The design and choice of a specific model should reflect the purpose of the research. Thus, characterization of the biomechanics of injury may require a different model from the evaluation of molecular mechanisms of tissue loss, or the efficacy of novel therapeutic interventions [1]. While earlier modeling efforts were focused on biomechanical aspects of brain injury [2, 3], recent models are more targeted towards improving the understanding of complex molecular destructive cascades initiated by injury [4, 5]. Regardless of the goals, it is imperative that the chosen experimental model fulfills the following requirements: (1) the mechanical force used to inflict injury is controlled, reproducible, and quantifiable; (2) the inflicted injury is reproducible, quantifiable, and parallels components of human conditions; (3) the injury outcome, measured by morphological, physiological, biochemical, or behavioral parameters, is related to the mechanical force causing the injury; and (4) the intensity of the mechanical force utilized to produce injury should predict the outcome severity [1]. Although in vivo models have many advantages over in vitro models, including the ability to study trauma-induced changes in behavior, physiology, metabolism, the blood/tissue interface, the blood brain barrier, and/or inflammatory- and immune-mediated responses, they inherently possess multiple interacting variables. The complexity of these interactions hinders research efforts to distinguish key mechanisms associated with neuronal cell death and the subsequent functional deficits. Therefore, a number of in vitro models have also been developed over the past two decades to address specific underlying mechanisms of posttraumatic cell loss. This chapter reviews in vivo and in vitro experimental models that have been developed to investigate physiological, cellular, and molecular mechanisms of TBI. Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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6.2

In Vivo Models

There are differing opinions regarding the preferred species used for modeling human TBI [6]. Many investigators have accepted rodent models as the most suitable choice for neurotrauma research; the relatively small size and cost of rodents enables repetitive measurements of morphological, biochemical, cellular and behavioral parameters that require relatively large numbers of animals. Such studies are less feasible in species that are phylogenetically higher than rodents because of ethical, technical and/or financial limitations. On the other hand, it has been argued that the lissencephalic rodent cortex is inappropriate for modeling the more complex changes in the anatomy of the human cortex. Moreover, differences between rodent and human systemic physiological and behavioral responses to neurotrauma have also raised concerns about the use of rodents [7]. Nonetheless, rodents remain the most frequently used animals for modeling human TBI. Several reviews [1, 7, 8] have presented somewhat differing classifications of experimental TBI models. The pioneering work by Denny-Brown and Russell [2] distinguishes two major categories of experimental brain injury: acceleration concussion and percussion concussion. Later classifications of experimental TBI models have often been based upon this work [7, 8]. In this chapter, we present a different classification (Fig. 6.1), based upon the biomechanical paradigms of TBI initially proposed by Ommaya and colleagues [9, 10]. As shown in Figure 6.1,

Figure 6.1 Schematic representation of in vivo experimental models of traumatic brain injury.

6.2 In Vivo Models

mechanical force causes either dynamic or static brain injury, depending on its the amplitude, duration, velocity and acceleration. 6.2.1

Static Brain Injury Models

In static models the amplitude and duration of the mechanical force is defined, whereas velocity and acceleration are irrelevant. For example, crushing a cranial nerve with a forceps for a defined period of time would be considered a static CNS injury model [11]. By their nature, the static models generally focus on morphological and functional processes involved in injury [11, 12]. 6.2.2

Dynamic Brain Injury Models

Mechanical force characterized by movement (i. e. amplitude, duration, velocity and/or acceleration) causes dynamic brain injury, which can be further subdivided into direct and indirect injury. Direct dynamic brain injuries are induced by a mechanical force being targeted at the head and/or brain, whereas in the case of indirect dynamic brain injury, the mechanical force is usually directed at the whole body with the kinetic energy of the oscillating pressure waves that traverse the body imparting their effects on brain tissue.

Indirect Dynamic Brain Injury Exposure to blast overpressure that is generated during an explosion is known to primarily damage organs that contain air or that consist of structures with different specific densities (ear, lungs, intestine) [13]. However, recent experimental studies have shown that peripheral blast trauma, without direct head injury, also initiates secondary alterations in the brain. Presumably, this brain injury is produced through afferent hyperexcitability, increased neurotransmitter and autacoid release, and/or kinetic energy transfer of the blast overpressure to the central nervous system (CNS) [14, 15]. The induction system used in blast exposure models consists of a cylindrical metal tube that is closed at one end. The anesthetized rats are fixed individually in special holders designed to prevent any movement of their body in response to the blast. The blast over- and under pressure waves are then generated by detonation of plastic explosive in the closed end of the tube. The animals are usually subjected to a blast wave with mean peak overpressure of between 154 and 340 kPa on the nearest surface of the animal’s body, with the pressure sustained for varying durations of time [15, 16]. With moderate blast injury in animals, swelling of neurons, an astroglial response, and myelin debris have been found in the hippocampus [15]. Immunohistochemical findings have shown significant damage to the neuronal cytoskeleton in layers II-IV of the temporal cortex, in the cingulated gyrus and the piriform cortex, the dentate gyrus and the CA1 region of the hippocampus up to 7 days following the blast exposure [16]. Develop6.2.2.1

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ment of oxidative stress, alterations in antioxidant enzyme defense systems [15], increased nitric oxide metabolism, and subsequent cognitive deficits [17] have also been reported. These results suggest that the pattern of blast-induced neuronal dysfunction is similar to that found in direct traumatic brain injury. Moreover, it has been subsequently shown that the rodent blast exposure model of indirect neurotrauma can produce many features of brain damage observed in humans injured by an explosion [18, 19]. Thus, the experimental rodent model can serve as useful tool to investigate mechanisms of prolonged neuronal dysfunction following blast exposure, and to test potential therapeutics.

Direct Dynamic Brain Injury Direct dynamic brain injuries can be classified as impact or non-impact/acceleration head injury models depending on the existence of a direct impact and/or acceleration. Both impact and non-impact injuries can be further sub-grouped depending on the head motion at the moment of injury; namely, whether the head motion is constrained to a single plane, or the head is unconstrained and permitted to move freely [1]. 6.2.2.2

Impact brain injury Penetrating head injury/direct brain deformation In penetrating head injury/direct brain deformation models, the impact energy is delivered to the brain parenchyma through a skull perforated by a missile or a craniotomy. a) Fluid Percussion Models

One of the most frequently used direct brain deformation models is fluid-percussion injury (FPI), found to be suitable for the study of injury pathology [20, 21], physiology [22, 23] and pharmacology [24, 25] in a wide range of species, including rats [26, 27], mice [28, 29], cats [30, 31], pigs [32, 33], rabbits [34], dogs and sheep [35]. The insult is initiated by application of a fluid pressure pulse to the intact dura either centrally (vortex) [36, 37], or laterally in combination with or without a contralateral skull opening [38–40]. Briefly, the anesthetized animals are placed in a stereotaxic frame and their scalp and temporal muscles reflected. A small craniotomy, usually located midway between the lambda and bregma sutures, is made to allow insertion of a Leur-Loc that is cemented into place. The fluid-percussion device consists of a Plexiglas cylindrical reservoir filled with sterile isotonic saline; one end includes a transducer that is mounted and connected to a tube that attaches through a male Leur-Loc fitting to the female Leur-Loc cemented on the animals skull at the time of surgery. A pendulum strikes a piston at the opposite end of the device, generating a pressure pulse that is delivered to the intact dura, and causes deformation of underlying brain. The degree of injury depends on the intensity of the pressure pulse [38, 41]. In general, central and lateral fluid percussion injuries have similar pathobiology, although distinct differrences have also been found between the two models

6.2 In Vivo Models

[7, 42]. While the lateral FP injury model causes primarily unilateral cortical damage, rarely involving the contralateral cortices and brain stem, central FP generates bilateral cortical alterations associated with direct axial movement of the lower brainstem [7, 8]. The immediate physiological response to both types of FP injuries includes alterations in blood pressure (mainly a transient hypertension) [26, 38], brief respiratory arrest [43, 44], increased craniocerebral pressure [32], reduced cerebral perfusion pressure [32], decreased cerebral blood flow [32] and elevated cerebral vascular resistance [32]. Changes in cerebral blood flow [32, 45, 46] and increased permeability of the blood-brain barrier also have been observed in both FP models [37, 38, 47]. Histopathological alterations reported in these models include petechial hemorrhage in the brain parenchyma, ranging from minor to fatal confluent hemorrhage, axonal damage, subarachnoid hemorrhage, tissue tears followed by focal necrosis and cell loss, and characteristic vascular damage at the gray/white interface defined as “gliding contusion” [36, 38, 43, 48–52]. Both necrotic and apoptotic types of neuronal cells have been shown in these FP models [52–54]. Central and lateral FP models have been associated with altered ionic homeostasis, including increases in intracellular calcium [55, 56] and tissue sodium [57–59], impaired potassium equilibrium [58, 60, 61], and decrease in intracellular free magnesium [25, 62–64]. Hyper- and hypometabolism have been reported in both experimental models [65, 66]. EEG depression [26, 38, 67, 68], motor [25, 36, 69], behavioral and cognitive deficits [70–73] have also been described following both types of fluid percussion injuries. It is noteworthy that the central FPI reproduces only limited injury dose-response because of death due to autonomic dysfunction at hypothalamic levels. Consequently, the resulted alterations in bioenergetics and cerebral blood flow are also limited, compared to the lateral FP method. Conceptually, this explains apparent controversies in the literature. The lateral fluid percussion model provides an injury that mimics clinical contusion without skull fracture, and manifests a direct relationship between the majority of pathological alterations and injury severity. Hence, it is widely utilized in neurotrauma research for both mechanistic studies and for drug screening. However, FP injuries of increased severity cause increased morbidity, mainly due to disproportional involvement of the brain stem and development of neurogenic pulmonary edema. These and other limitations, such as limited biomechanical control and inability to mimic the entire complexity of human TBI, initiated the search for alternative TBI models. b) Controlled Cortical Impact Models

Controlled cortical impact (CCI) models are sometimes described as rigid percussion models [7, 8]. This mode of injury has been used in the ferret [74], rat [75, 76], and mouse [77]. Since this model allows for better control over mechanical factors, such as velocity of impact and depth of resulting deformation, it offers potential advantages over the fluid percussion model for biomechanical studies of injury. The controlled impact is delivered to the intact dura by a compressed air-driven metallic piston producing deformation of the underlying cortex [75, 78]. Briefly,

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the device used in rodent CCI models consists of a pneumatic cylinder usually with a 4–5 cm stroke, which is mounted on a cross bar so that the position of the impactor can be adjusted. The impact velocity used in the majority of studies is between 0.5 and 10 m/s depending on the air pressure that drives the impactor. Duration of the impact (dwell time) can be adjusted between 25 and 250 msec, whereas the depth of cortical deformation controlled by vertical adjustment of the crossbar holding the cylinder can be varied between 1 and 3 mm. It has been shown that cerebral hemodynamic responses such as increased intracranial pressure, reduced blood and cerebral perfusion pressures [78], histological [76, 79] and cellular alterations [4, 80, 81], as well as functional deficits [77, 82] are related to both the depth of the deformation and the velocity of the impact. Impacts performed at a velocity higher than 4.3 m/s (4.3–8.0 m/s) and with a depth of cortical deformation from 1.0 mm demonstrate widespread acute and chronic neuronal injury [79, 83–85]. Pathoanatomical alterations, including diffuse axonal injury, have been shown in white matter of the subcortical region and cerebellar folia, internal capsule, thalamic nuclei, midbrain, pons and medulla [1, 8]. This model also causes coma of varying length [8]. In general, controlled cortical impact injury provides a more focused injury compared to lateral fluid percussion, which may have implications with respect to the behavioral suppression and/or functional changes resembling coma [86]. Taken together, the pathobiology of controlled cortical impact injury mimics alterations seen in clinical head injuries such as brain edema [87], increased intracerebral pressure, decreased cortical perfusion [78], decreased cerebral blood flow [88], neuroendocrine and metabolic changes [89], and coma [8]. Not surprisingly, this model, which mimics clinical brain injury with skull deformation and resulting cortical compression, is widely used to analyze complex molecular [90–93] and genetic [94, 95] mechanisms underlying neuronal cell death and subsequent neurological dysfunction following TBI. It is also a useful tool for developing novel therapeutic approaches for brain injuries [96–103]. c) Other Direct Brain-Deformation Models

Various other experimental models have been developed to produce dynamic cortical deformation, including: a vacuum pulse of clinically relevant duration (I100 ms) [104, 105]; microinjection of a fluid containing zymosan (an inert particulate macrophage activator), lipopolysaccharide, and latex microspheres into the brain parenchyma to induce progressive cavitation [106]; a mechanical suction force applied through the intact dura [107]; focal cortical cryolesion [108]; among others [109, 110] Although these models use different forms of a mechanical force to induce cortical injury, each of them essentially produces localized morphological alterations with corresponding metabolic effects. Because of the absence of extensive diffuse injuries, these models do not cause significant long-term deficits that would be of importance to clinical TBI. However, they are useful in studies analyzing pathoanatomy of localized contusions and/or for quality control of treatments aimed to reduce the size of focal lesions [1].

6.2 In Vivo Models

d) High-velocity Missile Injury

Experimental models of traumatic craniocerebral missile injury have been developed to study cerebral pathophysiology of a missile wound to the brain [111– 115]. The model developed by Carey et al. [115–117] used anesthetized mongrel cats placed in a stereotaxic frame, wherein the sloping outer wall of the right frontal sinus was removed, thus allowing the missile to penetrate the intact and vertically disposed posterior sinus wall. A 2-mm, 31-mg steel sphere fired from a distance of 80 cm at either 220 mps or 280 mps entered the right frontal bone and traversed the right cerebral hemisphere from anterior to posterior; the missile energy delivered to the brain was 0.9 J or 1.4 J, respectively. This model produced vasogenic edema around the missile wound track in the injured hemisphere [115, 116], increased intracranial pressure, reduced cerebral perfusion pressure, increase in blood glucose, transient rise in hematocrit, and respiratory arrest. These alterations appeared to be energy-dependent [117]. In Finnie’s model [113] head wounds were inflicted from a range of 3 meters by a 0.22 caliber firearm to a physically restrained sheep with the head held in an upright position. The bullet was fired at the temporal region of the skull and produced a right-to-left transverse wound to the brain through the temporal lobes. Morphological alterations included crushing and laceration of tissue, resulting in a hemorrhagic cavity, widespread stretch injuries to blood vessels, nerve fibers and neurons, and distortion and displacement of the brain. Although these models are useful in studies aimed at characterizing histopathological features of a missile wound, they offer less opportunity to analyze complex molecular responses and functional outcomes following brain injury due to the size of the animal. Non-penetrating/closed head injury a) Controlled Concussion Models

Various closed head impact models have been developed to reproduce the biomechanics and pathobiology of human concussive and diffuse brain injury [118–120]. It has been shown that this type of trauma is difficult to reproduce using experimental models such as fluid-percussion, cortical impact or focal brain contusion since injuries induced using these techniques are associated with focal axonal damage rather than inducing diffuse axonal injury. Early attempts to specifically modify the existing models were unsuccessful because of the difficulty associated with replicating the neuropathology of human concussive and diffuse TBI. Many of these animal models failed to produce a graded, reproducible range of injury outcomes through alteration of the mechanical force. Skull fracture, subdural, subarachnoid and intracerebral hemorrhages, as well as convulsions, frequently accompanied the injuries induced by these methods. Histopathological alterations produced by these techniques were restricted to the lower brainstem, and as such, were not comparable to morphological changes found in the human brain following injury [1, 121].

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The method developed by Tornheim and colleagues [122, 123] involved cats subjected either to an oblique lateral impact or to a blow, delivered to the coronal suture by a Remington humane stunner [122] [124]. The blunt craniocerebral trauma resulted in skull fractures, epidural hematoma, subdural and subarachnoid hemorrhages and brain contusions [124]. Injuries induced by this method have been associated with metabolic changes [125], edema [122] and decreased regional cerebral blood flow [123], and the model has been particularly useful for studies of cerebral contusion and consequent edema. However, it has not been broadly applied to research focusing on molecular and cellular mechanisms of post-traumatic neuronal cell death, mainly because of the frequent hemorrhages that accompany the injury. In Goldman’s rat model of moderate head concussion, the injury was induced by a pendulum falling on the skull midline of anesthetized animals, approximately 9 mm anterior to the coronal suture [126]. The optimum applied force and angle of the impact were selected from a nomogram interrelating the animal’s body weight and desired injury severity. For example, in rats weighing 330 to 430 grams, the selected angle values were in the range 60–85 h, creating an impact energy delivered to the skull varying from 1.62 to 1.89 Joules. This model, uncomplicated by skull fractures and contusions, produces a short (4–10 minutes) period of unconsciousness, early increases in cerebrovascular permeability, decreases in cerebral blood flow and increases in intracerebral pressure [126]. Histopathology included patchy neuronal degeneration in numerous forebrain locations at 48 h post trauma, the severity of which was linearly related to the duration of unconsciousness [127]. This model offers a reproducible range of mild to moderate severity of injury that depends upon pendulum stroke, impact load, and animal body weight. Absence of skull fractures or subdural, subarachnoidal and intracerebral hemorrhages, as well as the presence of physiological and morphological changes that are comparable to those shown in human TBI, recommend this model for investigating the mechanism of brain injury and corresponding therapy.

b) Impact Acceleration Models

Unconstrained Early impact acceleration models utilized primates that were injured by a 1-kg mass impact piston striking the head of an anesthetized animal at a designated location on the skull [128, 129]. Head motion in these models was constrained only by the neck, therefore closely reproducing the acceleration-deceleration force seen in human head injury. The injury severity depended on the velocity, mass and contact area of the impactor, as well as the type of interface material. These models successfully reproduced some of characteristic features of human TBI, such as short-term loss of consciousness, as well as histopathological, systemic and cerebral metabolic responses [3]. The ovine head impact model was developed to examine diffuse axonal injury resulting from a graded traumatic insult [130, 131]. In this model, the anesthetized sheep is placed in the sphinx position with the head resting on a support to allow

6.2 In Vivo Models

free rotational and lateral movement following impact. The impact is induced using a humane stunner aimed at the left temporal region of the unrestrained skull. A captive bolt is propelled from the muzzle of the stunner by the discharge of a blank cartridge inserted in a chamber behind the proximal end of the bolt, whereas the muzzle velocity depends on the predetermined charge [130]. This model produces widespread axonal injury in the hemispheric white matter, central gray matter, brainstem and cerebellum, as well as around cerebral contusions. The degree of axonal injury shows a strong correlation with systemic and cerebrovascular responses [130]. One of the difficulties with the fully unconstrained injury models is the reproducibility of the injury, particularly in terms of outcome. In an effort to increase the reproducibility of these outcome parameters, other studies have attempted to restrict the head motion during and after impact [132]. Typically, the head motion is simply confined to a single plane, thus improving outcome reproducibility without completely restricting head motion. Taken together, the unconstrained impact acceleration models show a high degree of variability due to lack of precise control over biomechanical forces related to impact and head dynamic response [1]. Because of the uncontrolled biomechanical factors, the pathophysiological response to injury in these models is inconsistent, albeit useful for morphologic characterization of injury. Constrained Marmarou’s weight drop model [133, 134] has become one of the most frequently used constrained models of impact acceleration head injury. The trauma device consists of a column of brass weights falling freely by gravity from a designated height through a Plexiglas tube. The skull of the animal is exposed by a midline incision and a stainless steel disc (10 mm in diameter and 3 mm in depth) is rigidly fixed with dental cement to the animal’s skull centrally between lambda and bregma. The rats are then placed on a 10-cm deep foam bed and the injury induced by dropping the brass weight onto the stainless steel disc. This model has been shown to produce graded brain injury in rodents depending upon the mass and the height from which the brass weight is released [133, 135, 136]. Impact with a 450-gram weight falling from a 2-meter height resulted in a mortality rate of 44 % with 12.5 % incidence of skull fracture. The death was primarily caused by respiratory depression, with mechanical ventilation after the impact significantly lowering the mortality rate [133]. Mathematical calculations confirmed that this mass-height combination produced a brain acceleration of 900 G and a brain compression gradient of 0.28 mm [133]. Morphological analysis showed an absence of supratentorial focal brain lesions, with petechial hemorrhages only being present in the brain stem after severe 2-meter level injury. Microscopically, the injury induced widespread and bilateral damage of the neurons, axons, dendrites and microvasculature [134, 137, 138]. It is noteworthy that this model produced massive diffuse axonal injury, particularly in the corpus callosum, internal capsule, optic tracts, cerebral and cerebellar peduncles, and the long tracts in the brainstem [134]. The pathomorphological alterations following 2-meter injury

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were more extensive than after 1-meter injury, indicating graded neuronal injury response caused by impact acceleration [134, 139]. Decrease in cerebral blood flow and increase of intracerebral pressure has been shown as a result of loss of cerebral autoregulation during the first four hours after the impact [140, 141]. Diffusion-weighted magnetic resonance imaging demonstrated that there is a predominantly vasogenic edema formation immediately after weight-drop impact and later a more widespread and slower edema formation due to a predominantly cellular swelling [142]. The impact acceleration model has been shown to produce motor and cognitive deficits [143–145] comparable to those seen with fluid percussion injury and controlled cortical impact. Cellular and molecular alterations produced by this model have been widely investigated. Changes in cerebral energy metabolism include a decrease in ATP, GTP, nicotinic enzymes (NAD, NADP) [146] and N-acetylaspartate (its reduction has been accepted a marker of neuronal dysfunction/loss) [147] starting at two hours post trauma. In addition, impact acceleration-induced injury caused a highly significant decline in free magnesium concentration, cytosolic phosphorylation ratio, and an increased rate of mitochondrial oxidative phosphorylation, without significant changes in pH [143]. Activation of pro-inflammatory mediators/modulators also have been reported in the rodent brain following a weight drop-induced TBI [148–150]. Calpain- and caspase-activation [151, 152], as well as release of cytochrome c [151] and pro-apoptotic bcl-2 family members [152] from the mitochondria into the cytosol have been documented to play an important role in apoptotic neuronal cell death induced by this model. The popularity of the weight drop model is based on the fact that it is inexpensive, easy to perform, and capable of producing graded diffuse axonal injury. However, the biomechanics of the impact produced by this model are not fully and strictly controlled. Namely, there is a possibility of a “second hit” induced by the weight after rebounding from the skull of the animal resting on the flexible sponge. Furthermore, although the weight is enclosed within a Plexiglas tube, a slight left-and-right movement occurs during its fall, which has the potential to cause lateralization of the impact, uneven distribution of the impact energy and increased variability of outcome measures. Non-Impact Head Acceleration Models The majority of human closed head injuries are not caused by direct impact to the head, but rather by movement of the brain within the skull; the aim of the non-impact head acceleration models is to reproduce this condition. Indeed, it has been demonstrated that rapid rotation of the head is one of the main causes of diffuse brain injury [153]. The rotational acceleration, through shearing mechanisms, causes diffuse axonal injury accompanied by a series of pathophysiological and behavioral changes [154]. The inertial effect, which depends upon the brain mass, has been reported to play a vital role in the magnitude of tissue deformations [155]. Hence, in designing acceleration head injury models, special attention has been placed on the scaling relationship between the brain mass and acceleration [156]. There are a number of head acceleration models involving nonhuman primates [157, 158], pigs [159, 160], rabbits [161] and rats [162]; some of them con-

6.2 In Vivo Models

strain the motion of the head, while others allow free head motion. Since the fully unconstrained head has been shown to increase the variability of the outcome parameters, the recently developed models restrict the head motion by using various fixation mechanisms [159–162]. The nonhuman primate model utilized a pneumatic shock tester to apply a non-impact, controlled, single rotation, which displaced the head 60 h within 10– 20 msec [157]. The resulting rotational acceleration was biphasic, with a long acceleration phase followed by a short deceleration phase. The duration of coma, degree of neurological deficit, as well as the intensity of diffuse axonal injury localized in the subcortical white matter, upper brainstem and cerebellum, were in direct correlation with the mechanical force applied via coronal rotation of the head [163]. This technique has been reported to replicate the spectrum of traumatic coma experienced in humans, and to produce widespread axonal damage comparable to that found in human brains after severe traumatic brain injury [158, 163]. The head acceleration model developed by Smith et al. [160] produced diffuse brain trauma in miniature swine through head rotational acceleration. The head was secured to a pneumatic actuator through a snout clamp. Triggering the pneumatic actuator generated linear motion, which was further converted to angular motion via a linkage assembly directly mounted to the device [160, 164]. The center of head rotation was positioned close to the skull base for coronal plane rotation, or at C-2 for axial plane rotation. Activation of the device rapidly rotated the animal’s head over the designated angular excursion of 110 h in 20 msec, with the peak acceleration at approximately 6 msec. The resulting rotational acceleration of the head demonstrated a biphasic course, with a predominant deceleration phase [160, 165]. Both coronal and axial rotations produced diffuse axonal injury, particularly in the hemispheric white matter and brainstem. Accumulation of amyloid precursor protein, which has been accepted a marker for diffuse axonal injury [166, 167], was found in the white matter of injured animals at 3–10 days postinjury [168]. Immediate prolonged coma occurred following axial rotation of the head, but not after coronal plate rotation [169]. Coma severity correlated with the applied kinetic energy, as well as with the extent of axonal injury in the brainstem [160, 169]. Nuclear magnetic resonance spectroscopy demonstrated acute reductions in the neuronal marker N-acetylaspartate, and in intracellular free magnesium levels, which persisted for up to 7 days post trauma [165, 170]. The alterations in intracellular metabolism induced by diffuse axonal changes in the white matter occurred in the absence of changes in pH and phosphocreatine/inorganic ratio; the concentrations of ATP and lactate also remained unaffected [170]. The non-impact acceleration injury models in nonhuman primates and miniature swine most closely reproduce the complex pathobiology of human traumatic brain injury. Although these techniques offer valuable information on morphological, cellular and molecular responses to diffuse brain injury, their use is difficult for most laboratories due to the cost and size of the animal, as well as the sophisticated technical requirements [7]. Additionally, the models of acceleration head injury using large experimental animals lack established functional outcome tests, which are critical to the preclinical assessment of neuroprotective strategies.

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6.2.3

Combined Neurotrauma Models

Hypoxia, ischemia, hypovolemia and hypotension have been reported to accompany traumatic brain injury in animals [171–173] and humans [174–176]. The combination of these pathological conditions exacerbates functional outcome and increases mortality [174–176]. Hence, experimental models combining traumatic brain injury and secondary insults have been developed using rats [171, 177, 178], mice [179], cats [31, 180] and swine [181, 182]. Generally, the TBI and hypoxia models utilize fluid percussion [171, 183, 184], controlled cortical impact [178, 185, 186] or impact-acceleration [187, 188] injuries to inflict neurotrauma, which is followed by hypoxic conditions (pO2 approximately 30–40 mmHg or FiO2 10–13 %) to stimulate secondary hypoxia. Models that combine mechanical brain injury and hypovolemia and/or hypotension use one of the abovementioned experimental TBI paradigms with subsequent hemorrhage, which reduces the mean arterial pressure to 30–50 mmHg [181, 182, 189]. Combined neurotrauma models may serve as valuable tools to assess the consequences and mechanisms of secondary insults after TBI. In conclusion, there is no single in vivo model of traumatic brain injury that can be recommended as a “gold standard” for neurotrauma research. Each animal model offers certain benefits, but also limitations. Thus, the choice must be made on the basis of specific research aims and objectives [1, 7]. Utilization of multiple animal models has also been suggested to characterize central mechanisms involved in the pathophysiology of traumatic brain injury [7].

6.3

In Vitro Models

Experimental in vitro models attempt to reproduce the stress (tension, compression and shear) and/or corresponding strains (deformations) that reflect the mechanical damage of neurons following traumatic brain injury. The strain depends upon the way in which the stress is applied; it is determined by the force per unit area, and not by the total force utilized. In these models, the intermolecular forces within the deformed tissue oppose the applied stress. Here, we review the most frequently used in vitro models categorized according to the main type of applied mechanical force: tension, compression or shear (Fig. 6.2). In clinical injury, these stress types generally coexist with a combination of stresses involved in the injury process [190–192]. Advantages of in vitro cell culture systems include: minimized numbers of experimental variables; the ability to study the role of specific receptors and intracellular events such as induction of second messenger systems; the ability to control the experimental environment; the potential to rapidly screen selective pharmacotherapies, and reduced cost. In contrast, a primary disadvantage is the inherent variability between models caused by the culture system (egs. acute slices, organotypic slice cultures, dissociated primary cultures or cell lines).

6.3 In Vitro Models

Figure 6.2

Schematic representation of in vitro experimental models of traumatic brain injury.

Acute slices maintain many aspects of the in vivo situation (egs. connectivity in three dimensions and age) and can provide key information regarding the effects of injury on three dimensional rather than two-dimensional cytoarchitecture. Furthermore, acute slices can allow evaluation of neuronal/glial signaling, which are lost during the preparation of dissociated cultures or altered during the establishment of organotypic cultures. However, the use of acute slices is usually limited to an 8-hour time span, beyond which cell viability becomes an issue. Organotypic slice cultures alleviate the time-dependent viability seen in acute slices and can provide an opportunity to investigate long-term effects of trauma upon cells which maintain connectivity similar to that seen in vivo and in acute slices. Furthermore, due to the nature of organotypic slice cultures, the ability to image such processes is enhanced. However, organotypic slices progressively lose their three-dimensional cytoarchitecture as they become two-dimensional over time, and undergo ontogenetic changes while developing in culture. Moreover, their recovery from the initial insult of preparation may impact such ontogenetic changes. Additionally, technical aspects of organotypic slice culture preparation are difficult. Dissociated cultures allow discrete control over which cell types (egs. neuronal, glial or neuronal/glial) are studied. Moreover, these cultures are relatively easy to prepare, cost -effective, and can be plated in either standard single Petri dishes or multiple well culture plates. This aspect makes the use of dissociated cultures

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in neuroprotective studies requiring large sample statistics preferential. However, dissociated cultures are influenced by multiple factors that potentially create confounding variables. For example, culturing methods, the presence or absence of serum, the age of the cells, and the presence or absence of glia cause dissimilarities in the expression of receptor and signal transduction molecules [193–197]. Moreover, the use of embryonic tissue to derive glial cells adds the confounding effect of having reactive astrocytes in culture [186, 198, 199]. The presence or absence of glia is important since glia as well as neurons express many of the same receptors [200]; furthermore, significant neuronal/glial interactions exist that can influence the outcome following trauma [201]. Indeed, it has been suggested that in regards to neuroprotection, mixed neuronal/glial cultures can respond opposite to neuronal cultures (Lea et al, 2003; J. Neurotrauma 20:1233–49). Nevertheless, dissociated cultures have been used extensively to study neuroprotective compounds and have withstood the test of time. Cell lines capable of differentiation into desirable phenotypes provide a costeffective way to study the effects of multiple neuroprotective compounds. In addition, the ease in which these cells can be transfected when compared with primary neuronal cultures make them a desirable system in which to study genes and gene products of interest. However, cell lines consist of immortalized or cancerous cells with the inherent ability to divide uncontrollably. This characteristic suggests that either gene and/or protein expression patterns may be significantly different than that found in functioning neurons. Despite the aforementioned disadvantages of the various culture systems used to date, in vitro trauma models have proved useful for delineating discrete mechanisms of cell death at both the cellular and molecular level that are similar across models used. Indeed, numerous extracellular and intracellular events have been implicated [202]. Such processes include the excessive release of excitatory amino acids, including glutamate, with concomitant receptor activation, increased neuronal excitability, alterations in intracellular calcium, activation of calcium-dependent proteases and phosphatases [24, 60, 203–207], and the activation of the extrinsic and/or intrinsic pathways of a class of cysteine proteases called caspases [208, 209]. An understanding of these processes in both the developing and adult brain is essential as key findings suggest significant differences between both the biochemical [210] and biomechanical [9] sequelae that can potentially impact the choice of therapy following traumatic brain injury. This review will focus on in vitro models of mammalian brain trauma that induce injury by primary mechanical stimuli. Although many of these models are currently used in conjunction with additional neurotoxic stimuli (egs. staurosporine, maitotoxin, etoposide, ceremide, hypoxia, etc.), an overview of these combinations is beyond the scope of this chapter. Nevertheless, our attempt to address the strengths and limitations of each of the given models should provide enough relative information to help in the development of future experimental paradigms using either a single or combined model approach. The reader is referred to the following reviews for more information regarding additional models of mechanical stimulation both related and unrelated to traumatic brain injury [190–192].

6.3 In Vitro Models

6.3.1

Tension Stress-induced Injury Stretch Injury Models A number of in vitro models have been developed to study stretch-induced deformation of a two-dimensional tissue culture [211–214]. The main principle of these methods is to culture cells on a deformable material; thus, the strain of this material will reflect the strain of the adhered cellular layer after application of a mechanical force. The model developed by Ellis and colleagues has been used to study axonal injury-induced molecular and biochemical responses in vitro [211]. It produces both necrotic and apoptotic pathways of cell death [215]. This model was characterized using rat cortical astrocytes from 1- to 2-day-old rats – grown to confluency on commercially available FlexPlatesr which have six 25-mm-diameter tissue culture wells with 2-mm-thick flexible silastic bottoms (Flexcell International) – but has more recently been used with septo-hippocampal, glial, astrocyte, neuronal, or mixed neuronal/glial cultures derived from embryonic and neonatal rat cortices. Rapid positive downward air pressure, which is applied via a cell injury controller, deforms the membrane and stretches the adhered cell layer. This induces strains up to 0.72 and cellular injury, both of which are directly correlated to the amplitude and duration of the air pressure pulse. Amongst primary benefits of this system is the utilization of standard microplates that allows analysis of cell viability, plus the ability to produce simultaneous injuries in multiple wells, as well as being able to remove the membrane for cell fixation, imaging or electrophysiology without significant perturbation of the culture. Indeed, this model has been successfully used to perform imaging of injury-induced calcium changes in cortical neuronal cultures [216]. Unfortunately, the thickness and opacity of the flexible membrane limits the ability to analyze the effects of stretch-injury to upright microscopes with water immersion lenses, and, perturbations of the plate during injury make imaging of real-time pathophysiological events rather difficult. Furthermore the injury controller is unable to measure real-time strain rates for each of the individual wells. The stretch model created by Cargill and colleagues was characterized using differentiated neuroblastoma cross glioma NG108–15 cells [212], cultured in custombuilt wells with thin silicone membranes. Deformations of the membrane and, by association, the cells is caused by a vacuum pulse with magnitudes up to 0.45 and rates between 10 s–1 and I 1 s–1, which is comparable to forces experienced by neural tissue in traumatic brain injury [212]. While at high rates of deformation (approximately 10 s–1) increases in intracellular free calcium concentration are exponentially related to the magnitude of the applied deformation, at low rates of deformation mild elevation in intracellular free calcium concentration is independent of the magnitude of the deformation. Studies using this technique have suggested that strategies for reducing severity of TBI should focus on minimizing the rate of neural cell deformation [212]. This model produces stretch injury of the cells at known strains and variable strain rates, and allows easy access to the cells for recording electrodes in electrophysiological studies. 6.3.1.1

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In an effort to overcome some of the shortcomings of the previous models, Morrison et al. designed an original injury device that incorporates needle valves and computer-controlled solenoid valves to regulate the duration and shape of the injury pulse. In addition, a laser displacement transducer was added to allow realtime imaging of the membrane deflection during injury [213]. This system generates a wide range of strains (up to 0.65) and strain rates (up to 15 s–1) and produces graded neuronal injury in vitro. The injury model developed by Smith and colleagues allows selective stretch of a designated region of the culture well, imaging of the effects of stretch on morphology, and acquisition of strain parameters in real-time. This model was characterized by selectively stretching axons bridging two populations of human neurons derived from the cell line N-Tera2 [217]. A computer-driven solenoid valve and pressure transducer regulates injury parameters and focuses mechanical force. The reported strain (0.58-0.77) and strain rates (26–35 s–1) fall within the range of traumatic neuronal injury. The limitations of this system include absence of a perfusion system necessary for minimizing the effects of neurotoxin accumulation during a study of longer duration, as well as absence of a controller to ensure consistent environmental temperature. The stretch injury model known as the Tissue Trainr is commercially available from Flexcell International. This model utilizes a computer-controlled vacuum manifold system to pull a deformable membrane downward, thereby imparting strain to the culture layer. Parameters controlled by the operator which influence injury severity are magnitude, frequency, waveform, and duty cycle of applied mechanical force [192]. Characterization studies performed with this device include measurements of strain, substrate strain anisotropy and heterogeneity, and combined computational and experimental characterization of the behavior of the substrate [192]. To date, this model has been used extensively to study the effects of tension strain on multiple non-neuronal cell types. However, recent efforts are underway to apply this system to in vitro neuronal injury. More importantly, such efforts plan to compare this model’s biomechanical properties and injury production to those models currently used (personal communication). 6.3.2

Compression Injury Models

A number of models have been developed to study the effects of compressive stress on tissue including direct platen abutment, axial compression, hydrostatic pressurization, weight-drop, and fluid percussion [191, 192]. The first three were recently reviewed elsewhere [192]. This review will focus on the weight-drop, hydrostatic pressurization and fluid percussion models, which are currently the most frequently used in vitro techniques.

6.3 In Vitro Models

Weight-drop Model In vitro devices that utilize weights to induce compression stress upon tissue [218– 220] have been designed using the principles involved in producing in vivo spinal cord injury [221]. In these models, a weight of known mass is dropped from a height of known distance onto cultured organotypic tissue typically derived from either immature or mature spinal cord. By varying the parameters of height and weight, changes in injury severity can be accomplished. Although this particular system is thought to duplicate the neuropathology of in vivo spinal cord injury, to date, this model has been used to primarily study cellular mechanisms underlying trauma-induced neurodegeneration and have not incorporated biomechanical measurements of strain or strain rate. Hence, the relative compatibility between these in vitro models and the in vivo situation remains to be determined. 6.3.2.1

Hydrostatic Pressure Models Over the last three decades, models of hydrostatic pressurization have been used to study effects of compressive stress on cells and tissue [220, 222]. These models, incorporating negative (vacuum) and positive pressurization, offer several benefits such as simplicity of equipment, spatial homogeneity of the stimulus, and easy management of loading paradigms. The model of quasistatic compression, developed by Murphy & Horrocks [222], applied increased pressure of up to 15 atm causing condensation of the cellular membrane of cultured cells and eliciting a mechanical-like trauma. The resulting injury has been shown to produce features similar to those seen in a compression-like spinal cord or brain injury [222]. The quasistatic model utilized several types of cell cultures such as ROC-1 oligodendroglia, N1E-115 neuroblastoma, and human umbilical vein endothelial (HUVE) cells. Technical shortcomings, such as alterations in pO2 and pCO2 levels of nutrient medium due to long periods of incubator gas pressure, could be compensated for by modification of the model [223]. 6.3.2.2

Fluid-percussion Models Shepard and colleagues developed a model of transient barotrauma in cell culture to analyze the consequences of injury on cell metabolism [224]. This model utilized the same fluid percussion device as used in in vivo brain injury studies. The compression applied using this device increased pressure gradients and generated strain fields. The effect of this injury was evaluated in monolayers of human glial cells, whose viability was measured by trypan blue exclusion and the production of leukotrienes. The authors reported direct correlation between reduced cell viability and the intensity of the applied mechanical force. 6.3.2.3

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6.3.3

Shear Injury Models

Shear stress applied to tissue incorporates a distortion, induced by two opposing parallel forces, resulting in the surfaces of the tissue sliding in opposite directions over an imaginary plane. Such in vivo events have been replicated by a number of in vitro models, and have involved the application of acceleration forces [225] using either a cone-and-plate system or a parallel plate flow chamber [192].

Acceleration Models Lucas and Wolf [225] designed an in vitro model of rapid acceleration injury to examine the effects of multiple impact (220 g/impact, 3–5 s intervals) injury on cultures of mammalian CNS cells. This model used an impacting pendulum to rapidly accelerate a culture flask. The multiple impacts, delivered tangential to the plane of growth, induced neuronal death, while normal (vertical) impacts were without effect. The threshold for neuronal death was 440 g (cumulative), and those neurons generally died within 15 minutes after injury. Histopathological analysis demonstrated that glia were not affected by any type of rapid acceleration, whereas somal swelling and increased nuclear prominence were often observed after tangential rapid acceleration injury; the frequency of these changes increased with the cumulative acceleration. Although this model is limited to multiple impact studies, the system is easy to use and has been shown to produce excitotoxic cell death [225]. However, there is no information on biomechanical parameters of shear stress or shear strain. 6.3.3.1

Hydrodynamic Model It is well known that a variety of physiological systems are influenced by fluid shear, including plasma membrane receptors, ion channels, protein kinase signaling, intracellular calcium, nitric oxide, cytoskeletal remodeling and others. LaPlaca and Thibault [226] developed a specialized flow configuration to examine the effects of traumatic mechanical loading on individual cells. The cell shearing injury device consisted of a parallel disk viscometer that applied fluid shear stress with a variable onset rate. The device was used in conjunction with microscopy and biochemical techniques to record and quantify the deformation and functional response of neurons to injury. Analytical and numerical approximations of the shear stress at the bottom disk were compared to determine the contribution of secondary flows [226]. A high rate (800 dyne cm2 s–1) shear stress applied to cultured neurons generated a range of cell membrane strains (0.01 to 0.5(3), suggesting inhomogeneity in cellular response. Increases in cytosolic calcium and extracellular lactate dehydrogenase levels occurred in response to high strain rate (i 1 s–1) loading. Furthermore, rapid deformation has been shown to induce cell death. In an improved version of the device, a cone replaces the parallel disk [227]. Since the relative local velocity and the cone and plate surface separations vary linearly with 6.3.3.2

6.3 In Vitro Models

radial position, this configuration is optimal to achieve spatially homogenous fluid shear stress. Furthermore, variations of the conic taper and the imposed angular velocity can produce a wide range of shear stresses [228]. Recently, the same lab has developed a new device to generate large shear strain (up to 50 %) and high strain rates (up to 350 Hz) in three-dimensional neural cultures [229–231]. This system utilizes two plates attached to the culture. While the bottom plate remains fixed, the top plate exerts controlled relative displacement to induce shear strain. Fundamental to this system is the use of a Linear Variable Differential Transformer, which measures displacement and provides feedback to the control system. Three-dimensional cell cultures are believed to resemble the in vivo situation more closely than the two-dimensional cultures [232]. Moreover, since acute slices with innate 3-D architecture cannot be used for long-term studies of secondary injury, 3-D cultures appear to be a better system to study complex interactions such as those seen during shear stress. Indeed, a recent shift in the neurotrauma field towards using 3-D cultures appears to be gaining impetus.

Primary Axotomy/Transection Models It is well established that primary axotomy is intimately associated with neuronal cell injury and death following traumatic brain injury [139, 233, 234]. In vitro primary axotomy models that are currently in use involve utilization of a stylet, scribe or laser to sever neuronal processes and soma. 6.3.3.3

a) Stylet Models

These models use a plastic stylet to mechanically disrupt processes and/or soma of neurons cultured in the presence of glia. Tecoma and colleagues [235] used the stylet to tear the soma and their processes from a small number of neurons and glia that had been adhered to the bottom of a standard culture dish, whereas Regan & Choi [236] used the stylet to scratch cellular monolayers. The stylet models have been used to illustrate the neuroprotective effects of NMDA antagonists [235– 237] and adenosine receptor agonist [237]. It has also been used to induce the release of a neurotoxic factor that produces cell death when applied to uninjured cells; this experiment demonstrated the ability of traumatized cells to propagate cell death to an uninjured cell population [237, 238]. However, the user-dependent nature of these models limits the evaluation of biomechanical parameters of a strain necessary to produce primary transection. b) Scribe/Punch Models

Mukhin and colleagues [239] modified the model developed by Regan & Choi [236], and used a rotating scribe, producing from one to six concentric circular cuts in the cell layer of neurons cultured on a standard 24-well plate. Changes in the number of circles (from 1 to 6) allowed variation in injury severity. Comparative studies of cell death induced by such trauma in glial and neuronal/glial cultures demonstrated that glial cells are relatively resistant to this type of injury, and that cell death after trauma to neuronal/glial cultures primarily reflects neuronal death

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[239]. This model has also been used for studying glutamatergic mechanisms involved in secondary injury [239]. Another in vitro model developed by Mukhin and colleagues [240] induced reproducible levels of injury, delivered by a punch device producing 28 parallel cuts in individual wells of 96-well microplates. Glial cultures demonstrated cell death that was restricted to the initial injury site, whereas neuronal/glial cultures showed significant spread of cell loss over time. This technique was used to examine the role of pH and NMDA receptors in delayed post-traumatic injury. Advantages of this model include: reproducible trauma induction; rapid measurements of cell injury; and use of 96-well microplates that reduces time and cost [240]. Indeed, this model appears to be well-suited for the study of selected mechanisms of post-traumatic neuronal injury as well as for screening potential neuroprotective agents [241, 242]. c) Laser Transection Models

In the laser transection models [243–245], the exposure of neuronal and glial cell processes to a large number (up to 300) of 12-nsec laser pulses, at a wavelength of 337 nm and energy densities below the threshold for nonlinear absorption, produced their gradual transection at the laser focus. Within 10 to 20 sec after cessation of firing, swelling of mitochondria, plasma membrane damage, and disappearance of microtubules occurred at either side of the process constriction. This method has been reported to offer a reliable approach to morphological alteration of single cells and to the tailoring of two-dimensional neuronal networks. However, there is no information on the ability of injured neurons to affect the survival of neighboring cells. Additionally, force, strain and strain rate data concerning the process of transaction cannot be measured in this model. The ability of this time- and labor-intensive technique appears limited for screening novel neuroprotective drugs. To date, a wide array of in vitro models have been developed to investigate the effects of tension, compression and shear forces comparable to those found in vivo following traumatic brain injury. Adequate measurement of the forces used to induce injury-related neurodegeneration in vitro is a prerequisite for correct translation of obtained results and their comparison with the features of in vivo pathobiology. These models have been utilized to evaluate mechanisms of secondary cellular injury/death as well as to evaluate pharmacological therapies.

6.4

Conclusion

Experimental models of traumatic brain injury have been developed to replicate selected aspects of human head injury, such as contusion, concussion and/or diffuse axonal injury [1, 7, 8]. Although earlier brain injury models utilized higher species such as primate, sheep, and cat, recent studies have favored the use of rodent models [7]. In the absence of an ideal experimental model, the choice of the model depends on the goal and the research objectives. Since rodent models

References

are inexpensive, easy to use, and generally involve highly inbred strains of one sex, they are useful tools to minimize intersubject variability and reduce outcome variability [246]. A variety of morphological, cellular, molecular and behavioral changes have been identified across experimental models and across laboratories such as alterations in ionic homeostasis (calcium, magnesium, sodium, potassium), free radical generation, inflammatory/immune response, release of excitatory amino acids, and alterations in multiple neurotransmitter/neuromodulator systems [247–249]. Experimental models of TBI have played an important role in the identification of the complex mechanisms leading to both necrotic and apoptotic neuronal cell death after injury [24, 53, 250]. These findings led to the development of various therapeutic strategies, including magnesium administration, glutamate antagonists, immune modulators, anti-oxidants, anti-apoptotic agents, amongst others [251–254]. Interestingly, pharmacokinetic and pharmacodynamic studies using animal models, as well as studies examining central penetration of systemically administered drugs following experimental TBI, are rarely conducted [246]. Further development of more complex animal models of traumatic brain injury incorporating hypoxia, ischemia, and other potentially relevant factors of clinical head injury, are necessary to reproduce the complete pathobiology of human TBI. Such models would be useful in testing combination therapies, which prevent various factors of the secondary injury cascade, or single compounds that modulate multiple mechanisms of the cascade [253, 255]. Additionally, there is a need for a reliable and reproducible animal model that replicates development-dependent responses to traumatic brain injury.

References Lighthall, J. W. and T. E. Anderson, In vivo models of experimental brain and spinal cord trauma, in The Neurobiology of Cenral Nervous System Trauma, S. K. Salzman and A. I. Faden, Editors. 1994, Oxford University Press: New York/Oxford. p. 3–12. 2 Denny-Brown, D. and W. R. Russell, Experimental cerebral consussion. Brain, 1941. 64: p. 93–164. 3 Ommaya, A. K. and T. A. Gennarelli, Cerebral concussion and traumatic unconsciousness. Correlation of experimental and clinical observations of blunt head injuries. Brain, 1974. 97(4): p. 633–54. 4 Clark, R. S., et al., Neutrophil accumulation after traumatic brain injury in rats: comparison of weight drop and 1

controlled cortical impact models. J Neurotrauma, 1994. 11(5): p. 499–506. 5 Rall, J. M., D. A. Matzilevich, and P. K. Dash, Comparative analysis of mRNA levels in the frontal cortex and the hippocampus in the basal state and in response to experimental brain injury. Neuropathol Appl Neurobiol, 2003. 29(2): p. 118–31. 6 Cenci, M. A., I. Q. Whishaw, and T. Schallert, Animal models of neurological deficits: how relevant is the rat? Nat Rev Neurosci, 2002. 3(7): p. 574–9. 7 Povlishock, J. T., et al., Workshop on animal models of traumatic brain injury. J Neurotrauma, 1994. 11(6): p. 723–32.

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Mukhin, A. G., S. A. Ivanova, and A. I. Faden, mGluR modulation of posttraumatic neuronal death: role of NMDA receptors. Neuroreport, 1997. 8(11): p. 2561–6. Movsesyan, V. A., et al., mGluR5 antagonists 2-methyl-6-(phenylethynyl)pyridine and (E)-2-methyl- 6-(2-phenylethenyl)-pyridine reduce traumatic neuronal injury in vitro and in vivo by antagonizing N-methyl-D-aspartate receptors. J Pharmacol Exp Ther, 2001. 296(1): p. 41–7. Gross, G. W., J. H. Lucas, and M. L. Higgins, Laser microbeam surgery: ultrastructural changes associated with neurite transection in culture. J Neurosci, 1983. 3(10): p. 1979–93. Kirkpatrick, J. B., et al., In vitro simulation of neural trauma by laser. J Neuropathol Exp Neurol, 1985. 44(3): p. 268–84. Lucas, J. H., et al., Neuronal survival or death after dendrite transection close to the perikaryon: correlation with electrophysiologic, morphologic, and ultrastructural changes. Cent Nerv Syst Trauma, 1985. 2(4): p. 231–55. Faden, A. I., Neuroprotection and traumatic brain injury: the search continues. Arch Neurol, 2001. 58(10): p. 1553–5. Faden, A. I., Experimental neurobiology of central nervous system trauma. Crit Rev Neurobiol, 1993. 7(3–4): p. 175–86. Yakovlev, A. G. and A. I. Faden, Molecular biology of CNS injury. J Neurotrauma, 1995. 12(5): p. 767–77. McIntosh, T. K., et al., Neuropathological sequelae of traumatic brain injury: relationship to neurochemical and biomechanical mechanisms. Lab Invest, 1996. 74(2): p. 315–42. Rink, A., et al., Evidence of apoptotic cell death after experimental traumatic brain injury in the rat. Am J Pathol, 1995. 147(6): p. 1575–83. Faden, A. I., Pharmacologic treatment of acute traumatic brain injury. JAMA, 1996. 276(7): p. 569–70. McIntosh, T. K., M. Juhler, and T. Wieloch, Novel pharmacologic strategies in the treatment of experimental trau-

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7 Spinal Cord Injury Poonam Verma and James W. Fawcett Spinal cord injury (SCI) is a major cause of disability; predominantly affecting young adults. The peak age of injury is amongst young males 19 years old [1, 2]. The number of spinal injuries that lead to paralysis is about 10,000 a year in the USA, and a similar number in Western Europe. The commonest cause of injury is automobile accidents, the next commonest being gunshot injuries in the USA, sporting injuries in Europe. As recently as 50 years ago, victims of spinal cord trauma rarely survived their injuries. Twentieth century advances in emergency care, diagnostic techniques, surgical intervention, and rehabilitation have greatly improved the prognosis of patients, to the extent that people with spinal injuries can now expect an almost normal lifespan. The turning point was during the Second World War, which generated large numbers of spinal injuries. The advances in rehabilitation and preventative treatment made by Sir Ludwig Guttaman at Stoke Mandeville Hospital, and other similar efforts in other countries laid down the basic methods on which modern care is based. However, those that do survive live with a devastating disability that affects all aspects of their life. The degree of disability following a spinal injury depends on whether the injury results in a complete functional transection of the cord, and on the level of the injury. Only one third of injuries completely transect the cord, but another third have an anatomically incomplete injury with no discernable function driven by the brain below the lesion. The final third have surviving axonal pathways passing through the injury which carry some function. A small number of axons can carry a large amount of function. There are patients who have lesions that appear to transect most of the spinal cord, yet have little functional loss. Indeed a number of patients are never classified as suffering a spinal injury, because after an initial period of weakness they appear to make a full neurological recovery. Yet an MRI scan of the spinal cord of some of these patients may reveal a cystic injury affecting a considerable proportion of the axonal tracts passing through it. The main determinant of the degree of disability of patients with a functionally complete injury is its level. From this point of view, spinal injury is many different conditions, because the condition of a patient with a high cervical injury is so different and the degree of disability so much greater than someone with a thoracic level injury. All patients suffer loss of function of their legs, and loss of control of bowel, bladder and sexual Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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function. Of these, patients usually place a much higher priority on developing a solution to their bowel, bladder and sexual problems than on walking. Patients may spend hours a day dealing with their bowel problems, and the unreliability of their bladders together with the infections and other issues associated with frequent bladder catheterization are a constant source of worry. Implanted stimulation devices can help considerably in some patients, but are far from providing complete control. It is now possible to obtain viable semen from many male patients, and females with cord injuries have gone through successful pregnancies, but restoration of sexual function remains a high priority for many. As the level of injury gets higher, it begins to affect upper limb function. The commonest level of injury is C6/7, and these patients have loss of hand function, although they retain control of the shoulders and elbows. Lack of hand function means that it is necessary to have at least one full-time carer to help with dressing, transfers, bladder and bowel function and other domestic tasks. For these patients, restoration of hand function is probably the highest priority. As the injury becomes yet higher, first all upper limb function is lost. This removes any ability to move in and out of bed, into cars and to perform household tasks. Patients now need almost full-time assistance. Even higher injuries remove control of the diaphragm, requiring full time mechanical ventilation or a phrenic nerve stimulator, and these patients are completely dependent on a team of carers for all aspects of their life. The large differences in the conditions of patients with different levels of spinal injury provides an opportunity to those attempting to develop treatments to repair spinal injuries. As described below, there are no treatments at present in prospect that will produce a complete repair of cord injuries. However, there are a number of treatments that have the ability to produce a partial return of function, and that functional return will probably be concentrated on the cord levels immediately below the injury. Axon regeneration for a maximum of 4 cm has been reported following a number of treatments. Each spinal level in the cervical cord is around 1 cm, so it is possible that treatments currently under development might bring about an improvement in function over two or more spinal segments. This would be sufficient to bring back some diaphragm control to patients with high lesions, some degree of hand and finger control to those with C6/7 lesions. SCI can be viewed as a summation of two processes: the initial traumatic disruption of tissue from direct impact and from shock waves, and a sequence of progressive secondary pathophysiological events. Successful recovery relies on the prevention and minimization of damage and the restoration of structure so as to return normal function to the patient. This requires injured axons to survive, regenerate, and reconnect with their original targets, processes integral to normal development, but abortive in the adult mammalian central nervous system. Recent discoveries suggest that Cajals’ concept of the central nervous system’s response to damage “once development was ended, the fonts of growth and regeneration of the axons and dendrites dried up irrevocably…Everything may die, nothing may regenerate”, may have been too fatalistic. As Cajal himself recognized, a more complete understanding of neurobiology has provided many insights into possible

7.1 Acute Neuroprotection (preventing neuronal death)

ways in which successful neuronal regeneration in the central nervous system may be encouraged. Basic research currently focuses on ways to improve the unsatisfactory cellular and molecular responses of central neurons to injury. Considerable advances have been made achieving sufficient regeneration to enable some functional recovery of motor ability. This research has brought us to the threshold of practical application along three lines of approach derived from animal models: acute neuroprotection, enhanced axonal regeneration or plasticity, neuronal transplantation and treatment of demyelination.

7.1

Acute Neuroprotection (preventing neuronal death)

In the event of acute SCI, neurological dysfunction at the level of the lesion is attributed to and consequent upon the force and nature of the primary insult [3–7] Although the spinal cord is rarely totally transected even after severe injury associated with complete paralysis [8,9], the primary mechanical impact and subsequent persisting compression on the spinal cord tissue initiates prolonged secondary pathophysiological alterations at both the cellular and molecular level, resulting in major destruction of residual tissue and conferring a complete transection-like pathology [10]. The sequence of pathological changes in the cord following the initial trauma include hemorrhage, edema, axonal and neuronal necrosis, metabolic perturbation, oxidative damage, release of catecholamines, excitotoxity, inflammation and demyelination followed by cyst formation and infarction 1 [1–17]. The developmental progression of this secondary insult is consistent with the gradual expansion of the initial lesion, both radially and longitudinally, from the epicenter of the initial force.4, [18, 19]. Subsequently, adjacent spinal segments are recruited into the successive secondary pathological cascade adding to an increase in tissue destruction that ultimately contributes to heightened functional impairment. Early studies highlighted these post-lesion molecular and cellular changes as targets in the treatment of traumatic pathologies of the central nervous system (CNS) [21, 22]. However, in spite of optimally designed protocols affecting specific targets of the neurochemical cascade, results have been mostly disappointing. In 1990, the National Acute Spinal; Cord Injury Study II (NASCIS II), a multi-center clinical trial involving 487 patients presenting with neurologically complete or incomplete lesions at intake, reported that the steroid drug, methylprednisolone sodium succinate (MPSS), when administered within 8 hours of injury, improved motor and sensory recovery of patients as compared to those who received naloxone or placebo [23]. Evaluation of patients in NASCIS II at 1-year follow-up confirmed the beneficial effects of MPSS [24]. Since the publication of the NASCIS II trial results and on the recommendations of NASCIS III, a multi-center trial designed to determine the dose-administration protocol for MPSS 25 the drug has been adopted as a standard of care for acute SCI in most North American centers.

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Although MPSS is the first accepted treatment strategy for acute SCI, criticisms regarding the drug’s therapeutic physiological effects and its appreciable benefits to recipients have been raised [26, 27]. In particular, the functional neurological improvement and independence measure scores reported in NASCIS II and III respectively, were only modestly enhanced in patients receiving MPSS within 8 hours of SCI, suggesting that most victims are still left devastated by the physical impairments of their injury. These criticisms have led to the questioning of MPSS’s therapeutic efficacy in the clinical setting and, as a result, to the recently disseminated professional advice to downgrade the clinical use of this drug from that of a standard of care to an option. Nonetheless, the ‘therapeutic window’ established by the second NASCIS trial showed that acute SCI is potentially amenable to drug therapy. Since these first trials, various other potentially neuroprotective treatments have emerged from animal experiments, including control of inflammation, prevention of neurotoxicity and other interventions. Of these, a trial of glutamate antagonists in recently injured human spinal injury patients failed to show clinical benefit.

7.2

Regeneration of Nerve Fiber Tracts (initiating growth following injury)

Traditionally, the approach to restoring function after spinal cord trauma focuses on the enhancement of axonal regeneration. Injured adult mammalian CNS axons display an extremely limited capacity to regenerate; a property based largely on a combination of extrinsic environmental factors and intrinsic neuronal factors. 7.2.1

Extrinsic Mechanisms

The CNS contains many inhibitory molecules, many of which are up-regulated following injury. Several experiments investigating the regenerative capacity of sensory nerves within PNS and CNS environments highlight the inhibitory nature of the CNS glial environment [28]. These findings have been further supported by transplantation experiments providing various glial environments for central neurons. In a classic experiment, David and Aguayo [29] transected the spinal cords of rats and then implanted a segment of peripheral nerve across the transection, with one end inserted into the brainstem and the other into the distal stump of the spinal cord. Brainstem axons were seen to regenerate into the peripheral nerve graft and to grow the full length of the graft. However, there was little or no growth of axons out of the graft back into the host CNS, and the grafts were therefore unable to return function to the damaged cord. This study clearly demonstrated that the cells of the CNS have the capacity to regenerate over long distances when confronted by a PNS environment, but not when the same axons meet a CNS environment.

7.2 Regeneration of Nerve Fiber Tracts (initiating growth following injury)

7.2.1.1

The Glial Scar Shortly after the initial trauma, the second phase of the injury response is characterized by the formation of a dense glial scar [30]. This glial scar is mostly composed of astrocytes which become hyperplastic and hypertrophic (the so-called ‘reactive astrocytes’) and a heterogeneous assembly of cells including microglia, oligodendrocytes, fibroblasts and other inflammatory cells. These changes in number and morphology of astrocytes are correlated with an increase in the expression of glial fibrillary acidic protein (GFAP) and GFAP-mRNA, resulting in the formation of a gliotic tissue or ‘glial scar’, which constitutes a major physical impediment to axonal regeneration. Coupled to this is the emergence of a biochemical barrier by either the down-regulation of growth-promoting or the up-regulation of growth inhibiting molecules [31, 32] Three experimental strategies have been developed to overcome the problem of this non-permissive environment; blockage of glial scar formation, modification of growth-inhibitory molecules, and selective removal of reactive astrocytes.

7.2.1.2

Blockage of Glial Scar Formation One experimental approach to manipulating the glial environment aims at blocking the initial formation of the glial scar. As reactive gliosis is characterized by hyperplasia one method consists of removing all proliferating cells generated in response to CNS injury. Non-invasive low dose X-ray irradiation has been shown to disrupt the non-permissive gliotic environment and prevent tissue degeneration around the lesion-site [33] in a SC transection model. Moreover, this reduction in scar formation is accompanied by the regeneration of numerous corticospinal axons and notable functional motor recovery [34, 35]. In addition to this non-invasive technique, attempts have been made to remove proliferating cells by injection of ethidium bromide (EtBr) in the lesion area. EtBr binds to DNA and RNA to destroy the cells through the disruption of numerous cellular functions. This treatment has been shown to be effective at clearing out CNS glia from the lesioned nigrostriatal tract following transection, producing a track through which axons can regenerate; probably in association with microglial cells, the other types of CNS glia soon re-invade to block further regeneration [36]. An alternative method for the modulation of glial scar formation is to remove those cytokines that promote it, as can be achieved by injection of specific antibodies [37]. Indeed, a neutralizing TGFb-1 antibody administered into the brain of injured rats significantly attenuates the formation of the fibrous scar tissue and the limiting glial membrane [38], but this was not associated with an increase in axon regeneration [39]. In addition, the systemic administration of IL-10 (an inhibitor of microglial cytokine synthesis) reduces the inflammatory response and results in a significant attenuation of the astroglial reaction [40], which is correlated with a reduction in neuronal damage after an SCI [41].

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7.2.1.3

Modification of Growth-inhibitory Molecules The glial reaction to injury is characterized by the recruitment of microglia, oligodendrocyte precursors, meningeal cells, astrocytes and stem cells. The damaged CNS also contains oligodendrocytes and myelin debris. Most of these cell types produce molecules that have been shown to be inhibitory to axon regeneration [42] Oligodendrocytes produce Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), versican and tenascin-R, oligodendrocyte precursors produce NG2 DSD-1/phosphacan, neurocan and versican, astrocytes produce tenascin, brevican, and neurocan, and meningeal cells produce NG2 and versican, and activated microglia produce free radicals, nitric oxide, and arachidonic acid derivatives. It is likely that many of these molecules participate in rendering the damaged CNS inhibitory to axon regeneration. Since the discovery that scar formation is associated with the creation of a nonpermissive biochemical environment containing specific inhibitory molecules that can block neurite outgrowth, there have been numerous attempts to ablate or neutralize these inhibitory factors in order to promote regenerative sprouting of the lesioned spinal cord. Chondroitin sulfate proteoglycans (CSPGs) are one class of components which represent a barrier to the re-growth of axons. Application of bacterial chondroitinase ABC at the site of injury digests the glycosaminoglycan side chains of these CSPGs, which are an important component of their inhibitory activity. Studies utilizing the contusion model of spinal cord injury have demonstrated a considerable reduction in CSPG immunoreactivity in response to chondroitinase ABC application [43]. Furthermore, when chondroitinase ABC was placed on top of the interface between the hemisectioned spinal cord and a nerve graft, it promoted substantial axonal growth into the grafts [44]. When partial spinal transections were treated with chondroitinase, it promoted both axon regeneration and functional recovery [45, 46] Subsequent experiments in crush injury models and other species have shown the efficacy of chondroitinase in promoting both regeneration and return of function. Removal of CSPGs with chondroitinase promotes both axon regeneration and plasticity [47], and it is not yet clear which is the more important for promoting return of function. CSPGs are not the only constituents of the extracellular matrix that play an important part in restricting axon regeneration. Studies carried out by Stichel and Muller [48–50] suggest that the impermeable nature of the scar is principally due to its basal membrane formed of collagen IV and laminin. Reducing collagen IV deposition by the iron-chelator 2–2’-dipyramidine after fimbria fornix lesion prevents the formation of a dense collagen network and allows extensive regeneration of the lesioned axons in the commissural track. However, this treatment is not sufficient to enhance corticospinal regeneration after a mid-thoracic spinal cord injury [51], although a more powerful chelator may be effective [52]. Over ten years ago, in vivo studies showed that preventing myelin formation or using a monoclonal antibody (mAb IN-1) that neutralized the activity of the inhibitory myelin protein Nogo-A promoted regeneration of corticospinal fibers in the lesioned spinal cords of adult rats5 3–55. These observations were confirmed by immunizing lesioned animals with CNS myelin, lysing myelin with antibody


and complement in vivo, or enhancing myelin clearance by injecting activated macrophages [56–58]. Functional improvements observed in such animals strongly indicate that nerve fibers allowed to re-grow in an adult spinal cord can make functional connections. However, inhibition of Nogo-A also leads to sprouting of unlesioned axons, so it is not clear at present what proportion of the return of function is due to the regenerated axons, what proportion to plasticity. Current efforts are aimed at blocking Nogo-A, as well as other growth-inhibitory molecules by the in vivo application of antibodies against these molecules, by blocking the receptors to which they bind, or by pharmacologically manipulating the downstream signaling pathways induced by these inhibitory signals in growing neurites [59, 60]. The discovery that the Nogo-A receptor exhibits neural specificity and is also a high-affinity receptor for other known myelin inhibitory proteins, in addition to Nogo-A (MAG and OMgp), suggests that there will be increased activity around this therapeutic target [61–63]. The convergence of three inhibitory influences (Nogo-A, MAG and OMgp) at the level of both cell surface receptors and intracellular signaling could be a reason for the devastating suppression of neuronal growth by CNS myelin after spinal cord injury. It will be important to find out whether blocking of Nogo-A, MAG, and OMgp will have additive effects, and produce a better effect than the increase in regeneration that is produced by blocking the activity of each individual growth-inhibitory protein [64]. Current research would indicate that the Nogo-A receptor, or the intracellular signaling cascades downstream of the receptor, are an attractive therapeutic target, rather than targeting Nogo-A, MAG, or OMgp independently. The biology of Nogo-A has been made somewhat uncertain by contradictory findings in parallel studies of knockout mice lacking Nogo A [65–67]. The reason for the varied results is unclear, but one possibility is that minor differences in the way that the genes were inactivated or the strain background of the different animals is responsible. In light of the evidence showing that three inhibitory mechanisms act via the Nogo receptor, Grandpre et al. have recently identified a competitive antagonist of the Nogo receptor, derived from amino-terminal peptide fragments of Nogo-66 [68]. They found that the Nogo-66 (1–40) antagonist peptide (NEP1–40) blocked the myelin inhibition of axonal growth in vitro. Intrathecal administration of NEP1–40 into rats with a mid-thoracic (T6–7 level) hemi-section induced significant growth of the corticospinal tract below the lesion. In addition, they observed a significant improvement in motor functioning. One major concern with this and other treatments that promote sprouting and plasticity, such as chondroitinase, is that it may enable maladaptive sprouting and growth of non-injured neurons, which could disrupt the highly ordered connectivity of the CNS established during development. Indeed, the functions of Nogo-A, MAG or OMgp may be related more to their possible physiological role in the preservation of the wiring of the CNS, by locking connections into place, than to the suppression of neuronal regeneration. In any case, myelin inhibition is not the only factor responsible for the lack of regeneration; astrocytes and other glial cell types also produce inhibitory molecules including chondroitin sulfate proteoglycan and tenascin [69].

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Yet another approach to counter the inhibitory effects of myelin-associated proteins in spinal cord injuries has been to modulate the intracellular signaling molecules that regulate the environmentally mediated inhibition of neurite outgrowth, namely cAMP levels. Qiu et al. reported that cAMP plays an important role in promoting extensive regeneration of cut dorsal root neuronal central projections into the dorsal columns of the spinal cord [70]. Previously, it was demonstrated that the central projections of dorsal root ganglia would regenerate if their peripheral projections were lesioned one week earlier [71]. The study by Qiu et al. suggests that this conditioning lesion effect manifests itself via a cAMP mechanism. An earlier study showed that elevated cAMP levels could allow axons to overcome myelin-specific inhibitors, myelin-associated glycoproteins (MAGs) and myelin in general [72]. The concept of targeting intracellular signaling molecules extends beyond cyclic nucleotides. Many of the inhibitory effects converge on the Rho pathway, especially relevant in relation to recent studies which have identified a potential co-receptor for the Nogo-A receptor as the neurotrophin receptor, p75NTR, and a possible second-messenger pathway involving RhoA [73–77]. Blockade of RhoA with a bacterial enzyme that ribosylates it has been shown to enable axons to overcome inhibitory environments in vitro and to regenerate after lesions to the optic nerve and spinal cord [78].

7.2.1.4

Selective Removal of Astrocytes Reactive astrocytes are the major cellular constituents of the glial scar. The tightly bound processes of these reactive astrocytes constitute a mechanical barrier, whilst their production of growth-inhibiting molecules up-regulated following injury induce a biochemical barrier to any neuronal repair. Attempts to limit the formation of the astrocytic scar, with 7B-hydroxycholesterol-oleate (a cholesterol derivative) [79], have proved successful not only in reducing astrocytic levels but also in enhancing local sprouting in serotonergic axons below the lesion. Also transgenic animals lacking the two main astrocytic intermediate filaments, GFAP and vimentin show enhanced axon regeneration after injury. There has been relatively little work in this area, and it is possible that future research might produce dividends. 7.2.2

Intrinsic Neuronal Control of Regeneration

Though the majority of axonal regeneration research focuses on the inhibitory effects of the CNS environment, the axons are not just passive bystanders whose regenerative ability is entirely dictated by their environment. Part of the reason for the failure of regeneration is to do with the nature of the axon itself. The three main properties affecting axonal regeneration are neuronal age, site of transection and neuronal type.


7.2.2.1

Neuronal Age Embryonic neurons grafted into the adult CNS will often extend axons considerable distances through the inhibitory glial environment [80–82]. The observed differences in the ability of embryonic and adult neurons to grow axons could be attributed to changes in the internal machinery for axon growth, or due to changes in the molecules by which axons respond to their environments, particularly adhesion molecules and receptors for inhibitory molecules. Modifications in molecules mediating interactions with the extracellular matrix (integrins), and those mediating cell-cell interactions, have been documented. For instance, the adhesion molecule N-CAM has several splice variants and, after development, an exon VASE is inserted. This peptide is inhibitory to axon growth, which may partially explain the poor regeneration of axons that express it [83]. In addition, there are differences in the molecules inside the axon, which are involved in axon growth. GAP-43 is present in all growth cones during development, and only expressed in regenerating axons. Microtubule-associated proteins stabilize the axonal cytoskeleton and are necessary for axonal growth [84], yet the type of MAPs present in axons changes late in development, and the embryonic forms are not re-expressed during regeneration [85]. Growth factors can also greatly increase the vigor of regeneration, yet their receptors are down-regulated in many axons after growth and may not return during regeneration [86]. Integrins mediate growth of axons on extracellular matrix molecules, and embryonic axons are able to adjust their cell surface integrins to suit their environment; adult axons appear to have lost this ability [87]. Whilst adult axons are critically dependent on the presence of autocrine survival factors, it appears that embryonic axons do not require such support, which may explain their more robust regeneration in hostile surroundings [88].

7.2.2.2

Site of Transection In stark contrast to axons of the PNS, the regenerative potential of CNS axons is related to the distance of transection from the cell body. The pioneering experiment of David and Agauyo, placing peripheral nerve grafts into the spinal cord, demonstrated that only axons whose cell bodies were close to the graft regenerated. This poses a major problem for spinal cord repair, since many of the vital axonal pathways that need to be repaired are long; an example being the pyramidal neurons of the motor cortex. Thus, there has been considerable interest in seeking to identify the mechanisms behind this phenomenon, and in finding methods to induce regeneration of axons severed far from the cell body. The most effective treatment to date has been trophic factors such as BDNF. CNTF, NT-3, GDNF. BDNF in particular, delivered to the cell bodies has been found to increase the ability of axons transected far from the cell body in the rubrospinal tract. This is associated with an increase in the expression of GAP-43 in the cell body [89].

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7.2.2.3

Neuronal Variability Different types of axons vary greatly in their ability to regenerate. Perhaps the most extreme example of this are the Purkinje cells of the cerebellum whose axons appear to have no regenerative response to axotomy. At the other extreme are spinal motor neurons. These axons are probably the best axons in the CNS at regenerating, especially when cut close to the cell body. This has been used as the basis of a technique for repairing injuries where the central roots are avulsed from the cord: peripheral nerve tissue is re-implanted into the cord, and the motor axons regenerate into it. The reasons for these differences are not established. Purkinje cells do not up-regulate growth-associated proteins such as GAP-43 or c-jun after axotomy unlike motor neurons, but transgenic animals in which these molecules are expressed in Purkinje cells do not regenerate their axons. Some up-regulation of these molecules in Purkinje cells is seen when animals are treated with Nogo-A blocking antibody, suggesting that myelination might have some part to play in suppressing regeneration [90].

7.3

Bridging Cysts and Scars and Cellular Replacement

Only around one third of injuries are complete transections, with most injuries having some preservation of white matter tracts [91]. Whilst some regeneration may take place through any tissue that remains in continuity across the injury, the human spinal cord injury is large, and injuries cause massive disruption of tissue and extensive scarring. Bridges that form a growth-permissive scaffold within the lesion site could greatly facilitate regenerative axon growth across the injured area, leading to the re-introduction of axons into the remaining intact parenchyma and the return of substantial neurological function. Many types of cells, tissues, or artificial materials have been implanted as bridges into the injured spinal cord [92]. However, the success of these experiments is limited, often because astrocytes “wall off” the foreign object, thus greatly restricting access of regenerating fibers to the implants, and preventing axons that have regenerated through the implant re-entering the host CNS. There is, however, one important exception to this rule, which are implants made of olfactory nerve glial cells, which support the growth of axons from the olfactory epithelium into the olfactory bulb throughout life. Unlike Schwann cell grafts, which are walled off by astrocytes, olfactory glia align themselves with the lesion site, migrate long distances into the anterior and posterior spinal cord stumps, and guide regenerating axons through the lesion [93, 94]. The migratory behavior of these specialized glial cells, which are also known to produce a variety of growth factors and growth permissive extracellular matrix molecules, may be part of their success. Several groups are currently exploring the potential use of other cell types as bridging material, including neural stem cells and cells genetically engineered to allow manipulation of their behavior or their ablation once their bridging function is complete [95]. Neural stem cells can be obtained by expanding a small biopsy of

7.4 Replacing Lost Neurons

brain or bone marrow tissue in culture. These cells could then be induced to form growth-permissive astrocytes, which could be implanted into the spinal cord lesion cavity. If such cells misbehave, for example, by inducing formation of dense scars or tumors, they could be ablated by the activation of an inbuilt suicide gene. The lengthy process of cell purification and expansion means this technique cannot be used in acute injuries. In principle, therefore, there are attractions in designing cell-free grafts of biocompatible bridging materials that use and control endogenous cell populations within the injury site in order to promote axonal regeneration, and control inflammatory and glial reactions [96] The substrate required will ideally be immuno-tolerant, possess a porous scaffold to enable nerve regeneration and cell repopulation, will have a molecular make-up that can be easily manipulated and will be easily absorbed by the CNS. Fibrin, or freeze-dried alginate, gels loaded with growth factors that attract regenerating neurites and keep scar formation under control show much promise in this respect and are attractive options because of their reabsorbable properties [97]. In addition, rapidly absorbable substrates, such as poly-a-hydroxyacids and vinyl chloride and acrylonitrile porous tubes, are currently being tested in the transected spinal cord [98] with some success, although there are some concerns over mechanical hindrance and their lack of absorptive properties. The principal obstacle to be overcome with all of these bridges is how to integrate them into the spinal cord tissue, preferably non-invasively, without inducing scar formation. In this case, the replacement of scar tissue with a more attractive cellular substrate might lead to a more functional repair. Either way, the use of any bridge will have to be part of an extremely complex series of treatments involving trophic factors and factors which neutralize inhibitory molecules, all of which together will allow significant axon regeneration in patients with spinal cord injury. Although there are many hurdles to overcome, reabsorbable substrates that are integrated with cells, guidance molecules or electric fields represent an exciting new approach for spinal cord repair.

7.4

Replacing Lost Neurons

When neurons are permanently lost, there are two potential ways of replacing them. The first is to transplant equivalent neurons taken from a donor brain or spinal cord. The second is to persuade stem or precursor cells, present or transplanted in the adult brain, to become neurons. 7.4.1

Neural Transplantation

When particular groups of neurons are lost, an obvious possibility is to transplant new neurons in their place. Although this technique appears attractive in conditions where cell loss is restricted to a localized site, successful neuronal transplan-

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tation has not yet been achieved, using neurons from adult donors. Neurons obtained from embryos of a certain age however, will survive transplantation. Furthermore, they will grow nerve fibers and make functional synaptic connections. 7.4.2

Replacing Lost Neurons from Stem Cells

Stem cells are undifferentiated cells that have the capability to divide and produce many other types of cell. Fetal cells represent the ideal source of stem cells due to their plasticity (pluripotentiality) and relative lack of immune excitation. The recent demonstration that the adult brain contains stem cells instead raises the attractive possibility that endogenous neurogenesis may be manipulated to therapeutic advantage after CNS injury [99]. Not only are the cells remarkably plastic: adult hippocampal stem cells can give rise to regionally-specific cell types not only in the hippocampus, but also in the olfactory bulb, cerebellum, and retina, and they can even migrate considerable distances. Thus, neural stem cells from both embryo and adult seem to be ‘re-programmable’; an idea drawn from hematopoiesis, that there is a genetically ‘naive’ brain stem cell. What is proving truly remarkable is the range of cell types that can be produced in a culture dish when the environment, growth or trophic factors, in combination with particular substrates, is configured appropriately. The contribution of developmental neurobiology to our understanding of neuronal regeneration and the implications for CNS cellular transplantation is clear. There may exist neuronal ‘stem’ cells in the adult CNS that may be cultured and used for CNS repair. If transplantation of cells into the CNS is to continue to progress, lessons from the developing CNS, such as the way in which a developing cell ‘integrates’ into its surrounding environment in a three-dimensional context and the factors which support this occurrence, must be applied.

7.5

Treating Demyelination

The concept of treating demyelination has arisen from detailed evaluation of histopathology and pathophysiology of spinal cord injuries, which has shown that local loss of myelin in surviving bundles of nerve fibers at sites of spinal cord injuries may lead to a disruption of electrical impulse conductivity and contribute to the permanent functional deficits seen. Myelin damage appears to result from inflammatory processes in the injured tissue and may involve delayed apoptotic death of oligodendrocytes, the glial cells that produce myelin in the CNS [100]. The ability of the spinal cord to replace lost myelin over time is uncertain. While studies of recently injured cords show extensive demyelination, cords injured two or more years previously have none, and also contain areas of Schwann cell myelination. Either the cord can remyelinate damaged axons over a period of time, or demyelinated axons gradually die [101].

7.6 Enhancing Plasticity

Two approaches are in clinical development. The first is pharmacological treatment with 4-aminopyridine, a potassium channel blocker that can restore action potential conduction in demyelinated, or partially myelinated, axons in the injured spinal cord [102]. Clinical studies in more than 200 patients, over 5 years, have reported modest beneficial effects on motor and sensory functions, significant reductions in spasticity and hyperreflexia and evoked potential evidence of enhanced conduction within spinal cord pathways [103, 104]. So far, the only significant side effect is a minimal risk of seizure, presumably because the compound also increases the excitability of healthy neurons. Large-scale human trials of the drug have already begun. The extent of benefit to an individual will be expected to depend on the particular surviving pathways within the spinal cord, as well as their state of myelination. A more challenging, though possibly permanent method of treating demyelination may be offered by cellular transplantation of either oligodendrocytes, or Schwann cells, (the PNS equivalent of oligodendrocytes responsible for myelination of axons). Neural stem cells, obtained from adult animals or human brain biopsies (and in the future possibly from adult bone marrow) and expanded in culture, could also be primed with the correct growth factor cocktail to become oligodendrocytes or Schwann cells, which could then be implanted into lesion sites to promote myelin production. Successful remyelination by such cells has been shown to occur in the spinal cord of adult rats, with restoration or enhancement in axonal conductivity [105–107] There are also data to indicate that olfactory ensheathing glial cells enhance functional axonal regeneration [108]. Repair of the myelin sheath and subsequent restoration of impulse conductivity in nerve fibers that have survived the lesion would enable patients to make much better use of their surviving nerve fiber tracts. The major issues, however, as with most other cellular therapeutic approaches revolve around source, delivery, control and immunogenicity.

7.6

Enhancing Plasticity

Although spontaneous regeneration of lesioned fibers is limited in the adult central nervous system, many people that suffer from incomplete spinal cord injuries show significant functional recovery. This recovery process can continue for several years after the injury and probably depends on the reorganization of circuits that have been spared by the lesion. Synaptic plasticity in pre-existing ‘silent’ pathways and the formation of new circuits through collateral sprouting of lesioned and unlesioned fibers are important components of this recovery process. Thus, extensive functional and anatomical reorganization has been demonstrated to occur in the spinal cord, both above and below the spinal cord injury, and in centers of the brain that have some input into spinal motor pools, which may, in fact, be responsible for paralysis recovery following spinal cord injury [109].

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Although pronounced before and just after birth, this plasticity (that is, the ability of one group of nerve fibers to take over the role of another injured group) is much less prominent in the adult CNS (the so-called ‘early lesion effect’ or ‘Kennard principle’) [110, 111]. Nevertheless, the fact that it occurs in adults at all provides the mechanism for the partial recovery of function that is seen after partial spinal cord injury, stroke, head injury and other pathologies. Antibodies against neurite growth-inhibitory factors have been shown to enhance compensatory sprouting of nerve fibers in the partially lesioned brain stem and spinal cord of the adult rat. This resulted in an almost complete functional restoration of fine paw movements in these animals, as demonstrated by the food pellet-reaching test [112, 113]. Several of the interventions that promote regeneration of damaged nerve fibers (neurotrophic factors, antibodies) will almost certainly also enhance compensatory growth of non-lesioned nerve fibers. In addition, functional and anatomical evidence exists that spontaneous plasticity can be potentiated by activity, as well as by specific experimental manipulations. In particular, the flow of electrical impulses in a particular neural circuit is known not only to strengthen connections, but may even induce the sprouting and formation of new connections. It is in this context that the application of electric fields may prove to be beneficial in the setting of spinal cord injury, and specific rehabilitation strategies may even come into play by this means [114]. The effects of activity on the molecular machinery of nerve fiber growth are currently under investigation, as are the effects of combining growth factors with specific training programs for treating spinal cord injuries. Studies in animal models should pave the way to a better understanding of rehabilitation treatments and to the development of new therapeutic approaches for people with injured spinal cords.

7.7

Future Expectations

The work of the past decade or more has finally produced a variety of treatments that can return partial function to experimental animals after spinal cord injury. The challenge for the coming decade is to translate these into treatments for human patients. It is not clear if rodent animal models are a sufficient testbed for determining whether treatments will work in humans. There has been extensive discussion about alternative large animal models in dogs, cats, primates and even pigs. Some treatments will be tested in these animals before reaching human trials, but some treatments will probably go directly from rodents to humans. Time will tell which is the correct route. It is also a consideration that many of the patients requiring treatment were injured some time ago, and have a stable if disabled neurological condition. The risk of making these patients worse rather than better is present, and only treatments that are well characterized should be tried in them. Present science has only produced partial cures. Future technological advances will lead to more and better tools, allowing us to address many unanswered ques-

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after spinal cord injury fails to augment corticospinal tract regeneration. Exp. Neurol. 160:40–50. Stichel CC, Lausberg F, Hermanns S, Muller HW. Scar modulation in subacute and chronic CNS lesions: Effects on axonal regeneration. Restor Neurol Neurosci. 1999;15(1):1–15. Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, Christ F, Schwab ME. (2000). Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature. 403: 434–9. Schnell L, Schwab ME. (1990). Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature. 343: 269–72. Bregman BS, Kunkel-Bagden E, Schnell L, Dai HN, Gao D, Schwab ME. (1995). Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature. 378: 498–501. Huang DW, McKerracher L, Braun PE, David S. (1999). A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord. Neuron. 24: 639–47. Dyer JK, Bourque JA, Steeves JD. (1998). Regeneration of brainstemspinal axons after lesion and immunological disruption of myelin in adult rat. Exp Neurol. 154: 12–22. Rapalino O, Lazarov-Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M, Solomon A, Gepstein R, Katz A, Belkin M, Hadani M, Schwartz M. (1998). Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nat Med. 4: 814–21. Brittis PA, Flanagan JG. (2001). Nogo domains and a Nogo receptor: implications for axon regeneration. Neuron. 30: 11–4. Brosamle C, Huber AB, Fiedler M, Skerra A, Schwab ME. (2000). Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1

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antibody fragment. J Neurosci. 20: 8061–8. Fournier AE, GrandPre T, Strittmatter SM. (2001). Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature. 409: 341–6. Wang KC, Koprivica V, Kim JA, Sivasankaran R, Guo Y, Neve RL, He Z. (2002). Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417: 941–4. Liu BP, Fournier A, GrandPre T, Strittmatter SM. (2002). Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science. 297: 1190–3. Bregman BS, Kunkel-Bagden E, Schnell L, Dai HN, Gao D, Schwab ME. (1995). Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature. 378: 498–501. Zheng B, Ho C, Li S, Keirstead H, Steward O, Tessier-Lavigne M. (2003). Lack of enhanced spinal regeneration in Nogo-deficient mice. Neuron. 38: 213–24. Kim JE, Li S, GrandPre T, Qiu D, Strittmatter SM. (2003). Axon regeneration in young adult mice lacking Nogo-A/B. Neuron. 38: 187–99. Simonen M, Pedersen V, Weinmann O, Schnell L, Buss A, Ledermann B, Christ F, Sansig G, van der Putten H, Schwab ME. (2003). Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron. 38: 201–11. GrandPre T, Li S, Strittmatter SM. (2002). Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature. 417: 547–51. McKeon RJ, Schreiber RC, Rudge JS, Silver J. (1991). Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. J Neurosci. 11: 3398–411.

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Schwann cell grafts to promote regeneration in the transected nude rat spinal cord. Exp Neurol. 1997 Dec;148(2):502–22. Shi R, Blight AR. (1997). Differential effects of low and high concentrations of 4-aminopyridine on axonal conduction in normal and injured spinal cord. Neuroscience. 77: 553–62. Potter PJ, Hayes KC, Segal JL, Hsieh JT, Brunnemann SR, Delaney GA, Tierney DS, Mason D. (1998). Randomized double-blind crossover trial of fampridine-SR (sustained release 4-aminopyridine) in patients with incomplete spinal cord injury. J Neurotrauma. 15: 837–49. Wolfe DL, Hayes KC, Hsieh JT, Potter PJ. (2001). Effects of 4-aminopyridine on motor evoked potentials in patients with spinal cord injury: a double-blinded, placebo-controlled crossover trial. J Neurotrauma. 18: 757–71. Liu S, Qu Y, Stewart TJ, Howard MJ, Chakrabortty S, Holekamp TF, McDonald JW. (2000). Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc Natl Acad Sci U S A. 97: 6126–31. Sasaki M, Honmou O, Akiyama Y, Uede T, Hashi K, Kocsis JD. (2001). Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia. 35: 26–34. Franklin RJ. (2002). Remyelination of the demyelinated CNS: the case for and against transplantation of central, peripheral and olfactory glia. Brain Res Bull. 57: 827–32. Ramon-Cueto A, Plant GW, Avila J, Bunge MB. (1998). Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants. J Neurosci. 18: 3803–15. Fouad K, Pedersen V, Schwab ME, Brosamle C. (2001). Cervical sprouting of corticospinal fibers after thoracic spinal cord injury accompanies shifts in evoked motor responses. Curr Biol. 11: 1766–70.

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II Cellular and Molecular Mechanisms


8 Apoptosis and Necrosis Laura Korhonen and Dan Lindholm

8.1

Introduction

The term apoptosis was introduced more than 30 years ago to describe a form of cell death exhibiting certain morphological characteristics. It is now considered that apoptosis occurs during development for the establishment of an optimized number of cells with correct morphology and function of most organs in the organism. In the developing nervous system, depending on the neuronal subtype, about 20–80 % of neurons born are eliminated during naturally occurring cell death which shows classical apoptotic morphology [1]. This developmental cell death is needed for correct morphology and proper function of the nervous system. This type of cell death has been shown to be regulated by the availability of neurotrophic factors which give trophic support for the developing neurons. However, neuronal death and degeneration occurs also in several human diseases, such as in Alzheimer’s and Parkinson’s disease (PD), Huntington’s disease (HD), in amyotrophic lateral sclerosis (ALS) and in spinal muscular atrophy as well as after more acute injuries occurring in brain trauma and ischemia/stroke. It has been shown that cell death occurring in different types of neurotrauma and neurodegenerative disorders show characteristics of apoptosis and that similar signaling events and proteins regulating developmental-induced neuronal death may also take part in neurodegeneration [2]. However there are also some clear differences between the two processes, for example with regard to timing of cell death, which in apoptosis is usually rapid, but in the more chronic degenerative disease occurs over a longer time span. In addition, our understanding about the mechanism and protein interactions triggering cell death in the different types of neurodegenerative diseases is still incomplete. Recent studies have shown the importance of protein aggregates and their toxicity for subsequent cell death in different neurological diseases. However, the relationship of disturbed protein metabolism and the regulation of cell survival and different signal cascades in the neurons are not fully understood. Increased knowledge in this field and about the role of apoptosis in neurodegenerative diseases is important, as it may reveal novel drug targets and foster developNeuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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ment of rational therapeutic approaches for intervention, based upon, for example, inhibition of death pathways or promoting neuronal survival. In the following, we will briefly discuss current concepts and cellular and molecular mechanisms of apoptosis/programmed cell death and how these differ from other types of cell death such as necrosis. The involvement of apoptosis in different human pathologies will also be shortly commented upon, but a closer description of the pathophysiology and neuronal vulnerability occurring in the different diseases will be given in other chapters in this book.

8.2

Apoptosis versus Necrosis

Apoptosis (Greek word meaning leaves falling off) is a strictly genetically controlled process that depends on both intrinsic and extrinsic factors. Many of the molecules and proteins underlying apoptosis have been revealed in recent years, together with the identification of cellular processes and signaling cascades regulating this type of cell death. The term programmed cell death has then often been used as a synonym to apoptosis and underscores the importance of cellular program in form of genes and proteins activated during the regulation and execution phase of this type of cell death. In contrast to this, death due to necrosis is thought to occur after a mechanical or pathological insult, with the cell passively dying off as a consequence of damage to membranes and cell organelles hampering vital functions. Apoptosis on the other hand is an active process which needs ATP, and which often, but not always, leads to caspase activation [3]. Necrosis is accompanied by an inflammatory reaction and the subsequent release of reactive cytokines, which, in apoptosis, is usually negligible [3, 4]. The morphological characteristics during apoptosis include chromatin condensation and DNA cleavage into 180bp internucleosomal fragments, due to an endonuclease activated by caspases [5]. In addition, high molecular weight DNA fragments (50–300kbp) are present, reflecting activation of different factors degrading DNA, such as Apoptosis Inducing Factor (AIF) [3, 6, 7]. In some cases, a variant type of cell death, exhibiting both signs of apoptosis and necrosis, occurs. Some authors have suggested the name paraptosis for a type of cell death that does not exhibit all the morphological characteristics of apoptosis [8]. Paraptosis was first described in epithelial 293T, and the cell death was neither affected by the anti-apoptotic protein Bcl-X nor by treatment with caspase inhibitors. However, the cell death was still mediated by caspase-9 activation in an Apaf-1-dependent manner [8]. In addition, many processes, which are called apoptosis, do not fulfill the morphological criteria of apoptosis as originally described. For example, a subset of neurons can have apoptotic characteristics after various necrotic insults [9–11]. Moreover, both necrotic and apoptotic signs, such as ruptured membranes and DNA fragmentation, can be found in the same neuron [12, 13]. The same chemical compound, such as kainic acid, stimulating glutamate receptor on neurons and causing excitotoxicity, can induce either apoptotic or

8.3 Genetics of Apoptosis and the Proteins Involved

necrotic cell death depending on time of exposure, concentration of the agent, age of animal or neurons treated [13]. Neuronal cells are also likely to undergo an apoptotic form of death when a greater amount of energy is available [14, 15]. Along this line, some authors have suggested that injured neurons first intend to undergo apoptosis, but can be halted, due to various factors such as the of availability of ATP and mitochondria function [4, 14, 15]. In addition, the levels of free radicals (ROS) play an important part in regulation of apoptosis through the involvement of cell organelles, including the mitochondria. In brain disorders, the production of ROS and oxidative stress are thought to contribute to the pathophysiology of different neurodegenerative diseases [16]. However, paradoxical effects of inhibition of ROS with an increased number of apoptotic cells have been described in brain trauma [17]. Recent reports on apoptosis have described a caspase-independent type of cell death, which nevertheless shows apoptotic morphology. For example, oxidative stress-induced death of retinal cells cannot be blocked by caspase inhibitors [18]. Studies with caspase-3 or caspase-9-deficient mice have shown that cell death occurs to a nearly normal extent within different populations of developing neurons [19]. However, the morphology of dying neurons differs from that seen in classical apoptosis, and the kinetics of cell death is also delayed. There is also evidence that, beside caspases, other proteases, such as calpains, serine proteases and cathepsins become activated during cell death [20]. In this respect, it has been shown that caspase-3 and calpain are synergistically activated in neonatal hypoxia-ischemia, which lead to accelerated cell death [21]. It is also possible that neurons may undergo both apoptosis and autophagic degeneration during the same disease process [22]. The simultaneous activation of different proteases and signaling pathways acting in concert in the cell is probably a common theme in many diseases causing neuronal dysfunction and death. As evident from the above discussion, the current terminology on the concept of cell death is not completely satisfactory. Therefore, it has recently been suggested that the term active cell death (ACD) should be used for the type of cell death that requires activation of intracellular processes, regardless of the strict morphology present in dying cells [23]. In this respect the term passive cell death (PCD) should be used instead of necrosis, a word that does mean bona fide only death.

8.3

Genetics of Apoptosis and the Proteins Involved 8.3.1

Caenorhabditis Elegans as a Model System

The nematode Caenorhabditis Elegans (C. Elegans) with its 1091 cells, of which 131 die via apoptosis, has served as a simple yet powerful genetic model for apoptosis [24]. There are at least 15 genes that function in programmed cell death [25], and they can be divided into following functional classes: (1) genes involved in

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decision-making (ces-1 and ces-2); (2) in the execution (ced-3, ced-4, ced-9 and egl-1); (3) the engulfment of dying cells (ced-1, ced-2, ced-5, ced-6, ced-7, ced-10, ced-12); and (4) genes involved in degradation of cell corpses within engulfing cells (nuc-1). Ces-1 and ces-2 (ces, cell death specification) regulate a subset of cell death in C. Elegans since mutations in these genes block the death of certain neural cells. However, mutations in ces-1 and ces-2 do not affect the death of all cells in the worm, indicating that several parallel mechanisms control the decision-making step of apoptosis [26]. Ces-2 encodes a transcription factor in the basic-leucine zipper family, indicating that the initiation of cell death is controlled at the level of gene expression [25]. Ced-3, ced-4 and egl-1 are required for cell death, and loss-of function mutations of the genes result in survival of nearly all cells in C. Elegans. Egl-1 (egl, egg laying defective) was initially discovered as a gain-of-function mutation leading to ectopic expression of cell death in HSN (hermaphrodite specific neurons), resulting in egg-laying defects [27]. Egl-1 binds to Ced-9 and is thought to act by releasing Ced-4 from Ced-9 [27]. The mammalian Egl-1 homologues, the so-called BH3only proteins (see below), also bind other Bcl-2 family proteins and are capable of inducing apoptosis [28]. Ced-3 (ced, cell death abnormal) encodes a member of cysteine-aspartases (caspases, see below), important for the execution of cell death, and acts in conjunction with ced-4 [29, 30]. Both of these genes are needed for the death process, since mutations in one or both of them largely inhibit cell death. Overexpression of Ced-3 can induce apoptosis independently of Ced-4 expression, whereas Ced-4 overexpression in Ced-3-negative cells results in very little cell death [25]. Thus, Ced-4 seems to potentiate the death-inducing activity of Ced-3. Contrary to these genes, ced-9 has been shown to inhibit apoptosis. Loss-of-function mutation in ced-9 causes death, which is inhibited by Ced-3 and Ced-4 mutations [31, 32]. Ced-9 thus seems to be a function of the other two gene products. The mammalian homologue of Ced-9, Bcl-2 protein also exhibits anti-apoptotic functions, as explained below [28]. The engulfment of dying cells is an important step during cell death. In C. Elegans, several genes have been identified to take part in this process, among them ced-2, ced-5 and ced-10, which are important for cytoskeleton rearrangement. There are also other genes involved, ced-1, ced-6 and ced-7, the functions of which are less understood [32]. Homologues genes have been found in mammals, which are involved in the alterations of the cytoskeleton during migration and engulfment processes of dying cells [33]. Apoptotic cells are recognized and phagocytosed by neighboring phagocytes, such as macrophages and dendritic cells [34]. The recognition step seems to be conserved and is mediated by surface receptors, among others scavenger receptors, integrins and phosphatidylserine receptor [34, 35]. Interestingly, during engulfment, some anti-inflammatory cytokines, such as transforming growth factor- b, are secreted that have many biological effects on neighboring cells. Following engulfment, the cell content must be degraded,


which is achieved in the worm by – among others – the activity of the nuc-1 gene (nuc, nuclease). Mutations in nuc-1 or the genes involved in engulfment do not prevent cell death, indicating that they are not required for cell-killing. Instead, Nuc-1 seems to regulate the activity of a nuclease required for DNA degradation. The homologues protein DNaseII degrades DNA during apoptosis of mammalian cells [36, 37]. 8.3.2

Caspases

Studies in different systems have shown that the activity of intracellular enzymes named cysteine aspartases or caspases are required for apoptosis and death of most cells. Identification of ced-3 in C. Elegans rapidly led to the discovery of the mammalian homologue interleukin-1b-converting enzyme (ICE), which is currently named caspase-1 [30]. Today, 14 mammalian caspases have been cloned and characterised. Caspases are synthesized as inactive precursors which are composed of four distinct domains: an N-terminal pro-domain, a large subunit, a short domain and a linker region [38]. The activation of caspases is induced by a proteolytic cleavage of the pro-domain and the linker region leading to a formation of a tetramer with two catalytic sites [38, 39]. Caspases are usually divided into two groups: one related to ICE (caspase-1) and the other to ced-3. The length of the prodomain that can be short, such as for caspase-3, -6, -7, or large as for the other caspases can further distinguish these enzymes. Caspases can also be arranged into groups, depending on their substrate specificity [39]. Group I consists of caspases-1, -4, -5 and -13 with a preference for hydrophobic amino acids in the position P4 of the cleavage site. In contrast, group II caspases (caspase-2, -3 and -7) require the amino acid, Asp in the same position. Group III caspases (caspase-6, -8, -9 and-10) prefer aliphatic amino acids in the position P4. These are found in many substrates including group II and III caspases, indicating that the latter one can be substrates for the group III enzymes. In this way the caspases belonging to the group III are thought to initiate a reaction that leads to apoptosis and cell demise via an amplification cascade [40]. To activate the initiator caspases, two domains mediating protein interactions have proven to be important: the death effector domain (DED) and the caspase recruitment domain (CARD). DED are found in the protein FADD, which is an adaptor protein linked to the Fas/APO-1/CD95 receptor upon Fas-ligand binding via its death domain (DD) [41]. Caspase-8 has one DED motif in its pro-domain, and Caspase-10 contains two DED domains [42]. Interaction between the DED domains and FADD connects the apoptotic signals from Fas/APO-1/CD95 receptor to the caspases. The CARD domain, which is present among others in Caspase-1, -2 and -9, is also thought to mediate the interaction between the adaptor and their respective caspase. For instance, CARD domains in APAF-1 binds CARD domains in Caspase-9 and in similar CARD domains of caspase-2 bind to RAIDD, which is an adapter molecule in tumor necrosis factor receptor 1 (TNF-R1) signaling [43, 44]. TNF signaling itself is important for – amongst others – the activation of

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microglia cells which mediate inflammation in the brain and are thought to be of importance in neurodegenerative disorders, such as in Alzheimer’s disease [45]. The importance of caspases for brain development has also been revealed by studies of mice that were gene-deleted for different caspases. In particular, caspase-3 and –9-deficient animals show a severe phenotype in the brain, indicating that they play a central role in regulating the death of brain cells. Caspase-3 knockout mice show malformations in brain at an early stage and in neural progenitor cells, suggesting that this caspase is required for normal brain development [46]. In addition, studies with animals and brain cells lacking different caspases have shown that caspase-1, -2 -11 -and -12 are involved in regulation of neuronal death [47]. Caspase-8-deficient mice have defects in the immune system, indicating that caspase-8 is required for proper function of immune cells [48], but this caspase may also be of importance in neurodegeneration in HD, and in many immune reactions in the brain. The amplification of the caspase cascade involving different downstream substrates ultimately leads to cellular destruction and the appearance of the cellular characteristics described above [40]. Among substrates described for caspase activity are different kinds of structural proteins, lamins, fodrin and gelsolin, proteins regulating DNA integrity, such as poly-ADP-ribose polymerase (PARP) and DNAPK, in addition to proteins involved in cellular signaling and survival reactions [49]. In the brain, both amyloid precursor protein (APP) and huntingtin with its polyglutamine repeats have been shown to be cleaved by caspases, which can result in toxic products aggravating the disease process in Alzheimer’s disease and in HD, respectively [50]. In addition, in ALS higher levels of activated caspase-1 and-3 have been found, and the inhibition of caspase-1 can delay the disease progression [51, 52]. However, the exact roles of different caspases and their activation in the disease processes causing neuronal dysfunction and death are so far not fully understood. Although the caspases are synthesized as pro-enzymes, which need to be proteolytically activated, the procaspase can also have some activities under certain conditions. It is therefore of ultimate importance for the cell to be able to control the activity of the caspases, with their potentially deleterious effects on cell survival. Studies of virally infected cells, which are usually prone to death, have shown that the virus can escape the cell death machinery by producing proteins inhibiting the caspases. Cowpox virus serves as an example of this: It produces a protein called cytokine response modifier, which binds and inhibits caspase-1, leading to the suppression of cell death in virally infected cells [53]. Proteins belonging to the inhibitor of apoptosis (IAP) protein family (see below) where also originally identified in baculoviruses, due to their ability to suppress cell death [54]. Based upon structural data, several synthetic peptides with the ability to inhibit caspases have been designed. The selectivity of the inhibitors has been developed based on the specific substrate cleavage sites, and they act as competitive pseudo substrates [40]. Depending on the chemical groups, they are either reversible or irreversible inhibitors. The former are often coupled to aldehyde (-CHO), ketone or nitrile groups, and the latter are linked to fluoro- or chloro-methyl ketones


(-CKM, -FMK). These inhibitors have been shown to increase survival and counteract death of both cultured and in vivo cells [55]. In view of their potential medical benefits the synthetic caspase inhibitors have also been used in studies of brain injuries. The results of these are promising and it has been shown that administration of caspase inhibitors in vivo can in most instances delay cell death after different types of brain trauma [56]. Beneficial effects have also been obtained studying caspase-deficient mice after stroke/ischemia [57]. Taken together, these studies indicate that the inhibition of the activity of caspases may be beneficial to consider in different types of brain trauma and in neurodegeneration. However, more data on the safety, selectivity and effects of potential blockers of caspases in vivo are clearly needed to resolve these questions. 8.3.3

Anti-apoptotic Proteins

In mammalian cells, including neurons, two large protein families, the Bcl-2 and IAP proteins regulate apoptosis, mainly at the mitochondrial and post-mitochondrial level. Bcl-2 is a mammalian homologue of ced-9, and belongs to a large gene family sharing structural similarity in the BH (Bcl-2 homology)-domain [28]. Some of these proteins, including Bcl-2, are shown in Fig. 8.1 The Bcl-2 family can structurally be divided into three classes: the Bcl-2, Bax and BH3only subfamilies. The Bcl-2 subgroup includes proteins such as Bcl-2, Bcl-XL, BCL-xs and BCL-w, which all have four BH-domains (BH 1–4) and exhibit, with the exception of Bcl-Xs, anti-apoptotic properties [58,59]. The pro-apoptotic Baxsubgroup consists of Bax, Bak, Bad and Bok, which only have the BH 1–3 domains

Figure 8.1 The Bcl-2 protein family. Note the occurrence of the homology regions in the different Bcl-2 subfamilies. (þ) denotes promotion of cell survival; (–), death inducing activity. See text for details.

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[28]. The third subfamily consists of proteins having only one BH domain, the so called BH3 – only proteins which include Bik, Bid and Bim, Hrk [28, 60]. These proteins are pro-apoptotic, showing that the BH3 domain alone can induce apoptosis under certain conditions [60]. It has also been shown that exchange of the BH3 region of Bcl-2 for that of Bax leads to a loss of the anti-apoptotic activity in Bcl-2, demonstrating the importance of this region in cell death regulation [61]. The genes encoding the different Bcl-2 family proteins also undergo alternative splicing, resulting in proteins with opposite functions in cells. For example Bcl-x exists in two alternative forms Bcl-x large (Bcl-xL) and Bcl-x small (Bcl-xs), of which Bcl-xL has a pro-survival and Bcl-xs a pro-apoptotic function [62]. The relative expression of the two forms in brain tissue or in disease processes has not been analyzed so far. Recently, studying Bak, a neuron-specific isoform of the protein was reported which shows anti-apoptotic properties in neurons, but is proapoptotic in glial cells [63]. A deeper understanding of the mechanism underlying these effects would add to our knowledge about the regulation of neuronal cell death by the Bcl-2 proteins. Some of the Bcl-2 family proteins have a hydrophobic transmembrane domain at the carboxyterminal end that can insert into membranes [64]. Bcl-2 and some other of the proteins have been found associated with the mitochondrial outer membrane, the nuclear membrane and the endoplasmic reticulum [64]. In the case of Bcl-2, membrane insertion is associated with its capability to antagonize apoptosis in most, but not in all cells [64,65]. In contrast, it has been shown that deletion of the transmembrane domain in Bcl-xL does not affect its anti-apoptotic properties [66]. Recent studies on the anti-apoptotic protein Bcl-w have shown that membrane integration may play a role in neutralizing the activity of Bcl-w [67]. It is thus possible be that the role of membrane association of the Bcl-2 proteins can vary in different cells and under different conditions. In addition to the BH regions, post-translational modification can influence the function of the Bcl-2 proteins. Bad has shown to be phosphorylated by growth factors through the activation of the downstream protein kinase, Akt [68]. Phosphorylation of Bad leads to its binding to the protein 14–3–3 in the cytoplasm, blocking its action [68]. Recent studies have shown that the BH3-only proteins like Bim can also be phosphorylated with an effect on their pro-apoptotic function in cultured neurons [60, 69]. The phosphorylation of Bcl-2 in the loop region between BH3 and BH4 can abrogate the function of Bcl-2 and lead to cell death as studied mostly in cancer cells [70]. Recent data on hippocampal neurons suggest that a similar mode of regulation of Bcl-2 through phosphorylation can also occur in neurons after injury [71]. It is likely that different modifications, such as phosphorylation of the Bcl-2 family proteins play a crucial role for their pro-or anti-apoptotic functions in cells. Future studies will reveal the importance of such changes in Bcl-2 protein in various neurodegenerative diseases of which little is known at the moment. The Bcl-2 family proteins exhibit a variety of biological actions in different systems. The Bcl-2 family members have been shown to form heterodimers, which can interfer with their mutual biological activities [72, 73]. Structural data using


model peptides has shown that the BH3 domain of the pro-apoptotic protein Bax and Bak inserts into a hydrophobic pocket formed by BH1, BH2 and BH3 regions in Bcl-xL and Bcl-2 [28]. The interaction between different Bcl-2 family proteins is of ultimate importance in the regulation of cell death upstream from mitochondria. Recent data on the BH3-only proteins in neurons as well as in other cells support this view. It has been shown that the expression of these proteins is up-regulated prior to induction of cell death. Sympathetic neurons deprived of the neurotrophic factor NGF have been studied as a model for developmental induced cell death and apoptosis [74, 75]. The deprivation of NGF causes an induction of the BH3-only proteins Bim and the related protein Hrk/DP5 that can promote the translocation of Bax to the mitochondria and lead to neuronal death [76, 77]. This mechanism of cell death induction is, however, not true for all types of neurons, as it has been recently shown that the levels of Bim and Hrk/DP5 actually decrease in hippocampal neurons after an excitotoxic injury [71]. Apart from binding other family members, Bcl-2 and partly also Bcl-x have been shown to interact with other proteins among others with Raf-1, R-Ras p23 and calcineurin [28]. The functional significance of these interactions, particularly in brain tissue, has not been thoroughly investigated. The determination of the three-dimensional structure of Bcl-XL gave a hint concerning the function of Bcl-2 proteins as ion channels [78]. The structure of Bcl-XL is very similar to that of some bacterial toxins e. g. diphtheria toxin, which contain a pore-forming domain. Subsequent studies have demonstrated that Bcl-2, Bcl-XL and Bax can form ion channels in synthetic lipid membranes [79, 80]. The pores formed by Bcl-2 and Bcl-XL are small and have a moderate cation preference and are formed only in non-physiological acidic conditions. In contrast, Bax forms larger pores, which prefer anions at physiological pH and, in some studies, but not all, it has been shown that Bcl-2 can prevent in vitro channel formation by Bax [79]. In the cellular context, Bcl-2, in addition to Bcl-xL and Bcl-w have been reported to inhibit cytochrome c release from mitochondria, whereas Bax can promote it [81–83]. Bak or BH3 oligopeptides, added to isolated mitochondria, induce cytochrome c release and lead to mitochondria dysfunction [81, 84]. It is thought that the Bcl-2 family members regulate cytochrome c release and the mitochondria permeability transition pore that is opened by the loss of the electrochemical gradient. Bcl-2 and Bcl-X overexpression have been reported to protect against different agents, such as cyanide, that directly act on the mitochondria PT pore [82]. Studies on gene-deleted animals have emphasized the importance of Bcl-2 in regulation of neuronal survival. Bcl-2-deficient mice can survive embryonic development, but die in early adulthood, accompanied by polycystic kidney disease and thymic and splenic abnormalities [85, 86]. On the other hand, transgenic mice overexpressing Bcl-2 in neurons show resistance against developmentally-induced neuronal death in addition to rescue of neurons following stroke/ischemia, and facial, sciatic and optic nerve axotomy [87, 88]. In contrast to Bcl-2-deficient animals, mice lacking Bcl-xL die during embryonic development with increased apoptosis and death of developing neurons and hae-

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matopoietic cells. This early-occurring neuronal death was prevented by Bax-deficiency, indicating that Bax binding to Bcl-xL critically regulates neuronal survival during this developmental period [89]. Bax-deficient mice show cellular hyperplasia in several tissues, including brain tissue [90]. Bax-deficient neurons survive better after facial nerve axotomy, and cultured cortical neurons from these animals are relatively more resistant to glutamate and DNA damage than controls. In addition, sympathetic neurons lacking Bax are less dependent on NGF for their survival [91]. Apart from the Bcl-2 family proteins, the IAP proteins are important regulators of cell death, which are known to inhibit caspases and promote cell survival [54, 92]. Different IAP proteins antagonize cell death upon a variety of signals, among others against treatment with chemotherapeutic agents, Fas activation and trophic factor withdrawal [93]. The IAP proteins act mainly downstream of mitochondria, although some of these proteins can also bind to death receptors, such as the cellular IAP proteins (c-IAP-1 and-2) [92, 93]. The latter proteins in addition to X-chromosome-linked inhibitor of apoptosis protein (XIAP) have been shown also to interfere with cellular signaling cascades. Survivin is shown to stimulate cell proliferation and is important in human cancer cells [94]. The five major human IAP proteins that have been mostly studied are shown in Fig. 8.2. Beside these, other homologues of IAP proteins have been characterized in different species where some have additional functions [92]. The IAP proteins share a well-conserved motif of approximately 65 residues, called the baculovirus IAP repeat (BIR) (Fig. 8.2). c-IAP-1 and –2, XIAP and neuronal inhibitor of apoptosis protein (NAIP) contain three BIR domains, whereas survivin has only one BIR region (Fig. 8.2). The structure of some of the BIR motifs has recently been revealed using nuclear magnetic resonance (NMR) spectroscopy [95, 96]. The NMR analyses of the second BIR region in XIAP, combined with a mutagenesis study, showed that this motif, in addition to some amino acids in the adjacent linker region between BIR1 and BIR2, is crucial for the binding of caspase-3 and -7. A similar analysis applied to the third BIR region in XIAP defined amino acids that are important for inhibiting caspase-9. The overall three-dimensional structure of all BIR domains in the IAP proteins is probably similar, with a classical zinc finger and five a-helices and three b-sheets [95–98].

Figure 8.2 The IAP protein family. Note the occurrence of the BIR and the RING domains in the different proteins. See text for details.


With the exception of NAIP and survivin, all other IAP proteins have a C-terminal RING motif, which can be found in over 200 different proteins. Typical for the RING domains are the histidine and cysteine rich regions with zinc binding capacity [99]. The RING domain is found in other proteins involved in among others RNA processing, organelle transport and viral replication [99]. The function of the RING domain has recently also been linked to ubiquitin-ligase activity and degradation of proteins via the proteasome system. The cellular IAP proteins, in addition to XIAP, can, through their RING domain, stimulate ubiquitination and mediate the proteasome-dependent degradation of caspase-3 [100]. However, the RING domain in XIAP is also able to cause self-degradation of XIAP [101]. The deletion of the RING domain in XIAP was shown to have a differential effect on the neuroprotective ability of XIAP in sympathetic neurons and in neuroblastoma cells [102]. The IAP proteins such as XIAP can also undergo degradation as the result of protease activation. Thus, it was shown that active caspase-3 cleave XIAP in the linker region between BIR2 and BIR3 in Jurkat T cells after irradiation and in hippocampal neurons after kainic acid treatment [103, 104]. This results in two XIAP fragments with altered cellular activities that are probably of functional importance in the cell including neurons. Recently it was shown that XIAP could also induce the degradation of the second mitochondrial activator of caspase (SMAC/Diablo) [105]. This protein is released from mitochondria during cell death along with cytochrome c and with the high-temperature requirement serine protease A2 (HtrA2/Omi). SMAC and HtrA2/Omi can bind and inhibit XIAP, whilst cytochrome c takes part in forming the apoptosome complex [92]. The mutual interaction between XIAP and its binding partners is thought to set the threshold for death in normal cells and after cell injuries. In the nervous system, XIAP is expressed mainly by neurons in different areas, both during development and in the adult brain [104]. Available data indicate that the relative level of XIAP expressed in the cell blocks death and improves survival. Studying transgenic mice overexpressing XIAP in neurons, it was shown that XIAP is protective against brain ischemia/stroke induced by middle cerebral arterial occlusion [106]. The neuroprotection observed with XIAP in these animals was correlated to a decrease in active caspase-3 following stoke [106]. Apart from ischemia, neuronal overexpression of XIAP protected dopaminergic neurons against death induced by MPTP in a model of Parkinson’s disease [107]. Overexpression of XIAP by adenoviral vectors was also neuroprotective in different types of brain injuries [108–110]. The roles played by XIAP in different neurodegenerative disorders, however, remain to be studied. In addition to XIAP, NAIP seems to be of particular importance in the nervous system. NAIP is widely expressed in the nervous system in motoneurons in the spinal cord, in the hippocampus, in the cortex, the amygdala and thalamus. NAIP was originally identified as a gene deleted in many patients suffering from Spinal Muscular Atrophy type 1 (SMA1), a devastating human disease, characterized by motor neuron degeneration in early life [111]. Subsequent studies have

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shown that mutations in the SMN gene are more important in the pathogenesis of SMA, whereas NAIP has a modulatory function in determining the severity of the disease [112]. NAIP can also delay motoneuron cell death in vivo and in vitro and suppress neuronal differentiation and apoptosis in PC12 cells [113, 114]. In addition, adenovirally overexpressed NAIP reduces ischemic damage in the hippocampus of rats and protects the nigrostriatal pathway in a rat PD model [115, 116]. Mice lacking NAIP are also more vulnerable to kainic acid-induced hippocampal cell death than control animals [117]. The mechanism for the neuroprotective function of NAIP may relate to the inhibition of different caspases. Recently, the BIR regions in NAIP were shown to bind caspase-3 and-7, albeit with a lower affinity than XIAP [118]. Studies searching for other proteins binding showed that NAIP can interact with hippocalcin which belongs to the neuronal calcium sensor protein family [119]. The results further showed that NAIP in conjunction with hippocalcin can protect neural cells against high intracellular calcium levels observed among others in excitotoxicity and in different types of neuronal disorders [119, 120]. The exact roles played by NAIP and its binding partners in SMA and in other human neurodegenerative diseases remain to be studied.

8.4

Cellular Mechanisms of Apoptosis

In general terms, one can distinguish two processes of death in mammalian cells; an extrinsic one, triggered by the activation of death receptors (Fas, TNF, TRAIL) residing on the cell membrane, and an intrinsic one, involving the mitochondria and possible other organelles in the cell, such as the endoplasmic reticulum. Both of these pathways lead to the activation of different caspases, e. g. TNF receptors activate caspase-8, whilst caspase-9 is activated downstream of the mitochondria. An outline of the molecular mechanisms and crucial proteins involved in apoptosis in mammalian cells is given in Fig. 8.3. In brain and in neurodegenerative diseases, the underlying mechanisms triggering cell death are not fully understood. Available data from numerous studies have shown that neuronal death may be caused among others by the assembly of abnormal protein aggregates, leading to the accumulation of toxic products. Likewise, an elevation of free radicals (ROS) and calcium, and alterations in intracellular pathways with the inhibition of survival signals can promote the death cascade in the neurons (Fig. 8.3). 8.4.1

The Role of Mitochondria and the Apoptosome Complex

Mitochondria play a decisive role in the regulation of apoptosis by controlling the release of cytochrome c and other pro-apoptotic factors. The exact mechanism controlling this is not fully understood, but several of the Bcl-2 family proteins are

8.4 Cellular Mechanisms of Apoptosis

Figure 8.3 Schematic view of major cell death pathways in neuronal cells. See text for details.

localized at the mitochondrial membrane. It has been shown that Bax promotes cytochrome c release, whereas the anti-apoptotic proteins Bcl-2 and Bcl-xL block the release [81, 82]. To accomplish this, Bax undergoes a change in conformation with translocation and binding to the mitochondria [121]. The BH3-only proteins are thought to act upstream of the anti-apoptotic Bcl-2 proteins and in conjunction with Bax inhibit their function leading to death [60]. Another characteristic of apoptosis is the loss of mitochondria membrane potential. That in itself could possibly cause cytochrome c release. One important structure in mitochondria is the permeability transition pore complex (PTPC), which consists of adenine nucleotide translocator (ANT) in the inner mitochondrial membrane, the cyclophilin D that binds to the ANT, and the voltage dependent channel (VDAC) [83, 122]. Several proteins have been described to bind to this large complex, among them Bax and Bcl-xL, which can form and inhibit channel formation respectively [84]. Recent data have shown that specifically the VDAC2 isoform is able to inhibit mitochondrial apoptosis by binding and regulating the activity of the pro-apoptotic protein BAK [123]. Once cytochrome c is released, it binds to the protein Apaf-1 with the formation of the apoptosome complex, consisting also of pro-caspase-9, and dATP. The activity of the complex leads to the activation of caspase-9 and an amplification of the caspase cascade via activation of caspase-3 [124, 125]. Apaf-1 (apoptosis protease activating factor) has a CARD domain, a central Ced-4 homology domain and carboxyterminal WD40 repeats, which are thought to mediate protein-protein interactions and negatively regulate Apaf-1 activity.

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Apaf-1 bound to cytochrome c hydrolyses dATP and undergoes oligodimerisation via its Ced-4 homology domain. At the same time, the CARD domain recruits and facilitates procaspase-9 processing. In addition, caspase-3 and -7 are recruited into the apoptosome complex where they are processed into smaller subunits [126] The size of the apoptosome complex has been reported to be approximately 1.4 MDa using recombinant proteins, but has a size of 700 kDa in native lysates [126]. Apaf-1 deficient mice die during embryonic development, and exhibit reduced apoptosis in brain tissue with craniofacial and eye alterations [127]. Similar changes have been also identified in caspase-9 deficient mice indicating that the apoptosome complex is important for the cell death process [128]. Apart from cytochrome c, other proteins are also released from mitochondria during cell death. Among this are Smac/DIABLO which can bind to XIAP and inhibits anti-apoptotic function [129, 130]. Structural studies have revealed that the very aminoterminal of Smac/DIABLO (AVPI sequence) is critical for the XIAP binding [98, 131]. Recently, it was shown that SMAC plays an important role in death of cultured sympathetic neurons by neutralizing the anti-apoptotic effect of XIAP [132]. The functional significance of SMAC and HtrA2/Omi and their binding to XIAP in other types of neurons and in neurodegeneration remains, however, to be studied. Recently, it was shown that the serine protease, Omi/ Hrt2A also contains a conserved IAP-binding motif (AVPS) at the aminoterminus [133–136]. Upon release into the cytosol, this motif is exposed, leading to IAP binding via the BIR3 domain and caspase activation. Omi/Hrt2A can also induce apoptosis in a caspase-independent manner through its protease activity. The functional role of Omi/Hrt2A in neuronal cell death and degeneration is so far unknown. AIF and endonuclease-G are other mitochondrial proteins released during cell death [137], and they translocate to the nucleus and induce DNA fragmentation independent of caspases. As shown recently, it is possible that AIF acts downstream of caspase activation [138]. In mice gene-deleted for AIF there is a decrease in programmed cell death of embryonic precursor cells and young neuroblasts [139]. AIF is a flavoprotein and could also play a role as a free radical scavenger, as shown in studies of the mutant mouse called harlequin (hq). In these animals, there is a decrease in the expression of AIF, due to proviral insertion into the aif gene, which leads to degeneration of differentiated retinal and cerebellar neurons [140]. The neurons were shown to be highly susceptible to death caused by peroxide, which could be abrogated by AIF expression [140]. 8.4.2

Cell Death in Neurodegenerative Diseases

As mentioned above, cell death occurring in different types of neurological disease has characteristics resembling that of apoptosis. Thus, many of the proteins discussed above are shown to be involved in developmental induced neuronal death, but they can also be activated and contribute to neuronal dysfunction and death in different neurodegenerative diseases. Studies of caspase-deficient mice and studies using caspase inhibitors in brain disorders have produced promising

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9 Inflammation Harald Neumann

9.1

Introduction

The tissue of the central nervous system (CNS) has a privileged status in regard to its immune milieu and shows a number of structural peculiarities (Fig. 9.1). The normal CNS tissue lacks most of the molecules that are required for an organized immune response (Lowenstein, 2002). First, a dense endothelial blood-brain barrier reduces immigration of immune cells and macromolecules to a necessary minimum (Huber et al., 2001). Second, there is no production and release of bioactive chemokines and cytokines that would attract and activate immune cells. Furthermore, most of the cell adhesion molecules that would be required to guide the migrant lymphocytes to the sites of lesions are missing on endothelium in the normal CNS. Finally, expression of major histocompatibility complex (MHC) molecules is very low or absent, which prevents presentation of antigens to specific T lymphocytes. The situation changes under pathological conditions (Neumann and Wekerle, 1998) during which the CNS mounts an organized innate immune response, which extends during bacterial and viral infections into an adaptive immune response (Fig. 9.1). In autoimmune brain diseases such as multiple sclerosis vivid inflammatory reactions and destructive immune responses are detected in the brain lesions. Furthermore, molecules of both the innate and adaptive immune response are induced in a wide diversity of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, stroke, Huntington’s disease and amyotrophic lateral sclerosis (ALS). The CNS acquires signals that attract T lymphocytes and monocytes, activate locally microglial cells and allow presentation of brainderived antigens to T lymphocytes. The CNS areas affected by the degenerative lesion become immune competent and show immune reactivity. The degenerative and inflammatory CNS processes lead to a rapid activation of immunologically relevant genes and structures, which allow bi-directional communication between the immune system and the nervous system. It appears that neurons have a central role in controlling the immune status of the CNS. Intact, electrically active neurons Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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9.2 Innate Immune Responses in the CNS m Figure 9.1

Innate and adaptive immune responses within the CNS. In the normal CNS the tissue growth factor and cytokine milieu maintain microglia in a quiescent state. Active neurons produce neurotrophins (NGF, BDNF and NT3), which have been shown to prevent expression of immunological molecules on glial cells. Astrocytes produce transforming growth factor-b having immunosuppressive activity. Only activated T cells can pass the bloodbrain barrier and undergo apoptosis after entering the brain tissue. Innate immunity is triggered by activation of microglia and macrophage invasion. Glial cells and neurons produce inflammatory cytokines such as IL-1b and TNF-a and chemokines such as CXCL10, CX3CL1, CCL2 and CCL5. Chemokines attract the motile microglia and macrophages to the lesion sites. During adaptive immunity endothelium cells produce chemokines (CCL19 and CCL21), which promote attraction and transmigration of lymphocytes into the CNS. Antigenspecific recognition locally stimulates lymphocytes and facilitates their full blown effector function including cytotoxictiy, cytokine secretion by T cells and antibody production by B cells.

prevent inducibility of immunologically relevant genes (Neumann, 2001). In turn, functional impairment of neurons results in activation of inflammation. In zones of neuronal degeneration, even weak pro-inflammatory signals appear to stimulate release of chemokines, up-regulation of cell adhesion molecules, production of soluble inflammatory mediators and expression of MHC molecules that are required to shape an organized innate and adaptive immune response in the CNS. The CNS immune response could be tissue-destructive in the development of most neurological diseases (Neumann et al., 2002). However, stimulation of the innate immune response in the CNS may also trigger the production of neurotrophic factors and promote repair and remyelination.

9.2

Innate Immune Responses in the CNS

Specific cells of the innate immune system sense microorganisms via specific receptors that bind to the so-called ‘pathogen-associated molecular patterns’ (PAMP). In particular, macrophages, dendritic cells and other phagocytic cells respond to such PAMP and induce an innate immune response. Lipopolysaccharide (LPS), an essential and unique component of the outer membranes of gram-negative bacteria, is a typical PAMP, which mounts a robust innate inflammatory response. The toll like receptor (TLR) family has received significant attention as signaling receptors of PAMP. The toll receptor was first described as a molecule for establishment of the dorsoventral axis in the Drosophila embryo. The mammalian homologue of toll is a receptor family, whose extracellular domains show multiple leucinerich repeats and intracellular domains similar to the cytoplasmic portion of interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a). Several members of the TLR family have been characterized so far (Akira, 2003). Toll like receptor-2 (TLR2) recognizes the PAMP that are derived from Gram-positive (Gramþ) bacterial cell wall components. TLR4, in association with CD14, is responsible for the recognition of lipopolysaccharide (LPS) from Gram-negative (Gram–) bacteria. Flagellin, the principal element of bacterial flagella, is recognized

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by TLR5. Unmethylated bacterial DNA is recognized by TLR9 and triggers an inflammatory response. TLR3 engages the innate immune response in the presence of viruses that produce double-stranded RNA (dsRNA). The CD14 and TLR4 receptors are constitutively expressed in the circumventricular organs of the CNS (Nguyen et al., 2002). Furthermore, circulating LPS induces expression of CD14 and several types of TLRs (TLR4, TLR2) on parenchymal microglial cells. Subsequently, this activation of microglial cells is associated with a robust microglial production of cytokines, chemokines and complement components. Inflammatory cytokines such as TNF-a are released by stimulated microglial cells (Nguyen et al., 2002). TNF-a itself acts in an autocrine and paracrine fashion and leads to activation and stimulation of the transcriptional factor NF-kB, which drives further cytokine gene transcription. Indeed, pre-treatment of mice with antiTNF antibodies significantly prevents LPS-induced cytokine gene transcription.

9.3

Dual Mechanism of Innate Immunity in the CNS

Although the innate immune response in the CNS might have a detrimental effect, it is clear that it also has a beneficial role. Pro-inflammatory cytokines are produced as early as 15 minutes after acute trauma to the CNS and are followed by temporal production of neurotrophic factors (Nguyen et al., 2002). This profile suggests a potential role of the inflammatory response in triggering the neurotrophic response phase. TNF-a and IL-1b are two of the main cytokines that are detected within parenchymal microglia along the lesion site, and a role for IL-1b in the induction of nerve growth factor expression by astrocytes has been demonstrated. Microglialderived IL-1b is also involved in astrocytic production of ciliary neurotrophic factor (CNTF) and insulin-like growth factor 1(IGF1), both of which promote repair of the injured CNS (Nguyen et al., 2002). TNF-a has a dual function in brain damage. It has been shown to either exacerbate or attenuate brain injury in distinct pathological situations. Furthermore, TNF-a has a crucial role in triggering remyelination via stimulation of TNF receptor-2 (TNFR2), but not TNFR1 (Arnett, 2001). Lack of TNF-a led to a significant delay in remyelination, which was associated with a reduction in the pool of proliferating oligodendrocyte progenitors and a reduction in the number of mature oligodendrocytes (Arnett, 2001). Cytokines such as TNF-a could directly act on neurons, and it was suggested that TNF-a and interferon-g (IFN-g) act synergistically to damage neurons. Transgenic mice that express IFN-g in the hippocampus show an enhanced microglial response to lesioninduced neuronal injury (Jensen et al., 2000). The activation and cytokine production of microglia occurs also in the CNS of patients with Alzheimer’s disease, Parkinson’s disease and Huntington’s disease and ALS (Nguyen et al., 2002). IL-1b and TNF-a are secreted by activated parenchymal microglial cells and can be potent inducers of cell death in models of neurodegeneration. However, the functional relevance of cytokine secretion in neurodegenerative diseases is not fully understood.

9.4 Antigen Presentation for Adaptive Immune Responses in the CNS

9.4

Antigen Presentation for Adaptive Immune Responses in the CNS

As mentioned before, the CNS is located in an immunologically privileged environment behind a blood-brain barrier (BBB), formed by tight junctions of endothelial cells (Fig. 9.1) (Huber et al., 2001). During inflammatory brain diseases, such as multiple sclerosis (MS) and viral or bacterial CNS disease, changes occur which allow specific and efficient defense against dangerous pathogens and healing (Neumann and Wekerle, 1998). Antigens derived from viruses or bacteria do not stimulate T lymphocytes directly. Instead, specialized cells named antigen-presenting cells (APCs) process and display the antigens on their cell surface to make them recognizable by specific T lymphocytes. The T lymphocytes recognize antigenderived peptide fragments bound to major histocompatibility complex (MHC) molecules. T cells carrying the CD4 antigen show cognate interaction between the T cell receptor (TCR) and MHC class II molecule displayed on the APC, while T cells carrying the CD8 antigen show cognate interaction between the T cell receptor (TCR) and MHC class I on the APC. The T lymphocytes, if they are in an activated status, possess the ability to pass the BBB and traffic through the CNS parenchyma in search for pathogens. However, the number of T lymphocytes surveying the normal CNS is very low compared to other organs (Yeager et al., 2000). The number of T cells surveying the CNS is substantially increased during inflammatory T cell reactions occurring in any part of the body outside the CNS (Hickey, 2001). One major reason for the delayed and suppressed local immune response in the CNS is the lack of ‘professional antigen-presenting cells’ professional APCs. Professional APCs are cells specialized to initiate or propagate the development of antigen specific T lymphocytes. Professional APCs are dendritic cells, macrophages and B lymphocytes. Professional APCs are not detectable in the normal CNS parenchyma, only a small sub-population of perivascular macrophages is found in the perivascular space. Furthermore, small numbers of dendritic cells are localized in the leptomeninges and dura mater (McMenamin, 1999) and accumulate in the perivascular areas of the CNS when strong inflammatory foci develop (Fischer and Reichmann, 2001), including experimental autoimmune encephalomyelitis (EAE, Appendix 1, p. 180ff) (Serafini et al., 2000) and cerebral toxoplasmosis (Fischer et al., 2000). Macrophages as professional antigen-presenting cells take up a variety of antigens and engulf and degrade particles and microorganisms via a process termed phagocytosis. They present antigens via their MHC class II molecules to CD4þ T cells. Macrophages home to a special area of the CNS, the perivascular space, termed Virchow-Robin space (Bechmann et al., 2001), the leptomeninges, the dura mater and the choroid plexus (McMenamin, 1999). The perivascular macrophages are bone marrow-derived and continuously enter this area of the healthy brain across the normal BBB (Bechmann et al., 2001). They show low expression of MHC class II molecules and can take up antigenic material from their local environment and present it to T lymphocytes (Ford et al., 1995).

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B lymphocytes, another professional antigen-presenting cell and a major effector cell type of the immune system, are capable of directly binding to antigen and participate as APCs in the activation of T lymphocytes and stimulation of immune responses. In contrast to perivascular macrophages, there is no evidence that B cells, which produce antibodies for neutralization uptake and presentation of viruses or antigenic material, home to specific CNS areas, patrol the CNS or enter the CNS under normal conditions (Hickey, 2001). However, B cells can be detected in inflammatory lesions of multiple sclerosis (MS) or experimental autoimmune encephalomyelitis (EAE, Appendix 1, p. 180ff), viral or bacterial meningitis or encephalitis, when the bloodbrain barrier is impaired (Hickey, 2001). In such diseases, the presence of ‘oligogoclonal bands’ – immunoglobulin bands in the cerebrospinal fluid detected by protein electrophoresis – indicates the entry of B cells into the CNS, clonal expansion, differentiation into plasma cells and secretion of antibodies (Hickey, 2001). A wide spectrum of brain tissue cells can present antigens for effector T cell function. Recognition of antigenic material presented on the cell surface of brain cells permits T cells, primed before by a professional antigen-presenting cell, to detect intracellular pathogen at an early stage before substantial replication of the microorganism can occur. Expression of MHC class I or MHC class II molecules is a pre-requisite for presenting antigens to CD8þ or CD4þ T lymphocytes, respectively. In most organs of the body, the tissue-resident cells do express MHC class I molecules, and subpopulations express MHC class II molecules. Due to its immune-privileged status, the CNS is exceptional in this respect, and MHC class I and class II molecules are not constitutively expressed on neural cells in the healthy CNS parenchyma (Neumann and Wekerle, 1998). CD8þ T lymphocytes do not find constitutive MHC class I-expressing target cells that present antigens in a recognizable manner in the normal parenchyma proper (Vass and Lassmann, 1990). However, pro-inflammatory cytokines such as IFN-g and TNF-a produced by brain cells and invading immune cells have been shown to induce de novo expression of MHC molecules on brain cells in vitro (Vass and Lassmann, 1990). All glial cell types, as well as neurons, can be stimulated by IFN-g to express MHC class I and therefore fulfill the basic requirement for interaction with effector CD8þ T lymphocytes (Neumann, 2001). However, inducibility of MHC class I in neurons is very strictly regulated (Neumann et al., 1995). In particular, cell membrane expression of MHC class I is only induced in a minority of cultured neurons by IFN-g, but is increased after additional blockade of neuronal activity with tetrodotoxin (Neumann et al., 1997).

9.5

Regulation of Adaptive Immune Responses in the CNS

The normal CNS parenchyma contains solely quiescent cells in regard to their antigen-presenting capacity. They are functionally inactivated and silenced. The local CNS microenvironment and the dense blood-brain barrier are the main players

9.5 Regulation of Adaptive Immune Responses in the CNS

in shaping the immunoprivileged milieu of the CNS (Neumann, 2001). The immune function of glial cells is under strict control of the neurochemical microenvironment (Fig. 9.1). Numerous receptors for neurotransmitters, neuropeptides and neurotrophins normally required for neuronal signaling are expressed on glial cells. In vitro studies have demonstrated that neuropeptides and neurotrophins released during normal neuronal activity counteract the effects of inflammatory cytokines. In particular, it was shown that intact neurons counter-regulate IFN-g-mediated induction of MHC class II molecules on surrounding astrocytes and microglia (Neumann et al., 1996; Neumann et al., 1998). Furthermore, it has been demonstrated that neurotrophins inhibit MHC class II inducibility in microglia (Neumann et al., 1998) and NGF directly acts on microglia and inhibits the expression of MHC class II and the co-stimulatory molecule CD86 (B7–2) (Neumann et al., 1998; Wei and Jonakait, 1999). The immunological microenvironment of the CNS is strongly shaped by antiinflammatory cytokines produced by astrocytes. Astrocytes produce the antiinflammatory mediators transforming growth factor- b (TGF-b) and interleukin-10 (IL-10) during autoimmune brain diseases such as multiple sclerosis (Aloisi et al., 2000). They are mainly involved in the inhibition of T cell responses and T cell activation. Therefore, it has been suggested that astrocytes have an important function in down-regulating T cell immune responses in the CNS. The immunological microenvironment in the CNS is also controlled by receptorligand interactions. Neurons and brain endothelium express the CD200 ligand. The corresponding receptor, CD200R, is a membrane glycoprotein of microglial cells (Hoek et al., 2000). Microglia of CD200-deficient mice spontaneously exhibit many features of innate immune activation, including a less ramified morphology and increased expression of CD11b and CD45. Following induction of experimental autoimmune encephalomyelitis (EAE), an autoimmune CNS animal model for multiple sclerosis (MS), CD200-deficient mice show increased expression of inducible nitric oxide synthase (iNOS) in microglia and macrophages. The data demonstrates that CD200 expressed in the CNS by neurons silence and down-regulate the innate immune function of microglia. The control of reactive T cells in the CNS is likely to have an important role in inflammatory CNS disease. Interestingly, most T cells that have entered the CNS parenchyma die rapidly via an apoptotic mechanism. Apoptosis of T cells has been studied mainly in the experimental model of multiple sclerosis, EAE. Apoptosis of T cells is mainly detected during the phase of spontaneous recovery from EAE (Schmied et al., 1993). The site of T cell apoptosis is the CNS parenchyma and not the perivascular area (Bauer et al., 1995). The T cell death is independent of antigen recognition and not selective to T cells directed against brain auto-antigens, as demonstrated for ovalbumin-specific and myelin basic protein (MBP)-specific T cells in EAE. The apoptotic T cells are rapidly phagocytosed by macrophages and glia (Nguyen and Pender, 1998). It was suggested that the peculiarities of the CNS environment stimulates apoptosis of T cells (Fig. 9.1). Glial cells and neurons might give signals that influence the susceptibility of T cells to apoptosis. In EAE, apoptotic inflammatory cells associate more closely with astrocytes than with

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microglia (Kohji et al., 1998). It has been suggested that Fas ligand/CD95 ligand constitutively expressed by astrocytes induces apoptosis of Fas/CD95 expressing T cells (Suvannavejh et al., 2000). Also, microglial cells do express CD95 ligand and can induce apoptosis by binding to the CD95 receptor of T cells (Frigerio et al., 2000). Recently, it has been demonstrated that neurons constitutively express CD95 ligand in vitro and in vivo (Flgel et al., 2000) and auto-reactive T cells rapidly undergo apoptosis in the vicinity of CD95 ligand expressing neurons in the facial nerve axotomy model (Flgel et al., 2000).

9.6

Immune Surveillance of the CNS by T Lymphocytes

As mentioned before, activated T lymphocytes patrol through the brain tissue. The entry of T cells appears to be random without any specific homing of certain antigen-specific T cells or subpopulations into the CNS (Flgel et al., 2001; Yeager et al., 2000). The number of T cells in the normal CNS is lower compared to other tissues. Indeed, the CNS has the lowest T-cell concentrations per gram of tissue (Yeager et al., 2000). It has been shown that pathogenic T cells, able to recognize their antigen in the CNS, accumulate and can reach relatively high numbers in inflammatory foci (Flgel et al., 2001), while those that cannot find their antigen in the CNS possibly disappear via apoptosis (Bauer et al., 1998). While T cells are low in number or virtually absent in the normal CNS, the situation changes dramatically, not only in the CNS infected by microbial organisms, but also in the lesioned CNS (Popovich et al., 1996; Raivich et al., 1998). Under these circumstances, the endothelium will be activated locally, and glial cells attract T lymphocytes via release of chemokines. Several signals determine whether a T cell is activated or not. T cell activation is influenced by the amount and avidity of the TCR-MHC/peptide interaction and the presence or absence of co-stimulatory signals and adhesion molecules including CD11a (LFA-1), CD58 (LFA-3) or CD54 (ICAM-1) on the APCs. Expression of co-stimulatory molecules such as CD80 (B7–1), CD86 (B7–2) on APCs supports TCR activation through binding to CD28 on T cells. In the absence of accessory and co-stimulatory molecules, APCs have been shown to anergize or induce apoptosis of T cells. In the normal CNS, expression of co-stimulatory molecules is restricted to a small subpopulation of perivascular macrophages (Ford et al., 1995). Microglia in their resting condition behave as poor APCs and express only low levels of CD14, CD45 and Fc receptors and no co-stimulatory molecules. In contrast, activated microglia in inflammatory CNS lesions including MS lesions acquire a macrophage-like phenotype and have been shown to express MHC class II and adhesion molecules (CD11a and CD54) and the co-stimulatory molecules CD80 and CD86 (De Simone et al., 1995). Such activated microglia (Gerritse et al., 1996; Issazadeh et al., 1998) could stimulate the full activation program of T cells in vitro (Aloisi et al., 2000). Furthermore, activated microglial release chemokines

9.7 Effector Mechanisms of Lymphocytes

including macrophage inflammatory protein 1a (MIP-1a), which recruit additional macrophages and T cells to the inflammatory lesions (Aloisi et al., 2000). In autoimmune diseases, such as MS and its animal model EAE, microglial cells and blood-derived macrophages express receptors for active endocytosis including Fc receptors and mannose receptors. These cells phagocytose myelin degradation products and present them via MHC class II in conjunction with co-stimulatory molecules to CD4þ T lymphocytes, indicating a major role of these APCs in stimulating the autoimmune T cell responses (Aloisi et al., 2000). In contrast, astrocytes are capable of producing MHC class II molecules in strong inflammatory lesions, but fail to express co-stimulatory molecules in vivo (Aloisi et al., 2000). Therefore, they cannot function as efficient APCs for CD4þ T cells in vivo. Astrocytes can promote inflammatory foci by producing and secreting chemotactic factors including monocytes chemotactic protein 1 (MCP-1) and IFN-g-induced protein-10, which recruit monocytes/ macrophages and T cells into the affected brain tissue (Ransohoff and Tani, 1998). Oligodendrocytes and neurons completely lack the expression of co-stimulatory molecules and are unable to trigger the full activation program of T cells.

9.7

Effector Mechanisms of Lymphocytes

CD4þ T cells regulate immune responses, exert immunoregulatory effects via secretion of soluble mediators and trigger repair in the CNS (Fig. 9.2). The CD4þ T lymphocytes appear as two major and distinct phenotypes according their cytokine profile. CD4þ T cells of the Th1 phenotype produce interleukin-2 (IL-2), IFN-g, TNF-a and lymphotoxin-a. They are essential in promoting the maturation of resting microglial cells into APCs that express adhesion molecules (e. g. CD54), co-stimulatory molecules (CD80, CD86 and CD40), pro-inflammatory cytokines (TNF-a, IL-1b) and chemokines (e. g. MIP-1a). The CD40 ligand (CD40L/ CD154) of activated CD4þ Th1 cells can bind to CD40 of activated microglia and stimulate microglial production of interleukin-12 (IL-12), nitric oxide (NO) and other cytotoxic and immunoregulatory mediators. The CD4þ Th1 T cells have been shown to be essential for clearance of neurotropic viruses by promoting differentiation of CD8þ T cells in cytotoxic effector T cells. Autoreactive myelin-specific CD4þ Th1 cells are the disease-inducing cell type in EAE, the animal model for multiple sclerosis. In contrast, CD4þ T cells of the Th2 phenotype produce interleukin-4 (IL-4), IL-5, IL-6, IL-10 and IL-13, cytokines that support humoral immune responses by stimulation of the differentiation of B cells and inhibit numerous macrophage inflammatory functions. CD4þ Th2 cells show immunosuppressive activity within the CNS. They down-regulate immune responses of Th1 cells and inhibit microglia activation (Aharoni et al., 2000). In particular, the Th2 cytokines IL-4 and IL-10 can inhibit CNS inflammation and suppress EAE (Mathisen et al., 1997). Isolated myelin-specific T cells geneti-

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Figure 9.2 Effector mechanisms of lymphocytes. CD8 positive T cells could have direct cytotoxic effects on neurons either via release of cytotoxic granules containing perforin and serine proteases or via ligand-receptor interaction (e. g. CD95– CD95 ligand). Antigen-specific recognition of the MHC I – peptide complex on the neuron via the T cell receptor (TCR) is a pre-requisite for the cytotoxic actions. CD4 positive T cells act via local release of inflammatory cytokines, which are known to have dual roles either protective or degenerative. B cells entering the CNS could produce antibodies directed against neuronal antigens, which either impair normal neuron function or activate the complement cascade and induce cell death.

cally engineered to over-express IL-10 inhibited the onset and clinical sings of established EAE (Mathisen et al., 1997). Surprisingly, CD4þ T lymphocytes, which induce a monophasic paralytic EAE after passive transfer, have been shown to protect injured neurons from secondary degeneration (Schwartz et al., 1999). Following partial lesion of a rat optic nerve, activated CD4þ T lymphocytes directed against the auto-antigen myelin basic protein were transferred and accumulated at the site of lesion (Moalem et al., 1999). The rats injected with the auto-reactive myelin peptide restricted CD4þ T lymphocytes showed less secondary degeneration of the retinal ganglion neurons (Moalem et al., 1999). The exact cytokine profile of these neuroprotective T cells was not analyzed in this study. Moreover, it is not understood how the auto-reactive T cells protect injured neurons from secondary degeneration. Recent data demonstrate that


T lymphocytes secrete significant amounts of neurotrophic factors that might be involved in the observed neuroprotective effect of CD4þ T cells (Kerschensteiner et al., 1999). In contrast to CD4þ T cells, which could exert protective and anti-inflammatory activity, the brain tissue is highly susceptible to the cytotoxic effects of CD8þ T cells (Fig. 9.2). Recognition of ‘their antigen’ presented via MHC class I molecules triggers three major pathways to destroy the target cells (Trapani et al., 2000; Shresta et al., 1998). They secrete cytotoxic granules containing perforin and serine proteases, express CD95 ligand that activates CD95 on target cells and secrete cytotoxic cytokines of the TNF family. The main cytotoxic T lymphocyte effector pathway is mediated via the vectoral release of cytotoxic granules (Trapani et al., 2000; Shresta et al., 1998). The major constituents of the granules are the lytic protein perforin and the granzymes. Perforin integrates and polymerizes in the plasma membrane of target cells to form cylindrical pores of 16 nm in diameter, which lead to rapid entry of ions and water into the target cell, resulting in disintegration of the cell membrane and uptake of granzymes. The granzymes (e. g. granzyme A and granzyme B) act by inducing apoptosis either by activating a cell membrane receptor (Motyka et al., 2000) or acting within the target cell having gained access with the help of perforin (Trapani et al., 2000; Shresta et al., 1998). CD95 ligand expressed in the membrane of activated CTLs binds to CD95 of the target cell. Activation of the CD95 can induce the apoptotic cascade from the target cell membrane (Siegel et al., 2000). TNF and other TNF-related molecules such as TRAIL (TNF-related apoptosis-inducing ligand) can also induce apoptosis in susceptible target cells (Ashkenazi and Dixit, 1999). Brain cells appear to possess protective mechanisms against unwanted attack by cytotoxic CD8þ T cells, which are still not well defined. As mentioned before, neurons constitutively express CD95 ligand, which could induce apoptosis in T cells. Furthermore, neurons stimulated to express MHC class I are partially and selectively protected against attack by CD8þ T cells. They only show segmental disruption of neurites and signs of CD95-mediated apoptosis at the cell body following attack by cytotoxic CD8þ T cells in vitro (Medana et al., 2000; Medana et al., 2001a; Medana et al., 2001b). The importance of CD8þ T cells in brain defense and has also been demonstrated in the clearance of viral infections (Murray et al., 1998b), (Murray et al., 1998a; Noske et al., 1998) the defense against brain tumors (Akasaki et al., 2001; Walker et al., 2000) and the rejection of transplants (Duan et al., 1995). In murine Theiler’s virus infection, CD8þ T cells remove the viruses from infected oligodendrocytes, but they are also responsible for demyelination and axonal damage of the spinal cord (Rivera-Quinones et al., 1998; Murray et al., 1998b). Mice deficient for b2-microglobulin, which show impaired function of CD8þ T cells, have preservation of axons despite extensive virus-mediated demyelination (Rivera-Quinones et al., 1998). Cytotoxic effects of CD8þ T cells have been observed in a variety of animal models and human CNS diseases. In the model of transfer EAE, CD8þ T lymphocytes

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have a dual role. First, they have a regulatory function and might be involved in the induction of disease remission (Sun et al., 1988; Jiang et al., 1992; Koh et al., 1992). Second, they can induce tissue damage following injection into the CNS or after systemic transfer (Sun et al., 2001; Huseby et al., 2001). In Rasmussens’s encephalitis and patients with paraneoplastic diseases of the central nervous system close apposition of CD8þ T cells to MHC class I expressing, degenerating neurons have been observed (Bien et al., 2002; Panegyres et al., 1993; Hainfellner et al., 1996). In adreno-leukodystrophy, CD8þ CTLs dominate the immune cell infiltrate and appear to be involved in tissue destruction and lysis of oligodendrocytes (Ito et al., 2001). Antibodies produced by B cells are directed against neuronal antigens in paraneoplastic neurological diseases, Rasmussen encephalitis and Stiff-man syndrome (Table 9.1). Paraneoplastic neurological diseases are characterized by the presence of tumors outside the CNS. The patients show high-titer peripheral antibodies directed against their tumors, but also recognizing neuronal antigens. These antigens are termed ‘onconeural’ owing to their cross-reactivity with tumor tissue.

Table 9.1 Auto-antibodies directed against neurons.

Disease

Neuronal auto-antigen

Functional impairment

Reference

Paraneoplastic neurological diseases

NOVA1

Aberrant RNA splicing of GABA and glycine receptor subunits

Buckanovich RJ et al.; 1993, Neuron 11: 657

hu

Deficient RNA binding

Musunuru K et al.; 2001, Annu Rev Neurosci 24: 239

Cdr2

Disruption of cell-cycle signaling

Musunuru K et al.; 2001, Annu Rev Neurosci 24: 239

mGluR1

Impaired plasticity

Solimena M et al., 2000, Neuron 28: 309

GluR3

Excitotoxicity

Rogers SW et al.; 1994, Science 265: 648

Munc-18

Impaired synaptic function

Yang R et al.; 2000, Neuron 28: 375

Gephyrin

Impaired neurotransmission

Butler MH et al.; 2000, Neuron 26: 307

GAD

Impaired GABA synthesis

Li L et al.; 1994, J Immunol 152: 930

Rasmussen encephalitis

Stiff-man syndrome

Amphiphysin Impaired presynaptic activity

De Camilli P et al.; 1993, J Exp Med 178: 2219

Abbreviations: Cdr2 – cerebellar-degeneration-related protein 2; GABA – g-aminobutyric acid; GAD – glutamic acid decarboxylase; GluR3 – ionotropic glutamate receptor 3; mGluR1 – metabolotropic glutamate receptor 1; NOVA1 – neuro-oncological ventral antigen 1.


The antibodies recognize the neuronal antigens and disrupt the normal function of neurons and might lead to cell death. Neuro-oncological ventral antigen 1 (NOVA1) is such an onconeural antigen, which is recognized in neurons of the ventral horn and hindbrain in patients with paraneoplastic opsoclonus-myoclonus ataxia. The antibodies prevent the normal function of NOVA1, which is required for correct alternative splicing of the glycine receptor and GABA receptor subunits (Buckanovich et al., 1993).

Acknowledgement

The Neuroimmunology Group at the European Neuroscience Institute Gttingen is financed by the University Gttingen and supported by the DFG, Hertie-Stiftung and Boehringer Ingelheim Fonds. Appendix 1 Experimental Autoimmune Encephalomyelitis – the prototypic autoimmune brain disease model Experimental Autoimmune Encephalomyelitis (EAE) is the most popular and widely used animal model for autoimmune CNS inflammation. In particular, it is as a suitable experimental model of multiple sclerosis (MS). Furthermore, EAE is commonly used as a therapeutic paradigm to validate anti-inflammatory and immunosuppressive treatments. It is known for many years that immunization of experimental animals with brain white matter, or its protein components, together with immunostimulatory adjuvant causes a reproducible inflammatory brain disease, named EAE. EAE is a paralytic autoimmune disease, typically having a monophasic course either leading to death, or showing fluctuations with interchanging relapses and remissions. Induction of EAE by immunization with myelin components triggers an autoimmune response with large-scale destruction of myelin, and of the myelin-forming glial cells, the oligodendrocytes and axonal injury. The particular clinical picture and the characteristic histological changes mimic important aspects of human MS (Kornek et al., 2000). EAE can be induced either by active immunization with myelin component, or by the transfer of CD4þ T lymphocytes, which were propagated in vitro and directed against myelin auto-antigens. The transfer of auto-reactive CD4þ T lymphocytes definitely confirmed that EAE is caused by autoimmune mechanisms. As it turned out, the immunization of experimental animals with myelin proteins also results in the stimulation of myelin-autoreactive T cells, mainly in the lymph nodes next to the injection site. Surprisingly, such autoimmune T cells are normal components of the healthy immune repertoire and must have escaped negative immune selection during development. Such autoimmune myelin-specific T cells appear to stay in the lymphoid tissues throughout a lifetime, without ever attacking the brain tissue. The auto-reactive myelin-specific T cells become only

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auto-destructive upon activation, either by confrontation with myelin autoantigen in immunostimulatory adjuvant. The immunostimulatory adjuvant creates a pro-inflammatory milieu mimicking microbial infections. After stimulation, the myelin-specific T cells must enter the CNS to launch the pathogenic immune attack. Detailed studies of the cellular events involved in the development of EAE showed that these self-reactive T cells invade the CNS only upon fresh activation. After intravenous injection, the T lymphocytes interact with brain cells and APCs of secondary immune organs during an initial phase of 3–4 days. The cells then accumulate in perivascular brain regions leading to a second wave of T lymphocyte recruitment, monocyte brain invasion and transient neurological deficits. In the CNS, the invading auto-reactive T cells form round perivascular cell infiltrates in the white matter. The infiltrates are composed of CD4þ T cells, along with macrophages. Furthermore, the CNS parenchyma is invaded by CD4þ, CD8þ T cells and some rare B cells. EAE is often regarded as the perfect animal model of MS. It should be pointed out that this is an over-simplification, since there are numerous different variants of EAE, exhibiting highly diverse clinical and histopathological features. The type of EAE variant depends on the experimental animal, on the brain auto-antigen, and on the mode of immunization.

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Medana, I., Martinic, M. A., Wekerle, H., and Neumann, H. (2001b). Transection of major histocompatibility complex class I-induced neurites by cytotoxic T lymphocytes. Am. J. Pathol. 159, 809–815. Medana, I. M., Gallimore, A., Oxenius, A., Martinic, M. M., Wekerle, H., and Neumann, H. (2000). MHC class I-restricted killing of neurons by virus-specific CD8þ T lymphocytes is effected through the Fas/FasL, but not the perforin pathway. Eur. J. Immunol. 30, 3623–3633. Moalem, G., Leibowitz-Amit, R., Yoles, E., Mor, F., Cohen, I. R., and Schwartz, M. (1999). Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat. Med. 5, 49–55. Motyka, B., Korbutt, G., Pinkoski, M. J., Heibein, J. A., Caputo, A., Hobman, M., Barry, M., Shostak, I., Sawchuk, T., Holmes, C. F., Gauldie, J., and Bleackley, R. C. (2000). Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell 103, 491–500. Murray, P. D., McGavern, D. B., Lin, X., Njenga, M. K., Leibowitz, J., Pease, L. R., and Rodriguez, M. (1998a). Perforin-dependent neurologic injury in a viral model of multiple sclerosis. J. Neurosci. 18, 7306–7314. Murray, P. D., Pavelko, K. D., Leibowitz, J., Lin, X., and Rodriguez, M. (1998b). CD4(þ) and CD8(þ) T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis. J. Virol. 72, 7320–7329. Neumann, H. (2001). Control of glial immune function by neurons. Glia 36, 191–199. Neumann, H., Boucraut, J., Hahnel, C., Misgeld, T., and Wekerle, H. (1996). Neuronal control of MHC class II inducibility in rat astrocytes and microglia. Eur. J. Neurosci. 8, 2582–2590. Neumann, H., Cavalie, A., Jenne, D. E., and Wekerle, H. (1995). Induction of MHC class I genes in neurons. Science 269, 549–552. Neumann, H., Medana, I. M., Bauer, J., and Lassmann, H. (2002). Cytotoxic T lymphocytes in autoimmune and degenerative CNS diseases. Trends Neurosci. 25, 313–319. Neumann, H., Misgeld, T., Matsumuro, K., and Wekerle, H. (1998). Neurotrophins inhibit major histocompatibility class II inducibility

References of microglia: involvement of the p75 neurotrophin receptor. Proc. Natl. Acad. Sci. U. S. A 95, 5779–5784. Neumann, H., Schmidt, H., Cavalie, A., Jenne, D., and Wekerle, H. (1997). Major histocompatibility complex (MHC) class I gene expression in single neurons of the central nervous system: differential regulation by interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. J. Exp. Med. 185, 305–316. Neumann, H. and Wekerle, H. (1998). Neuronal control of the immune response in the central nervous system: linking brain immunity to neurodegeneration. J. Neuropathol. Exp. Neurol. 57, 1–9. Nguyen, K. B. and Pender, M. P. (1998). Phagocytosis of apoptotic lymphocytes by oligodendrocytes in experimental autoimmune encephalomyelitis. Acta Neuropathol. (Berl) 95, 40–46. Nguyen, M. D., Julien, J. P., and Rivest, S. (2002). Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat. Rev. Neurosci. 3, 216–227. Noske, K., Bilzer, T., Planz, O., and Stitz, L. (1998). Virus-specific CD4þ T cells eliminate borna disease virus from the brain via induction of cytotoxic CD8þ T cells. J. Virol. 72, 4387–4395. Panegyres, P. K., Reading, M. C., and Esiri, M. M. (1993). The inflammatory reaction of paraneoplastic ganglionitis and encephalitis: an immunohistochemical study. J. Neurol. 240, 93–97. Popovich, P. G., Stokes, B. T., and Whitacre, C. C. (1996). Concept of autoimmunity following spinal cord injury: possible roles for T lymphocytes in the traumatized central nervous system. J. Neurosci. Res. 45, 349– 363. Raivich, G., Jones, L. L., Kloss, C. U., Werner, A., Neumann, H., and Kreutzberg, G. W. (1998). Immune surveillance in the injured nervous system: T-lymphocytes invade the axotomized mouse facial motor nucleus and aggregate around sites of neuronal degeneration. J. Neurosci. 18, 5804–5816. Ransohoff, R. M. and Tani, M. (1998). Do chemokines mediate leukocyte recruitment in post-traumatic CNS inflammation? Trends Neurosci. 21, 154–159. Rivera-Quinones, C., McGavern, D., Schmelzer, J. D., Hunter, S. F., Low, P. A., and Ro-

driguez, M. (1998). Absence of neurological deficits following extensive demyelination in a class I-deficient murine model of multiple sclerosis. Nat. Med. 4, 187–193. Schmied, M., Breitschopf, H., Gold, R., Zischler, H., Rothe, G., Wekerle, H., and Lassmann, H. (1993). Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell death as a mechanism to control inflammation in the brain. Am. J. Pathol. 143, 446–452. Schwartz, M., Moalem, G., Leibowitz-Amit, R., and Cohen, I. R. (1999). Innate and adaptive immune responses can be beneficial for CNS repair. Trends Neurosci. 22, 295–299. Serafini, B., Columba-Cabezas, S., Di Rosa, F., and Aloisi, F. (2000). Intracerebral recruitment and maturation of dendritic cells in the onset and progression of experimental autoimmune encephalomyelitis. Am. J. Pathol. 157, 1991–2002. Shresta, S., Pham, C. T., Thomas, D. A., Graubert, T. A., and Ley, T. J. (1998). How do cytotoxic lymphocytes kill their targets? Curr. Opin. Immunol. 10, 581–587. Siegel, R. M., Chan, F. K., Chun, H. J., and Lenardo, M. J. (2000). The multifaceted role of Fas signaling in immune cell homeostasis and autoimmunity. Nat. Immunol. 1, 469–474. Sun, D., Qin, Y., Chluba, J., Epplen, J. T., and Wekerle, H. (1988). Suppression of experimentally induced autoimmune encephalomyelitis by cytolytic T-T cell interactions. Nature 332, 843–845. Sun, D., Whitaker, J. N., Huang, Z., Liu, D., Coleclough, C., Wekerle, H., and Raine, C. S. (2001). Myelin antigen-specific CD8þ T cells are encephalitogenic and produce severe disease in C57BL/6 mice. J. Immunol. 166, 7579–7587. Suvannavejh, G. C., Dal Canto, M. C., Matis, L. A., and Miller, S. D. (2000). Fas-mediated apoptosis in clinical remissions of relapsing experimental autoimmune encephalomyelitis. J. Clin. Invest 105, 223–231. Trapani, J. A., Davis, J., Sutton, V. R., and Smyth, M. J. (2000). Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo. Curr. Opin. Immunol. 12, 323–329. Vass, K. and Lassmann, H. (1990). Intrathecal application of interferon gamma. Progressive appearance of MHC antigens within the rat

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10 Metabolic Dysfunctions Konstantin-A. Hossmann

10.1

Introduction

Metabolism is the entirety of chemical and physical processes that go on continuously in living organisms and which comprise processes in which assimilated nutrients are built up into protoplasm (anabolism) as well as processes in which these are used and broken down to produce energy and to eliminate waste products (catabolism). In neurological disorders, dysfunctions of two metabolic pathways are of particular importance: the energy metabolism, required to fuel the considerable energy needs of the living brain, and protein synthesis, needed not only to build up and to preserve the structural integrity and the enzymatic homeostasis of the brain, but also to translate the consistently changing genomic information required to control the integrated function of this complex organ. Other metabolic dysfunctions, notably those associated with the metabolism of amino acids, mono- or disaccharides, purines or various fatty acids and lipids are of interest mainly for genetically determined hereditary diseases, and will not be discussed in this context. Obviously, the vast literature on this topic cannot be covered in this chapter. Readers interested in more details are, therefore, referred to several excellent review articles that appeared during the past years (DeGracia et al. 2002; Dirnagl et al. 1999; Fiskum 2000; Ginsberg 2003; Gjedde 2003; Heiss et al. 2001a; Hou and MacManus 2002; Lipton 1999; Mattson 2000; Paschen 2003; Siesj et al. 1999). For the same reason, references to original investigations will only be given where necessary to document recent developments or specific findings.


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10.2

Disturbances of the Energy Metabolism 10.2.1

Energy Production and Turnover

Under physiological conditions, the human brain consumes about 10 mmol, and that of smaller mammals up to 20 mmol energy-rich phosphate bindings per g per min for the maintenance of its normal function. 50 % of the energy requirements are used to maintain structural and ion homeostasis, notably for the preservation of membrane polarization, and the other 50 % for supporting spontaneous and evoked electrical activity. Endergonic processes, required to maintain structural integrity, include the biosynthesis and controlled degradation of proteins, lipids and carbohydrates, intracellular transport, chaperone functions, DNA repair and a multitude of phosphorylations involved in cell regulation and signal transduction. ATP, the currency of both structural and functional energy requirements, is almost exclusively generated by the oxidation of glucose and relies on the steady delivery of glucose and oxygen by the arterial circulation. Oxidation of glucose proceeds in two phases: the glycolytic pathway involves degradation of glucose to pyruvate, and the tricarbonic acid cycle (TCA) mediates the oxidation of pyruvate to carbon dioxide and water. ATP is formed mainly in the mitochondria by oxidative phosphorylation of ADP, using the electrochemical proton gradient across the inner mitochondrial membrane to drive mitochondrial ATPase (Fig. 10.1). Under physiological conditions, this pathway results in the net production (after subtraction of transport energy) of about 30 mol ATP per mol glucose, but this high energy yield is only maintained when both, the arterial blood supply of glucose and oxygen, and mitochondrial function are unimpaired. Otherwise, glucose is metabolized anaerobically, which, however, dramatically reduces the ATP yield by a factor of 15 from 30 to 2 mol per mol glucose, and which results in the production of potentially harmful lactic acid. 10.2.2

Reduction of Oxygen Supply

The powerful mechanisms of blood flow regulation protect the brain well against systemic hypoxia, notably hypoxia induced by respiratory failure or anemia. In fact, under most experimental conditions of systemic hypoxia, it is the heart and not the brain that first exhibits functional disturbances. Interruption of cerebral blood flow, in contrast, results almost instantaneously in tissue hypoxia and primary breakdown of the energy metabolism. When blood flow declines to below 45 % of control, anaerobic glycolysis is stimulated, tissue lactate content begins to increase and pH declines (Paschen et al. 1992). At flow values of 30 %, the glycolytic rate reaches a maximum, followed at even lower values by a sharp decline due to limitation of the glucose supply. The tissue ATP content remains remarkably stable down to a

10.2 Disturbances of the Energy Metabolism

Energy production by oxidative glucose utilization. Glucose is first degraded in the cytosol by the glycolytic pathway to pyruvate, resulting in the generation of 2 mol ATP per mol glucose. The next step involves the oxidation of pyruvate in the mitochondria to CO2 and water. To this end pyruvate is converted by oxidative decarboxylation to acetyl coenzyme A (acetyl CoA) which enters the tricarbonic acid (TCA) cycle for further oxidation to CO2. Oxidation is coupled mainly to the reduction of NADþ to NADH which along the electron transport chain reduces molecular oxygen to water. The electron transport chain generates an electrochemical gradient across the mitochondrial inner membrane that drives the mitochondrial ATPase. ATP is delivered from the mitochondria to the cytosol in exchange for ADP. The total energy yield of oxidative glucose utilization is close to 36 mol ATP per mol glucose from which about 6 mol ATP have to be deducted for transport processes. The resulting net ATP production of about 30 mol ATP is 15 times higher than that obtained by anaerobic glycolysis. Figure 10.1

flow rate of 15–20 % of control, demonstrating that over the range from 15–45 %, the declining oxidative energy production is compensated by anaerobic glycolysis (Fig. 10.2). The biochemical changes correlate closely with the electrophysiological alterations and are the basis of the viability thresholds of cerebral ischemia (Hossmann 1994). Given the low energy yield of anaerobic glycolysis, maintenance of a normal energy state under such conditions is only possible by reducing energy consumption, as in the peri-infarct penumbra, where evoked potentials and spontaneous electrocortical activity are suppressed (Touzani et al. 2001). Likely mechanisms of functional suppression are tissue acidosis, or the increase in cellular osmolality. The latter is due to the accumulation of lactate and so-called idiogenic osmoles, producing dendritic swelling with possibly adverse effects on synaptic transmission. The severity of lactacidosis depends on the degree of tissue hypoxia, glucose availability and the residual rate of blood flow. At high flow rates, some of the lac-

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Figure 10.2 Dependency of metabolic and functional disturbances of the brain on changes of blood flow. At declining flow values protein synthesis is first inhibited, followed – at distinctly lower flow values – by a stimulation of anaerobic glycolysis, as reflected by the increase of glucose utilization and the development of lactacidosis. At flow values below 15–20 % of control, the energy metabolism breaks down and cell membranes depolarize. The amplitudes of spontaneous EEG activity and of cortical evoked potentials (EP) begin to decline below 50 % of control in parallel with the stimulation of anaerobic glycolysis.

tate formed may be cleared into the circulating blood, but with declining blood flow, lactate increasingly accumulates in the brain tissue. Under oligemic conditions with some residual blood flow, the increase in tissue lactate, and hence tissue acidosis, may be more severe than during complete cerebrocirculatory arrest where the formation of lactate is limited by the endogenous glucose and glycogen stores of the brain (Siesj and Siesj 1996).

10.3

Disturbances of Flow-coupling

An impairment of brain energy metabolism may also arise from disturbances of the regulation of blood flow, resulting in a mismatch between blood oxygen supply and the oxygen requirements of the brain. Such disturbances can be caused by the decline of the local perfusion pressure below the auto-regulatory range, or by derangements of the molecular mechanisms responsible for the coupling between the functional activity of the brain and blood flow. A decline of perfusion pressure to or below the lower limit of the auto-regulatory range is a consistent finding in the penumbra of an ischemic infarct. In this situation, blood oxygen delivery may still be adequate to cover the basal oxygen consumption of the tissue, but, as the supplying vessels are fully dilated, any increase in the metabolic rate is not coupled to an appropriate increase in blood flow. This is most evident during ischemia-induced depolarizations, the reversal of which requires a substantial increase in metabolic activity (see below). However, even

10.4 Mitochondrial Dysfunction

lesser increases of the metabolic rate, as associated with functional brain activation or biochemical repair processes, may result in the transient mismatch of oxygen supply and the activation of anaerobic glycolysis. Disturbances of the coupling between functional activity and blood flow also occur under pathophysiological conditions which are characterized by a transient phase of hyperemia, followed by a more prolonged state of hypoperfusion. Examples are the flow alterations induced by transient hypoxia-ischemia, head trauma or epileptic seizures. During hyperemia, vessels are fully dilated, and oxygen supply exceeds the oxygen requirements of the tissue, but during the state of hypoperfusion, the cerebrovascular tone is increased and does not respond to mediators of vasodilatation, notably nitric oxide or carbon dioxide. As a result, any increases of metabolic activity during normal or pathological activation are not coupled to a parallel rise in oxygen supply and may result in relative tissue hypoxia and the stimulation of anaerobic glycolysis. Coupling disturbances may, therefore, be responsible for some of the delayed disturbances following transient pathological impacts (Hossmann 1997).

10.4

Mitochondrial Dysfunction

The high energy yield of oxidative glucose utilization is closely linked to mitochondrial respiration (Fig. 10.1). Electrons are passed along a series of respiratory enzymes – the electron transport chain - located at the mitochondrial inner membrane, and the energy released by this transfer is used to pump protons across the membrane. The resultant electrochemical gradient is used by ATP synthase to phosphorylate ADP to ATP at a rate which accounts to more than 80 % of the cellular ATP turnover, rendering the brain particularly sensitive to mitochondrial dysfunction. However, the importance of the mitochondria in brain pathophysiology goes beyond their critical role for the generation of energy. As the primary intracellular source of free radicals, their involvement in the regulation of calcium homeostasis and the association with apoptosis-promoting pathways, dysfunction of mitochondria is a central mediator of cellular injury. An important cause of mitochondrial dysfunction is the pathological rise of the intracellular calcium activity, as induced by toxic levels of glutamate or other excitatory neurotransmitters (Fig. 10.8). In the attempt to reverse this increase, mitochondria actively accumulate calcium via the Ca2þ uniporter located in the mitochondrial inner membrane. As this uniporter is driven by the same electrochemical gradient as that used by mitochondrial ATPase, part of the respiratory capacity of the electron transport chain is uncoupled from oxidative phosphorylation, resulting in the decline of ATP production. Moreover, calcium increases the generation of reactive oxygen species (ROS) which interfere with energy metabolism in a dual way: directly by inhibiting complex I of the electron transport chain, or indirectly by producing oxidative membrane alterations that result in further increase of intracellular calcium (Fiskum 2000).

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Another consequence of calcium accumulation is the promotion of an increase in the permeability of the mitochondrial inner membrane, known as the mitochondrial permeability transition (MPT). The transition is mediated by the opening (“assembly”) of an MPT pore which is indiscriminately permeable to solutes with a molecular weight of up to 1.500 Da and which can be inhibited by cyclosporin A. The opening of this pore has three potentially deleterious consequences: the breakdown of the electrochemical gradient across the mitochondrial inner membrane; the release – and hence reflux – of the accumulated calcium back into the cytosol; and a massive osmotic swelling, resulting in the physical disruption of the outer mitochondrial membrane and the release of pro-apoptotic proteins, notably cytochrome c, into the cytoplasm. Obviously, the MPT-induced breakdown of the electrochemical gradient directly affects the energy metabolism by stopping the activity of ATP synthase, but the release of pro-apoptotic proteins may impair energy metabolism also indirectly. In fact, the apoptosis-related fragmentation of DNA strands causes an activation of the repair enzyme, poly(ADP-ribose)polymerase, the substrate of which is NAD. Consumption of NAD may deplete the cellular NAD pool which, in turn, results in the impairment of oxidative phosphorylation. This opinion has been challenged by demonstrating, in a model of transient brain ischemia, that the NAD pool begins to decline after ATP is depleted, and not the other way round (Paschen et al. 2000). A causal relationship between NAD loss and energy failure is, therefore, unlikely. However, even in the absence of this particular mechanism, mitochondrial insufficiency affects the energy metabolism in so many different ways that the preservation of its function is a major target of all neuroprotective interventions.

10.5

Importance of Disturbed Energy Metabolism for Injury Evolution 10.5.1

Global Brain Ischemia

Under normothermia, the sudden interruption of cerebral blood flow, as induced by cardiac arrest, produces an energy metabolism breakdown and the depletion of all energy reserves within a few minutes (Fig. 10.3). Creatine phosphate (CrP) and adenosine triphosphate (ATP) sharply decline, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) rise, and the adenylate energy charge (EC), defined as the fraction of phosphorylated to total adenine nucleotides, is reduced from close to 1 to below 0.1. Energy depletion is delayed by deep anesthesia, hypothermia, membrane-stabilizing agents such as local anesthetics, or interventions that increase the primary and secondary energy reserves of the brain, such as creatine infusions or hyperglycemia. The combination of deep hypothermia with membrane-stabilizing agents is routinely used in cardiac and aneurysm surgery to protect the brain against induced circulatory arrest.

10.5 Importance of Disturbed Energy Metabolism for Injury Evolution

Effect of 1 hour global brain ischemia on blood flow and energy metabolism. During ischemia, lactate increases and energy metabolites are rapidly used up. After restoration of blood flow, glucose utilization is resumed before oxidative metabolism recovers, resulting in a further transient increase of lactate. However, the adenylate energy charge (EC) rapidly recovers to nearnormal. The incomplete recovery of ATP is due to the ischemic deamination of adenine nucleotides which requires some time for reversal (data from Hossmann et al. 1976 and Kleihues et al. 1974).

Figure 10.3

The interruption of the blood oxygen supply to the brain leads to the stimulation of anaerobic glycolysis and lactacidosis, the degree of which depends on the glucose and glycogen stores of the tissue. At normoglycemia, tissue lactate rises to between 12 and 15 mmol/g wet weight, and pH drops to about 6.2. Under hyperglycemic conditions and in the presence of some residual blood flow, as maintained after cardiac arrest by extrathoracal cardiac massage, larger amounts of glucose are available, and lactate may rise above 20 mmol/g, associated with a decline of pH to below 6.0 (Siesj 1988).

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Within a few minutes of ischemia, the total adenylate content of the brain (AMP þ ADP þ ATP) begins to decrease, and, within one hour, declines to about one third of the pre-ischemic value, mainly by dephosphorylation of AMP to adenosine. Adenosine, in turn, is deaminated to inosine and further degraded to hypoxanthine, which can be salvaged during recirculation for the replenishment of the adenine nucleotide pool. However, as some of the hypoxanthine is irreversibly degraded to xanthine and uric acid, part of the adenine nucleotide pool has to be restored by purine de novo synthesis (Kleihues et al. 1974). With the restoration of blood flow – and hence the re-supply of glucose and oxygen – these changes can be reversed, but recovery depends not only on the duration of ischemia, but also on several general physiological variables. Low reperfusion rate, hyperthermia or severe lactacidosis (induced by hyperglycemia or some residual blood flow) shorten the revival time, whereas fast reperfusion, hypothermia, mild lactacidosis (resulting from sudden complete circulatory arrest under normo- or hypoglycemia) have the opposite effect. The loss of adenosine from the adenylate pool during ischemia limits the recovery of ATP, the maximum possible concentration of which declines accordingly. However, the calculated adenylate energy charge may return close to normal within minutes after circulatory arrest lasting as long as one hour, which indicates that recovery of energy delivery matches the energy needs of the tissue (Fig. 10.3). This is the reason that even after prolonged ischemia, energy-dependent ion exchange pumps rapidly recover, leading to the restoration of membrane potentials and neuronal excitability. However, with the gradual return of spontaneous electrical activity, the energy requirements of the tissue increase, and as the cerebral blood flow remains uncoupled from brain metabolism for some time, the resulting mismatch between energy turnover and energy production gives rise to an unstable energy equilibrium which may easily collapse. Such decompensation can be evoked by even minor fluctuations of metabolic activity or oxygen supply, resulting in the stimulation of anaerobic glycolysis with the consequences of secondary lactacidosis, brain swelling and delayed energy failure. These secondary disturbances can be detected by NADH fluorometry and may evolve in circumscribed regions of the brain, notably in the watershed areas at the borders of the main vascular supplying territories where the local blood perfusion pressure is lowest (Welsh et al. 1977). They are not responsible, however, for delayed cell death in the so-called selectively vulnerable areas where the dominant post-ischemic metabolic disturbance is a failure of protein synthesis (see below). 10.5.2

Focal Brain Ischemia

The main pathophysiological difference between global and focal brain ischemia is the gradient of blood flow which declines from the peripheral to the central parts of the occluded vascular territory. As a consequence, tissue hypoxia and the associated metabolic changes also progress from the periphery to the center. The metabolic changes can be used to differentiate between the core and the penumbra of


brain infarction (Fig. 10.2): the core is the central region in which glucose utilization and energy metabolism have ceased, and the penumbra is the peripheral zone which exhibits metabolic disturbances, such as suppressed protein synthesis or lactacidosis, but still normal ATP (Fig. 10.4). With ongoing ischemia time, the infarct core gradually expands into the penumbra until, after 6 to 12 hours, the penumbra has disappeared. A likely reason for infarct growth are peri-infarct depolarizations which are evoked by local releases of glutamate from the infarct core and which spread over the entire ipsilateral hemisphere (Mies et al. 1991). In the penumbra, the associated increase in metabolic activity is not coupled to a similar increase in blood flow, leading to the stimulation of anaerobic glycolysis and secondary energy failure. In fact, the combination of electrophysiological recordings with in vivo NMR revealed not only sharp rises of lactate but also stepwise increases of the infarct volume with each depolarization (Gyngell et al. 1994). When blood flow – and hence oxygen supply – is resumed within the first hour after vascular occlusion, energy metabolism recovers (Fig. 10.4). A reliable non-invasive predictor of post-ischemic survival is the restoration of oxygen utilization, as reflected by the increase of the blood oxygen extraction fraction (Heiss et al. 2001b). However, with ongoing recirculation time, the energy metabolism may undergo secondary failure. The latency of this delayed injury depends on the duration of the preceding ischemia. After one hour of MCA occlusion, secondary energy failure becomes apparent between three and six hours of recirculation, but after 30 minutes of vascular occlusion, the delay may be as long as three weeks (Du et al. 1996). Secondary energy failure appears first in the core of the affected vascular territory and slowly expands into the more peripheral parts (Fig. 10.4). The final

Figure 10.4 Effect of focal brain ischemia on energy metabolism. Pictorial measurements of ATP at various times after permanent (above) and transient (below) middle cerebral artery (MCA) occlusion in rat. During permanent ischemia the core region of ATP depletion gradually expands from the central to the more peripheral parts of the MCA territory. If vascular occlusion is reversed within 1 hour, ATP rapidly recovers, but a few hours later, this is followed by secondary energy failure, progressing again from the core to the periphery of the MCA territory (data from Hata et al. 2000a,b).

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size correlates inversely with the recovery of cerebral protein synthesis which progresses from the periphery to the core (Fig. 10.7). Obviously, suppression of protein synthesis does not immediately result in the breakdown of the energy metabolism. It is, therefore, likely that another – possibly hemodynamic – factor is the common cause for this interdependence. 10.5.3

Epileptic Seizures

Experimental seizures induced by topical or systemic application of excitatory amino acids or GABA antagonists produce massive neuronal excitation with greatly enhanced neurotransmitter turnover and transmembrane ion fluxes that lead to the activation of membrane-bound Naþ-Kþ-ATPases, an increase in oxygen and glucose metabolism and a coupled increase in blood flow. Similar to functional activation under physiological conditions, the rise in blood flow exceeds the increased oxygen requirements of the tissue, resulting initially in hyperoxia. At the onset of seizures, the energy state may be briefly disturbed but shortly later it returns to near-normal (Siesj and Wieloch 1986). Despite the high tissue oxygen pressure, lactate markedly rises and remains increased for some time even after seizures have ceased. Typical values recorded shortly after the onset of bicuculline-induced seizures are an increase in the metabolic rate of glucose by more than 100 %, a transient decrease of phosphocreatine – but not ATP – down to 50 %, maintenance of the adenylate energy charge at 98 % of control and an increase of lactate from 1 to about 8 mmol/g (Folbergrova et al. 1981). Pyruvate also increases, but to a lesser degree, resulting in the rise of the lactate/pyruvate ratio from about 10 to similar to 35. The increase in the metabolic rate of glucose is most pronounced in cerebral cortex and hippocampus and least in the cerebellum, in accordance with the different regional involvement of seizure activity (Siesj and Wieloch 1986). An absolute requirement for the preservation of a normal energy state at increased functional and metabolic activity is the unimpaired oxygen supply. However, epileptic seizures are frequently complicated by respiratory hypoxia due to restriction of respiratory movements, airway obstruction or the development of neurogenic pulmonary edema. Under these conditions oxidative respiration is readily disturbed, leading to the stimulation of anaerobic glycolysis, further accumulation of lactate and breakdown of the energy state. But even without such complications, the energy metabolism may gradually decline, as evidenced by the progressive fall of CrP and ATP, and the gradual shift from hyper- to hypometabolism. These disturbances are most severe in the substantia nigra pars reticularis which, despite adequate oxygenation, develops severe lactacidosis and which eventually may undergo infarction (Inamura et al. 1989). The transition from hypermetabolism at the onset of seizures to hypometabolism after cessation of seizures is of practical importance for the detection of epileptogenic foci in patients suffering from focal epilepsy. In fact, measurements of local glucose metabolism by positron emission tomography revealed interictal foci


of reduced glucose metabolism in more than 70 % of such patients, i. e. a diagnostic sensitivity which by far exceeds that of magnetic resonance imaging or computed tomography (Wieser 1994). 10.5.4

Head Trauma

Head trauma produces greatly varying alterations in the energy metabolism, depending on the kind, severity and time of the traumatic impact. Obviously, direct mechanical destruction leads to primary necrosis, but mild to moderate injury results in remarkably little initial disturbances of energy metabolism, even if the injury is severe enough to produce lasting brain damage. 30 minutes after lateral cortical brain impact injury, CrP concentration is decreased by about 40 % at the primary site of injury but not in the surrounding, and ATP does not change at all (Prasad et al. 1994). This has been related to an increased mitochondrial capacity and ATP turnover (Fink et al. 1999), or to the down-regulation of cortical energy consumption, as reflected by the low metabolic rate of glucose (Dietrich et al. 1994). The absence of hypoxia is also supported by the fact that cytochrome a, a3 is transiently oxidized (Duckrow et al. 1981) and that different from ischemia hyperglycemia does not aggravate post-traumatic injury (Vink et al. 1997). Nevertheless, lactate rises for at least 24 hours (Bentzer et al. 2000), but as pH at this time tends to be in the alkaline range, this may be due to the development of vasogenic edema (Hermann et al. 1999). The paucity of early disturbances of energy metabolism is in sharp contrast to the gradual evolution of mitochondrial damage. Already 1 hour after injury, cytochrome c is released from the mitochondria into the cytosol, followed a few hours later by DNA-laddering as an early indicator of apoptosis (Morita-Fujimura et al. 1999). Mitochondria isolated from both experimental animals and patients with head trauma revealed progressing dysfunction which could be partly associated with the assembly of mitochondrial permeability transition (MPT) pores. Possible mediators of mitochondrial injury are the massive release of glutamate at the time of injury and, to a lesser degree, during post-trauma spreading depressions, the associated calcium overload, mechanical stress, free radical-induced peroxidation of the mitochondrial membranes and inflammatory reactions. Lipid peroxidation products, such as malondialdehyde can be detected within minutes after trauma, whereas energy deficiency does not show up before 2 hours and reaches its maximum only after 6 hours (Vagnozzi et al. 1999). This difference suggests that mitochondrial insufficiency is a slowly progressing process that takes some time for completion. Another pathophysiological factor leading to delayed energy failure is a mismatch between metabolic activity and blood flow which, a few hours after injury, may markedly decline. This mismatch may be further increased by functional activation which, after trauma, is not coupled to an adequate increase in blood flow. It has, therefore, been suggested that functional activation constitutes a secondary injury to the post-traumatic brain which, in the worst case, may lead to delayed cell death (Ip et al. 2003).

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10.6

Disturbances of Protein Synthesis 10.6.1

Mechanisms of Disturbed Protein Synthesis

The translation of the nucleotide sequence of a messenger RNA molecule into the corresponding protein is carried out on ribosomes in a sequel of complex biochemical reactions that can be differentiated into the initiation, elongation and termination of the polypeptide chains (Fig. 10.5). In small rodents, the incorporation of leucine into brain proteins amounts to 10–16 nmol/g/min (Smith 1991). If the other amino acids are translated at a similar rate, total amino acid incorporation is about 260 nmol/g/min; the corresponding energy demand is 0.8 mmol GTP/g/ min or 3 % of the total energy requirements of the brain. Disturbances of protein synthesis may arise from energy failure, amino acid deficiency, disturbances of gene transcription or malfunctions of the translational machinery. Energy-dependent failure of protein synthesis is an obvious consequence of all pathological conditions in which the energy metabolism is compromised. However, in many neurological diseases, protein synthesis is impaired despite a preserved or restored energy state. First indications of such an association came from electron microscopical studies which demonstrated disaggregated ribo-

Figure 10.5 Schematic representation of the translation of the nucleotide sequence of a messenger RNA into the corresponding protein. Translation is initiated by the binding of the ternary complex, consisting of the eukaryotic initiation factor eIF2, GTP and methionine initiator transfer RNA, to the small ribosomal subunit which scans the mRNA for the initiation codon AUG. When AUG is recognized, eIF2 is released and the large ribosomal subunit joins the small unit to form a complete ribosome that begins protein synthesis. For elongation of the nascent polypeptide chain, the ribosome moves to the next codon, attaching the appropriate aminoacyl-tRNA. Finally, the protein molecule is terminated and the large and small ribosomal subunits are detached from the mRNA. Each of these steps requires GTP and various differentially controlled initiation, elongation and release factors. Breakdown of the energy metabolism results in the interruption of protein synthesis at all 3 steps, leading to the “freeze” of ribosomal aggregation. Inhibition of protein synthesis at the initiation step, in contrast, does not prevent the continuation of elongation and termination and, therefore, produces ribosomal disaggregation.

10.6 Disturbances of Protein Synthesis

somes under various pathological conditions, notably after ischemia (Kleihues and Hossmann 1971) or seizures (Wasterlain 1972). As ribosomes disaggregate only in the presence of some residual and/or restored energy metabolism, the suggestion has been made that disaggregation is an active process that results from the dissociation between an inhibition of polypeptide chain initiation, on the one hand, and an orderly functioning polypeptide chain elongation and termination, on the other. Biochemical studies confirmed this interpretation and demonstrated that inhibition of protein synthesis in the absence of energy failure is mainly due to the phosphorylation and inactivation of the eukaryotic polypeptide chain initiation factor eIF2alpha (DeGracia et al. 1996). An important mediator of this process is the endoplasmic reticulum stress response (ERS) which, in turn, is closely linked to disturbances of the intracellular calcium homeostasis (Paschen 2003). Under physiological conditions, the ER calcium activity is several orders of magnitudes higher than that in the cytosol, requiring active calcium pumping across the ER membrane. Many pathological conditions, including brain ischemia, neurodegenerative diseases or seizure activity, cause a disturbance of the ER calcium homeostasis. The resulting decline of ER calcium activity induces a signal transduction cascade which culminates, among others, in the expression of stress proteins, the activation of the PKR-like ER kinases (PERK) and the phosphorylation of eIF2alpha (DeGracia et al. 2002). It has been speculated that this evolutionarily highly conserved response serves to prevent misfolding of newly synthesized proteins and, therefore, is a self-protective mechanism against otherwise lethal stress situations (Paschen 2003). However, as a shut-down of protein synthesis may occur both in reversibly and irreversibly damaged tissue, this response alone does not suffice to prevent injury. An important consequence of CPS inhibition is the failure of the tissue to respond to alterations in gene regulation. With the only exception of a few mRNAs, which are translated by bypass mechanisms, gene regulation at the transcriptional level is dissociated from the synthesis of the corresponding protein products. This is frequently ignored when differential expression displays and in situ hybridizations are used to analyze the effect of pathological disorders on genetically mediated injury cascades. In fact, depending on the half-life of regulated proteins, inhibition of protein synthesis causes a decline of the protein concentration, which, in turn, may lead to the superinduction of the corresponding messenger RNA. The importance of gene regulation for the evolution of brain injury must, therefore, be evaluated at the translational level by measuring the concentration of the protein product rather than that of its message.

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10.7

Importance of Disturbed Protein Synthesis on Injury Evolution 10.7.1

Global Brain Ischemia

Complete interruption of brain circulation causes cessation of protein synthesis within a few minutes, in unison with the breakdown of the energy metabolism. The simultaneous suppression of all steps of mRNA translation, i. e. polypeptide chain initiation, elongation and termination, results in the preservation of ribosomal aggregates, a phenomenon that has been referred to as “ischemic freeze” (Kleihues et al. 1975). When blood flow and energy metabolism are restored, protein synthesis is briefly resumed but then dissociates from the energy state of the brain. In fact, as polypeptide chain elongation and termination recover faster than chain initiation, the synthesis of those chains that have been initiated prior to ischemia is properly completed. However, with the ongoing inhibition of polypeptide chain initiation, polyribosomes disaggregate and protein synthesis ceases. Reversal of this disturbance depends on the duration and severity of ischemia on one hand, and on the anatomical location, on the other (Fig. 6). In the so-called selectively vulnerable regions, notably hippocampal CA1 sector and the dorsolateral striate nucleus, protein synthesis is irreversibly suppressed after an ischemia only lasting 5 minutes (Thilmann et al. 1986). In other brain regions protein synthesis slowly recovers, the delay of recovery increasing with the duration of ischemia. After 5 minutes bilateral common carotid artery occlusion in the gerbil, recovery to normal takes about 6 hours, but this delay is prolonged to about 24 hours after 30 minutes four-vessel occlusion in rats, and even longer after 60 minutes complete cerebrocirculatory arrest in cats or primates (Hossmann 1993). Obviously, permanent failure of CPS recovery results in irreversible injury but the delay between the onset of protein synthesis inhibition and the manifestation of cell death may be quite long. For example, in the selectively vulnerable CA1 sector of hippocampus, neuronal death following brief periods of global ischemia may be delayed for several days, although protein synthesis never recovers. Conversely, restoration of protein synthesis, as induced by hypothermia or deep barbiturate anesthesia, salvages CA1 neurons even if recovery may take as long as 24 hours (Xie et al. 1989). These observations demonstrate that neurons are able to survive inhibition of protein synthesis for extended periods, and suggest that restoration of protein synthesis, even if delayed for some time, is able to salvage the tissue from impending death.

10.7 Importance of Disturbed Protein Synthesis on Injury Evolution

Effect of global brain ischemia on protein synthesis. Autoradiographic measurements of amino acid incorporation into brain proteins before and at various times after 5 min global brain ischemia in gerbil (above) and after 1 hour ischemia in a baboon (below). During ischemia protein synthesis is inhibited in unison with the breakdown of energy metabolism, but after the onset of recirculation, protein synthesis is further suppressed for some time despite the rapid recovery of energy metabolism (see Figs. 11.3 and 11.4). The reason is an inactivation of the polypeptide chain initiation factor eIF2, the duration of which correlates with the duration of the preceding ischemia. In the selectively vulnerable regions of the brain, notably the pyramidal CA1 sector of hippocampus (arrows), inhibition of protein synthesis is irreversible, resulting in delayed neuronal death (data from Bodsch et al. 1986 and Xie et al. 1989).

Figure 10.6

10.7.2

Focal Ischemia

According to the threshold concept of cerebral ischemia, protein synthesis breaks down at much higher flow values than the energy state of the tissue (Fig. 10.2). In analogy to lactacidosis, protein synthesis can therefore be used to identify the outer margin of the peri-infarct penumbra (Fig. 10.7). In fact, during infarct evolution, bioluminescence imaging of ATP and autoradiographic evaluation of CPS have been extensively used to differentiate between the normal tissue on the one hand, and the core and penumbra, on the other: the normal tissue being characterized as a region with undisturbed CPS and ATP, the penumbra as a region in which CPS is suppressed, but ATP is preserved, and the core region as the area in which both CPS and ATP are lost (Hossmann 1994). The functional significance of this differentiation is reflected by the co-localization of selective gene expression: hsp70 mRNA which is a marker of the endoplasmic reticulum stress response, co-localizes precisely with the penumbra whereas immediate-early genes such as c-jun or c-fos which respond to peri-infarct depolarizations, are also up-regulated in the intact tissue, in agreement with the known spread of depolarizations over the entire ipsilateral hemisphere (Hata et al. 1998).

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Figure 10.7 Effect of focal brain ischemia on protein synthesis. Autoradiographic measurements of leucine incorporation into brain proteins at various times after permanent (above) and transient (below) middle cerebral artery (MCA) occlusion in rat (same experiments as in Fig. 10.4). According to the threshold concept of brain ischemia, protein synthesis ceases at higher flow levels than the energy metabolism. To illustrate this difference, the outlines of preserved ATP (taken from Fig. 10.4) are projected on adjacent autoradiograms of protein synthesis (green: ATP; red: protein synthesis). During permanent ischemia, the penumbra – defined as the region of suppressed protein, but preserved energy metabolism – slowly disappears due to the growth of the ATP-depleted infarct core. If blood flow is restored 1 hour after MCA occlusion, protein synthesis recovers only in the peripheral parts of the MCA territory, corresponding to the ischemic penumbra, but not in the infarct core in which secondary energy failure evolves (see Fig. 10.4. Data from Hata et al. 2000a,b).

Similar to global ischemia, protein synthesis predicts the further fate of the tissue. In permanent focal ischemia, penumbral protein synthesis remains severely suppressed, in association with the gradual deterioration of energy metabolism and, eventually, cell death. This is reflected by the progressie increase of the flow threshold of ATP depletion from 19 ml/100 g/min shortly after the onset of ischemia to 23 ml/100 g/min after 6 hours and to 32 ml/100 g/min after 12 hours (Mies et al. 1991). Eventually, the threshold of ATP merges with that of protein synthesis, leading to the expansion of the infarct core into the penumbra. Recovery of protein synthesis following transient focal ischemia, and hence tissue survival, depends on the delay and the efficacy of blood recirculation. After 1 hour ischemia or longer, protein synthesis recovers only in the penumbra, i. e. the region in which ATP is preserved (Hata et al. 2000b). In the core, ATP may transiently return, but as in this region the inhibition of protein synthesis is irreversible, a delayed type of injury evolves in analogy to delayed neuronal death in the selectively vulnerable brain regions after global ischemia (Fig. 10.6). If ischemia lasts shorter than 1 hour, protein synthesis may also recover in the peripheral parts of the infarct core and – after ischemia of less than 15 minutes – even throughout the ischemic territory, provided the reperfusion rate is in the normal range. Recovery of protein synthesis thus progresses from the peripheral to the more central parts of the ischemic territory and is the more efficient, the shorter the duration of ischemia and the higher the post-ischemic reperfusion rate.

10.7 Importance of Disturbed Protein Synthesis on Injury Evolution

10.7.3

Epileptic Seizures

Inhibition of cerebral protein synthesis is a common response to all forms of experimental seizures. In focal seizures induced by the topical application of convulsants, protein synthesis is markedly inhibited at the site of application (Willmore et al. 1981) and, to a lesser degree, also in regions connected ortho- and antidromically to this area (Collins and Nandi 1982). Generalized seizures, induced by bicuculline or kainic acid, result in depression of protein synthesis, mainly in the cerebral cortex and the pyramidal cell layer of the hippocampus, but not or much less in the cerebellum or the dentate gyrus (Kiessling et al. 1984). The decrease of protein synthesis is closely associated with a disaggregation of ribosomes (Wasterlain 1972), pointing to an inhibition at the polypeptide chain initiation step. The regions of disturbed protein synthesis co-localize with the areas of increased glucose metabolism. These areas are particularly sensitive to a mismatch in the coupling of blood flow and, therefore, are susceptible to impairment of oxidative respiration. It has been suggested that the resulting reduction of the adenylate energy charge may cause an increase of the GDP/GTP ratio and, as a consequence, an inhibition of protein synthesis (Dwyer et al. 1986). An alternative explanation is the inactivation of the eukaryotic initiation factor eIF2alpha, either in response to the seizure-induced activation of nitric oxide synthase (Petrov et al. 2003) or to an endoplasmic reticulum stress response (Wang et al. 1993). The latter is supported by the parallel up-regulation of the heat stress protein Hsp70 which is a common response of ER stress and which also accompanies the inhibition of protein synthesis under ischemic conditions. 10.7.4

Head Trauma

Contrary to ischemia and epilepsy, cerebral protein synthesis is only marginally inhibited after head trauma, provided, of course, that the tissue has not been mechanically destroyed. In the vicinity of cortical necrotic lesions, cerebral protein synthesis does not change at all (Hermann et al. 1999), and following fluid percussion, it declines only by about 20 %, which, however, is not reversed during the following days (Petrov et al. 2001). A transient inhibition of protein synthesis has also been observed after cortical puncture, but this disturbance recovers within 15 minutes, probably in parallel with the repolarization of the tissue after the passage of a lesion-induced spreading depression (Dunlop et al. 1984). A remarkable finding is the preservation of cerebral protein synthesis in the surroundings of a circumscribed cortical lesion. This is different from the penumbra around a focal ischemic infarct where protein synthesis is severely suppressed. The obvious reason for this difference is the blood flow which, in the vicinity of a traumatic lesion, is increased, in contrast to the peri-infarct penumbra, where it is reduced. Only at longer intervals after trauma, the gradual deterioration of blood flow may result in a flow-metabolism mismatch (Ginsberg et al. 1997), but it

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has not been established whether this situation is associated with disturbances of cerebral protein synthesis. The preservation of protein synthesis after brain trauma may have implications for the evolution of post-traumatic apoptosis. There is substantial evidence that brain trauma activates the extrinsic and intrinsic pathways of caspases, leading to the initiation of programmed cell death (Eldadah and Faden 2000). As apoptosis is an active process requiring the maintenance of protein synthesis, the metabolic scenario after head trauma favors the execution of this injury pathway, quite contrary to ischemia, where protein synthesis is severely disturbed. Another interesting aspect of post-traumatic protein synthesis is its failure to respond to central axonal injury. This is different from transection of peripheral or cranial nerves where protein synthesis markedly rises in the corresponding cell bodies (Sun et al. 1993). It has, therefore, been speculated that the failure of central regeneration following trauma may be due to the cell’s inability to increase its rate of protein synthesis in response to axonal injury (Kameyama et al. 1983).

10.8

Therapeutic Implications

In the pathophysiological situations reviewed here, metabolic disturbances are induced mainly by the following mechanisms: primary reduction of blood flow below the thresholds of protein synthesis and energy metabolism; uncoupling of flow and metabolism, leading to intermittent episodes of tissue hypoxia; disturbance of mitochondrial function, resulting in protracted energy failure and the initiation of apoptosis; and an endoplasmic reticulum stress response, causing the inactivation of polypeptide chain initiation factors and, hence, a down-regulation of protein synthesis. Each of these injury mechanisms requires a different therapeutic approach. The primary reduction of blood flow can only be treated by flow-promoting interventions, notably vascular recanalization and collateralization-enforcing procedures. Examples of this approach are rtPA-induced thrombolysis or the stimulation of collateral growth by activation of monocytes. Disturbances of the coupling between blood flow and metabolic activity can be alleviated symptomatically by either improving blood flow or reducing metabolic activity, or more causally, by interfering directly with the coupling mechanisms. Symptomatic interventions include rheological and vasoactive approaches in order to improve flow, and hypothermia or barbiturate anesthesia for reducing the oxygen consumption rate. A reliable medication for the restoration of coupling deficiencies is not yet available, but some positive effects have been reported using endothelin antagonists or prostacyclin. A much greater repertoire of interventions is available for the treatment of the molecular injury cascades that lead to mitochondrial insufficiency and apoptosis (Fig. 10.8). Glutamate antagonists, calcium channel blockers, nitrous oxide

10.8 Therapeutic Implications

Schematic representation of molecular injury pathways leading to mitochondrial failure and the endoplasmic reticulum stress response. Injury pathways can be blocked at numerous sites, providing multiple approaches for the amelioration of both necrotic and apoptotic tissue injury.

Figure 10.8

synthase antagonists and free radical scavengers interfere at different steps of this cascade, resulting in the alleviation of mitochondrial membrane peroxidation and calcium overload. Mitochondrial function can also be improved by preventing the assembly of a mitochondrial permeability transition (MPT) pore, e. g. by cyclosporin A (CsA). All these interventions improve the energy metabolism and, in consequence, those pathological disorders which are sensitive to energy deficits. A great number of drugs is also available that interfere with apoptosis. In particular, drugs blocking the extrinsic and intrinsic caspase pathways or antagonizing the final execution of cell death have been tested under various pathological conditions with promising results. An indirect effect of apoptosis on energy metabolism is the activation of poly(ADP-ribose)polymerase (PARP), which is a repair enzyme that reverses caspase-induced DNA strand breaks. This reaction consumes NAD, which is required for the proper functioning of the oxidative respiratory chain. PARP antagonists have, therefore, been proposed to prevent apoptosis-induced disturbances in the energy metabolism. To sum up, metabolic disturbances arising in the course of various brain disorders and contributing to the evolution of permanent brain damage can be attacked

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in numerous ways, although not always with the anticipated effect. More detailed descriptions of these specific neuroprotective pathways are given in the respective chapters of this book.

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10 Metabolic Dysfunctions Paschen W, Olah L, Mies G. (2000) Effect of transient focal ischemia of mouse brain on energy state and NAD levels: No evidence that NAD depletion plays a major role in secondary disturbances of energy metabolism. Journal of Neurochemistry 75:1675–1680. Petrov T, Rafols JA, Alousi SS, Kupsky WJ, Johnson R, Shah J, Shah A, Watson C. (2003) Cellular compartmentalization of phosphorylated eIF2alpha and neuronal NOS in human temporal lobe epilepsy with hippocampal sclerosis. Journal of the Neurological Sciences 209:31–39. Petrov T, Underwood BD, Braun B, Alousi SS, Rafols JA. (2001) Upregulation of iNOS expression and phosphorylation of eIF-2alpha are paralleled by suppression of protein synthesis in rat hypothalamus in a closed head trauma model. Journal of Neurotrauma 18:799–812. Prasad MR, Ramaiah C, McIntosh TK, Dempsey RJ, Hipkens S, Yurek D. (1994) Regional levels of lactate and norepinephrine after experimental brain injury. Journal of Neurochemistry 63:1086–1094. Siesj BK. (1988) Acidosis and ischemic brain damage. Neurochem. Pathol. 9:31–88. Siesj BK, Elmer E, Janelidze S, Keep M, Kristian T, Ouyang YB, Uchino H. (1999) Role and mechanisms of secondary mitochondrial failure. Current Progress in the Understanding of Secondary Brain Damage from Trauma and Ischemia 73:7–13. Siesj BK, Siesj P. (1996) Mechanisms of secondary injury [Review]. European Journal of Anaesthesiology 13:247–268. Siesj BK, Wieloch T. (1986) Epileptic brain damage: pathophysiology and neurochemical pathology. Advances in Neurology 44:813–847. Smith CB. (1991) The measurement of regional rates of cerebral protein synthesis in vivo. Neurochem. Res. 16:1037–1045. Sun Y, Deibler GE, Smith CB. (1993) Effects of axotomy on protein synthesis in the rat hypoglossal nucleus - examination of the influence of local recycling of leucine derived

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11 Protein Misfolding Milene Russelakis-Carneiro, Claudio Hetz, Joaquin Castilla and Claudio Soto

Abstract

Compelling evidence support the hypothesis that diverse human disorders – including the most common neurodegenerative diseases – arise from the misfolding and aggregation of an underlying protein. In spite of good evidence for the involvement of protein misfolding in disease pathogenesis, the mechanism by which protein conformational changes participate in the disease is still unclear. One of the best-studied diseases of this group are the Transmissible Spongiform Encephalopathies or prion-related disorders, in which misfolding of the normal prion protein plays a key role. In this chapter, we outline the data relating prion protein misfolding to the pathogenesis of spongiform encephalopathies and discuss the potential mechanisms by which the accumulation of the misfolded protein may damage the brain.

11.1

Introduction

In the last few years, protein misfolding has been proposed to be a central aspect of diverse diseases, which are now classified as protein misfolding disorders [1–4]. This group includes transmissible spongiform encephalopathies (TSEs), Alzheimer’s disease, Diabetes type 2, Huntington’s disease, serpin-deficiency disorders, Parkinson’s disease, amyloid polyneuropathy, hemodialysis-related amyloidosis and several others. Despite the very different clinical manifestations of diverse protein misfolding disorders, there are many similarities at the molecular level. In general, the pathological conformation is rich in b-sheet structure, and shows a capacity to oligomerize or aggregate due to protein stabilization by intermolecular b-sheet interactions [2]. This leads to the accumulation of different forms of protein aggregates in diverse tissues of affected individuals. One of the illnesses that have been extensively studied in terms of the involvement of protein misfolding in the pathogenesis of the disease is TSE. In the present chapter, we discuss the putative Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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mechanism by which protein misfolding and aggregation is associated with tissue damage and organ dysfunction, using TSEs as a model. Transmissible Spongiform Encephalopathies (TSEs), also called prion diseases, are a group of disorders that affect human and animals and have a long incubation period. Once the first clinical signs appear, the disease progresses relatively fast, and death occurs in a short time. Prion diseases are characterized by neurological dysfunction that may include dementia, ataxia and psychiatric disturbance. The term “transmissible” refers to the infectious origin of some TSEs and to the fact that almost all TSEs can be transmitted by experimental inoculation. Spongiform refers to the most typical neuropathological feature, which is the vacuolation of brain tissue. TSEs present a complex etiology that includes inherited, sporadic and infectious forms. Human familial TSEs are all associated with different mutations in the PrP gene, and include around 10 % of Creutzfeldt-Jacob disease (CJD) cases, and 100 % of the cases of Gertmann-Straussler-Sheinker (GSS) syndrome and fatal familial insomnia (FFI) [5]. Sporadic CJD has not been associated with any known mutation and occurs world-wide with an incidence of 0.5–1.5 new cases per one million people each year [6]. Infectious human TSEs include kuru, which was propagated by ritualistic cannibalism, and iatrogenic CJD, which is spread by tissue transplantation, contamination of surgical tools or inoculation with materials derived from CJD-infected tissues [7]. New variant CJD (vCJD) is a new infectious disease, which was first described in 1996 [8]. In animals, the most common disease is scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE) or “mad cow disease” in cattle [7]. There is strong evidence linking the appearance of vCJD in humans to exposure to the BSE agent, most possibly through dietary contamination with BSE tissue [9, 10].

11.2

Disease Propagation by Replication of Prion Protein Misfolding

During the disease process, the normal cellular prion protein (PrPC), a nearly ubiquitous constituent of mammalian cells, undergoes a conformational change that results in the formation of the abnormal isoform of PrP, here named PrP scrapie (PrPSc). The central event in the pathogenesis of prion diseases is the conversion of PrPC into PrPSc (Fig. 11.1A). PrPSc is not only a typical marker for the disease, but seems to be the major (if not only) component of the infectious agent [11]. The name prion was introduced to denote the novel nature and properties of the TSE transmissible agent as small proteinaceus infectious material and for this reason, TSEs are also known as prion diseases [12]. PrPC presents a high content of a-helical structure, is detergent-soluble and sensitive to proteolysis. In infectious forms of prion diseases, the normal cellular prion protein binds to the abnormal PrPSc, preferentially from a homologous species. As a consequence of the interaction between PrPC/PrPSc, PrPC undergoes conformational transition from a- helical to b-sheet structure, resulting in the formation of PrPSc (Fig. 11.1A). The high b-sheet content of PrPSc confers to this protein

11.3 Prion Biology

distinct physicochemical properties from PrPC, reflected in its insolubility in nondenaturing detergents, partial resistance to proteolysis, and its ability to form fibrillar structures in vitro [11]. The hypothesis that PrP structure plays a role in prion propagation is supported by experiments in which treatment of prion infectious material with compounds that selectively destabilize b-sheet structure of PrPSc, resulted in a delay in the appearance of the clinical symptoms and a 90–95 % reduction of infectivity [13]. Similarities between the primary sequence of PrPC and PrPSc are necessary for efficient PrPSc formation and constitute a pre-requisite for crossing the barrier between different species. A great deal of effort has been made in the last few years to understand the remarkable biology of the prion conversion process. PrPC is essential for the development of prion diseases since ablation of the gene which codifies PrPC renders mice resistant to prion infection [14]. Under certain conditions, the conversion of PrPC into PrPSc can be achieved in a cell-free conversion assay [15]. However, in vitro conversion does not progress beyond a certain level, even when PrPC is not limiting, suggesting that other factors are likely to be involved in this reaction. In fact, some in vitro and in vivo data have demonstrated that the conversion of PrPC into PrPSc required the presence of other factor(s), probably a protein, not yet identified and referred as factor or protein X (Fig. 11.1B) [16,17]. A highly efficient in vitro PrPSc replication system has been reported in which minute amounts of brain derived PrPSc can be used to direct high extent of PrPC conversion in a cyclical process [18].

11.3

Prion Biology

PrPC is a glycophosphatidylinositol-anchored protein located in membrane subdomains called detergent resistant-membranes (DRMs), also known as lipid rafts (Fig. 11.1C) [19, 20]. The high concentration of receptors and other signaling molecules in these structures provided a first hint of a potential signaling role of PrPC. Support for this idea came from antibody-mediated cross-linking experiments showing a caveolin-1-dependent coupling of PrPC to Fyn, a member of the Src family of tyrosine kinases [21]. Additional evidence to support the hypothesis that PrPC plays a role in neuroprotective signaling comes from recent studies in which stress-inducible protein 1 (STI1) was identified as a potential PrPC-interacting protein in the plasma membrane [22]. It was shown that the interaction between PrPC and STI1 protects neurons from anisomycin-induced apoptosis [23]. In addition, PrP-null neuronal cell lines are more susceptible to serum deprivation-induced apoptosis, and PrPC over-expression protects human neurons against Bax-induced apoptosis [24]. Alterations on the cholesterol or sphingolipid composition or replacement of the PrP GPI signal, rendering the protein soluble, alter the ability of chronically prioninfected cells to replicate prions [25, 26]. In vitro studies using a cell-free system have shown that the insertion of PrPSc into the DRMs is required for the conver-

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11.3 Prion Biology m Figure 11.1

Prion biology and pathology. (A) Schematic representation of the primary structure of PrP and the three-dimensional structures of the two alternative forms, PrPC and PrPSc. The image of PrPC corresponds to the experimentally determined structure of the recombinant protein by NMR [99] while PrPSc represents a computational model, based on secondary structure estimations. (B) A model for the progression of transmissible spongiform encephalopathy. The hallmark event in the disease is the misfolding of the normal PrPC to form the b-sheet-rich PrPSc. The conversion of PrP is believed to be dependent of the presence of a yet unidentified factor termed protein-X. The misfolded protein has a high tendency to aggregate, forming amyloid prion rods, which might be the precursor of cerebral amyloid plaques seen in some TSE cases. The final cause of clinical symptoms associated with TSEs is the extensive neurodegeneration of the brain. (C) PrP is a GPI-anchored protein located in membrane fractions enriched in glyco-sphingolipids, cholesterol and sphingomyelin, termed lipid rafts or DRM. The conversion of PrPC into PrPSc is believed to occur in DRMs where PrPSc has also been shown to accumulate. (D) PrP pattern on Western blot before and after PGNase treatment and the proportion of PrP glycoforms in the brain, optic nerve and retina of Syrian hamsters. D, M and N correspond to the di-, monoand non-glycosylated PrP, respectively. NBH – normal brain homogenate, ON – optic nerve, Ret – retina.

sion of PrPC into PrPSc [27]. More recently, transgenic mice expressing a dimeric, soluble form of PrP in a PrP knockout background were shown to be resistant to prion infections [28]. When this soluble form of PrP was expressed in a wild type background, the mice showed a delay in the evolution of scrapie. This effect was correlated with a shift in the subcellular location of this soluble PrP, into a complex with PrPSc in DRMs that arrest, probably via a competitive mechanism, the conversion of PrPSc. Altogether, these data highlight the importance of the interaction of PrPC and PrPSc in the context of the membrane DRMs in the pathogenesis of prion diseases. A better understanding of the structure and components of DRMs may shed light on the prion conversion mechanism. Depending on the species and the scrapie strain involved, different proportions of three distinct bands of PrP (including the mono- (30KDa), di-glycosylated (33– 35KDa) and a non-glycosylated band (27KDa)) can be observed (Fig. 11.1D). In the neuronal cell bodies of mice, the non-glycosylated form of PrPC is virtually absent, while in axons, the 3 forms of PrP have been detected [29], suggesting that PrP profile can vary between different subcellular locations. Distinct proportions of the three isoforms of PrP have been used as criteria to identify different strains of prions and may be involved in the phenomenon of species barrier observed in these diseases [30]. Glycosylation is not required for the conversion of PrPC into PrPSc [31]. On the contrary, several lines of evidence indicate that the absence of glycosylation facilitates PrP conversion. Mutation of the glycosylation sites or the inhibition of glycosylation induced accumulation of non-glycosylated PrP within cells and acquisition of some of the PrPSc properties, such as detergent insolubility and protease resistance [32–34]. Using a cell-free conversion assay, the non-glycosylated isoform of PrPC binds more efficiently homologous or heterologous PrPSc, increasing the possibility of conversion [35]. Therefore, glycosylation seems to influence the conversion of the PrPC into PrPSc and the crossing of species barrier in prion diseases.

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11.4

Prion Pathogenesis

PrPSc is the only component so far associated to infectivity in prion diseases. PrPC expression is necessary for the neuronal spread of the prion agent and the pathological changes associated with prion diseases [36, 37]. Infectivity propagates along defined neuro-anatomical pathways concomitant with histopathological changes typical of prion diseases [38, 39]. The speed of propagation of the disease in the peripheral and central nervous system was calculated, by titration of an infectivity assay, in 1–2 mm/day coincident with the slow component of the axonal transport [40, 41]. The transport of prions within neurons is not known, but has been proposed to occur in a PrPC-dependent way, in which PrPC would be converted by adjacent PrPSc in a domino-like effect [42]. The mechanism by which the disease is propagated from the periphery to the brain is still not fully understood. During the incubation period, replication of PrPSc first takes place in lymphoid organs, independent of the route of infection [43]. Splenectomy causes an increase in the incubation period after intraperitoneal injection of prions, and folicullar dendritic cells have been suggested to play an important role in the transmission of the disease from the periphery to the nervous system [44, 45]. In addition to dendritic cells, the complement system and B-lymphocytes have been shown to influence the progression of prion diseases initiated by peripheral infection [46, 47]. Following oral infection, the pathogenesis of prion diseases seems to be influenced by the number of Peyer’s patches, from where infectivity seems to be propagated by M-cells [48, 49]. The peripheral nervous system certainly plays a major role in the propagation of the disease to the central nervous system. Following intraperitoneal injection of prions in PrP knockout mice, it was shown that reconstitution of the bone marrow tissue with PrPC-expressing cells restored infectivity in lymphoid organs, but still, the animals did not develop scrapie [50]. Chemical and immunological sympathectomy from peripheral lymphoid organs prevented or delayed scrapie in mice after intraperitoneal injection of prions [51]. Thus, after an initial phase of replication in lymphoid organs, PrPSc propagates to the brain via peripheral nerves. One interesting aspect of the peripheral propagation of prions is that it does not cause pathological changes in these organs. Remarkably, no morphological or functional abnormalities are found in the peripheral nervous system, despite the fact that this tissue expresses PrPC and propagates the disease [52, 53]. The passage of PrPSc accross the blood-brain barrier (BBB) and the contribution of this pathway to prion neuroinvasion has not been studied. However, recent data suggest that PrPSc can indeed penetrate the BBB, arguing that direct transmission from blood to brain could play a role in PrPSc brain uptake (Banks and Soto, unpublished observations).

11.6 Neuronal Apoptosis in Prion Diseases

11.5

Neuronal Targeting

One intriguing feature of prion diseases is that histopathological changes are restricted to the central nervous system, despite the trafficking of PrPSc along the peripheral nervous system and the detection of PrPSc in lymphoid organs [54]. Common histopathological findings in prion diseases are vacuolation, astrocytosis, microglial activation, PrPSc deposition and neuronal death [55]. Besides deposition of PrPSc, all others histopathological features of prion diseases can be found in other acute and chronic central nervous system injuries. An inflammatory response, characterized by microglia activation and T-lymphocyte infiltration, mainly CD8þ lymphocytes is present in the brain parenchyma at early stages of murine scrapie [56]. The inflammatory response follows the propagation of the disease along neuronal pathways and precedes the deposition of PrPSc, as shown by immunohistochemistry [57]. Microglial activation and apoptosis are common histopathological findings. Therefore, the role of the inflammatory response in the brain during prion diseases remains unclear. At early stages of scrapie infection in mice, synaptic abnormalities associated with the deposition of abnormal PrP have been described in mice, long before neuronal death [58]. Recently, synaptic dysfunction has been linked to behavioral changes, but the mechanisms underlying it remain to be established [59]. A role for PrP in synaptic function has been suggested in studies performed in PrP knockout mouse [60, 61]. PrPC has been shown to interact with other pre-synaptic proteins, including synaptophysin, and hence an interference of this interaction, due to conversion of PrPC into PrPSc, may play a role in the disease [62]. Our recent findings indicate that at early stages of the disease, PrPSc accumulates in DRMs, producing a detachment of caveolin and synaptophysin from these membrane domains (Russelakis-Carneiro et al, unpublished observations). The identification of early events in the pathogenesis of prion diseases is crucial for the development of therapeutic strategies aimed to avoid neuronal death.

11.6

Neuronal Apoptosis in Prion Diseases

There are several observations indicating that neuronal death in prion diseases occurs by apoptosis. In humans, apoptosis has been described in the brain of patients affected by CJD [63, 64] and Fatal Familial Insomnia [65]. It has also been described in scrapie-infected hamsters, mice and sheep [66, 67]. In addition, transgenic mice expressing mutant forms of PrP, associated with inherited prion diseases, exhibit neuronal cell death by apoptosis (review in [68]). Although a considerable effort has been made to understand the molecular basis of prion conversion, little is known about the mechanism by which this process is associated with neuronal apoptosis.

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Attempts to understand the molecular basis of neuronal dysfunction in prion diseases have led to the search for in vitro models to analyze the role of PrPSc in cellular death. The prion protein fragment spanning the sequence 106–126 (PrP106–126), corresponding to a putative transmembrane region of PrPC, has been extensively used to induce cell death in neuronal cultures [69–71]. Apoptotic-cell death mediated by this peptide is associated with the activation of caspase-3 due to intracellular calcium release and the induction of mitochondrial dysfunction [69–71]. The major drawback of using PrP106–126 as a model is that although this sequence is present in several longer peptides isolated from cerebral amyloid plaques of patients suffering from GSS [72], the PrP106–126 peptide as such has never been found in vivo. Therefore, the relevance of the findings obtained with this peptide remains to be determined. It has been reported earlier that purified PrPSc leads to cell death [73]. In addition, as described below, we have recently identified the signaling pathways involved in neuronal apoptosis induced in vitro by highly purified PrPSc [74]. Most of the research in the field has focused on understanding the nature of the infectious agent and the mechanism by which a possible gain of neurotoxic activity of PrPSc contributes to TSE neurodegeneration. However, a more complete understanding of PrPC-mediated signaling and its participation in the maintenance of neuronal activity is the key to understanding whether a loss-of-function of PrPC may be implicated in the pathogenesis of prion disorders. Resolving this question is crucial for the development of therapeutic approaches for TSE, because if reduced PrPC physiological function is involved in disease progression, then strategies directed towards maintaining or replacing this activity could provide novel opportunities for treatment. The interpretation of the current data is that although a number of studies have suggested that PrPC may be implicated in neuronal function and survival, it is unlikely that TSE neurodegeneration is explained exclusively by a loss of PrPC activity [75].

11.7

ER-Stress and Apoptosis in Prion Diseases

Apoptosis is a programmed form of cell death, which plays a central role during the development and in the homeostasis of multicellular organisms and has been implicated in a number of pathological conditions [76–78]. The central executioner molecules of apoptosis are a large family of cysteine proteases known as caspases [79, 80]. Based on structural similarities and substrate preference, caspases have been divided into initiators (such as caspase-8 and caspase-9), downstream executor caspases (such as caspase-3) and inflammatory caspases (such as caspase-1). Activation of caspase-dependent apoptosis may be initiated by cell surface receptors or by mitochondrial stress [81]. Recently, another apoptotic-regulatory pathway has been described, in which the induction of endoplasmic reticulum (ER) stress, due to alteration of calcium homeostasis or accumulation of misfolded proteins, triggers the activation of an ER-resident caspase, termed caspase-12 [82].

11.7 ER-Stress and Apoptosis in Prion Diseases

We have shown recently that low concentrations (nano-Molar) of highly purified PrPSc induce apoptosis in vitro [74]. This effect appears to be very specific to the pathological conformation of the protein, since no toxicity was observed using recombinant PrPC, synthetic PrP peptides or PrPSc treated with a peptide that disrupt b-sheet conformation [74]. Apoptotic-induction by PrPSc was shown to be dependent on caspase-3 activation, evidenced by the detection of caspase-3 activity after incubation with PrPSc, and the inhibition of toxicity by pre-treatment of N2A cells with caspase-3 inhibitors. Upstream of caspase-3 activation, there is a rapid and sustained release of calcium from the ER in response to PrPSc treatment [74]. Perturbation of calcium homeostasis and accumulation of misfolded proteins in the ER have been shown to elicit a cell-stress response that can lead to apoptosis [82]. Consistent with this model, we found that PrPSc treatment leads to the induction of ER-chaperones of the glucose regulated family proteins (Grps). Experiments with N2A cells chronically infected with scrapie prions further support the relationship between PrPSc and ER-stress [74]. Infected cells exhibit increased sensitivity to the ER-stress induced apoptosis mediated by several stimuli, including exogenous PrPSc itself. ER-stress has been associated to the pathogenesis of other neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis [83, 84]. The molecular mechanism linking ER stress and apoptosis has been shown to be mediated by the ER-resident caspase12 (caspase-4 in humans), which upon activation leads to caspase-3 cleavage and cellular death [82]. It seems that the main function of caspase-12 is to facilitate apoptosis in cells irreversibly damaged by stress signals from the ER [85]. Treatment of neurons with b-amyloid peptide [82], or the expression of polyglutamine aggregates associated with Huntington’s disease induce ER stress and activation of caspase-12 in vitro [86]. These diseases are characterized by misfolding and accumulation of proteins in the brain [4], suggesting that the activation of this pathway could be a general mechanism associated with cellular toxicity induced by the accumulation of misfolded proteins. Our results clearly show that activation of caspase-12 was part of the apoptosis pathway induced by PrPSc because the caspase-12 active form was detected in cells treated with PrPSc, and cellular death was inhibited by overexpression of a caspase-12 catalytic mutant [74]. These in vitro results were supported by biochemical and histological studies in scrapie-infected mice, which show a good temporal and spatial correlation between neuronal death and caspase-12 activation [74]. In addition, we found a direct relationship between up-regulation of ER-stress inducible protein Grp58 and the levels of PrPSc in different brain areas of scrapie-infected mice. Grp58, also called ER-60 or Erp-57, is a disulfide isomerase that forms part of the ER-chaperone family designated as glucose regulated proteins (Grps) [87]. More recent results revealed that abundant PrPSc accumulation and Grp58 up-regulation are the first abnormalities detected in the brain of scrapie-infected mice, several weeks before onset of clinical signs (Hetz and Soto, unpublished data). As the disease progresses, caspase-12 is activated, and this is followed by neuronal death.

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Additional evidence linking prion pathogenesis to ER alterations has been found in experiments showing that stimulation of retrograde transport toward ER increases the accumulation of PrPSc in prion-infected neuroblastoma cells [88]. Moreover, several PrP mutants associated with inherited prion diseases are accumulated in this organelle [89–92]. Finally, several ER-chaperones including Bip/ Grp78, calnexin and protein disulphide isomerase have been shown to interact with mutant PrP [93]. The overexpression of Grp chaperones has been described as a protective cellular response against different forms of ER-stress [87, 94, 95]. In the case of TSE and other protein misfolding processes, it is likely that ER-chaperones are up-regulated in an attempt to correct the misfolded conformation, or, in case this is not successful, to send the abnormal protein down the ER-associated degradation pathway (ERAD). This process may have a double-edged sword effect when a large accumulation of misfolded proteins reach the ERAD, leading to dysfunction of the proteasome. This “traffic jam” might activate cell death pathways related with the pathological accumulation of misfolded and immature proteins in the ER, killing in an indirect way the cells that presented initially PrP accumulation. The first evidence for a role for the proteasome system in the pathogenesis of prion diseases came from studies using cell lines expressing a mutated form of PrP associated with Gerstmann-Strussler-Scheinker Syndrome (GSS), a human inherited form of prion disease [91]. The authors described that mutation PrPY145stop leads to an accumulation of PrP in intracellular compartments and proteasome inhibition, inducing an increase in the amount of mutated PrP intracellularly, mainly in the ER, Golgi and nucleus [91]. It has been described that after proteasome inhibition with different compounds, fully mature PrP is accumulated in the cytoplasm and exhibits some of the biochemical properties of PrPSc, such as insolubility in non-ionic detergents and resistance to protease degradation [96, 97]. Interestingly, the accumulation of PrP in the cytoplasm, following proteasome inhibition in PrP-over-expressing cells, was shown to cause cellular death by apoptosis [32]. After removal of the proteasome inhibitors, the accumulation of PrP in the cytosol continued, suggesting that a self-perpetuating (infectious-like) mechanism could be involved [32]. However, it has been suggested that these results could be produced by an artificial increase in PrP mRNA levels because of an unspecific effect of those inhibitors in the viral promoter used to over-express PrP [98]. Transgenic mice expressing a cytosolic PrP mimicking the PrP form generated through the ERAD process, developed severe ataxia with cerebellar degeneration and gliosis. It was postulated that in conditions in which the proteasome ability to degrade PrP is compromised, like cellular stress and events associated with aging, accumulation of cytosolic PrP may promote neuronal degeneration.

11.8 Concluding Remarks

11.8

Concluding Remarks

In this chapter, we have described a general overview of the biochemical and cellular events involved in the pathogenesis of protein misfolding disorders using TSEs as a model (Fig. 11.2). Several evidences suggest that, independent of the protein involved in each particular disease, a central hallmark of these illnesses is alteration in protein folding, associated with increases in b-sheet secondary structure [2]. This structural change is directly associated with the concomitant changes in the physicochemical properties of the misfolded protein, reflected, for example, in changes in the aggregation state and sometimes subcellular distribution. Interestingly, the late consequence of protein misfolding is the occurrence of cellular dysfunction that can trigger cell death by apoptosis. Based on the results discussed here, it seems that ER-stress induced by treatment with PrPSc is most likely the cause of neuronal death in TSE. ER-stress leads to activation of caspase-12, which in turn activates the apoptosis executer, caspase-3. The cellular protective signaling pathways normally involved with the elimination of misfolded protein during synthesis and maturation of proteins plays a key role in controlling the cellular fate. This response consists in the specific up-regulation of ER-chaperones to attempt correcting or removing the misfolded protein. However, this mechanism of defense may turn to have a negative consequence by overloading the

Figure 11.2 ER-stress and neuronal apoptosis induced by protein misfolding. Protein misfolding might be induced by a variety of factors, including mutations, oxidative stress, changes in the levels of calcium or other cations, prion infection, heat shock, trauma, etc. A double response is triggered, including an anti-apoptotic mechanism involving the upregulation of ER-chaperones and a pro-apoptotic pathway consisting in the activation of caspase-12. If the anti-apoptotic pathway is not successful in removing the misfolded protein, a protein traffic jam is produced, leading to proteasomal failure that further contributes to cellular apoptosis.

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proteasomal degradation system, resulting in dysfunction of this important cellular process, contributing to cell damage. Similar findings have been reported in other neurodegenerative diseases associated with cerebral accumulation of misfolded proteins, including Alzheimer’s, Parkinson and Huntington diseases, suggesting that ER-stress, caspase-12 activation and proteasomal dysfunction might be key players in brain degeneration.

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12 Axonal Growth Inhibition Anne D. Zurn and Christine E. Bandtlow

12.1

Introduction

Traumatic injuries to the adult mammalian central nervous system (CNS) cause severe and irreparable disabilities. Even limited local insults to the brain or the spinal cord have devastating consequences due to the inability of the CNS – in contrast to the peripheral nervous system – to regenerate injured nerve fibers or to react with structural plasticity. Nerve fibers initially start to sprout, but they fail to regenerate over long distances and cannot contact appropriate target cells, resulting in a permanent loss of function. This lack of regeneration appears to be due to both the intrinsic inability of central fibers to grow, and an inhospitable environment. The present review focuses on the cellular and molecular aspects of intrinsic differences in growth potential and of axonal growth inhibition, in particular on the recent advances in the characterization of growth-inhibitory molecules and their mode of action. Additional details may be found in other recent reviews (De Winter et al., 2002a; Hunt et al., 2002; Morgenstern et al., 2002; Bareyre and Schwab, 2003; Filbin et al., 2003; Huber et al., 2003).

12.2

Intrinsic Properties of CNS Neurons 12.2.1

Survival of Injured Neurons

Injury or trauma of the nervous system can vary considerably in extent and severity, from minor contusions or crush injuries to open injuries associated with partial or even complete disruption of the nerves. Any type of axonal injury induces an array of dramatic molecular and cellular responses at the level of the cell bodies and at the site of the injury that have a direct impact on the potential for successful regeneration. One of the most immediate changes is the destruction of tissue at Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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the site of injury, which can vary greatly depending on the type of injury. Damage to the axons may consist in the complete transection of the nerve fibers, followed in some cases by the death of the neuronal cell body. However, the most important requirement for regeneration of the injured axons to occur is the survival of the nerve cell body and its capacity to induce gene expression, leading to axonal re-growth and re-innervation of the original targets (for a review see Goldberg and Barres, 2000). The closer the axonal insult occurs to the cell body, the less likely it is that the neuron can be maintained in a healthy state. Injuries that sever axons relatively distally to the cell body produce a severe reduction in neuronal size (atrophy), but the neurons survive the lesion (Sofroniew and Isacson, 1988). They undergo morphological and molecular changes that involve major rearrangements of the endoplasmic reticulum and Golgi apparatus, a process referred to as chromatolysis. Chromatolysis is often accompanied by an increase in the expression of a set of genes associated with axon growth. However, even if the neuron ultimately survives the injury, its nerve terminals and the entire segment distal to the lesion will degenerate, a process called Wallerian degeneration (Stoll et al., 2002). 12.2.2

Developmental Loss of Regenerative Ability

A number of studies have demonstrated that the intrinsic growth ability of mammalian CNS neurons is developmentally regulated. While embryonic CNS axons can regenerate quite readily, they lose this capacity with age (Steeves et al., 1994; Chen et al., 1995, Dusart et al., 1997; Gianola and Rossi, 2001). For instance, successful regeneration and sprouting of nerve terminals following lesion gives rise to functional recovery in the CNS of young, but not adult mammals (Bates and Stelzner, 1993; Hasan et al., 1993). In addition, defined spinal cord lesions in chicken embryos or newborn mammals lead to extensive regeneration of fibers, whereas no such growth is observed in the adult (Kalil and Reh, 1982; Saunders et al. 1992; Tolbert and Der, 1987; Treherne et al., 1992; Bernstein-Coray and Bregman, 1993, Saunders 1998). Adult neurons seem to loose their ability to grow during postnatal development, and their regenerative response becomes abortive. The loss in capacity to grow appears to coincide with the maturation of CNS glial cells, both astrocytes and oligodendrocytes, and with the onset of myelination. Delaying spinal cord myelination, for instance, delays the end of the permissive period in chick embryos (Keirstead et al., 1992). However, myelin and associated molecules are not the only candidates that account for the developmental loss of regenerative capacity. Indeed, in an in vitro model system devoid of myelin, axotomized Purkinje cells retain their age-dependent decrease in ability to regenerate, despite the absence of myelin (Bouslama-Oueghlani et al., 2003). In the retina, amacrine cells appear to be responsible for the loss of intrinsic growth capacity of retinal ganglion neurons (Goldberg et al., 2002b). Neurons can thus send signals to other neurons, inducing them to lose their axonal growth ability. Developmental loss of regenerative ability is also correlated with an intrinsic decrease in the neuronal expression of growth-associated proteins such as GAP-43

12.2 Intrinsic Properties of CNS Neurons

and SCG10 (Jacobson et al., 1986; Sugiura and Mori, 1995; for review, see Mori and Morii, 2002) and a decrease in endogenous cAMP levels (Cai et al., 2001). Interestingly, there may also be a developmental increase in the capacity to respond to extrinsic inhibitory cues, since increases in intracellular calcium concentrations in response to the myelin protein Nogo are small and transient in cultured embryonic chick retinal ganglion neurons compared to mature neurons (Bandtlow and Loschinger, 1997). Similarly, a difference in receptor levels or responsiveness to trophic or guidance signals could explain these differences. Exogenously elevating TrkB levels, however, fail to revert postnatal axonal growth rates to embryonic levels (Goldberg et al., 2002a). Down-regulation of the expression of the anti-apoptotic protein Bcl-2 was also proposed to be responsible for the decreased axonal growth rates of retinal ganglion cells (Chen et al., 1997). However, Bcl-2 overexpression in these neurons in vitro and in vivo neither promotes axon growth nor enhances growth in response to neurotrophic factors (Goldberg et al., 2002a). In summary, both the intrinsic neuronal growth state (developmental expression of genes involved in axonal growth and/or in limiting axonal growth), as well as the neuronal environment (oligodendrocytes and myelin or astrocytes) appear to account for the age-related switch from a successful to an abortive regeneration (Goldberg and Barres, 2000). 12.2.3

Intrinsic Differences in Growth Potential

Differences in the intrinsic growth potential among CNS neurons themselves have also been described. For example, peripheral nerve grafts implanted in the CNS promote regeneration of inferior olivary axons from deep cerebellar nuclei, but not from Purkinje cells (Rossi et al., 1995) (reviewed in Anderson et al., 1998). In addition, distinct classes of neurons react differently to the same non-permissive CNS environment since biotin-dextran-labeled ascending sensory axons, but not the subgroup labeled with CGRP, can regenerate after spinal cord crush in mice (Inman and Steward, 2003). 12.2.4

Transient Expression of Growth-associated Molecules

Injured nerve fibers in the CNS of adult mammals undergo a reaction characterized by Wallerian degeneration in the distal portion of the nerve, and a spontaneous, but short-lived and ultimately abortive attempt at repair called regenerative sprouting (Ramon y Cajal, 1928). These sprouts extend for only a few mm before growth is aborted and the new sprouts are gradually resorbed. The reasons for this unsuccessful regenerative process remain largely unknown, but they indicate that injured CNS neurons have not necessarily lost their intrinsic property to grow, but that they fail to initiate and/or maintain a specific growth program required for axonal elongation. Important regulators of axonal growth that are expressed during nervous system development (GAP-43, cytoskeletal proteins, transcription factors,

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neuropeptides, integrins, growth factors and neurotrophin receptors) are re-expressed upon nerve injury (for review see Fernandes and Tetzlaff, 2000). However, in contrast to the PNS, this event is often only transient or completely absent in the CNS. These observations suggest that prolonged expression of growth-associated genes may be required for successful regeneration. Additional evidence that expression of growth-associated molecules may be a prerequisite for axonal regeneration comes from experiments on sensory neurons. Primary sensory neurons with their cell bodies in the dorsal root ganglia (DRG) possess two axonal branches, a peripheral axon that can regenerate, and a central axon, located in the spinal cord, that cannot regenerate upon injury. Interestingly, lesion of the central ascending sensory axons, in contrast to lesion of the peripheral branches, does not lead to the up-regulation of GAP-43, c-Jun, and JAK in the DRG neurons (Schreyer and Skene, 1993; Chong et al., 1994; Broude et al., 1997; Schwaiger et al., 2000). Overexpression of GAP-43 alone, however, allows only sprouting and not regeneration of CNS neurons (Aigner et al., 1995; Buffo et al., 1997; Mason et al., 2000). Only the combined expression of GAP-43 with another growth cone protein, CAP-23, permits regrowth of sensory axons from the spinal cord lesion site into a peripheral nerve graft (Bomze et al., 2001). In addition to a reactivation of growth associated genes expressed during development, peripheral axotomy leads to the up-regulation of regeneration-associated proteins (RAGs) including SPRR1A, a small proline-rich protein that associates with F-actin, and fibroblast growth factor-inducible-14 (Bonilla et al., 2002; Tanabe et al., 2003). These two proteins are not expressed during development, but promote axon growth when overexpressed in neurons and PC12 cells, respectively (Bonilla et al., 2002; Tanabe et al., 2003). RAGs, in addition to growth-associated proteins, may therefore also be required to trigger successful nerve regeneration. Up-regulation of regeneration-associated genes upon injury in the CNS is influenced by the distance between the cell body and the site of the lesion (Hll and Bhr, 1994; Fernandes et al., 1999). This is illustrated by the observation that corticospinal cell bodies respond to intracortical, i. e. proximal, axotomy by up-regulating a range of growth-associated molecules, while they fail to do so in response to spinal axotomy (Mason et al., 2003). This may in part explain the lack of regeneration of corticospinal axons into peripheral nerve grafts placed in the spinal cord (Richardson et al., 1980). 12.2.5

Conditioning Lesion

As described in the previous chapter, peripheral, but not central processes of primary sensory neurons can regenerate after injury. This discrepancy is due in part to the non-permissive character of the CNS environment, but also to the failure to induce growth-associated molecules such as GAP-43, CAP-23, and SCG10 upon injury of central, in contrast to peripheral sensory processes (Schreyer and Skene, 1993; Chong et al., 1994; Mason et al., 2002). Evidence for the contribution of the intrinsic growth state of central sensory processes to their capacity to

12.3 Extrinsic Factors: The CNS as A Non-conducive Growth Environment

regenerate comes from the demonstration that central sensory processes show improved regeneration through a peripheral nerve graft if their peripheral axons have been previously cut (conditioning lesion) (Richardson and Issa, 1984; Chong et al., 1996). In addition, a conditioning peripheral nerve lesion prior to dorsal column injury results in some growth of ascending sensory axons into the spinal cord above the lesion (Neumann and Woolf, 1999). Interestingly, regenerating ascending sensory axons appear to grow across areas with strong expression of tenascin-C and CSPG, but avoid areas containing scar-associated fibroblasts expressing Semaphorin3A, as well as areas with CNS myelin (Pasterkamp et al., 2001). Thus, there is a balance between intrinsic and extrinsic factors, but also a balance between distinct environmental factors themselves that control the final outcome of regeneration.

12.3

Extrinsic Factors: The CNS as A Non-conducive Growth Environment 12.3.1

Cellular Components of Inhibition

Besides the intrinsic growth state of CNS neurons that may determine a neuron’s commitment to growth, CNS regeneration failure may also be attributed to extrinsic factors present in CNS tissue and/or produced upon injury. For instance, peripheral neurons that grow well in peripheral tissues often show only limited growth when transplanted into the brain or spinal cord of adult vertebrates. On the other hand, some injured CNS neurons can grow through peripheral nerve grafts transplanted as bridges into the brain or spinal cord of adult rats, but they cease to grow when they re-enter the CNS tissue (Richardson et al., 1980; David and Aguayo, 1981; Pettigrew and Crutcher, 1999). These experiments demonstrate that (1) adult CNS tissue is unfavorable for axonal growth and (2) some adult CNS neurons can extend long processes when provided with a permissive environment. Traumatic spinal cord injury results in many cellular and molecular changes that extend over months and years after injury. During the acute phase of injury (first few days), there is disruption of the blood-brain barrier, ischemia, edema, leading to neuronal death, degeneration of axons, astrocyte proliferation, and infiltration of immune cells and meningeal fibroblasts. The subsequent secondary injury extends over a time course of several weeks and results in reactive gliosis, formation of a glial scar, strong inflammation, formation of a cystic cavity, and apoptotic cell death (for review see Schwab and Bartholdi, 1996).

The Glial Scar Traumatic injury to the adult central nervous system results in a rapid response from resident glial cells around the injury site, leading to as reactive astrogliosis or glial scarring. Although the functional role of glial scarring is not completely 12.3.1.1

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understood, it has been suggested to be an attempt made by the CNS to restore homeostasis through isolation of the damaged region (for review see Stichel and Muller, 1998a). Astrogliosis may have other beneficial effects, since astrocytes contribute to the clearance of glutamate, restoration and maintenance of ion concentrations, and provide anti-inflammatory cytokines as well as neurotrophic support (Costam et al., 1992; Muller et al., 1995; Cui et al., 2001). However, glial scarring can also be detrimental, since it may impede axonal regeneration and thus interfere with neuronal repair. Reactive gliosis involves astrocytes, oligodendrocyte precursor cells, microglia/macrophages, leptomeningeal fibroblasts, and eventually forms a scar that is a major impediment to axonal growth (Berry et al., 1983; reviewed in Fawcett and Asher, 1999; Hirsch and Bahr, 1999). Reactive astrocytes are characterized by a number of cellular changes, including hypertrophy, generation of long and tightly packed cytoplasmic processes, increased production of the intermediate filament proteins GFAP and vimentin, and up-regulation of a number of cell surface and extracellular matrix (ECM) molecules, in particular chondroitin sulfate proteoglycans (CSPGs) and tenascin (McKeon et al., 1991; SmithThomas et al., 1994; for review see Hoke and Silver, 1996; Morgenstern et al., 2002). The glial scar thus forms both a mechanical and molecular barrier to axon growth. Upon injury, microglial cells also rapidly divide, transform into phagocytic cells that remove neuronal and myelin debris, and release inflammatory cytokines, glutamate, and free radicals (reviewed in Kreutzberg, 1996; Raivich et al., 1999). In addition, another type of glial cell is recruited to the site of injury and participates in the formation of the glial scar, the oligodendrocyte precursor cell (OPC), believed to be an immature cell of the oligodendrocyte lineage (McTigue et al., 2001). OPCs are present throughout the CNS and respond to injury by dividing, hypertrophy, and increase in chondroitin sulfate proteoglycan NG2 expression (Levine, 1994; for review see Levine et al., 2001). Furthermore, if the injury disrupts the meninges and blood vessels, meningeal fibroblasts invade the lesion area (Carbonell and Boya, 1988; Ness and David, 1997; Shearer and Fawcett, 2001). They contribute to the inhibitory nature of the glial scar by synthesizing semaphorin class III chemorepellent molecules, tenascin-C, and NG2 (De Winter et al., 2002b; Niclou et al., 2003; reviewed in Pasterkamp and Verhaagen, 2001).

Oligodendrocytes and Myelin (Wallerian Degeneration) Following CNS lesion, the entire distal segment of the nerve undergoes Wallerian degeneration, with disintegration of the distal axonal segment, degradation of myelin sheaths and the axon cytoskeleton, and the subsequent apoptotic death of oligodendrocytes around the lesion site. This process occurs within the first few days after lesion and is probably the result of protease, such as calpain, activation (Coleman and Perry, 2002). While axonal injuries in the PNS trigger the rapid recruitment of macrophages from the peripheral blood to remove axonal and myelin debris (Shamash et al., 2002), the number of circulating macrophages that invade the CNS lesion site is considerably smaller (Stoll and Jander, 1999). Instead, resident CNS microglia are activated, migrate into the lesion site, and progressively 12.3.1.2


acquire properties of fully competent macrophages to finally phagocytose axon and myelin debris. CNS injury therefore results in the long-term persistence of myelin debris at the lesion site (George and Griffin, 1994). Cell culture experiments have shown that both CNS myelin and myelin-producing oligodendrocytes can inhibit axonal growth in vitro, indicating that myelin debris may participate in the failure of nerve fiber re-growth after lesion (Caroni and Schwab, 1988; Bandtlow et al., 1990). Consistent with this view are the observations that CNS injuries in very young mammals, i. e. before the onset of fiber myelination, allow axonal regeneration and often lead to full functional restoration. Thus, the onset of myelination and the presence of myelin-associated inhibitory proteins are considered to be involved in the developmental switch that marks the end of the growth permissive period.

Secondary Injury: Inflammation, Cell-mediated Immunity, and Glial Cell Loss Traumatic spinal cord injury leads to hemorrhage, glial activation, ischemia, and edema, and results in inflammation, cyst formation, and secondary loss of neurons and glia (Liu et al., 1997; Fitch et al., 1999; Grossman et al., 2001; for review see Beattie et al., 2002 (Beattie et al., 2002). These secondary events may be mediated by microglia/macrophage activation and the subsequent recruitment of cells of the immune system, leading to the production of free radicals and the release of pro-inflammatory cytokines such as Il-1, Il-6, TNFa, and TGFb (Bartholdi and Schwab, 1997; Rostworowski et al., 1997; Carlson et al., 1998; McTigue et al., 1998; Schwab et al., 2001; reviewed in Allan and Rothwell, 2001; Raivich et al., 2003). Inflammatory mediators do not appear to be sufficient to cause oligodendrocyte loss in white matter tracts that are spared after contusion lesion of the spinal cord, although many macrophages and neutrophils are present (Frei et al., 2000). However, oligodendrocyte death has been observed primarily associated with degenerating fiber tracts, i. e. where axons have also been damaged (Crowe et al., 1997; Liu et al., 1997). This death is apoptotic and depends on the activation of p75 and on the expression of Bax (Casha et al., 2001; Dong et al., 2003). Astrocytes may also be severely damaged and lost following CNS injury prior to the development of reactive gliosis (Grossman et al., 2001). The most common early response to injury is astrocyte swelling (Kimelberg, 1992). Astrocytic edema develops as early as 3h following trauma, and persists for as long as 3 days (Bullock et al., 1991). Cellular brain edema results from an entry of water into the intracellular compartment due to a dysregulation of ion homeostasis, leading to impaired neurotransmitter uptake and release of excitotoxic amino acids such as glutamate by reactive astrocytes (Kimelberg, 1992). This contributes to neuronal and oligodendroglial death as well as to a transient loss of astrocytes (Grossman et al., 2001). However, inflammation, if properly controlled, may also have beneficial effects on secondary neuronal and glial cell loss, axonal regrowth, and remyelination, thus promoting CNS repair (Arnett et al., 2001; for more detail, see recent reviews by WyssCoray and Mucke, 2002; Schwartz et al., 2003). 12.3.1.3

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Several inflammatory cytokines have been suggested to be involved in mediating glial scar formation and thus impede regeneration. For instance, interleukin-1, TGFa, TGFb, TNFa, and IFN-g all have been shown to be increased in scar tissue after brain lesion, to promote astrocyte proliferation in vitro, and/or to augment gliosis in vivo (Giulian and Lachman, 1985; Yong et al., 1991; Logan et al., 1994; Rostworowski et al., 1997; Rabchevsky et al., 1998; Lagord et al., 2002). Moreover, a poor or slow appearance of inflammatory mediators in the CNS compared to the PNS may also contribute to the inefficient removal of myelin debris and thus add to the failure of regeneration in the brain (Shamash et al., 2002). Interestingly, the expression of tenascin and of neurocan, a CNS-specific chondroitin sulfate proteoglycan with growth-inhibitory properties, is increased in highly purified rat astrocytes treated with TGFb (Smith and Hale, 1997; Asher et al., 2000). However, the potential regulation of these and other myelin-associated growth-inhibitors by inflammatory mediators remains to be evaluated further.

Genetic Influences on Cellular Reactions to CNS Injury Among other mammals examined to date, mice exhibit a unique pathological response to spinal cord injury. This is probably due to a species-specific neuroinflammatory response that contributes to the formation of distinct tissue environments at the lesion site (Sroga et al., 2003). For instance, in contrast to rats and humans in which spinal cord lesion leads to tissue loss and the formation of large cystic cavities (Fitch et al., 1999; Sroga et al., 2003), the lesion site in mice is filled with dense connective tissue matrix made up of macrophages and fibroblasts, leading to revascularization of the tissue over time (Zhang et al., 1996). In addition, the genetic background also appears to be important. For example, the lesion area and cavitation following spinal cord injury are larger in FVB/N strains that are sensitive to kainate toxicity than in the kainate-resistant strains C57Bl/6 and Balb/c (Inman et al., 2002; reviewed in Steward et al., 1999). Interestingly, C57Bl/6 mice are deficient in peripheral axonal regeneration after sciatic nerve lesion, compared to Balb/c and other strains of mice (Xin et al., 1990). 12.3.1.4

12.3.2

Molecular Components of Inhibition Axon growth Inhibitors in the Glial Scar Astrocyte-associated inhibitory molecules are possible candidates responsible for growth-inhibition in the glial scar. This is supported by the demonstration that astrocytes, depending on culture condition, source, and age, have growth-inhibiting properties in vitro (Fawcett et al., 1989; Bahr et al., 1995). Similarly, various astrocytic cell lines have been obtained that are either permissive or inhibitory to axonal growth in vitro (Fok-Seang et al., 1995). ECM components of reactive astrocytes in the glial scar include laminin, fibronectin, matrix metalloproteases and their inhibitors, chondroitin sulfate proteoglycans (CSPGs) and tenascins (Snow et al., 1990; Laywell et al., 1992; McKeon et al., 1995; Muir et al., 2002). Oligoden12.3.2.1


drocyte precursor cells, oligodendrocytes, and meningeal cells that also make up the glial scar synthesize inhibitory molecules as well, among which CSPGs, the chemorepulsive axon guidance molecules semaphorins, and collagen (Stichel et al., 1999; Liesi and Kauppila, 2002; Niclou et al., 2003; Moreau-Fauvarque et al., 2003; for reviews see Stichel and Muller, 1998a; Levine et al., 2001; Pasterkamp and Verhaagen, 2001; Morgenstern et al., 2002). Many of these molecules have been shown to restrain neurite outgrowth of a variety of neurons in vitro and are therefore strong candidates as inhibitors of regenerative responses in the CNS in vivo. CSPGs

Chondroitin sulfate proteoglycans (CSPGs) appear to be the main class of inhibitory molecules produced by activated astrocytes. Indeed, CSPGs are up-regulated in the glial scar upon injury (Snow et al., 1990), and axon growth on astrocytic scars is inhibited in the presence of injury-induced proteoglycans (McKeon et al., 1995). In addition, axon growth can be increased on inhibitory astrocytic cell lines treated with chondroitinase ABC (Smith-Thomas et al., 1994) or grown in the presence of proteoglycan synthesis inhibitors (Smith-Thomas et al., 1995). Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix glycoproteins carrying varying degrees of covalently bound sulfated glycosaminoglycan (GAG) chains (for further details see reviews by Bandtlow and Zimmermann, 2000; Hartmann and Maurer, 2001). The CSPGs brevican, neurocan, and versican are a subgroup of proteoglycans called lecticans/hyalectans which share similar globular N-terminal hyaluronan-binding domains and C-terminal selectin-like domains, with a central region which is not conserved among lectican family members (reviewed in Margolis and Margolis, 1994; Yamaguchi, 2000). While the N-terminal domain of hyalectans interacts with hyaluronan, the C-terminal globular domains interact with tenascin C and tenascin R (Aspberg et al., 1995; Hagihara et al., 1999), surface molecules such as L1 and N-CAM (Grumet et al., 1994), integrins (Wu et al., 2002), and the ECM proteins fibulins (Olin et al., 2001); for review see Timpl et al., 2003). The brain-specific link proteins BralI and BralII probably serve to stabilize hyaluronan/versican and hyaluronan/brevican aggregates, respectively, by binding to both molecules and forming hyaluronan/Bral/hyalectan complexes (Oohashi et al., 2002; Bekku et al., 2003). Brevican is the most abundant lectican in the adult CNS (Yamaguchi, 1996). It is present in different isoforms, a secreted form primarily produced by neurons and deposited in perineuronal nets where it interacts with hyaluronan and tenascin-R (Hagihara et al., 1999), and a GPI-linked form mainly produced by oligodendrocytes (Seidenbecher et al., 1995; Seidenbecher et al., 1998; Ogawa et al., 2001). Brevican may also be expressed by adult astrocytes (Yamada et al., 1997; Ogawa et al., 2001) and has been isolated from brain myelin (Niederst et al., 1999). Neurocan is primarily expressed by CNS neurons (Rauch et al., 1992; for a detailed review see Rauch et al., 2001). It undergoes proteolytic processing and is deposited in perineuronal nets where it binds to tenascin C and tenascin R (Grumet et al., 1994; Milev et al., 1998). Versican exists in 4 isoforms resulting from differential splicing, V0, V1, V2, and V3 (reviewed in Bandtlow and Zimmermann, 2000; Wight, 2002).

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Versican V2 is exclusively present in the CNS where it appears to be expressed both by oligodendrocyte precursor cells and mature oligodendrocytes (Schmalfeldt et al., 1998; Niederst et al., 1999; Asher et al., 2002). Versican V2 has also been isolated from bovine CNS myelin (Schmalfeldt et al., 1998; Niederst et al., 1999). Versican V2 appears to be co-localized with the brain-specific link protein BralI to the CNS white matter, especially at the nodes of Ranvier (Oohashi et al., 2002). While Brevican, Neurocan, and Versican V2 are all CNS-specific, Versican V1 has a wider tissue distribution, being expressed along neural crest pathways and in the adult aorta (Yao et al., 1994; Landolt et al., 1995). The NG2 proteoglycan is a unique transmembrane CSPG that can also undergo proteolytic cleavage to produce a secreted form (Nishiyama et al., 1995). NG2 is expressed mainly by oligodendrocyte precursor cells during brain development as well as throughout the adult brain (Levine and Nishiyama, 1996; Dawson et al., 2000). Its possible expression on microglia and subpopulations of activated astrocytes remains to be confirmed. Like versican V1, NG2 is not CNS-specific, since it has recently been described to be present on Schwann cells and/or perineurial fibroblasts in peripheral nerve and to be increased at the site of sciatic nerve lesion (Levine and Nishima, 1996; Jones et al., 2003; Morgenstern et al., 2003). Brevican, neurocan, versican V2, and NG2 have all been described to be potent growth inhibitors in vitro (Friedlander et al., 1994; Dou and Levine, 1994; Yamada et al., 1997; Niederst et al., 1999; Schmalfeldt et al., 2000; Ughrin et al., 2003). Furthermore, neurocan, brevican, and NG2 are up-regulated in the glial scar after various types of CNS lesions (Levine, 1994; Haas et al., 1999; Jaworski et al., 1999; McKeon et al., 1999; Thon et al., 2000; Jones et al., 2002; Jones et al., 2003; Tang et al., 2003). Whether Versican V2 expression is modulated upon CNS injury is still controversial since a knife lesion in the cerebral cortex appears to increase versican V2 expression (Asher et al., 2002), whereas unilateral transection of the spinal cord leads to a decrease in versican V2 production (Tang et al., 2003). CSPGs may also be important growth-inhibitors for sensory neurons, since grafts of adult sensory neurons can regenerate axons in degenerating white matter tracts of the spinal cord, but not through a glial scar containing CSPGs (Davies et al., 1999). Evidence that CSPGs are key players in preventing CNS regeneration has also recently been brought by the demonstration that extensive regeneration occurs after nigrostriatal axotomy or spinal cord lesion in rats treated with chondroitinase ABC, the enzyme which cleaves the GAG side chains of CSPGs (Moon et al., 2001; Bradbury et al., 2002). CSPGs may therefore, together with other inhibitory molecules of CNS myelin, contribute to growth inhibition in the brain and spinal cord. Tenascins

Tenascins are a family of large extracellular matrix glycoproteins including tenascin-C and tenascin-R that are expressed during development, down-regulated upon maturation, and re-expressed after injury (for more details see Pesheva and Probstmeier, 2000; Joester and Faissner, 2001; Chiquet-Ehrismann and Chiquet, 2003). Tenascin-C is present both in the PNS and CNS, while tenascin-R is


predominantly expressed in the CNS. Both tenascin-C and R, and the recently described new family member, tenascin-N, are repulsive for neurite growth in vitro (Dorries et al., 1996; Xiao et al., 1996; Neidhardt et al., 2003). Enhanced expression of tenascin C by activated astrocytes after CNS lesion may contribute to failed regeneration through the glial scar (Laywell et al., 1992; Ajemian et al., 1994). Tenascin-R is expressed by oligodendrocytes and by subpopulations of neurons where it is deposited in perineuronal nets (Bartsch et al., 1993; Hagihara et al., 1999). Upon injury, it accumulates around the lesion site but is down-regulated in motoneurons of the spinal cord (Probstmeier et al., 2000; Lindholm et al., 2001). Semaphorins

Semaphorins are one of the largest families of axon guidance molecules (for recent reviews see Raper, 2000; Yu and Bargmann, 2001; Pasterkamp and Kolodkin, 2003). Sema3A, a chemorepellent during nervous system development, is up-regulated in meningeal fibroblasts in the glial scar and is therefore likely to contribute to the inhibitory properties of this tissue (Pasterkamp et al., 1999; De Winter et al., 2002b; reviewed in Pasterkamp and Verhaagen, 2001; De Winter et al., 2002a). Sema3A, but not Sema3C, has recently been demonstrated to inhibit neurite outgrowth of embryonic DRGs in vitro (Niclou et al., 2003). While class 3 semaphorins are secreted molecules, class 4 semaphorins are membrane-bound. Sema4D has recently been shown to induce the collapse of growth cones of CNS axons and to be up-regulated upon CNS injury (Swiercz et al., 2002; Moreau-Fauvarque et al., 2003). However, in contrast to Sem3A, it is expressed by oligodendrocytes and myelin (Moreau-Fauvarque et al., 2003). Sema4D is therefore a newly identified myelinassociated growth inhibitor.

Myelin-associated Growth Inhibitors Growth inhibitors present in myelin seem to be involved particularly during the early phase that follows CNS injury, before the glial scar forms (Caroni and Schwab, 1988). Several myelin-associated proteins have been identified that are thought to contribute to a hostile environment for regenerating nerve fibers. These proteins include Nogo/RTN-4, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), tenascin-R, and chondroitin-sulfate proteoglycans (CSPGs). The relative contribution of these proteins to the inhibitory activity of myelin has been a subject of intense study. The main in vivo evidence for an inhibitory effect of myelin on CNS regeneration comes from experiments using either antibodies that neutralize myelin-inhibitory molecules (Huang et al., 1999; Sicotte et al., 2003; Schnell et al., 1990; Bregman et al., 1995), or a peptide that specifically acts as a NgR antagonist (GrandPre et al., 2002). Both approaches have improved the regeneration of corticospinal tract axons and have shown some functional recovery after dorsal hemisection of the adult spinal cord (for recent reviews see Hunt et al., 2002; Filbin, 2003; Grados-Munro and Fournier, 2003). 12.3.2.2

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12 Axonal Growth Inhibition MAG

MAG belongs to the family of immunoglobulin-type lectins (I-type lectins) called siglecs, a sub-family of the immunoglobulin superfamily of cell adhesion molecules (Salzer et al., 1987). While all other siglecs play important roles in the immune system, MAG/siglec-4 is expressed by oligodendrocytes and Schwann cells of the central and peripheral nervous system, respectively. In the CNS, MAG is found exclusively in the periaxonal myelin membrane, while in the PNS, it is also found in the outermost membrane of the myelin sheath (Willison et al., 1987). MAG was shown in a variety of in vitro studies to act as a bifunctional protein that either promotes or inhibits neurite outgrowth, depending on the age and type of neuron. For example, neurite outgrowth in response to MAG is enhanced in early postnatal dorsal root ganglion (DRG) (Johnson, et al., 1989) and embryonic spinal cord neurons (Turnley and Bartlett, 1998). In contrast, growth is inhibited in older DRG neurons and in neurons such as cerebellar granule cells, retinal, spinal and hippocampal neurons, i. e. cells that are myelinated in vivo (Mukhopadhyay et al., 1994). Changes in endogenous cAMP levels are thought to account for the developmental switch in the neuronal response to MAG in vitro (Qiu et al., 2002; Neumann et al., 2002). To what extent MAG contributes to growth failure in vivo is less clear. The presence of MAG in the PNS, where regeneration can occur, appears to question the function of MAG as an important inhibitor in vivo. Several studies, however, have demonstrated that peripheral nerve myelin is cleared rapidly and efficiently by macrophages after injury to the PNS (reviewed in Hirata and Kawabuchi, 2002). PNS regeneration is restored in Wlds mice that have a slow myelin clearance and regenerate poorly, when they are crossed with MAG-deficient mice (Schfer et al., 1996). These studies support a role for MAG as an in vivo inhibitor. However, experiments in MAG-null mice suggest that its inhibitory contribution in the CNS may be minor, as the extent of axonal regrowth in lesioned optic nerve and corticospinal tract is not, or only moderately enhanced in MAG-deficient mice (Bartsch et al., 1995; Li et al., 1996). Nogo

Perhaps the most widely studied myelin-associated inhibitor is Nogo. Nogo was independently cloned in different laboratories (Chen et al., 2000; GrandPr et al., 2000; Prinjha et al., 2000) following the publication of a partial protein sequence of a high molecular weight inhibitory myelin protein, bovine NI-220 (Spillmann et al., 1998). Three distinct isoforms of Nogo, Nogo A, B, and C, are generated by alternative splicing (Nogo-A/B) and promoter usage (Nogo-C). All three Nogo isoforms share a common C-terminal domain of 188 amino acids that is homologous to the reticulon homology domain of reticulon family proteins (RTNs). Reticulon proteins were originally identified as proteins anchored to the endoplasmic reticulum (ER; hence the name reticulons) via a C-terminal ER retention signal. With the exception of Nogo/RTN 4, little is known about the possible function of the other three known mammalian paralogues (RTN1, -2, -3) (for more details see Oertle and Schwab, 2003). Nogo-A contains at least two domains with distinct specificities that inhibit neurite outgrowth and cause growth cone collapse in vitro:


the amino terminal portion of Nogo-A (termed amino-Nogo) and a sequence of 66-amino acids spanning the two putative transmembrane domains of Nogo (Nogo-66) (Chen et al., 2000; GrandPr et al., 2000; Fournier et al., 2001; Oertle et al., 2003). In the adult brain, Nogo-A is mainly expressed by white matter oligodendrocytes and by a subpopulation of large projecting neurons, but not or little in Schwann cells in the PNS (Josephson et al., 2001; Huber et al., 2002; Wang et al., 2002b). Consistent with the characteristics of a reticulon family protein, Nogo-A mainly associates with the endoplasmic reticulum, with a small fraction also present on the surface of differentiated oligodendrocytes and on the innermost axonal and outermost myelin membranes (Oertle et al., 2003; Huber et al., 2002). Several lines of evidence demonstrate the relevance of Nogo to axon regeneration in vivo. Antibodies directed against Nogo-A can promote axon growth and plasticity in the adult brain (for a detailed review see Schwab, 2002). In addition, a Nogo66 peptide antagonist increases axonal sprouting and functional recovery after spinal cord injury (GrandPre et al., 2002; Li and Strittmatter, 2003). Furthermore, overexpression of Nogo-A in PNS Schwann cells delays regeneration following sciatic nerve lesion (Pot et al., 2002). Similar results were obtained with transgenic animals overexpressing Nogo-C, the smallest Nogo isoform, supporting a general inhibitory role for Nogo (Kim et al., 2003a). Analysis of Nogo knockout mice, however, has complicated the picture. Three different laboratories independently generated Nogo deletion mutants using different strategies. In the laboratory of Strittmatter, insertion of a gene trap vector created a Nogo A/B mutant line that disrupted Nogo-A and -B expression, but Nogo-C expression remained unaffected (Kim et al. 2003b). These mice show substantial corticospinal sprouting proximal to a spinal cord dorsal hemisection, and regenerating corticospinal axons below the lesion. The regenerative effects seem to be restricted to young mice. A second Nogo-A mutant line was generated in the laboratory of Schwab using conventional gene targeting disrupting the large Nogo-A-specific exon 3 (Simonen et al., 2003). Although these mice lack Nogo-A, a compensatory increase in Nogo-B was detected in this strain. Compared to the Strittmatter line, the regenerative effects in these mice are significant, but more modest. The upregulation of Nogo-B, which contains the Nogo-66 inhibitory domain, may explain the more modest recovery. Finally, two Nogo knockout strains were produced by Tessier-Lavigne and collaborators: one disrupted in Nogo-A/B without affecting Nogo C, and the other one disrupted in all three Nogo isoforms by deletion of their common C-terminal region (Zheng et al., 2003). Surprisingly, neither of these strains showed improved axonal regeneration after dorsal spinal cord hemisection. To date, there is no clear explanation for the disparate findings from the various strains of Nogo knockout mice. One may speculate that differences in genetic background or compensatory up-or down-regulation of unidentified inhibitory influences may be responsible. Clearly, more in-depth analysis is required to resolve these discrepancies.

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12 Axonal Growth Inhibition Oligodendrocyte-Myelin glycoprotein (OMgp)

The most recently identified myelin-associated protein that inhibits nerve fiber growth in vitro is the oligodendrocyte-myelin glycoprotein (OMgp) (Wang et al., 2002; Kottis et al., 2002). This GPI-linked protein has been previously described as a relatively minor component of CNS myelin, being expressed by myelinating oligodendrocytes and localized to the glial-axonal interface of myelinated axons (Mikol and Stefansson 1988; Mikol et al. 1990; 1993). The OMgp gene is placed within an intron of the neurofibromatosis type I gene on the opposite strand (Habib et al., 1998), but until recently, its precise function has remained unknown. Mouse and human OMgp are structurally very similar. The protein has a series of tandem leucine-rich repeats, like those in a variety of adhesion molecules and receptors, including the Nogo receptor NgR. Like Nogo, OMgp in the mouse brain is not only expressed by white matter oligodendrocytes, but also in diverse groups of neurons (Wang et al., 2002) such as the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, motoneurons in the brainstem and spinal cord. Whether and to what extent neuronally expressed OMgp contributes to axon growth inhibition remains to be determined. 12.3.3

Transduction of Axon Growth-inhibitory Signals Receptors for Myelin-associated Inhibitors A key step in understanding how axons respond to inhibitory influences is the identification of axonal receptors that bind myelin-associated inhibitory molecules. So far, NgR is the only known high affinity neuronal receptor originally identified as a binding molecule for Nogo-66 (Fournier et al., 2001). NgR is a GPI-linked neuronal protein that consists of eight consecutive leucine-rich-repeat (LRR) domains followed by a carboxy-terminal LRR. NgR is not only necessary to render neurons sensitive to Nogo-66, but its axonal expression pattern is consistent with NgR acting as a functional Nogo receptor (Wang et al., 2002). Unexpectedly, recent studies have identified NgR as a component required also in the signaling of the inhibitory effects of MAG and OMgp (Liu et al., 2002; Domeniconi et al., 2002; Wang et al., 2002). Thus, the same molecule acts as a high affinity binding receptor for three different myelin-associated ligands. This finding is somewhat surprising since MAG, Nogo-66, and OMgp are structurally unrelated proteins. Based on the crystal structure of the NgR ligand-binding domain, it was proposed that the highly symmetrical arrangement of conserved aromatic residues on the concave face of the curved ectodomain could provide generic binding sites for multiple ligands (Barton et al., 2003; He et al., 2003). Future studies employing site-directed mutagenesis of NgR will be necessary to gain further insight into the promiscuous nature of the NgR binding protein. Although NgR is essential for the binding of the myelin-associated inhibitors, the lack of a cytoplasmic domain in the NgR molecule suggested the participation of a transmembrane co-receptor involved in the transduction of inhibitory signals. The recent discovery that the low affinity neurotrophin receptor p75NTR transduces 12.3.3.1


the inhibitory signal of MAG, together with the finding that MAG is a functional ligand of NgR, raised the possibility that p75NTR is the NgR co-receptor (Yamashita et al., 2002). Subsequently, p75NTR was recognized to interact with NgR in a liganddependent fashion and to be required for mediating the inhibitory effects of MAG, Nogo-66 and OMGp, but not of amino-Nogo or CSPGs (Wang et al., 2002a; Wong et al., 2002; Yamashita et al., 2002; Schweigreiter and Bandtlow, unpublished results). The knowledge about the molecular interaction of the three main inhibitors of myelin with the same NgR/p75NTR receptor complex implies that there is functional redundancy between these inhibitors. This redundancy is likely to be even greater if the three inhibitors have the same or overlapping binding sites on NgR. This may explain previous findings in MAG or Nogo deficient mice, where no or only a small amount of spontaneous long distance axonal regeneration was observed. Possibly the presence of other, unblocked ligands for NgR/p75NTR, or of other unidentified receptors, can still prevent most axons from regenerating. It is unknown whether other functional receptors for Nogo, MAG, and OMgp exist. In fact, NgR/p75NTR does not interact or mediate the in vitro effects of amino-Nogo-A, implying the existence of another, yet unidentified receptor or receptor complex for Nogo (Oertle et al., 2003). This receptor seems to be more widely expressed, since amino-Nogo-A, in contrast to Nogo-66, can also inhibit spreading of non-neuronal cells (Oertle et al., 2003). Whether this effect has any physiological relevance has yet to be determined. In addition, a direct, NgR-independent interaction of Nogo-66 with Caspr was recently described at axonal paranodes (Nie et al, 2003). MAG has also been described to bind to neurons via a sialic acid linkage involving the gangliosides GT1b and GD1a, a subclass of glycosphingolipids containing one or more sialic acid residues. These gangliosides have therefore been proposed to act as functional MAG receptors involved in inhibition (Vinson et al., 2001; Vyas et al., 2002). Further evidence that gangliosides may be involved in mediating growth inhibition is supported by the finding that clustering of ganglioside GT1b with antibodies in the absence of ligand is sufficient to inhibit neurite outgrowth (Vinson et al., 2001; Vyas et al., 2002). However, sialic-aciddependent binding of MAG to neurons is neither necessary nor sufficient for MAG-mediated inhibition of axonal regeneration, since binding of MAG to NgRexpressing cells is unaffected by removal of sialic acid residues, and since the interaction between MAG and NgR does not require sialic acid (Domeniconi et al., 2002). Nevertheless, MAG binding to gangliosides may potentiate its inhibitory effects by clustering signaling molecules at the intracellular membrane face.

12.3.3.2

Signaling Pathways of Myelin-associated Inhibitors

Rho-GTPases and Rho kinase

Consistent with the redundancy of myelin-associated growth inhibitors is the finding that the effects of all three inhibitors can be overcome by altering certain intracellular signaling molecules. One such molecule is the small GTPase, Rho. Rho family GTPases transduce extracellular signals to the actin cytoskeleton to modu-

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late growth cone motility. The most intensely studied members are Rho, Rac, and Cdc42. Rho GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. This transition is positively or negatively controlled by several GTPase activating proteins (GAPs), by guanine nucleotide exchange factors (GEFs), and by guanine nucleotide dissociation inhibitor (GDI). These modulators maintain Rho GTPases in their inactive GDP-bound state (Etienne-Manneville and Hall, 2002). In general, activation of Cdc42 and Rac is associated with growth cone attraction via promotion of F-actin polymerization in filopodia and lamellipodia. In contrast, activation of RhoA causes growth cone repulsion and collapse, possibly by enhancing retrograde F-actin flow (Ng et al., 2002). Involvement of RhoA in myelin-dependent outgrowth inhibition and growth cone collapse is supported by various findings. For instance, inactivation of RhoA by the clostridial toxin C3 transferase protects CNS neurons from myelin-induced growth cone collapse and outgrowth inhibition in vitro (Lehmann et al., 1999; Niederst et al., 2002). A problem with C3 transferase however, is that its membrane permeability is poor. C3 could penetrate injured axons soon after spinal cord injury, but this may be limited to a short period after the trauma. Application of C3 in vivo has been described to promote regeneration after optic nerve lesion or spinal cord injury (Lehmann et al., 1999; Dergham et al., 2002). However, the marked improvement in function was seen already two days post-injury, i. e. too early to be accounted for by axonal regeneration. Whether these early effects are the consequence of an early effect of C3 on neuronal survival remains to be confirmed. Biochemical studies have shown that amino-Nogo-A, Nogo-66, MAG, Versican V2 and presumably other CSPGs directly activate RhoA (Niederst et al., 2002; Yamashita et al., 1999; Monnier et al., 2003; unpublished results) and concomitantly inactivate Rac1 (Niederst et al., 2002). Although it is reasonable to assume that a specific GEF is recruited by activated RhoA during neurite inhibition, the identity of such a guanine nucleotide exchange factor remains speculative. However, the guanine nucleotide dissociation inhibitor Rho-GDI seems to directly interact with p75NTR, and is enhanced by binding of myelin-associated inhibitors to NgR (Yamashita and Tohyama, 2003). This suggests a model whereby Rho-GDI is sequestered by p75NTR, leading to Rho activation. This model is consistent with previous results, demonstrating that p75NTR activation stimulates Rho activity (Yamashita et al., 1999). RhoA acts on several downstream effectors to regulate the underlying cytoskeleton (Bishop and Hall, 2000). One of these effectors, termed Rho kinase, or ROCK, has been implicated in neurite outgrowth inhibition by CNS myelin components (Niederst et al., 2002). Treatment with Y-27632, an ATP competitive antagonist that blocks ROCK activation, promotes neuronal outgrowth on myelin-associated inhibitory substrates in vitro as well as in in vivo models of spinal cord injury (Dergham et al., 2002; Niederst et al., 2002; Borisoff et al., 2003; Fournier et al., 2003). Moreover, glial-scar derived inhibitors such as CSPGs, can also be blocked by inhibitors of ROCK (Borisoff et al., 2003). How ROCK induces neurite retraction is a complex issue because it has been shown to phosphorylate and signal through multiple downstream effectors (for review see Riento and Ridley, 2003).

12.4 Experimental Strategies to Overcome Growth Inhibition

In summary, regardless of its mechanism of activation, RhoA seems to be the point of convergence for all presently identified myelin and glial scar derived inhibitors and might therefore be an attractive molecular target for small molecules that interfere with its activation. One caveat, however, is that small GTPases and their effector proteins are universally expressed and involved in regulating many distinct cellular functions, such as cell migration, proliferation, and adhesion (reviewed in Etienne-Manneville and Hall, 2002). Thus, therapeutically manipulating RhoA and/or ROCK to improve axonal regeneration may negatively influence other physiological events. cAMP/PKA

Neuronal cAMP levels decrease spontaneously and sharply with development at a time that correlates with their loss of regenerative ability and the appearance of an inhibitory response to MAG and myelin (Cai et al., 2001). Furthermore, elevation of intracellular cAMP levels by intraganglionic administration of cAMP overcomes inhibition by MAG and myelin and results in extensive regeneration of injured dorsal column axons (Qiu et al., 2002; Neumann et al., 2002). This process initially involves phosphorylation of PKA, but eventually becomes PKA-independent (Qiu et al., 2002). Elevated cAMP levels up-regulate arginase I, a key enzyme involved in polyamine synthesis. Subsequent studies showed that both overexpression of arginase I, and exogenous addition of polyamines, can overcome MAG and myelin inhibition (Qiu et al., 2002.). Conversely, blocking either ArgI or subsequent enzymes in polyamine synthesis can abrogate the ability of increased cAMP levels to overcome inhibition. How polyamines exert their effects on inhibition remains to be investigated (reviewed in Filbin, 2003).

12.4

Experimental Strategies to Overcome Growth Inhibition

Several experimental strategies have been developed to overcome inhibition after CNS injury. They involve the use of antibodies to neutralize growth inhibitors, Nogo peptides binding to NGR and blocking its activity, and blockers of the intracellular pathways mediating growth inhibition (for recent reviews see Ellezam et al., 2002; Hunt et al., 2002; Filbin, 2003). In addition, as an alternative to blocking inhibitors, receptors, or their signaling pathways, the growth of dorsal column ascending sensory axons has been improved by intraganglionic administration of cAMP (Neumann et al., 2002; Qiu et al., 2002). To decrease its inhibitory properties, the biochemical composition of the ECM has also been altered by treatment with chondroitinase ABC, the enzyme which cleaves the GAG side chains of CSPGs (for review see Morgenstern et al., 2002). Furthermore, attempts to reduce glial scarring have been made using pharmacological agents to decrease collagen deposition in the ECM (reviewed in Stichel and Muller, 1998b). Matrix and ADAMs metalloproteinases, enzymes that can degrade ECM proteins and CSPGs, are also potential candidates for remodeling the glial scar (reviewed in

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Yong et al., 2001). Other strategies to promote CNS repair, including application of neurotrophic factors, implantation of olfactory bulb ensheathing cells, or grafting of peripheral nerve, have recently been reviewed elsewhere (Blesch et al., 2002; Blits et al., 2002; Santos-Benito and Ramon-Cueto, 2003). The discovery that the NgR/p75NTR complex binds and mediates the effects of the three most prominent myelin-derived ligands has made this receptor complex a very attractive target for therapeutic drug development. Therapeutic candidates for targeting NgR include protein antagonists and small-molecule antagonists, but an antagonist of NgR that blocks binding of all three myelin proteins has not yet been found. One likely explanation is the lack of compelling evidence for the existence of a single molecular binding site on NgR for all myelin inhibitors. Recently, inhibition of NgR was achieved utilizing a peptide corresponding to residues 1–40 of Nogo-66 (NEP1–40) that acts as a competitive antagonist of the receptor in vitro and, more importantly, promotes axonal regeneration and functional recovery following CNS injury in vivo (GrandPre et al., 2002; Li and Strittmatter, 2003). A soluble version of a recombinant NgR could also be a possible solution to effectively inhibit the action of all three myelin proteins interacting with NgR. Several independent reports have shown that the soluble full length NgR ectodomain lacking the GPI anchor, and an C-terminally truncated NgR ectodomain comprising the ligand binding domain alone (NgR-310), are potent antagonists of neurite outgrowth inhibition mediated by Nogo-66, MAG and whole myelin in vitro (Fournier et al., 2002; Domeniconi et al., 2002; see Lee et al., 2003). Similarly, preventing the interaction of NgR with its co-receptor p75NTR could be an alternative site for intervention to promote axonal regeneration. The recent finding that the interaction between p75NTR and Rho-GDI requires the fifth alpha helix of p75NTR, has opened additional strategies. A peptide ligand, Pep5, for instance, has been shown to bind this helix and has been used to inhibit the interaction between Rho-GDI and p75NTR (Yamashita and Tohyama, 2003). This peptide blocks both Rho activation and the growth inhibitory effects of MAG and Nogo on cerebellar granule cells. Pep5 therefore has potential as a therapeutic agent to block inhibitory cues that impede regeneration in the CNS. Increased deposition of a number of ECM molecules such as collagen IV and CSPGs during glial scar formation is considered to contribute to the local impediment of axonal growth. Two main in vivo approaches have been taken to reduce the inhibitory properties of the ECM (1) reduction of collagen deposition, and (2) enzymatic digestion of CSPGs. Injection into a fimbria fornix lesion of the iron chelator 2,2-dipyridine (DPY), an inhibitor of collagen synthesis and triple helix formation, significantly reduced lesion-induced collagen deposition. This effect was correlated with a pronounced regeneration of the lesioned fimbria fornix axons (Stichel et al., 1999). However, in a spinal cord lesion model in which basement membrane deposition is more extensive, injections of DPY did not result in sufficient reduction in collagen deposition to allow regrowth of corticospinal tract axons (Weidner et al., 1999; Hermanns et al., 2001). Furthermore, because basal membrane ECM proteins such as collagen IV are structural elements of blood vessels, it is uncertain whether the vascular blood supply is compromised by the

12.5 Conclusion

application of DPY to the injured area. To remove additional inhibitory components of the glial scar, the bacterial enzyme Chondroitinase ABC (ChABC) was used to digest CSPGs. ChABC hydrolyzes glycosaminoglycan side-chains of CSPGs and has been shown to block the inhibitory activity of CSPGs in vitro (Zuo et al., 1998). Application of ChABC via a gelfoam to the contused dorsal thoracic cord resulted in decreased CSPG immunoreactivity, but no axonal re-growth was reported (Lemons et al., 1999). Some growth of spinal cord neurons into a peripheral nerve graft was achieved when gelfoam soaked with ChABC was placed on the interface between the thoracic hemisection and a peripheral nerve graft (Yick et al., 2000). More recently, Bradbury and collaborators have reported that intrathecal administration of ChABC after dorsal column lesion in rats, could successfully promote regeneration of corticospinal tract axons as well as functional recovery of locomotor and proprioceptive function (Bradbury et al., 2002).

12.5

Conclusion

In the last few years, enormous advances have been made to revise the dogma that axons in the adult mammalian CNS cannot regenerate after injury. In various experimental CNS, and in particular spinal cord lesion models, administration of neurotrophic factors, implantation of olfactory ensheathing cells, blockade of myelin-associated growth inhibitors and/or their receptors, or removal of inhibitory extracellular matrix molecules by enzymatic treatment has had very encouraging success, leading to some functional recovery. Several of the experimental interventions that promote axonal regeneration have also enhanced compensatory sprouting of non-lesioned nerve fibers, resulting in innervation of denervated targets (see review Raineteau and Schwab, 2001). This type of plasticity may therefore contribute to functional recovery after injury. Aberrant sprouting of sensory fibers, for instance, may, however, lead to debilitating pain. Hence, a delicate balance between beneficial versus detrimental sprouting/regeneration needs to be achieved for optimal outcomes. Although the final aim for the treatment of human spinal injuries is to promote long fiber regeneration, restore myelination, and to achieve appropriate connectivity, a more limited repair may already improve the quality of life of paraplegic patients. Thus, the many promising results obtained in the last decade offer new strategies for developing clinically effective therapies, either alone or in combination, for many patients.

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12 Axonal Growth Inhibition transducers and activators of transcription (STAT). Eur J Neurosci 12, 1165–1176. Schwartz, M., Moalem, G., Leibowitz-Amit, R., and Cohen, I. R. (1999). Innate and adaptive immune responses can be beneficial for CNS repair. Trends Neurosci 22, 295–299. Schwartz, M., Shaked, I., Fisher, J., Mizrahi, T., and Schori, H. (2003). Protective autoimmunity against the enemy within: fighting glutamate toxicity. Trends Neurosci 26, 297–302. Seidenbecher, C. I., Gundelfinger, E. D., Bockers, T. M., Trotter, J., and Kreutz, M. R. (1998). Transcripts for secreted and GPI-anchored brevican are differentially distributed in rat brain. Eur J Neurosci 10, 1621–1630. Seidenbecher, C. I., Richter, K., Rauch, U., Fassler, R., Garner, C. C., and Gundelfinger, E. D. (1995). Brevican, a chondroitin sulfate proteoglycan of rat brain, occurs as secreted and cell surface glycosylphosphatidylinositolanchored isoforms. J Biol Chem 270, 27206–27212. Selmaj, K. W., Farooq, M., Norton, W. T., Raine, C. S., and Brosnan, C. F. (1990). Proliferation of astrocytes in vitro in response to cytokines. A primary role for tumor necrosis factor. J Immunol 144, 129–135. Shamash, S., Reichert, F., and Rotshenker, S. (2002). The cytokine network of Wallerian degeneration: tumor necrosis factor-alpha, interleukin-1alpha, and interleukin-1beta. J Neurosci 22, 3052–3060. Shearer, M. C. and Fawcett, J. W. (2001). The astrocyte/meningeal cell interface–a barrier to successful nerve regeneration? Cell Tissue Res 305, 267–273. Sicotte, M., Tsatas, O., Jeong, S. Y., Cai, C. Q., He, Z., and David, S. (2003). Immunization with myelin or recombinant Nogo-66/MAG in alum promotes axon regeneration and sprouting after corticospinal tract lesions in the spinal cord. Mol Cell Neurosci 23, 251–263. Simonen, M., Pedersen, V., Weinmann, O., Schnell, L., Buss, A., Ledermann, B., Christ, F., Sansig, G., van der Putten, H., and Schwab, M. E. (2003). Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron 38, 201–211. Smith, G. M. and Hale, J. H. (1997). Macrophage/Microglia regulation of astrocytic tenascin: synergistic action of transforming

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13 Neurogenesis Sebastian Jessberger and Gerd Kempermann

13.1

Introduction

Many neurological diseases result in the death of neurons in the adult brain, leading to severe and often terminal failure of brain function. A variety of strategies has been devised to reduce the incidence of neurodegenerative disorders by neuroprotective measures and to slow disease progression, but to date none of these approaches has lead to satisfying results. Given this, the discovery that new nerve cells are born in various brain regions throughout adulthood opened a novel approach to restore brain function in neurological disorders. During development, the central nervous system is built from neural stem cells that differentiate into neurons, oligodendrocytes and astrocytes. The long held belief that cells with the capacity to divide and generate new neurons only exist during development was overcome by the demonstration that cells with such properties persist throughout adulthood in the mammalian brain (Reynolds and Weiss, 1992; Ray et al., 1993; Kirschenbaum and Goldman, 1995; Palmer et al., 1995; Palmer et al., 1999; Kondo and Raff, 2000; Tropepe et al., 2000). Already in the 1960s data were obtained which suggested the continuous generation of neurons in the subventricular zone (SVZ) and the hippocampal dentate gyrus in adult animals (Altman and Das, 1965b; Hinds, 1968). About 10 years later, Kaplan and Hinds confirmed these data using electron microscopy (Kaplan and Hinds, 1977). Nevertheless, it took another 20 years and the introduction of the thymidine-analogue bromo-deoxy-uridine (BrdU) to reliably label dividing cells in greater number until these findings were accepted. Are the dividing cells “stem cells”? During the last fifteen years there have been discussions about the use of stem cells in medicine, often misusing the biological nomenclature. In general, stem cells have the capacity of theoretically unlimited self-renewal, and they are able to generate cells different from themselves through asymmetric cell division. Stem cells can be divided in several levels of potency: (1) the “totipotent” zygote, capable of generating a complete organism when implanted into the uterus of a living animal (but this is the only “stem cell” without self-renewing), (2) the Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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“pluripotent” embryonic stem cell, which can give rise to all body tissues, except the trophoblasts of the placenta and perhaps the gonads, (3) “multipotent” stem cells such as the “neural stem cells” (NSCs) that can be found in the developing and also adult brain. “Multipotency” means that these cells can self-renew but give rise to only tissue-specific cell types, which in the case of “neural stem cells” are neuronal and glial cells. It has been suggested that some neural stem cells may be more than multipotent because it has been claimed that they could also give rise to cell types from other tissues, which would make them essentially “pluripotent” (Bjornson et al., 1999; Johansson et al., 1999; Clarke et al., 2000; Rietze et al., 2001). These studies have challenged the assumption of a lineage-restriction of organ-specific progenitors and proposed a model of transdifferentiation where progenitors overcome lineage restrictions when exposed to a novel cellular environment (Anderson et al., 2001; Morrison, 2001). To date, it remains unclear if this “pluripotency” occurs under normal conditions or is merely due to specialized in vitro situations. In addition, cell fusion is a potential alternate explanation of these results (Terada et al., 2002; Wagers et al., 2002). Furthermore, there is only little evidence of functional transdifferentiation because the majority of the cited studies concerning the “pluripotency” of isolated stem cells were based on the morphology of newly generated cells and the expression of marker proteins. Nevertheless, cells with stem cell properties have been isolated from almost all brain areas in rodents (Richards et al., 1992; Cameron et al., 1993; Morshead et al., 1994; Palmer et al., 1995; Kuhn et al., 1996) and also humans (Kirschenbaum et al., 1994; Eriksson et al., 1998; Pincus et al., 1998; Kukekov et al., 1999; Arsenijevic et al., 2001). However, there are only two brain areas, in which new neurons are added spontaneously during adulthood: the subgranular zone (SGZ) of the hippocampal dentate gyrus (Altman and Das, 1965a; Cameron et al., 1993; Kuhn et al., 1996; Cameron and McKay, 1999; van Praag et al., 2002) and the subventricular zone (SVZ) which gives rise to neurons that migrate into the olfactory bulb (Luskin, 1993; Doetsch et al., 1997; Pham et al., 1997; Carlen et al., 2002; Carleton et al., 2003). What makes these two regions special in their ability to be “neurogenic” versus other “non-neurogenic” brain areas is not fully understood. However, the local environment seems to dictate the fate choice decision of progenitors (Gage et al., 1995; Takahashi et al., 1998; Shihabuddin et al., 2000). In the hippocampus astrocytes provide essential clues for local progenitors to generate new neurons (Song et al., 2002a). This effect seems to be mediated by the direct contact of dividing cells with astrocytes but also by soluble factors that are secreted by surrounding glial cells (Song et al., 2002a). The discovery that the adult brain could continuously produce new neurons added a completely new level of complexity to the understanding of brain structure and plasticity and opened up chances for restoring brain function and neuronal repair. The application and use of neural stem cells (NSCs) to the diseased human brain are manifold: (1) replacement of dead or dysfunctional endogenous neurons in a circumscribed neuronal population (e. g. in Parkinson’s disease), (2) cell restor-

13.2 Neural Stem- and Precursor Cells in the Adult Brain

ing in more diffuse brain damage (e. g. stroke), (3) therapeutic targets where disturbed neurogenesis may be the cause of disease (e. g. depression, seizure, brain tumor). However, to make use of NSCs in human disease it is necessary to understand endogenously occurring adult neurogenesis in all its aspects (development, regulation and function).

13.2

Neural Stem- and Precursor Cells in the Adult Brain 13.2.1

Neural Stem Cells in the Adult Brain: In vitro

Cells with stem cell properties can be isolated from various brain regions from the fetal, early postnatal and adult brain (Reynolds and Weiss, 1992; Palmer et al., 1997; Palmer et al., 1999; Uchida et al., 2000). As a standard method, the region of interest is mechanically dissected and the tissue is disaggregated (Fig. 13.1). Dissociated cells are then exposed to potent mitogens such as epidermal growth factor (EGF) with or without the addition of fibroblast growth factor-2 (FGF-2). Cells can be further expanded in vitro as floating aggregates, so-called “neurospheres” (Reynolds and Weiss, 1992; Morshead et al., 1994; Palmer et al., 1999) or as monolayers attached to a coated culture dish surface (Palmer et al., 1995; Palmer et al., 1999). After induction of differentiation – which is normally done by withdrawing the mitogens with or without the addition of differentiation factors (e. g. forskolin, retinoic acid) – these cells can give birth to all three neural cell types (Reynolds and Weiss, 1992; Palmer et al., 1999; Song et al., 2002b). Culturing neural progenitors is routinely done from rodent brain tissue (Reynolds and Weiss, 1992; Palmer et al., 1999) but also from human fetal (Fricker et al., 1999; Svendsen et al., 1999; Uchida et al., 2000) and post-mortem adult human brain (Palmer et al., 2001). Stem cells that are isolated from various brain regions (and also the spinal cord) largely differ in their ability to produce neurons or glial cells. For example, isolated precursor cells from white matter formed a pure glial progeny under optimized conditions for glial cells (Mayer-Proschel et al., 1997; Rao et al., 1998; Yakovlev et al., 1998; Palmer et al., 1999; Kondo and Raff, 2000). But only little changes within the culture conditions, such as the addition of FGF-2, made cells isolated from white matter indistinguishable from neural stem cells derived of the SVZ or the SGZ of the dentate gyrus (Palmer et al., 1999; Kondo and Raff, 2000). Technical differences in stem cell culturing, concerning the method and region of dissection (Palmer et al., 1995; Seaberg and van der Kooy, 2002), age of the donor animal, substrates used and plating method (Palmer et al., 1995; Gritti et al., 1996; Johe et al., 1996) potentially influence the behavior of isolated progenitors (Gage, 2000). Importantly, isolated “stem cells” from the adult hippocampus give rise to functionally competent neurons (Song et al., 2002a; Song et al., 2002b), making the in vitro expansion of purified precursor cells an attractive source for re-populating the damaged or degenerated adult brain (Roy et al., 2000).

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Figure 13.1 Schematic overview of neural stem cell preparation. The region of interest is isolated and mechanically dissected. Dissociated cells are cultured in the presence of EGF and FGF-2. Cells can be grown as free floating “neurospheres” or attached to the culture dish surface as “monolayers”. With the withdrawal of the mitogens and the addition of differentiation factors like retinoic acid, neurons, astrocytes and oligodendrocytes are generated.

13.2.2

Neural Stem Cells In The Adult Brain: In vivo

There have been many attempts to find and define markers that characterize “neural stem cells” in the adult brain (Geschwind et al., 2001; Luo et al., 2002; Ramalho-Santos et al., 2002; Luo et al., 2003) without succeeding in describing a unique set of expressed genes. But there is growing evidence that in the adult hippocampus GFAP-positive cells give rise to neurons and glial cells (Seri et al., 2001), reflecting a situation earlier in development (Leavitt et al., 1999; Malatesta et al., 2000). Lendahl and colleagues suggested that stem cells in the neural tube of the developing brain express nestin, an intermediate filament protein (Lendahl et al., 1990). There are nestin-expressing cells within the adult hippocampus (Wei et al., 2002; Kronenberg et al., 2003; Filippov et al., 2003). Nestin-positive

13.2 Neural Stem- and Precursor Cells in the Adult Brain

cells in the dentate gyrus can be further subdivided regarding their morphology and the expression of additional marker proteins such as doublecortin (DCX) and the homeobox-transcription factor prox-1 (Fig. 13.2; (Kronenberg et al., 2003). We suggested a classification of hippocampal stem or progenitor cells into four subtypes. Type-1 cells have a large triangular-shaped cell body and a long, tree-like branched process (Fig. 13.3). Type-1 cells co-label with GFAP and show some, but lack other properties of classical astrocytes, as they were consistently negative for S100b – which is expressed in mature astrocytes (Savchenko et al., 2000). However, they had an endothelial endfoot and astrocytic electrophysiological properties (Kronenberg et al., 2003; Filippov et al., 2003). Type-1 cells rarely divide and it seems that they are not influenced by neurogenic stimuli (Kronenberg et al., 2003). Type-2 cells are consistently negative for GFAP and have a small round morphology. They can be further subdivided into type-2A cells and type-2B cells, the latter expressing the immature neuronal marker DCX. Type-3 cells that are positive for DCX lost nestin-expression but still possess the ability to divide (Kronenberg et al., 2003). Whether type-1 to type-3 cells are already restricted to one neural cell lineage remains unclear to date.

Suggested developmental steps from the dividing precursor to the mature granule cell in the adult hippocampus. The presumed stem cell in the adult dentate gyrus is nestin-positive. Nestin-positive cells can be further subdivided by their morphology and the expression of additional marker proteins. Type-1 cells have a large triangular cell body within the subgranular zone and a long tree-like process reaching into the molecular layer. In contrast, type-2 cells have a small round-shaped cell body with only short, mostly horizontal processes. They can be further subdivided into type-2A cells and type-2B cells, the latter also express doublecortin and prox-1, but still possess the ability to divide. Type-3 cells lost nestin-expression, but still divide. Calretinin expression in newborn granule cells defines the step from the dividing precursor to a post-mitotic yet immature neuron. During this developmental step, newborn granule cells also co-express the neuronal marker NeuN. The mature adult-generated granule cells in the adult dentate gyrus switch their Ca2þ -binding protein from calretinin to calbindin and send long, branched dendrites to the molecular layer.

Figure 13.2

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Regarding the SVZ, several concepts have been proposed to describe the “neural stem cell” of this region. One set of experiments found that ependymal cells of the adult lateral ventricles are the stem cells of the SVZ (Johansson et al., 1999). In contrast to that, there is growing evidence that the stem cell of the SVZ are – comparable to the data later obtained for the dentate gyrus – astrocytic cells that express GFAP and are located in the subependymal layer of the lateral ventricles (Doetsch et al., 1999b; Laywell et al., 2000). As with the search for the hippocampal stem cell, multi-lineage potency and self-renewal has not been shown directly in vivo, as at the moment dividing cells cannot be followed over time in the adult brain due to technical difficulties.

13.3

Maturation and Migration of Adult-generated Neurons in the Adult Brain

At the moment knowledge rapidly increases concerning the development from the dividing precursor to a neuron or glial cell using immunohistochemical and electrophysiological approaches. The characterization of developmental steps from the dividing cell to a fully mature neuron will make it possible to describe fate decisions, vulnerable phases and the mechanisms of functional integration. In the hippocampus, van Praag and colleagues showed that adult-generated neurons become physiologically integrated into preexisting networks and that the newborn granule cells mature into functional neurons with electrophysiological properties very similar to older granule cells (van Praag et al., 2002). This study used a retrovirus expressing GFP in infected cells, which visualized them for electrophysiological studies. Other experiments had shown that newborn neurons extend their axons into area CA3 several days after division (Stanfield and Trice, 1988; Hastings and Gould, 1999; Markakis and Gage, 1999) and build synaptic connections with surrounding neurons (Carlen et al., 2002). Furthermore, adult-generated neurons become capable of activity-dependent gene expression, one criterion of cellular plasticity in the adult brain (Jessberger and Kempermann, 2003). But what are the steps from a nestin-positive, dividing precursor cell to a fully matured and functional neuron? Shortly after cell division, many cells express markers of immature neurons, such as doublecortin (DCX), the polysialated form of the neural cell adhesion molecule (PSA-NCAM) or b-III-tubulin (Jin et al., 2001; Nacher et al., 2001; Nacher et al., 2002; Brandt et al., 2003; Kronenberg et al., 2003). The neuronal marker NeuN (Mullen et al., 1992) is expressed early during maturation, and the numbers of new cells expressing NeuN seems to remain highly stable (Kempermann et al., 2003). As an additional marker defining the transition from a dividing cell to a postmitotic but yet immature neuron, the Ca2þ -binding protein calretinin was found (Brandt et al., 2003). Calretinin is transiently expressed in newborn neurons and strictly identified an early post-mitotic step in neuronal differentiation. Mature granule cells then switch their Ca2þ -binding protein and express calbindin.

13.3 Maturation and Migration of Adult-generated Neurons in the Adult Brain

A proposed model of the developmental steps from the dividing cell to a mature newborn hippocampal granule cells is given in Fig. 13.2. The precursor cells that give rise to new granule cells in the adult dentate gyrus are located in the SGZ. For a long time, the hypothesis was held that after division newborn neurons migrate into the granule cell layer in an inside-outside fashion with the oldest cells near the molecular layer (Wang et al., 2000) as it occurs during development of the hippocampal formation (Altman and Bayer, 1990). However, adult-generated neurons in the dentate gyrus reach their final position very early and migrate only a small distance without a foreseeable direction (Fig. 13.4; Kempermann et al., 2003). The situation of newly-generated neurons in the adult SVZ is different from the SGZ because the immature cells travel over a long distance to their final destination in the olfactory bulb. Migration starts at the SVZ where precursors divide at the lateral walls of the ventricle and young neurons are aligned in long chains (Lois and Alvarez-Buylla, 1994; Lois et al., 1996). Many of these chains merge into the rostral migratory stream (RMS), which leads the newborn cells into the core of the olfactory bulb (Luskin, 1993). Once there, newborn cells disperse radially into the bulb and fully differentiate into periglomerular and granular interneurons (Fig. 13.3). There have been several genes identified that seem to be involved in the migration of SVZ-generated neurons into the olfactory bulb. Among them are cytoskeleton-associated molecules such as integrins (Murase

Figure 13.3 Dividing cells in the adult dentate gyrus. Bromodeoxyuridine (BrdU) is integrated in the DNA during the S-phase and can be visualized using a specific antibody. In this example, animals received seven daily BrdU-injections and were killed four weeks after the last injection (A). Panel B illustrates a 3Dreconstructed picture of a nestinpositive progenitor cells in the adult dentate gyrus (for details see Fig. 13.2). Type-1 cells with their treelike processes reach into the inner parts of the molecular layer. In this example, the inner molecular layer was visualized with a staining against calretinin.

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and Horwitz, 2002), neural-cell-adhesion-molecule (Cremer et al., 1994; Jenkins et al., 2001) and tenascins (Cremer et al., 1994). Furthermore, the secreted glycoprotein reelin might be critically involved in the migration of newly generated neurons from the SVZ to the olfactory bulb (Hack et al., 2002; Kim et al., 2002a) again reflecting mechanisms that are involved during the development of the CNS (D’Arcangelo et al., 1995; Curran and D’Arcangelo, 1998). In an elegant study that used a retrovirus to label dividing cells and their progeny, the laboratory of Pierre-Marie Lledo studied the maturation of neurons generated in the SVZ and traveling to the olfactory bulb (Carleton et al., 2003). Using electrophysiological and immunohistochemical techniques they characterized in detail the properties of migrating neurons throughout the rostral migratory stream to the fully mature olfactory bulb interneuron (Carleton et al., 2003).

Figure 13.4 Schematic overview of the two spontaneously neurogenic regions in the adult brain. Neural stem cells divide in the subventricular zone (SVZ) and newly generated neurons migrate along the rostral migratory stream (RMS) into the olfactory bulb. In the hippocampal dentate gyrus, neural progenitors reside within the subgranular zone and give rise to new granule cells that send their axons to area CA3.

13.4 Neurogenesis in the Adult Brain – Age Dependency, Persistence and Genetic Determinants

13.4

Neurogenesis in the Adult Brain – Age Dependency, Persistence and Genetic Determinants 13.4.1

Neurogenesis in Aged Animals

Many neurodegenerative disorders that might be linked to impaired endogenous neurogenesis tend to occur in the elderly human. Thus, it is of particular interest to study adult neurogenesis in the aged animal. Adult hippocampal neurogenesis dramatically decreases with increasing age in the adult animal (Kuhn et al., 1996; Kempermann et al., 1998a; Cameron and McKay, 1999). Nevertheless, the aged brain still possesses the ability to respond to various stimuli with an increase in the number of newborn neurons (Kempermann et al., 1998a; Cameron and McKay, 1999; Kempermann et al., 2002). Environmental enrichment of aged mice resulted in a 5-fold increase of net neurogenesis compared to controls, and this increase was also correlated with improved performance in a spatial learning task (Kempermann et al., 2002). In contrast to the younger animals that are routinely used to study adult neurogenesis, this increase in newborn neurons was not observable in the number of dentate gyrus BrdU- positive cells, but only became apparent when analyzing the phenotypes of the newly generated cells (Kempermann et al., 2002). Thus, the mechanism by which environmental enrichment increases net neurogenesis appeared to be different between young and old animals. Whether these differences are due to altered inherent stem cells properties or to changes in the local environment of dividing cells is not known. 13.4.2

Long-term Persistence of Adult-generated Neurons in the SVZ and Hippocampus

The demonstration that newborn cells survive and are persistent is one important prerequisite to show the functional role newly-generated neurons actually play in the adult brain. To date, there are only long-term data for physiologically generated neurons in the hippocampal granule cell layer and the SVZ and olfactory bulb (Winner et al., 2002; Kempermann et al., 2003). Most of the newborn cells die within the first several days after division (Biebl et al., 2000; Kempermann et al., 2003), suggesting that there is – mimicking the situation during brain development – a surplus of cells and that cells which are not actively recruited become eliminated by apoptosis (Blaschke et al., 1996). However, once adult-generated cells in the hippocampus expressed the mature, neuronal marker NeuN, they seemed to survive for a long time (Kempermann et al., 2003). This implies that early after cell division the decision is made if a particular cell survives or dies. The critical phase seems to be identified by calretinin-expression because cells that have reached this post-mitotic step remain highly stable (Brandt et al., 2003).

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In analogy to the above experiments, Winner and colleagues showed that newborn cells that are generated in the SVZ and subsequently migrate into the olfactory bulb are comparably stable in terms of their survival as adult-generated hippocampal granule cells (Winner et al., 2002). To date, studies that examine the persistence and stability of neurons that were generated in response to pathologic stimuli, such as stroke and seizure (see below), are missing. 13.4.3

Genetic Influence on Adult Hippocampal Neurogenesis

Adult neurogenesis is strongly influenced by genetic determinants. Mouse strains widely differed in the number of proliferative cells in the dentate gyrus, and there were also large variations in net neurogenesis (Kempermann et al., 1997b; Kempermann and Gage, 2002a). Similar data were obtained by the comparison of two different rat strains (Perfilieva et al., 2001). Interestingly, the number of newborn neurons correlated with the performance in a hippocampus-dependent learning task (Kempermann and Gage, 2002b). This may be suggestive of a functional connection between the birth of new neurons and hippocampal learning (as it will be discussed in detail below). Additionally, strain differrences could be found after stimulating neurogenesis (Kempermann et al., 1998b). These data further indicated that there is a differential impact of inheritable traits on the regulation of adult hippocampal neurogenesis. Similarly, inherited traits will also affect the brain’s ability for endogenous repair.

13.5

Regulation of Adult Neurogenesis

Adult neurogenesis is not a static condition, but is in fact dynamically influenced by a variety of stimuli. Experimental paradigms regulating adult neurogenesis range from specific stimuli such as learning (Gould et al., 1999) and the complexity of environment (Kempermann et al., 1997a; Young et al., 1999) to rather non-specific stimuli such as hormonal changes (Cameron and Gould, 1994; Gould, 1994) and physical activity (Van Praag et al., 1999a), to pathologic stimuli such as epilepsy (Bengzon et al., 1997; Parent et al., 1997; Scott et al., 1998), ischemia (Liu et al., 1998; Arvidsson et al., 2002; Parent et al., 2002) and brain trauma (Magavi et al., 2000; Chirumamilla et al., 2002). The main parameter which is analyzed to describe adult neurogenesis is the number of BrdU-labeled cells expressing neuronal markers such as NeuN. This, however, will often not be precise enough to capture the complexity of adult neurogenesis. Furthermore, many studies of adult neurogenesis only analyzed proliferation of precursors, but not net neurogenesis (Parent et al., 1997; Chen et al., 2000). Assuming that adult neurogenesis is functionally relevant, it would be surprising if so many different stimuli led to an identical reaction, which is a plain increase in the number of dividing cells. Proliferative

13.5 Regulation of Adult Neurogenesis

cells in the dentate gyrus, but also the subventricular zone are a heterogeneous group of cells which are differentially activated by different stimuli (Doetsch et al., 1999a; Seri et al., 2001; Kronenberg et al., 2003). Furthermore, maturation can be subdivided into several developmental stages (Brandt et al., 2003; Steiner et al., 2003). Therefore we postulate that adult neurogenesis should not only be seen as a quantitative process. Rather, the qualitative differences, such as dividing cell subtypes and the quality of neuronal development may be as critical as counting numbers. With respect to neurorepair in the diseased brain, a differentiated view of neurogenesis seems mandatory. Neurons which are generated in response to unspecific stimuli may not fulfill all demands for a functional brain repair. 13.5.1

Learning

As it has been proposed that adult neurogenesis is involved in normal hippocampal function and processes underlying certain forms of learning and memory, Gould and colleagues examined the effects of several behavioral tasks on adult neurogenesis (Gould et al., 1999). They found that the number of adult-generated neurons in the dentate gyrus doubled after associative, hippocampus-dependent learning tasks (Gould et al., 1999). They found that this effect was only observable when dividing cells were labeled with BrdU one week prior to training. In contrast, increased numbers of newly-generated neurons were not observed when BrdU was injected during the training period of a hippocampus-dependent learning task (Van Praag et al., 1999a). Importantly, the increase in net neurogenesis did not occur if the behavioral task was independent of proper hippocampal function (Gould et al., 1999). To date, it remains unclear which factors mediate the net increase of newly-generated neurons, but many genes are known to be induced following hippocampus-dependent learning tasks. Among them are growth factors such as BDNF and NGF (Kesslak et al., 1998; Hall et al., 2000; Tyler et al., 2002) and effector proteins such as arg3.1/arc (Link et al., 1995; Montag-Sallaz and Montag, 2003) and Homer1A (Guzowski et al., 1999; Vazdarjanova et al., 2002). Which activity-induced genes are involved in the increase of newborn neurons following hippocampus-dependent learning tasks is currently not known. 13.5.2

Environmental Enrichment

Adult neurogenesis is induced when animals are kept under conditions which are environmentally enriched (Kempermann et al., 1997a; Young et al., 1999). “Enriched” means that these animals have a larger cage with several variable toys and live in bigger social groups (about 8–10) compared to standard-caged animals (2–4). Thus, these animals have an environment which is “a combination of complex inanimate and social stimulation” (Rosenzweig et al., 1978; Rosenzweig and Bennett, 1996). Adult mice that lived for forty days in such an environment did not show any significant differences in the proliferation of dentate gyrus precur-

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sors, but the number of surviving neurons four weeks after labeling dividing cells with BrdU was robustly increased (Kempermann et al., 1997a; Young et al., 1999). What is the cellular correlate of this net increase and where may be differences compared to standard caged animals? Interestingly, there was no increase in the number of type-1 and type-2 cells both positive for the presumed precursor marker nestin (Kronenberg et al., 2003). In contrast, the number of type-3 cells expressing the early granule cell marker prox-1 were significantly increased compared to controls (Kronenberg et al., 2003). Thus, the neurogenic effect of enriched environment may act on a distinct population of dividing precursors in the adult brain. One could suspect that in type-3 cells the decision to become a granule cell is already made. Thus, environmental enrichment not only results in an enhancement of the survival of new neurons, but also in an increase of proliferation in this specific subset of dividing precursors. It has been proposed that growth factors such as BDNF, NGF or GDNF may be involved in the increased survival of newborn neurons, as their expression is up-regulated in animals that are kept in an enriched environment (Falkenberg et al., 1992; Rasika et al., 1999; Young et al., 1999). Additionally, alterations in neurotransmitters in enriched animals may mediate effects of living in an enriched environment (Rosenzweig and Bennett, 1996; Rasmuson et al., 1998). In general, changes in the environment can have a strong impact on brain architecture and behavior of adult animals (Barnea and Nottebohm, 1994; Barnea and Nottebohm, 1996; Kempermann et al., 1997a). However, there has always been discussion whether enriched environment really is an “enrichment” or if normally caged mice are merely impoverished (Cummins et al., 1977). We and many other researchers in the field argue that laboratory animals in standard cages are not deprived, as these strains have been kept under these conditions for more than 80 years: the laboratory has become their natural habitat. 13.5.3

Physical Activity

Adult mice that are kept in a cage with free access to a running wheel run several kilometers during their active night period (Van Praag et al., 1999b; Van Praag et al., 1999a). When dividing cells were labeled with BrdU, the number of proliferating cells in the subgranular zone of the dentate gyrus was strongly increased (Van Praag et al., 1999a; Kitamura et al., 2003). This increase also resulted in an approximate doubling of net neurogenesis four weeks after BrdU-injections. Additionally, the ratio of newly generated neurons out of all newborn cells was increased compared to control animals. In parallel to environmental enrichment, activity did not affect the number of type-1 cells, but increased the number of dividing type-2 cells (Kronenberg et al., 2003). Not surprisingly, the number of type-3 cells positive for granule cell marker prox-1 were also elevated in mice with free access to a running wheel (Kronenberg et al., 2003). Physical activity is a physiologic but rather general stimulus affecting other organ systems besides the brain (Natali et al., 2002; Droste et al., 2003; Elphick et al., 2003). Nevertheless, the effect on adult hippocampal


neurogenesis seemed to be surprisingly specific to the dentate gyrus as the number of proliferating cells in the SVZ and newborn neurons in the olfactory bulb remained stable following physical activity (Brown et al., 2003). Free access to a running wheel also layer-specifically increased the proliferation of dividing cells in the neocortex (Ehninger and Kempermann, 2003). In contrast to the dentate gyrus where most of the newborn cells turned into neurons newborn cells in the neocortex were mainly microglia. The mechanisms by which running leads to an increase in cell proliferation are yet unidentified. A variety of genes are induced in their expression following wheel running, including again numerous growth factors such as BDNF, IGF-1, FGF-2 and GDNF (Neeper et al., 1995; Gomez-Pinilla et al., 1997; Carro et al., 2000; Gomez-Pinilla et al., 2002). Other studies suggested that increased neurogenesis following physical activity may be mediated by the activation of NMDA-receptors (Kitamura et al., 2003). Thus, there seems to be an overlap in gene expression between physical activity on the one hand and environmental enrichment and learning on the other hand, suggesting that these factors may actually be involved in stimulating neurogenesis. It is also discussed that physical activity increases neurogenesis via changes in the vascularization of the hippocampal region which seems to play a role in adult neurogenesis (Palmer et al., 2000). But again, further studies will have to determine the definite mechanisms and working points of physical activity regarding its role in adult neurogenesis. 13.5.4

Stress and Depression

Additionally to the above mentioned positive regulators of adult neurogenesis, hormones of the adrenal gland that are secreted in response to stress decrease the proliferation of dentate gyrus precursor cells in the adult hippocampus (Gould, 1994). Corticosteroids that were applied to adult mice decreased the number of newly formed granule cells. Consistent with this, the removal of the adrenal gland leads to an increase in proliferation of residing precursors, further suggesting that corticosteroids regulate stem cell division in a negative fashion (Gould, 1994). This effect was also described in adult monkeys that were exposed to a stressful experience (Gould et al., 1998). The mechanisms by which stress leads to a decrease of cell division in the adult hippocampus are yet unidentified, but there is some evidence that downstream of corticosteroid action NMDA-receptors may be involved, as the blockage of NMDA-receptors prevents the negative effects of corticosteroids (Cameron et al., 1995). The effects of elevated corticosteroid-levels are well-fitting into concepts that impaired adult neurogenesis may be involved in the origin or maintenance of depression (D’Sa and Duman, 2002; Jacobs, 2002; Kempermann and Kronenberg, 2003). Stress is believed to be one important factor and a causal trigger in the etiology of depression in humans (Kendler et al., 1999). Furthermore, corticosteroid levels are elevated in the serum of patients suffering from major depression (Starkman et al., 1992). Post-mortem and imaging studies demonstrated that the prefrontal, but also

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the hippocampal region showed a relative atrophy in depressed patients (Gurvits et al., 1996; Sheline et al., 1996; Sheline et al., 1999). Indeed, treatment with anti-depressive drugs such as fluoxetine that increase serotonergic neurotransmission resulted in an increase of adult-generated cells in the dentate gyrus, an effect which seemed to be mediated by the activation of serotonin-receptors (Malberg et al., 2000). Furthermore, inhibition of serotonin synthesis resulted in a decrease of hippocampal neurogenesis in the adult brain (Brezun and Daszuta, 1999). Santarelli et al. blocked neurogenesis by whole brain irradiation and using an behavioral paradigm testing anxiety (novelty-suppressed-feeding test) showed that the positive effects of fluoxetine on anxiety treatment were absent in irradiated animals (Santarelli et al., 2003). They speculated that blocking of neurogenesis by irradiation and the subsequent failure of fluoxetine to decrease anxiety were causally linked (Santarelli et al., 2003). Several methodological issues make this study difficult to interpret: for example, the chosen test may also depend on proper hippocampal function and only partially reflects depressive behavior. Furthermore, irradiation itself may cause other damage to the hippocampus besides blocking neurogenesis, which is too subtle for gross anatomical and electrophysiological examination. For example, Monje and colleagues showed significant alterations in the hippocampal microstructure after irradiation – an increase in microglial activation and altered vascularization (Monje et al., 2002). Thus, it seems plausible that neurogenesis is related to depression, but further studies will have to explore the exact connection. 13.5.5

Epilepsy

The generation of dentate granule cells in the adult CNS is greatly increased in rodent models of limbic epilepsy or acute seizures (Bengzon et al., 1997; Parent et al., 1997; Scott et al., 1998; Madsen et al., 2000). Chemo-convulsants such as pilocarpine or kainic acid (KA) were applied to mimic human temporal lobe epilepsy (TLE) which represents the majority of chronic, focal epilepsies in adult humans (Helmstaedter, 2002). After a latency of several days seizures increased the proliferation of dividing cells in the SGZ 5-to 10-fold (Bengzon et al., 1997; Parent et al., 1997; Gray and Sundstrom, 1998). Pentylenetetrazole (PTZ) also induced neurogenesis through inhibiting inhibitory pathways (Jiang et al., 2003) and so did electrical stimulation of the hippocampal afferents (Bengzon et al., 1997; Parent et al., 1997; Scott et al., 1998). Most of the seizure-generated neurons mature into dentate granule cells and migrate into the granule cell layer, comparable to “normal” adult neurogenesis. What causes the dramatic increase of cell proliferation in the dentate gyrus after seizure activity? There is dramatic damage to the hippocampus of epileptic rodents. Most prominent, the CA1-region shows widespread neuronal loss, whereas the granule cells of the dentate gyrus seem relatively resistant to excitotoxic cell death (Ben-Ari, 2001). Cell death itself may cause an increase in cell proliferation (Gould and Tanapat, 1997). Nevertheless, until now it has not been shown that cell


death is mandatory to induce neurogenesis following seizures. There are even data that found a more pronounced induction of neurogenesis if excitotoxic cell death was prevented (Ekdahl et al., 2001). Many factors, among them numerous growth factors that are up-regulated in response to epileptic activity, may mediate increased proliferation of neural precursors in the subgranular zone (Gall et al., 1994; Ferhat et al., 1996; Jankowsky and Patterson, 2001; Minami et al., 2002). To date, it remains unclear which subtype of stem or progenitor cells within the adult dentate gyrus actually divides in response to epileptic activity. Importantly, seizure-generated granule cells migrate not only into the granule cell layer, but also a few newly generated granule cells ectopically migrate into the hilus/CA3 border of the hippocampal region where they are electrically synchronized with CA3 pyramidal neurons (Scharfman et al., 2000). These aberrant connections may trigger spontaneous epileptiform bursts and by that may be an important factor in epileptogenesis after the first epileptic discharge. But there is also data arguing against a pro-ictogenic role of adult neurogenesis. Parent and colleagues blocked neurogenesis with whole brain irradiation and found that recurrent seizures following pilocarpine also occurred in these animals (Parent et al., 1999). But again, it cannot be ruled out that irradiation of the brain itself caused damage besides blocking the proliferation of residing precursors (Monje et al., 2002). In clear contrast to an ictogenic role of newborn neurons in the epileptic hippocampus, there is also the hypothesis that increased neurogenesis following seizures may represent an attempt to repair the injured brain. Besides above-mentioned aberrant migration patterns, the vast majority of seizure-generated neurons matures into “normal” dentate gyrus granule cells with regular antigenic features and synaptic connections (Bengzon et al., 1997; Parent et al., 1997; Scharfman et al., 2000). Given this, increased neurogenesis following seizures could try to overcome excitotoxic cell death. Without doubt, mechanisms and molecular events resulting in increased neurogenesis after seizures in the adult brain remain largely unclear to date (Parent, 2002; Parent and Lowenstein, 2002). Furthermore, the most important question, if increased neurogenesis is a profit (substitution of lost cells) or a disprofit (epileptogenic connections) for the epileptic brain, will require extensive studies. 13.5.6

Ischemia and Cortical Trauma

There is also an increase in the proliferation of neurogenic precursors after different forms of ischemia to the adult brain. In experimental studies mainly two models of CNS ischemia are used. The thread occlusion model of the middle cerebral artery (MCAO) mimics focal brain ischemia, whereas global forebrain ischemia (GFI) results in a fast and abrupt loss of cerebral blood flow, mimicking pathophysiologic changes after cardiac arrest in humans. Ischemia increases the proliferation of precursors in the hippocampal SGZ (Liu et al., 1998; Yagita et al., 2001). It is not clear what stimulates the precursors to

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divide, but cell death does not seem to be the cause of increased cell genesis (Liu et al., 1998). Following GFI there is under certain conditions a selective loss of CA1 pyramidal cells, and Nakatomi et al. showed that after ventricular infusion of the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2), dead cells in CA1 were replaced by newborn neurons (Nakatomi et al., 2002). Most likely, precursors in the posterior paraventricular region gave rise to these structure-restoring neurons that also seemed to restore synaptic properties, such as LTP, which was impaired in ischemic control animals without growth factor infusion (Nakatomi et al., 2002). Secondly, MCAO, but not GFI lead to an increased cell proliferation in the SVZ, ipsilateral to the injured side (Jin et al., 2001; Arvidsson et al., 2002; Parent et al., 2002). Few immature neurons subsequently migrated from the lateral walls of the ventricle into the damaged striatum where they fully matured into striatal, medium-sized spiny neurons, many of them expressing DARP-32 and calbindin (Arvidsson et al., 2002; Parent et al., 2002). But the number of cells that was replaced in the ischemic brain was very low, and only about 0.02 % of the killed striatal neurons became substituted (Arvidsson et al., 2002). Whether ischemia-induced newborn striatal neurons remain stable over time is not known. Additionally and in contrast to normally generated granule cells in the dentate gyrus (van Praag et al., 2002; Jessberger and Kempermann, 2003), there is no direct evidence that striatal or pyramidal neurons become fully functional in vivo and play a significant role in the recovery after stroke. Concerning cortical neurogenesis after stroke there is contradictory data. Only one group found BrdU-positive cells expressing neuronal markers in the cortical penumbra (Gu et al., 2000), whereas several other studies could not confirm this finding (Arvidsson et al., 2002; Parent et al., 2002). In contrast to this, precursors in the cortex give rise to new nerve cells after targeted apoptosis of layer VI cortico-thalamic projection neurons (Magavi et al., 2000). To induce apoptosis a chromophore was injected into the thalamus, and the anterior cortex was exposed to 674nm wavelength light two weeks later. The neurons were targeted in the anterior neocortex, as this region is spatially near to the SVZ. Indeed, single newborn cells that were labeled with BrdU expressed NeuN (Magavi et al., 2000).

13.6

Possible Functional Relevance of Neurogenesis in the Adult Brain

To date, the function of naturally occurring neurogenesis in the adult brain is not clear. Most of the theories why adult neurogenesis may play a role in certain brain functions derive from correlational data, and causal links are rare. This paragraph will mainly deal with the hypothetical function of adult generated neurons in the hippocampal dentate gyrus and will discuss possible roles in learning and memory and neuropsychiatric disorders.

13.6 Possible Functional Relevance of Neurogenesis in the Adult Brain

Experimental paradigms that increase neurogenesis, such as environmental enrichment or physical activity, ameliorate at the same time spatial memory (Kempermann et al., 1997a; Van Praag et al., 1999b; Van Praag et al., 1999a; Young et al., 1999). Additionally, rapidly learning mouse strains tend to have a greater extent of spontaneously occurring neurogenesis in the adult dentate gyrus (Kempermann and Gage, 2002b). Furthermore, increased neurogenesis is paralleled by an enhancement of synaptic plasticity (Van Praag et al., 1999b). Mice that were kept in an enriched environment, leading to an enhanced survival of newborn neurons, also showed increased long-term potentiation (LTP) which is thought to be an electrophysiological correlate of processes underlying certain forms of learning and memory (Bliss and Collingridge, 1993). Cell proliferation and net neurogenesis were increased following hippocampus-dependent learning tasks (Gould et al., 1999). Together, these data suggested that adult neurogenesis might be involved in certain forms of hippocampal learning. Shors and colleagues blocked neurogenesis using a cytostatic drug and showed that a hippocampus-dependent learning task was impaired, suggesting a causal relationship between neurogenesis and learning (Shors et al., 2001). However, this study could not finally settle the question because the drug used to block neurogenesis was methylazoxymethanol acetate (MAM) which also blocks protein synthesis (Lundeen et al., 1971; Grab et al., 1979). De-novo synthesis of protein is needed for proper hippocampal function (Davis and Squire, 1984). Also, the used behavioral task (eye blink conditioning) probably relies in parts on accurate hippocampal function, but is not appropriate to appreciate the complexity of the hippocampus. A later study by the same authors showed that the Morris water maze performance was not impaired in animals that received MAM (Shors et al., 2001), weakening the conclusions drawn from the first study. The addition of new neurons in the adult brain probably does not lead to acute and fast changes in hippocampal function, and new neurons rather optimize the strength of the mossy fiber connection between the dentate gyrus and area CA3 in an activity- and experience-dependent way (Kempermann, 2002). Compatible with this assumption is the observation that it takes at least 14 days until newborn neurons seem to be functionally integrated (van Praag et al., 2002; Jessberger and Kempermann, 2003). A speculative, yet important theory proposes that the generation of new neurons in the adult brain might be involved in the development or maintenance of depression (D’Sa and Duman, 2002; Jacobs, 2002; Kempermann and Kronenberg, 2003; Santarelli et al., 2003). The idea is mainly based on correlational data: stress and elevated corticosteroid-levels decrease proliferation of dentate gyrus precursors, and neurotransmitters involved in depression such as serotonin are potent regulators of adult neurogenesis. Two studies have linked the generation of new neurons in the adult hippocampus with the action of the antidepressive drug fluoxetine (Malberg et al., 2000; Santarelli et al., 2003).

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13.7

Therapeutic Strategies for Neural Cell Replacement 13.7.1

Endogenous Neurogenesis

The main focus on cell replacement is currently on the transplantation of stem or progenitors cells, which are derived from embryonic, fetal or adult tissue. Comparatively less attention has been paid on the ability of the adult brain to produce new neurons from endogenous resources. It seems that the generation of new neurons can occur in several brain regions after pathologic stimuli such as ischemia (Arvidsson et al., 2002; Parent et al., 2002) and targeted cell ablation (Magavi et al., 2000). To date, it is unknown which factors could turn a normally “non-neurogenic” region into a “neurogenic” region. As described above, for hippocampal progenitors is has been demonstrated that astrocytes provide essential clues for the generation of new neurons (Song et al., 2002a), but what exactly constitutes the stem cell niche in the adult brain remains to be shown. Following global forebrain ischemia and the subsequent massive infusion of EGF and FGF-2 into the ventricles, an extensive cell replacement was seen within hippocampal subfield CA1 (Nakatomi et al., 2002). This study showed that in general the substitution of lost cells in certain brain areas might be possible. In addition, newly generated cells in area CA1 seemed to have a positive effect on the functional outcome (Nakatomi et al., 2002). Despite these promising findings in rodent models, there is a large amount of work to do until endogenous neurogenesis can be used for brain repair in humans. The region-specific microenvironment rather than the stem or progenitor seems to provide crucial clues for the generation of new neurons (Gage et al., 1995; Suhonen et al., 1996; Takahashi et al., 1998; Shihabuddin et al., 2000). Much has to be learnt about how to manipulate the local microenvironment or progenitor cell to transform normally “non-neurogenic” regions into “neurogenic” regions. 13.7.2

Transplantation of Stem- or Precursor Cells into the Damaged Brain

For some exemplary neurological disorders, clinical trials for the transplantation of immature neurons or of precursor cells into the adult brain have begun. Cells used for transplantation (in rodent models and some cases also in humans) range from embryonic stem cells, fetal precursors to stem cells which were isolated from adult brains (Isacson et al., 1995; Studer et al., 1998; Wagner et al., 1999; Bjorklund and Lindvall, 2000; Kim et al., 2002b). Transplanted cells are required to integrate into existing networks to fulfill their functional tasks. Many studies indicated that fully matured neurons with long neuritic branches lost the ability to integrate into preexisting circuits. Furthermore, the age of the host seems to play a role in the integration of the transplant (Sortwell et al., 2001). Human fetal tissue which has been successfully used in clinical trials with patients suffer-

13.7 Therapeutic Strategies for Neural Cell Replacement

ing from Parkinson’s disease matches requirements for a proper integration (Olanow et al., 1996; Lindvall, 1999). Explants were taken from the fetal ventral midbrain containing developing dopamine-producing neurons. Animal models of Parkinson’s disease as well as human patients seem to benefit from the striatal transplantation through a significant improvement of movement deficits (Herman and Abrous, 1994; Olanow et al., 1996; Lindvall, 1999). In contrast, double-blinded, sham-controlled studies saw benefits after the transplantation of fetal mesencephalic cells into the brains of Parkinson’s disease patients only in specific patient groups (Freed et al., 2001; Olanow et al., 2003). Additionally, severe side effects as dyskinesia were reported after the transplantation of fetal tissue (Hagell et al., 2002). Besides the difficulty to obtain enough material from human fetal tissue for transplantation, this source is not perfect for several reasons. It seems difficult to set up standardization regarding the culture conditions and explantation procedure, both influencing the viability of cells. Secondly, the medical use of aborted fetuses also brought up controversial discussions and serious ethical concerns. For that reason, other sources to generate neural progenitors are extensively studied (Gottlieb, 2002). Among them are embryonic stem cells (Kim et al., 2002b) but also neural stem cells (Svendsen et al., 1999; Englund et al., 2002). Furthermore, if stem cells from different tissue may be able to cross lineage boundaries, it might be possible to use easily accessible stem cells (such as hematopoetic stem cells) to generate, for example, neural stem cells (Anderson et al., 2001). Many hopes rest on the rare “multipotent adult precursor cell” (MAPC) as described by Verfaillie and co-workers that may be an attractive source for stem cell-based therapy (Jiang et al., 2002; Keene et al., 2003). Comparable to transplantation strategies in Parkinson’s disease, there are currently clinical trials under way with patients suffering from Huntington’s disease. The target area of transplantation is the damaged striatum where animal models and data obtained from human post-mortem studies showed that transplanted cells differentiate into GABA-ergic neurons. Transplant-derived neurons seemed to reduce pallidal output, resulting in a decrease of locomotor activity, one of the major symptoms of this devastating disease (Dunnett, 1995; Bachoud-Levi et al., 2000; Freeman et al., 2000a; Freeman et al., 2000b). The transplantation of restoring cells has been also extensively studied in different animal models of brain ischemia. Many types of donor cells – ranging from porcine fetal cells, neural progenitors, bone marrow-derived cells and embryonic stem cells – have been used (Dinsmore et al., 1996; Svendsen et al., 1996; Borlongan et al., 1998; Chen et al., 2001; Hoehn et al., 2002). Within these studies, sites of implantation, time-point after ischemia and host animals largely differed. Nevertheless, there seems to be a positive effect in the outcome of transplanted animals compared to controls (Modo et al., 2002; Zhao et al., 2002). If this benefit is due to transplanted cells and their functional integration or is mediated by trophic factors released or evoked by the transplant is not clear. Given that the local microenvironment seems to play a pivotal role in differentiation and integration, the benefit by simply transplanting neural stem or progenitor

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cells into the damaged brain area may be limited (Gage et al., 1995; Suhonen et al., 1996; Takahashi et al., 1998; Shihabuddin et al., 2000). As a summary, there is a definite need for carefully designed studies to use transplantation of stem or progenitor cells into the adult brain to treat neurological disease. They have to show that transplants become functionally integrated and definitely improve the outcome. Furthermore, it is mandatory to determine the benefits in relation to the risk that a transplantation of any material into the human brain still is.

Acknowledgements

We would like to thank Harish Babu for helpful comments on the manuscript and Golo Kronenberg for the preparation of the nestin-GFP image in Figure 13.3.

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of the adult mammalian central nervous system [see comments]. Science 255:1707–1710. Richards LJ, Kilpatrick TJ, Bartlett PF (1992) De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci U S A 89:8591–8595. Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett PF (2001) Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 412:736–739. Rosenzweig MR, Bennett EL (1996) Psychobiology of plasticity: effects of training and experience on brain and behavior. Behav Brain Res 78:57–65. Rosenzweig MR, Bennett EL, Hebert M, Morimoto H (1978) Social grouping cannot account for cerebral effects of enriched environments. Brain Res 153:563–576. Roy NS, Wang S, Jiang L, Kang J, Benraiss A, Harrison-Restelli C, Fraser RA, Couldwell WT, Kawaguchi A, Okano H, Nedergaard M, Goldman SA (2000) In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat Med 6:271–277. Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancio O, Belzung C, Hen R (2003) Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301:805–809. Savchenko VL, McKanna JA, Nikonenko IR, Skibo GG (2000) Microglia and astrocytes in the adult rat brain: comparative immunocytochemical analysis demonstrates the efficacy of lipocortin 1 immunoreactivity. Neuroscience 96:195–203. Scharfman HE, Goodman JH, Sollas AL (2000) Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis. J Neurosci 20:6144–6158. Scott BW, Wang S, Burnham WM, De Boni U, Wojtowicz JM (1998) Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci Lett 248:73–76. Seaberg RM, van der Kooy D (2002) Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J Neurosci 22:1784–1793. Seri B, Garcia-Verdugo JM, McEwen BS, Alvarez-Buylla A (2001) Astrocytes give rise to

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13 Neurogenesis new neurons in the adult mammalian hippocampus. J Neurosci 21:7153–7160. Sheline YI, Sanghavi M, Mintun MA, Gado MH (1999) Depression duration but not age predicts hippocampal volume loss in medically healthy women with recurrent major depression. J Neurosci 19:5034–5043. Sheline YI, Wang PW, Gado MH, Csernansky JG, Vannier MW (1996) Hippocampal atrophy in recurrent major depression. Proc Natl Acad Sci U S A 93:3908–3913. Shihabuddin LS, Horner PJ, Ray J, Gage FH (2000) Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci 20:8727–8735. Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001) Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372–376. Song H, Stevens CF, Gage FH (2002a) Astroglia induce neurogenesis from adult neural stem cells. Nature 417:39–44. Song HJ, Stevens CF, Gage FH (2002b) Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat Neurosci 5:438–445. Sortwell CE, Camargo MD, Pitzer MR, Gyawali S, Collier TJ (2001) Diminished survival of mesencephalic dopamine neurons grafted into aged hosts occurs during the immediate postgrafting interval. Exp Neurol 169:23–29. Stanfield BB, Trice JE (1988) Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Exp Brain Res 72:399–406. Starkman MN, Gebarski SS, Berent S, Schteingart DE (1992) Hippocampal formation volume, memory dysfunction, and cortisol levels in patients with Cushing’s syndrome. Biol Psychiatry 32:756–765. Steiner B, Kronenberg G, Jessberger S, Brandt MD, Reuter K, Kempermann G (2004) Differential regulation of gliogenesis in the context of adult hippocampal neurogenesis in mice. Glia 46:41–52. Studer L, Tabar V, McKay RD (1998) Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1:290–295. Suhonen JO, Peterson DA, Ray J, Gage FH (1996) Differentiation of adult hippocampusderived progenitors into olfactory neurons in vivo. Nature 383:624–627.

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14 Excitotoxicity Stuart A. Lipton, MD, PhD

Abstract

Cerebrovascular insults, including stroke, are responsible for the greatest number of mortalities among neurologic disorders. Alzheimer’s disease is the most common form of dementia, at least in western countries. Taken together, it is estimated that the cost of these disorders to society will consume the entire gross national product of the U. S.A. by the middle of this century if left unabated. To date, only tissue plasminogen activator (tPA), which dissolves blood clots, has been approved for acute stroke therapy in the U. S.A. where clinical trials were positive, but the European experience has been more equivocal. No neuroprotective drugs have yet been approved for the treatment of stroke, and in fact many such drugs tested in clinical trials have proven to be poorly tolerated. Concerning dementia, to date only one neuroprotective drug, memantine, has been shown to be both effective and clinically tolerated. Memantine was recently approved by the European Union and the U. S. Food and Drug Administration (FDA) for the treatment of Alzheimer’s disease. Previously used cholinergic treatments symptomatically enhanced the cognitive state to some degree, but were not neuroprotective like memantine. Recent phase 3 clinical trials have shown that memantine is effective in the treatment of moderate-to-severe Alzheimer’s disease and possibly vascular dementia. Here we review the molecular mechanism of memantine’s action, as first described by my laboratory, and also the basis for the use of the drug in these neurological diseases, which are mediated at least in part by excitotoxicity. Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. Excitotoxic neuronal cell death is mediated in part by overactivation of N-methyl-d-aspartate (NMDA)-type glutamate receptors, which results in excessive Ca2þ influx through the receptor’s associated ion channel. Physiological NMDA receptor activity, however, is also essential for normal neuronal function. This means that potential neuroprotective agents that block virtually all NMDA receptor activity will very likely have unacceptable clinical side effects. For this reason, many previous NMDA receptor antagonists have disappointingly failed advanced clinical trials Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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for a number of diseases, including stroke and neurodegenerative disorders. In contrast, studies in our laboratory have shown that the adamantane derivative, memantine, preferentially blocks excessive NMDA receptor activity without disrupting normal activity. Memantine does this through its action as a low-affinity, open-channel blocker; it enters the receptor-associated ion channel preferentially when it is excessively open, and, most importantly, its off-rate is relatively fast so that it does not substantially accumulate in the channel to interfere with normal synaptic transmission. Clinical use has corroborated the prediction that memantine is thus well tolerated. Besides Alzheimer’s disease, memantine is currently in trials for additional neurological disorders, including other forms of dementia, depression, glaucoma, and severe neuropathic pain. Memantine has not yet been tested in detail in humans with acute stroke, although preclinical studies have been encouraging. A series of second-generation memantine derivatives are currently in development and may prove to have even greater neuroprotective properties than memantine. These second-generation drugs take advantage of the fact that the NMDA receptor has other modulatory sites in addition to its ion channel that potentially could also be used for safe but effective clinical intervention.

14.1

Introduction

Acute and chronic neurologic diseases are among the leading causes of death, disability, and economic expense in the world. For example, cerebral ischemia (stroke) and Alzheimer’s disease rank third and fourth as causes for mortality in the U. S.A. In fact, it has been estimated that as the population continues to age, treatment of patients with stroke and dementia will consume our entire gross national product by the latter decades of this century. Excitotoxic cell death, as described below, is thought to contribute to neuronal cell injury and death in these and other neurodegenerative disorders. Excitotoxicity is due, at least in part, to overactivation of N-methyl-d-aspartate (NMDA)-type glutamate receptors, permitting excessive Ca2þ influx through the receptor’s associated ion channel. On the other hand, glutamate-mediated synaptic transmission is critical for the normal functioning of the nervous system. Glutamate is the major excitatory neurotransmitter in the brain. There are three classes of glutamate-gated ion (or ionotropic) channels, known as AMPA, kainate, and NMDA receptors. Among these, the ion channels coupled to classical NMDA receptors are generally the most permeable to Ca2þ. Excessive activation of the NMDA receptor in particular leads to production of damaging free radicals and other enzymatic processes contributing to cell death [1, 2]. With the disruption of the energy metabolism during acute and chronic neurodegenerative disorders, glutamate is not cleared properly and may even be inappropriately released. Moreover, energetically compromised neurons become depolarized (more positively charged) because in the absence of energy they cannot maintain their ionic homeostasis; this depolarization relieves the normal Mg2þ block of NMDA receptor-coupled channels because the relatively positive charge in the cell

14.2 The Search for Clinically-Tolerated NMDA Receptor Antagonists

repels positively-charged Mg2þ from the channel pore. Hence, during periods of ischemia and in many neurodegenerative diseases, excessive stimulation of glutamate receptors is thought to occur. These neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, HIV-associated dementia, multiple sclerosis, amyotrophic lateral sclerosis, and glaucoma, are caused by different mechanisms but may share a final common pathway to neuronal injury due to the overstimulation of glutamate receptors, especially of the NMDA subtype [1]. Hence, NMDA receptor antagonists could potentially be of therapeutic benefit in a number of neurologic disorders manifesting excessive NMDA receptor activity, including stroke, dementia, and neuropathic pain. NMDA receptors are made up of different subunits: NR1 (the presence of which is mandatory), NR2A-D, and, in some cases, NR3A or B subunits. The receptor is probably composed of a tetramer of these subunits. The subunit composition determines the pharmacology and other parameters of the receptor-ion channel complex. Alternative splicing of some subunits, such as NR1, further contributes to the pharmacological properties of the receptor. The subunits are differentially expressed both regionally in the brain and temporally during development. For this reason, some authorities have suggested developing antagonists selective for particular subunits, such as NR2B, which is present predominantly in the forebrain (reviewed in reference [3]).

14.2

The Search for Clinically-Tolerated NMDA Receptor Antagonists

Excitotoxicity is a particularly attractive target for neuroprotective efforts because it is implicated in the pathophysiology of a wide variety of acute and chronic neurodegenerative disorders [1]. The challenge facing those trying to devise strategies for combating excitotoxicity is that the same processes which, in excess, lead to excitotoxic cell death are, at lower levels, absolutely critical for normal neuronal function. Until recently, all drugs that showed promise as inhibitors of excitotoxicity also blocked normal neuronal function and consequently had severe and unacceptable side effects, so clinical trials for stroke, traumatic brain injury, and Huntington’s disease failed [4–6]. It has been suggested that successful NMDA receptor antagonists in animal models of stroke fail in human trials because they protect neuronal cell bodies (gray matter) and not the white matter (cell processes and glia), which is more prominent in the human brain. However, a careful perusal of the stroke trials with NMDA receptor antagonists to date reveals that the amount of drug administered in advanced trials was below the neuroprotective concentration because neuroprotective levels of drug proved not to be clinically tolerated in the phase 1 doseescalation trials. Hence, in my view the true test of an NMDA receptor antagonist in a human clinical stroke trial has not yet been achieved, i. e., testing efficacy of a clinically-tolerated drug at neuroprotective doses within a few hours of the insult. Recently, however, the well-tolerated, but under-appreciated drug, memantine, has been re-discovered to be not only capable of blocking excitotoxic cell death

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[7], but, most importantly, also of doing it in a clinically-tolerated, non-toxic manner [8–11]. Memantine was recently approved by the European Union and the U. S. FDA for the treatment of moderate-to-severe Alzheimer’s disease and may also show efficacy for vascular dementia [12, 13]. Previous cholinergic drugs only offered symptomatic relief from Alzheimer’s disease, while memantine is believed to be the first neuroprotective drug to achieve clinical approval. The drug is also under investigation as a potential treatment for other neurodegenerative disorders, including HIV-associated dementia, neuropathic pain, and glaucoma, as well as depression and movement disorders. Hopefully, since several trials suggest that memantine may prove effective in vascular dementia, it will also be tested in acute stroke trials as well. The purpose of the present review is to provide a brief and perhaps somewhat surprising primer on excitotoxicity as a promising target of neuroprotective strategies and to present a scientific and clinical overview of the excitotoxicity blocker memantine. Some preliminary information on second-generation memantine derivatives, termed NitroMemantines, is also provided.

14.3

Excitotoxicity 14.3.1

Definition and Clinical Relevance

The ability of the nervous system to rapidly convey sensory information and complex motor commands from one part of the body to another, and to form thoughts and memories, is largely dependent on a single powerful excitatory neurotransmitter, glutamate. There are other excitatory neurotransmitters in the brain, but glutamate is the most common and widely distributed. Most neurons (and also glia) contain high concentrations of glutamate (Z10 mM) [1]; after sequestration inside synaptic vesicles, glutamate is released for very brief amounts of time (milliseconds) to communicate with other neurons via synaptic endings. Because glutamate is so powerful, however, its presence in excessive amounts or for excessive periods of time can literally excite cells to death. This phenomenon was first documented when Lucas and Newhouse [14] observed that subcutaneously injected glutamate selectively damaged the inner layer of the retina (representing primarily the retinal ganglion cells) [14]. John Olney later coined the term “excitotoxicity” to describe this phenomenon [15, 16]. A large variety of insults can lead to the excessive release of glutamate within the nervous system and, thus, excitotoxic cell death. When the nervous system suffers a severe mechanical insult, as in head or spinal cord injury, large amounts of glutamate are released from injured cells. These high levels of glutamate reach thousands of nearby cells that had survived the original trauma, causing them to depolarize, swell, lyse, and die by necrosis. The lysed cells release more glutamate, leading to a cascade of autodestructive events and progressive cell death that

14.3 Excitotoxicity

can continue for hours or even days after the original injury. A similar phenomenon occurs in stroke; the ischemic event deprives many neurons of the energy they need to maintain ionic homeostasis, causing them to depolarize, lyse, die, and propagate the same type of autodestructive events that are seen in traumatic injury (reviewed in [1, 17]). This acute form of cell death occurs by a necroticlike mechanism, although a slower component leading to an apoptotic-like death can also be present, as well as a continuum of events somewhere between the two (see below). A slower, subtler form of excitotoxicity is implicated in a variety of slowly progressing neurodegenerative disorders as well as in the penumbra of stroke damage. In disorders such as Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, HIV-associated dementia, amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease), and glaucoma, it is hypothesized that chronic exposure to moderately elevated glutamate concentrations or glutamate receptor hyperactivity for longer periods of time than occur during normal neurotransmission trigger cellular processes in neurons that eventually lead to apoptotic-like cell death, a form of cell death related to the programmed cell death that occurs during normal development [2, 18–24]. More subtle or incipient insults can lead to synaptic and dendritic damage in the early stages of disease. This process may be reversible and so is of considerable therapeutic interest. Importantly, elevations in extracellular glutamate are not necessary to invoke an excitotoxic mechanism. Excitotoxicity can come into play even with normal levels of glutamate if NMDA receptor activity is increased, e. g., when neurons are injured and thus become depolarized (more positively charged); this condition relieves the normal block of the ion channel by Mg2þ and thus abnormally increases NMDA receptor activity [25]. Increased activity of the enzyme nitric oxide synthase (NOS) is associated with excitotoxic cell death. The neuronal isoform of the enzyme is physically tethered to the NMDA receptor and activated by Ca2þ influx via the receptor-associated ion channel, and increased levels of nitric oxide (NO) have been detected in animal models of stroke and several neurodegenerative diseases. 14.3.2

Links between Stroke and Vascular Dementia to Excitotoxic Damage

As stated earlier, the glutamate content of whole brain is approximately 10 mM. Because of the activity of glutamate transporters, most of this glutamate is intracellular. The extracellular glutamate concentration in brain has been estimated to be approximately 0.6 mM. The sensitivity to excitotoxicity of cultured cortical neurons isolated away from astrocytes or of hippocampal neurons in intact tissue is approximately 2 to 5 mM glutamate. Therefore, the ambient concentrations of glutamate are close to those that can cause neuronal death, and it is important that extracellular glutamate concentration and compartmentalization be exquisitely controlled to prevent excitotoxicity. On the other hand, with 10 mM glutamate present in cells, the potential for disaster is obviously great.

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Extracellular glutamate levels have been shown to rise in the face of hypoxicischemic insults. There will probably prove to be several mechanisms for the excess accumulation of glutamate, even in a single disorder such as ischemia (for a review, see [1]). Energy failure might cause abnormal accumulations of glutamate either by impairment of uptake (into neurons and especially astrocytes) mediated via glutamate transporters or by reversal of the direction of transport. This series of events would be followed by injury to some neurons and abnormal potentiation of glutamate release from others. With glutamate release from injured neurons, and excess physiological release from otherwise intact neighboring neurons, the process might then develop a self-propagating character that extends the area of neuronal damage. Deprivation of oxygen and glucose, for example during ischemia, cause a decrement in the production of high-energy phosphate compounds and “energy failure”. However, short-term energy failure per se is not particularly toxic to neurons. What does make energy failure highly neurotoxic is the activation of glutamate receptordependent mechanisms. If these are blocked by suitable glutamate antagonists, then neurons can survive a period of oxygen and metabolic substrate deprivation.

Possible Links between Excitotoxic Damage and Alzheimer’s Disease There are several potential links between excitotoxic damage and the primary insults of Alzheimer’s disease, which, based on rare familial forms of the disease, are believed to involve toxicity from misfolded mutant proteins (recently reviewed by [26]). These proteins include fibrillar b-amyloid peptide (Ab) and hyperphosphorylated tau proteins [27]. For example, oxidative stress and increased intracellular Ca2þ generated by Ab have been reported to enhance glutamate-mediated neurotoxicity in vitro. Additional experiments suggest that Ab can increase NMDA responses and thus excitotoxicity [28–30]. Another potential link comes from recent evidence that glutamate transporters are down regulated in Alzheimer’s disease and that Ab can inhibit glutamate reuptake or even enhance its release [31, 32]. Finally, excessive NMDA receptor activity has been reported to increase the hyperphosphorylation of tau, which contributes to neurofibrillary tangles [33]. The NMDA receptor antagonist memantine has been found to offer protection from intrahippocampal injection of Ab [34]. Moreover, memantine improved performance on behavioral tests (T-maze and Morris water maze) in a transgenic mouse model of Alzheimer’s disease consisting of a mutant form of amyloid precursor protein and presenilin 1 [35]. Additionally, memantine was recently found to reduce tau hyperphosphorylation, at least in culture [36]. 14.3.2.1

14.3.3

Pathophysiology of Excitotoxicity: Role of the NMDA Receptor

Apoptotic-like excitotoxicity is caused in part by excessive stimulation of the NMDA subtype of glutamate receptor (Fig. 14.1). When activated, the NMDA receptor opens a channel that allows Ca2þ (and other cations) to move into the cell. In

14.3 Excitotoxicity

Figure 14.1 Schematic illustration of the apoptotic pathways triggered by excessive NMDA receptor activity. The cascade of steps leading to neuronal cell death include: (1) NMDA receptor (NMDA-Rc) hyperactivation; (2) activation of the p38 mitogen activated kinase (MAPK) – MEF2C (transcription factor) pathway (MEF2 is subsequently cleaved by caspases to form an endogenous dominant-interfering form that contributes to neuronal cell death [71]; (3) toxic effects of free radicals such as nitric oxide (NO) and reactive oxygen species (ROS); and (4) activation of apoptosis-inducing enzymes including caspases and apoptosis-inducing factor. nNOS: nitric oxide synthase; Cyt c: cytochrome c. (Adapted from the Lipton website at www.burnham.org).

some areas of the brain, this activity is important for long-term potentiation (LTP), thought to be a cellular/electrophysiological correlate of learning and memory formation. Under normal conditions of synaptic transmission, the NMDA-receptor channel is blocked by Mg2þ sitting in the channel and only activated for brief periods of time. Under pathological conditions, however, overactivation of the receptor causes an excessive amount of Ca2þ influx into the nerve cell, which then triggers a variety of processes that can lead to necrosis, apoptosis, or dendritic/synaptic damage. These processes include Ca2þ overload of mitochondria, resulting in oxygen free radical formation, activation of caspases and release of apoptosis-inducing factor, Ca2þ -dependent activation of neuronal NOS, leading to increased NO production and the formation of toxic peroxynitrite (ONOO-); and stimulation of mitogen-activated protein kinase p38 (MAPK p38), which activates transcription factors that can go into the nucleus to influence neuronal injury and apoptosis [18, 37–43]. As mentioned above, conventional NMDA receptors consist of two subunits (NR1 and NR2A-D), and more rarely NR3A or B subunits. There are binding sites for glutamate, the endogenous agonist, and glycine, which is required as a co-agonist for receptor activation (Fig. 14.2) [44]. NMDA is generally not thought to be an endogenous substance in the body; it is an experimental tool that is highly selective for this subtype of glutamate receptor and therefore became the source of its name. When glutamate and glycine bind and the cell is depolarized to remove Mg2þ block, the NMDA-receptor channel opens with consequent influx of Ca2þ and

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Figure 14.2 NMDA receptor model illustrat-

ing important binding and modulatory sites. Glu or NMDA: glutamate or NMDA binding site. Gly: glycine binding site. Zn2þ : zinc binding site. NR1: NMDA receptor subunit 1. NR2: NMDA receptor subunit 2A. SNO: cysteine sulfhydryl group (-SH) reacting with nitric oxide species (NO). X: Mg2þ, MK-801, and memantine binding sites within the ion channel pore region.

Naþ into the cell, the amount of which can be altered by higher levels of agonists and by substances binding to one of the modulatory sites on the receptor. The two modulatory sites that are most relevant to this review are the magnesium (Mg2þ) site within the ion channel and an S-nitrosylation site located toward the N-terminus (and hence extracellular region) of the receptor. Note that S-nitrosylation reactions represent transfer of NO to a thiol or sulfhydryl group (-SH) of a critical cysteine residue. This reaction modulates protein function, in this case decreasing channel activity associated with stimulation of the NMDA receptor. Each of these sites can be considered as targets for therapeutic intervention to block excitotoxicity, as explained below. Moreover, other modulatory sites also exist on the NMDA receptor and may in the future prove to be of therapeutic value. These include binding sites for Zn2þ, polyamines, the drug ifenprodil (the endogenous ligand remains unknown), and a pH (i. e., proton)-sensitive site – for a review see reference [45]. Additionally, 3 pairs of cysteine residues can modulate channel function by virtue of their redox sensitivity [46]. To be clinically acceptable, an anti-excitotoxic therapy must block excessive activation of the NMDA receptor while leaving normal function relatively intact in order to avoid side effects. Drugs that simply compete with glutamate or glycine at the agonist binding sites block normal function and therefore do not meet this requirement, and have thus failed in clinical trials to date because of side effects (drowsiness, hallucinations, and even coma) [1, 8, 12, 47–51]. Competitive antagonists compete one for one with the agonist (glutamate or glycine) and therefore will block healthy areas of the brain (where lower, more physiological levels of these agonists exist) before they can affect pathological areas (where higher levels of agonist accumulate). Thus, in fact, such drugs would preferentially block normal activity and would most likely be displaced from the receptor by the high concentrations of glutamate for prolonged periods that can exist under excitotoxic conditions.

14.3 Excitotoxicity

The Importance of Off-Rate from Channel Block As a useful analogy, the NMDA receptor can be thought of like a television set. The agonist sites are similar to the “on/off” switch of the television. Drugs that block here cut off all normal NMDA receptor function. What we need to find is the equivalent of the volume control (or in biophysical terms, the gain) of the NMDA receptor. Then, when excessive Ca2þ fluxes through the NMDA receptorassociated ion channel, we would simply turn down the “volume” of the Ca2þ flux more towards normal. A blocker that acts at the Mg2þ site within the channel could act in such a manner. However, in the case of Mg2þ itself, the block is too ephemeral, a so-called “flickery block,” and the cell continues to depolarize until the Mg2þ block is totally relieved. Hence, in most cases Mg2þ does not effectively block excessive Ca2þ influx to the degree needed to prevent neurotoxicity. If, on the other hand, a channel blocker binds too tightly or works too well at low levels of receptor activation, it will block normal as well as excessive activation and be clinically unacceptable. Following the television set analogy, turning the volume all the way down is as bad as turning off the “on/off” switch in terms of blocking normal functioning of the television. This is the case with MK-801; it is a very good excitotoxicity blocker, but because its “dwell time” in the ion channel is so long (reflecting its slow “off-rate”) due to its high affinity for the Mg2þ site, it also blocks critical normal functions. A human taking a neuroprotective dose of MK-801 would not only become drowsy, but would lapse into a coma. Drugs with slightly shorter but still excessive dwell times (off-rates) make patients hallucinate (e. g., phencyclidine, also known as Angel Dust), or so drowsy that they can serve as anesthetics (e. g., ketamine). A clinically tolerated NMDA receptor antagonist would not make a patient drowsy, hallucinate, or comatose, and in fact should spare normal neurotransmission while blocking the ravages of excessive NMDA receptor activation. In fact, one type of drug that would do this and block preferentially higher (pathological) levels of glutamate over normal (physiological) levels would be an “uncompetitive” antagonist. An uncompetitive antagonist is distinct from a noncompetitive antagonist (which simply acts allosterically at a noncompetitive site, i. e., a site other than the agonist-binding site). An uncompetitive antagonist is defined as an inhibitor whose action is contingent upon prior activation of the receptor by the agonist. Hence, the same amount of antagonist blocks higher concentrations of agonist better than lower concentrations of agonist. This uncompetitive mechanism of action coupled with a longer dwell time in the channel (and consequently a slower “off-rate” from the channel) than Mg2þ but a substantially shorter dwell time (faster off-rate) than MK-801 would yield a drug that blocks NMDA receptor-operated channels only when they are excessively open while relatively sparing normal neurotransmission. Our experiments suggested that memantine is such a drug [1, 8–11]. Moreover, memantine blocks all NMDA receptor-oriented channels that are comprised of NR1 and NR2A-D subunits. Note that our principal discovery that led to the testing of a clinically successful NMDA receptor antagonist was the kinetics of the drug in the NMDA receptorassociated ion channel [1, 8–11]: we found that the dwell time in (or off-rate 14.3.3.1

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from) the channel is the major determinant of clinical tolerability of an open-channel blocker because excessive dwell time (associated with a slow off-rate) causes the drug to accumulate in the channels, interfere with normal neurotransmission and produce unacceptable adverse effects (as is the case with MK-801). In contrast, too short a dwell time (reflecting too fast an off-rate) yields a relatively ineffectual blockade, especially with membrane depolarization, which relieves the block of positively charged molecules (as seen with Mg2þ). The apparent affinity of a positively charged channel blocker like memantine at a particular membrane voltage is related to its off-rate divided by its on-rate (derived from Scheme 1, in the Appendix). The on-rate is a property of the channel’s open probability and the drug’s diffusion rate and concentration. On the other hand, the off-rate is an intrinsic property of the drug-receptor complex, unrelated to drug concentration (see reference [9] for a detailed quantitative discussion of these points). A relatively fast off-rate is a major contributor to a drug like memantine’s low affinity for the channel pore. Thus, memantine represents a class of relatively low-affinity, open-channel blockers, i. e., these drugs only enter the channel when it is opened by agonist. In the case of memantine, at concentrations administered to patients, the drug appears to enter the channel preferentially when it is (pathologically) activated for long periods of time, e. g., under conditions of excessive glutamate exposure. The relatively fast off rate prevents the drug from accumulating in the ion channels and interfering with normal synaptic transmission. Hence, as we showed previously [9], memantine has favorable kinetics in the channel to provide neuroprotection while displaying minimal adverse effects (occasional restlessness or, in rare cases, slight dizziness at higher dosages) [1, 8].

14.4

Memantine 14.4.1

Background and Pharmacology: Uncompetitive Open-channel Block

Memantine was first synthesized by Eli Lilly and Company and patented in 1968, as documented in the Merck Index, as a derivative of amantadine, an anti-influenza agent. Memantine has a three-ring (adamantane) structure with a bridgehead amine (-NH2) that under physiological conditions carries a positive charge (-NH3þ) and binds at or near the Mg2þ site in the NMDA receptor-associated channel (Fig. 14.3) [8–11, 52]. Unlike amantadine, memantine has two methyl (-CH3) side groups that prolong its dwell time in the channel (hence, slowing the offrate and increasing the affinity for the channel somewhat compared to amantadine). The reported efficacy of amantadine and memantine in Parkinson’s disease, which was discovered by serendipity in a patient taking amantadine for influenza, led scientists to believe that these compounds were dopaminergic or possibly anticholinergic drugs. It wasn’t until the late 1980s and early 1990s that memantine was found to be neither dopaminergic nor anticholinergic at its clinically used do-

14.4 Memantine

Figure 14.3 Chemical structure of memantine. Several important features are – (1) the three-ring structure; (2) the bridgehead amine (-NH2 group), which is charged at the physiological pH of the body (-NH3þ) and represents the region of memantine that binds at or near the Mg2þ binding site in the NMDA receptor-associated ion channel; and (3) the methyl group (-CH3) side chains (unlike amantadine), which serve to stabilize memantine’s interaction in the channel region of the NMDA receptor.

sage, but rather, an NMDA receptor antagonist. Work at a small German company named Merz first suggested that the drug was a quite “potent” NMDA receptor inhibitor [53], which in fact it is not. Instead, my laboratory found that under physiological conditions the drug is of such weak potency at the NMDA receptor (affinity in the micromolar range rather than nanomolar or higher) that big Pharma initially thought that it was a poor candidate as a neuroprotective drug. However, one should not confuse affinity with selectivity; as long as a drug acts selectively and specifically on the target of interest and the effective concentration can be achieved, a high affinity per se is not the key issue. In fact, work in my laboratory first showed why memantine could be clinically tolerated as an NMDA receptor antagonist; namely, it was an uncompetitive open-channel blocker with a dwell time/off-rate from the channel that limited pathological activity of the NMDA receptor while sparing normal synaptic activity [9–11]. These findings led to a number of U. S. and world-wide patents on the use of memantine for NMDA receptor-mediated disorders and spurred on several successful clinical trials with the drug, as discussed below. (By way of disclosure, my laboratory was then at Harvard Medical School, so patents on which I am the named inventor are assigned to Harvard-affiliated institutions, including Children’s Hospital of Boston). To illustrate the blockade of NMDA-induced ionic currents by memantine, a sample experiment is shown in Fig. 14.4 in which the membrane voltage of a neuron was held at the resting potential. The concentration of memantine used in this experiment is similar to the level that can be achieved in human brain when the drug is used clinically. At such concentrations, memantine greatly reduces pathologically high levels of NMDA-induced current to near zero within approximately 1 second. Once the memantine application stops, the NMDA response returns

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Figure 14.4 Blockade of NMDA current by memantine. At a holding potential of approximately –50 mV, whole-cell recording of NMDA-evoked current from a solitary neuron revealed that the on-time (time until peak blockade) of micromolar memantine was approximately 1 sec, while the off-time (recovery time) from the effect was Z5.5 sec. The application of memantine produced an effective blockade only during NMDA receptor activation, consistent with the notion that its mechanism of action is open-channel block (after ref. [10]).

to previous levels over a period of about 5 seconds. This indicates that memantine is an effective, but temporary, NMDA receptor blocker. Perhaps the most astonishing property of memantine is illustrated in Fig. 14.5 [8, 10]. In this experiment, the concentration of memantine was held constant (at a clinically achievable level of 1 mM) while the concentration of NMDA was increased over a wide range. It was found that the degree to which this fixed concentration of memantine blocked NMDA receptor activity actually increased as the NMDA concentration was increased to pathological levels. In fact, memantine was relatively ineffective at blocking the low levels of receptor activity associated with normal neurological function, but became exceptionally effective at higher concentrations. This is classical “uncompetitive” antagonist behavior. Further studies indicate that memantine exerts its effect on NMDA receptor activity by binding at or near the Mg2þ site within the ion channel [8–11, 52]. This information and the pharmacological/kinetic data presented above suggest that memantine preferentially blocks NMDA receptor activity if the ion channel is excessively open. During normal synaptic activity, the channels are open on average for only several milliseconds, and memantine is unable to act or accumulate in the channels; hence, synaptic activity continues essentially unabated. In technical terms, the component of the excitatory postsynaptic current due to activation of NMDA receptors is inhibited by only 10 % or less [11]. During prolonged activation

14.4 Memantine

Paradoxically, a fixed dose of memantine (i. e., 1 mM) blocks the effect of increasing concentrations of NMDA to a greater degree than lower concentrations of NMDA. This finding is characteristic of an uncompetitive antagonist (after ref. [10]).

Figure 14.5

of the receptor, however, as occurs under excitotoxic conditions, memantine becomes a very effective blocker. In essence, memantine only acts under pathological conditions without much affecting normal function, thus relatively sparing synaptic transmission, preserving long-term potentiation and maintaining physiological function on behavioral tests such as the Morris water maze [11]. The kinetics of memantine action in the NMDA receptor-associated ion channel explain the favorable clinical safety profile that has been seen to date. 14.4.2

Voltage Dependence, Partial Trapping, and Other Possible Effects of Memantine

We were the first to find that memantine block is voltage-dependent like Mg2þ [9, 10] (recall that memantine is positively charged at physiological pH). Hence, during the depolarization caused by excitatory neurotransmission (excitatory postsynaptic potentials), memantine block might be partially relieved. Nonetheless, we have shown that even when voltage-clamped to avoid such depolarization, memantine does not block synaptic activity to any substantial degree [11]. Hence, the voltage-dependence of memantine is not the major factor for its lack of effect on synaptic activity. Rather, the relatively fast off-rate from the ion channel is the cri-

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tical feature, as explained above. Additionally, our group [9] and Blanpied et al. [54] demonstrated that memantine can be trapped in the NMDA receptor-associated ion channel (i. e., the channel could shut behind memantine and trap it in the closed channel). However, since memantine does not block a significant proportion of the synaptic activity (and thus is not in the channels for a prolonged period during normal neurotransmission), we do no feel that trapping in the channel is a major factor under physiological conditions. Additionally, closed-channel block (entering the channel before it has opened) has been proposed as a mechanism of action of memantine [55], but this occurs only at very high concentrations of the drug, so we feel that this action is not pathophysiologically relevant under the conditions that memantine is used clinically [9]. Finally, memantine has provisionally been found to block 5-HT3 receptor-channels at concentrations similar to those that block the NMDA receptor-channels and, at somewhat higher concentrations, memantine may also block nicotinic receptor-channels [9–11, 56, 57, reviewed in 26]. Memantine’s effect on 5-HT3 receptors may possibly further enhance cognitive performance. 14.4.3

Neuroprotective Efficacy

The neuroprotective properties of memantine have been studied in a large number of in vitro and in vivo animal models by several laboratories (for a recent review, see reference [58]). Among neurons protected in this manner both in culture and in vivo are cerebrocortical neurons, cerebellar neurons, and retinal neurons [8–11, 21, 59–62]. Additionally, in a rat model of stroke, memantine, given as long as 2 hours after the ischemic event, reduces the amount of brain damage by approximately 50 % [10, 11]. A series of human clinical trials have been launched to investigate the efficacy of memantine for the treatment of Alzheimer’s disease, vascular dementia, HIV-associated dementia, diabetic neuropathic pain, depression, as well as glaucoma. Some of these studies have only recently been completed and remain unpublished at this time except in abstract form. One very recent, high-profile publication reported the results of a U. S. phase 3 (final) clinical study, showing that memantine (20 mg/ day) is efficacious for moderate-to-severe Alzheimer’s disease [12]. Another study reported in abstract form that, in combination with Aricept, memantine treatment actually improves memory and function in moderate-to-severe Alzheimer’s patients. The results of clinical studies for Alzheimer’s disease were sufficiently positive to convince the European Union to approve memantine for the treatment of this form of dementia last year, and the U. S. FDA also recently granted approval to the drug. One full-length European publication of a multi-center, randomized controlled trial reported that memantine was beneficial in severely demented patients, probably representting both Alzheimer’s disease and vascular dementia [63]. Still another recent publication of a randomized, placebo-controlled clinical trial described significant benefit from memantine therapy (20 mg/day) in mild to moderate vascular dementia [13]. These clinical trials are summarized in

14.4 Memantine Table 14.1

Clinical Trials with Memantine Y Y Y Y Y Y

German/Merz phase 3 trial for Vascular Dementia and Alzheimer’s Disease Karolinska/Italian phase 3 trial for Vascular Dementia Three USA Multicenter phase 3 trials for Alzheimer’s Disease United Kingdom phase 3 trial for Vascular Dementia French phase 3 trial of Vascular Dementia USA phase 2 trials for Neuropathic Pain and HIV-Associated Dementia

+ + + + + +/–

Table 14.1. Most trials have reported minimal adverse effects of memantine. In those trials reporting adverse effects, the only memantine-induced side effects encountered were rare dizziness and occasional restlessness/agitation at higher doses (40 mg/day), but these effects were mild and dose-related. Importantly, as outlined above, the anti-intuitive aspects of memantine action that we discovered show that MORE glutamate receptor activity is blocked better than less activity at a fixed dose of memantine. Hence, memantine would be expected to work better for severe conditions, e. g., excessive glutamate receptor activity to the point of causing cell death, than more mild conditions manifest by slightly elevated synaptic transmission. Bearing this out, recent studies suggest that memantine may have a larger effect in moderate-to-severe dementia than in mild dementia. Another case in point is neuropathic pain, which is currently thought to be mediated at least in part by excessive NMDA receptor activity. Given the mode of action that we described for memantine, more severe pain, e. g., the nocturnal pain of diabetic neuropathy, might be expected to benefit from memantine to a greater extent than more mild forms of neuropathic pain. In fact, a phase 2B clinical trial suggested that this is indeed the case, whereas more mild pain conditions were not statistically benefited by memantine in phase 2/3 clinical trials. Along these same lines, one would predict that a higher concentration of memantine would be needed to combat pain than to prevent neuronal cell death because greater NMDA receptor activity is associated with cell death (recall that the on rate of channel block by memantine can be increased by increasing the drug’s concentration. Then, a greater proportion of channels will be blocked even with less total channel activity). Again, clinical trials have suggested that this is indeed the case because 40 mg/day of memantine have been needed in successful pain studies but only 20 mg/day in dementia studies. However, further clinical trials will be necessary to prove the efficacy of memantine for severe neuropathic pain. The data thus far suggest that mild cases of neuropathic pain may not be helped by the drug because of its uncompetitive mechanism of action, whereby more intense NMDA channel activity is blocked to a relatively greater degree than mild increases in activity. As promising as the results with memantine are, we are continuing to pursue ways to use additional modulatory sites (the “volume controls”) on the NMDA receptor to block excitotoxicity even more effectively and safely than memantine alone. New approaches in this regard are explored below.

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14.5

NitroMemantines

NitroMemantines are second-generation memantine derivatives that were designed to have enhanced neuroprotective efficacy without sacrificing safety. As mentioned earlier, a nitrosylation site is located on the N-terminus or extracellular domain of the NMDA receptor, and S-nitrosylation of this site (NO reaction with the sulfhydryl group of the cysteine residue) down-regulates receptor activity (Fig. 14.2). The drug nitroglycerin, which generates NO-related species, can act at this site to limit excessive NMDA receptor activity. In fact, in rodent models, nitroglycerin can limit ischemic damage [64], and there is some evidence that patients taking nitroglycerin for other medical reasons may be resistant to glaucomatous visual field loss [65]. From crystal structure models and electrophysiological experiments, we have found that NO binding to the NMDA receptor at the major S-nitrosylation site apparently induces a conformational change in the receptor protein that makes glutamate and Zn2þ bind more tightly to the receptor. The enhanced binding of glutamate and Zn2þ in turn cause the receptor to desensitize and, consequently, the ion channel to close [46]. Electrophysiological studies have demonstrated this effect of NO on the NMDA channel [39, 66, 67]. Unfortunately, nitroglycerin is not very attractive as a neuroprotective agent. The same cardiovascular vasodilator effect that makes it useful in the treatment of angina could cause dangerously large drops in blood pressure in stroke, traumatic injury, or glaucoma patients. Consequently, we carefully characterized S-nitrosylation sites on the NMDA receptor in order to determine if we could design a nitroglycerin-like drug that could be more specifically targeted to the NMDA receptor. In brief, we found that 5 different cysteine residues on the NMDA receptor could interact with NO. One of these, located at cysteine residue #399 on the NR2A subunit of the NMDA receptor, mediates approximately 90 % of the effect of NO under our experimental conditions. Using this kind of information, we created modified memantine molecules called NitroMemantines that will interact with both the memantine site within the NMDA receptor-associated ion channel and the nitrosylation site. Two sites of modulation are analogous to having two volume controls on your television set for fine-tuning the audio signal. Preliminary studies have shown NitroMemantines to be highly neuroprotective in both in vitro and in vivo animal models. In fact, it appears to be substantially more effective than memantine. Moreover, because the memantine portion makes these drugs specific for the NMDA receptor and serves to target the NO group to the nitrosylation site on the receptor, NitroMemantines appear to lack the blood pressure lowering effect typical of nitroglycerin. More research still needs to be performed on NitroMemantine drugs, but the fact that they chemically combine two clinically tolerated drugs (memantine and nitroglycerin) enhances their promise as second-generation memantine-derivatives that are both clinically tolerated and neuroprotective.

14.6 Summary

14.6

Summary

Necrosis- and apoptosis-mediated excitotoxic cell death is implicated in the pathophysiology of many neurologic diseases, including stroke, CNS trauma, dementia, glaucoma, polyglutamine (triple repeat) disorders, and other neurodegenerative conditions. This type of excitotoxicity is caused, at least in part, by excessive activation of NMDA-type glutamate receptors. Intense insults, such as that occurring in the ischemic core after a stroke, triggers massive stimulation of NMDA receptors because of increased glutamate and energy failure, leading to membrane depolarization, relief of Mg2þ block of NMDA channels, and disruption of ionic homeostasis. The fulminant buildup of ions results in neuronal cell swelling and lysis (necrosis). In contrast, more moderate NMDA-receptor hyperactivity, such as that occurring in the ischemic penumbra of a stroke and in many slow-onset neurodegenerative diseases, results in moderately excessive influx of calcium ions into nerve cells which, in turn, triggers free radical formation and multiple pathways leading to the initiation of apoptotic-like damage [18, 68]. Blockade of NMDA receptor activity prevents to a large degree both necrosis- and apoptosis-related excitotoxicity. NMDA receptor activity is also required for normal neural function, however, so only those NMDA blockers that selectively reduce excessive receptor activation without affecting normal function will be clinically acceptable. We have shown that memantine is such a drug. Laboratory tests have demonstrated that memantine blocks excessive NMDA receptor activation but not normal, low-level activation. Importantly, the author, in collaboration with Vincent Chen (a graduate student at the time and currently a faculty member at The Burnham Institute and UC San Diego in La Jolla) reported that memantine had a relatively short dwell time in (and hence fast off-rate from) the NMDA-associated ion channel, in part explaining the drug’s relatively low apparent affinity as an antagonist [8–10]. We realized that the relatively short dwell-time/rapid off-rate from the channel was the predominant factor in determining the drug’s clinical tolerability as well as its neuroprotective profile [8]. Most importantly, this mode of action meant that memantine blocked high (pathological) levels of glutamate at the NMDA receptor while relatively sparing the effects of low (physiological) levels of glutamate seen during normal neurotransmission because the drug does not accumulate in the channel during normal synaptic activity. The discovery that memantine, a low-affinity but still highly selective agent with a mechanism of uncompetitive antagonism, is neuroprotective yet clinically tolerated triggered a paradigm shift in the history of drug development by the pharmaceutical industry [8–11]. Prior to that discovery, low-affinity drugs were thought to be inferior and not clinically useful. In particular, the relatively rapid off-rate from the NMDA receptor-associated ion channel of the memantine class of drugs largely accounts for its clinical tolerability as well as its low affinity. Clinical studies have borne out our hypothesis that low-affinity/ fast off-rate memantine is a safe NMDA receptor antagonist in humans and beneficial in the treatment of neurological disorders mediated, at least in part, by excitotoxicity.

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The NitroMemantines are second generation NMDA receptor antagonists that may work even better than memantine by using the memantine binding site for the targeted delivery of NO to a second modulatory site on the NMDA receptor. Work is progressing rapidly in this area of investigation. Further clinical studies of the efficacy of memantine in the treatment of Alzheimer’s disease, vascular dementia, HIV-associated dementia, glaucoma, depression, and severe neuropathic pain, are currently underway, and there is every reason to expect the results to be positive although this is, of course, not yet proven except in the case of moderate-to-severe Alzheimer’s disease and possibly vascular dementia in which phase 3 clinical trials have proven successful (Table 14.1) [12, 13, 69]. Hopefully, since memantine has shown promise for vascular (or multi-infarct) dementia, trials for acute stroke will also be undertaken. The efficacy of memantine in neurodegenerative diseases and its ability to protect neurons in animal models of both acute and chronic neurologic disorders suggest that memantine and drugs acting in a similar manner could become very important new weapons in the fight against neuronal damage [70].

Acknowledgments

I would like to thank my colleagues for their contributions to this work. I am especially grateful to Drs. Joachim Bormann, H.-S. Vincent Chen, Yun-Beom Choi, and Jonathan S. Stamler for their discussions or collaborations. This work was supported in part by NIH grants P01 HD29587, R01 EY50477, and R01 EY09024.

Appendix

kon Channel þ MEM lm Channel-MEM (blocked channel) koff

(1)

This simple bimolecular scheme predicts that the macroscopic blocking and unblocking actions of memantine (MEM) proceeds with exponential relaxation. The macroscopic pseudo-first order rate constant of blocking (kon) depends linearly on memantine concentration (as well as a constant, A), and the macroscopic unblocking rate (koff ) is independent of memantine concentration ([MEM]). kon ¼ A p [MEM]

(2)

koff : [MEM] independent

(3)

These predictions were borne out experimentally [9]. Both the macroscopic blocking and unblocking processes could be well fitted by a single exponential function. The macroscopic on-rate constant is related to the reciprocal of the measured time constant for onset (ton), which is the sum of the pseudo-first order blocking rate constant (kon), which unblocking constant (koff ). The unblocking rate constant

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(koff ) is the reciprocal of the measured macroscopic unblocking time constant (toff ). These transformations lead to Eqs. (4) and (5): kon ¼ 1/ton – 1/toff

(4)

koff ¼ 1/toff

(5)

The kon calculated from Eq. (4) increased linearly with memantine concentration with a slope factor of 0.4 e 0.03 106 M–1s–1 (mean e s. d.), while the koff from Eq. (5) remained relatively constant with a Y-axis intercept of 0.44 e 0.1 s–1 [9]. A rapid method to validate this result was obtained by estimating the equilibrium apparent dissociation constant (Ki) for memantine action from the following equation: Ki ¼ koff/(kon/[MEM])

(6)

Here we found empirically that memantine was a relatively low-affinity (apparent affinity Z 1 mM) open-channel blocker of the NMDA receptor-coupled ion channel, and a major component of the affinity was determined by koff at clinically relevant concentrations in the low micromolar range.

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15 Spinal Cord Trauma V. Dietz

Abstract

Paraplegia is due to a lesion of the spinal cord or the cauda. The neurological deficits concern motor, sensory and autonomic functions. The consequence is a dysfunction of the motor (spastic, para- or tetraplegia, flaccid paraplegia in cauda lesions), sensory (hyp- or anesthesia – algesia below the level of lesion), and autonomic (neurogenic, bladder, bowel and sexual dysfunction, cardiovascular dysfunction) spinal nervous systems. This chapter concerns the rehabilitation of sensorymotor systems. Recent progress in the understanding of movement control allows us to define more precisely the requirements for the successful rehabilitation of patients with motor deficits following a spinal cord injury (SCI). There is increasing evidence that a defective utilization of afferent input in combination with secondary compensatory processes is involved in typical movement disorders after central motor lesions. This has implications for therapy that should be directed to take advantage of the plasticity of the CNS. When signs of spinal shock disappear in patients with incomplete and complete paraplegia, a locomotor pattern can be induced and trained under conditions of body unloading using a moving treadmill. Load- and hip joint position-related afferent input seems to be of crucial importance for the generation and for the success of a locomotor training. In these patients, an increase of gastrocnemius electromyography activity occurs during the stance phase of a step cycle with a daily locomotor training, coincident with a signifycant decrease of body unloading. The locomotor pattern depends on the level of lesion, i. e. the higher the level of spinal cord lesion, the more ‘normal’ is the locomotor pattern. In the future a combination of various approaches, including interventions to induce some regeneration, will provide an optimal base for functional locomotor recovery also in severely affected para-/tetraplegic patients. For the assessment of the effectiveness of any new interventional therapy, standardized functional tests have to be established.


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15.1

Epidemiology

The prevalence of acute spinal cord injury (SCI) is estimated to be 30–50 cases per million population per year [1]. Paralysis of the upper and lower extremities, socalled quadriplegia or tetraplegia, accounts for 54 % of the injured patients; the remaining 46 % are paraplegic. Traumatic spinal cord injury predominantly occurs in young men (61 % are between 16 and 30 years old); 20 % or less of the patients are women. About half the patients have neurologically complete injuries when they are first seen.

15.2

Clinical Aspects 15.2.1

Acute Stage

An overview about clinical aspects of spinal cord lesion can be found elsewhere [2]. After a traumatic spinal cord injury, it is essential immediately to differentiate with certainty between para- and quadriplegia. If the level of injury within the cervical spinal cord ascends even a few millimeters, it can lead to a change in functional status from relative self-independency to complete dependency. History/anamnesis concerning the trauma is important to get information about the forces which acted upon the patient. In an unconscious patient, a cervical cord lesion can be recognized by breathing which is exclusively diaphragmatic. More caudal spinal cord lesions may eventually be detected only at a later stage. During the acute stage, the principal symptoms include loss of voluntary movements and sensory perceptions as well as dysfunction of bladder and bowel. When paresis of muscles in all four limbs is present, associated with absent sensation to pinprick within dermatomes C6 to Th1 [thumb (C6), little finger (C8), elbow (Th1)] and from the thorax downward, the lesion is at the level of the cervical cord causing quadriplegia. When only voluntary leg movements are impaired and sensation is lost in the legs, paraplegia can be assumed. In 1992, the American Spinal Cord Injury Association (ASIA) published standards for neurological and functional classification of spinal cord injuries that have been revised twice [3, 4]. Depending upon the motor and sensory deficits, a patient can be categorized according to the ASIA impairment if the deficits have been rated according to the standard ASIA chart. Reflex examination during the early stage after trauma provides information about the presence of spinal cord shock (which may last for weeks) during which tendon reflexes are absent. The initial traumatic lesion is in most cases associated with hemorrhage and edema within the spinal cord. Soon, at the site of the lesion, cellular infiltrates and ischemia occur, associated with tissue necrosis. Later, degenerative changes

15.2 Clinical Aspects

of bones and vertebral disks may occur, which can lead to secondary compression of the cord. Early after the trauma, spinal concussion should be distinguished from a spinal cord lesion. Spinal concussion represents an impairment of spinal cord functions with clinical symptoms that are completely reversible within 72 hrs [5]. A spinal cord lesion is associated with morphological changes and persistent clinical symptoms–incomplete or complete paraplegia or quadriplegia. Nevertheless, neurological deficits may recover during the first 3 months after a traumatic lesion. Interestingly, total unilateral transection of descending motor tracts, combined with 85–90 % transection of the contralateral tracts in human beings leads to total paralysis of the lower limbs that can, however, recover so that walking, albeit with spastic paraparesis, may return after 2 months [6]. The similarity of this recovery to effects of partial lesions in rats and cats suggests a common mechanism; that is, plasticity of local spinal circuits with adaptation to inputs by a small number of remaining descending axons. Recently, the injured rat spinal cord was shown to spontaneously form new intraspinal circuits [6a]. 15.2.2

Dysfunction of the Autonomic Nervous System

Spinal cord injuries, particularly lesions above T6, compromise sympathetic nervous system activity with consequences for cardiovascular function. Because sympathetic outflow from the cord occurs between T1 and L2, sympathetic tone throughout most of the body in quadriplegics and high-level paraplegics is severely or completely impaired (depending on the completeness and level of the injury). This results acutely in hypotension and bradycardia or, in the extreme, with increases in parasympathetic tone, in asystole. The latter tends to occur particularly during manipulations of the patientls oropharynx which produce increased vagal activity. Regulation of body temperature is usually impaired with lesions above T10 and, when the lesion is above C8, affects the whole body. This leads, for example, to hyperthermia when the patient is in a hot environment because equilibration of body temperature by peripheral vasodilatation and perspiration is impossible. Similarly, hypothermia results when the patientls environment is cold because vasoconstriction and shivering are no longer possible. Respiration is impaired with a lesion above T6 when most intercostal muscles are paralyzed. With a lesion above C5, diaphragmatic breathing also is impaired, so these patients usually require a respirator for several weeks to avoid prolonged atelectasis and pneumonia. Contractility of the bladder is lost after spinal cord injury and the bladder may remain areflexic for weeks to months, even in patients with upper motoneuron lesions in whom return of such reflexes is anticipated. Advances in management of the neurogenic bladder have constituted some of the most important developments in the care of patients with spinal cord injury. Following spinal shock, the

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form of bladder dysfunction depends on the level of lesion. Usually reflex bladder function develops, which, in some cases, can be used to empty it sufficiently. Following spinal cord injury, peristalsis ceases within 24 hr as a result of spinal shock and this produces gastrointestinal problems that usually persist for 3–4 days. Severe spinal cord lesions are always associated with impaired sexual functions. With lesions above the level of T9/10, reflex erections and ejaculations can eventually develop. 15.2.3

The Evolution of Signs and Symptoms

A severe spinal cord lesion is initially associated with spinal shock below the level of lesion. Spinal shock is characterized by flaccid paresis, absent tendon reflexes and failure of the autonomic nervous system (paralytic ileus and loss of bladder function). Some conus reflexes are preserved (e. g., the bulbocavernosus reflex). Spinal shock can last several (3–6) weeks. As it disappears, exaggerated tendon reflexes develop [7]. Persistent areflexia may result from myelomalacia below the level of lesion. Non-neuronal factors (e. g. transformation of muscle and connective tissue) contribute to the development of spastic hypertonia and the occurrence of muscle spasms (Fig. 15.1). This might also result in nociceptive input to the cord. Partial recovery of spinal cord function can take place during the first 2–3 months after trauma. In addition to the clinical examination, electrophysiological recordings improve predictions about the prognosis of motor and sensory deficits even at an early stage after the trauma and even in unconscious patients. By using nerve conduction studies, any contribution of peripheral lower motoneuron impairment can be assessed within the first few weeks after injury [8]. With these recordings, the outcome in terms of a flaccid versus spastic hand can be predicted in patients with cervical injuries and, consequently, appropriate physical and occupational therapy can be applied at an early stage. With a combination of neurographic

Figure 15.1 Schematic diagram

of the mechanisms that contribute to spastic paresis and spastic movement disorder. A central motor lesion leads to impaired reflex control by the CPG and a loss of supraspinal drive. The consequence is hyperexcitability of short latency reflexes and loss of long latency reflexes, as well as changes in muscle properties. The combination of all sequels of the primary lesion leads to spastic movement disorder. CNS: central nervous system; CPG: central pattern generator (from [39]).

15.3 Principles of Therapy

and somatosensory evoked potential (SSEP) recordings from ulnar and median nerves, eventual hand function (i. e., passive versus active hand function) can be predicted [9]. With early SSEP studies stimulating nerves in the legs combined with recordings of leg muscle potentials evoked by transcranial magnetic stimulation, prognosis for locomotor capability can be made [10, 11]. Spastic muscle tone develops over weeks and increases for several months; these changes in muscle tone are not reversible. When tendon reflexes reappear following spinal shock, a reflex bladder usually develops which makes special urologic management necessary.

15.3

Principles of Therapy 15.3.1

General Concepts

Treatment concepts for spinal cord injury fall into three separate time frames. Treatment of acute spinal cord injury is directed at interrupting the cascade of secondary injury processes thus limiting tissue damage. This approach seeks to arrest or even reverse sensory-motor dysfunction. The second therapeutic phase deals with consequences of spinal cord injury. Rehabilitative efforts aim to stabilize the current status and train reflexes and residual circuits to achieve optimal living conditions for the patient who has a given deficit. In the future, a third treatment approach will be directed toward enhancement of axonal regeneration. Many substances have been tested in experimental neurotrauma models, but few have been evaluated in extensive dose-response studies. Lack of such data has made it difficult for certain promising drugs to be taken into clinical trials so this exciting approach remains experimental at this time. Steroids have been used extensively in animal studies as well as in clinical trials [11, 12]. Treatment of spinal cord injury with steroids was initially based on their anti-inflammatory actions and their effectiveness in treating cerebral edema. There are several experimental studies on spinal cord injury with steroids (for review see [13]. Biochemical studies showed that very high doses of the synthetic steroid methylprednisolone also exert non-specific free radical scavenging effects [14]. A series of animal experiments, conducted while the first U. S. national acute spinal cord injury study (NASCIS 1) was in progress, suggested that higher doses of methylprednisolone than those used in that study might be needed in order to achieve a therapeutic response. Therefore, NASCIS 2 began in 1985 using high dose methylprednisolone, naloxone, or placebo [15]. Those patients who received methylprednisolone within 8 hrs following injury eventually achieved significantly better sensory and motor function than those who received placebo. Delayed treatment with methylprednisolone was, however, associated with decreased neurological recovery [16]. Because of its systemic effects, methylprednisolone may not be suitable for multiply traumatized patients.

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According to the NASCIS 3 trial [12], steroid therapy should be maintained for 48 hours if it is initiated 3 to 8 hrs after injury. In contrast to the results reported in NASCIS 2 and 3, recent studies in the rat failed to show a protective effect of methylprednisolone during the early phase of ischemic necrosis [17]. Instead, a pronounced anti-inflammatory effect with massive reduction of the recruitment of neutrophils and macrophages was observed. Recently, use of high dose methylprednisolone has been questioned because the initial evidence that it improves neurological recovery seems not to be supported by a systematic meta-analysis [18, 19]. Gangliosides are supposed to play an important role in normal neuronal development and differentiation [20]. A study testing the efficacy of combinations of GM-1 with high dose methylprednisolone [21] did not provide convincing evidence of a beneficial effect of this treatment [22]. The opiate receptor antagonist naloxone has been shown to facilitate sensorimotor recovery in cats after spinal cord contusion and to prevent the development of post-traumatic ischemia [13, 23]. However, a randomized clinical trial in spinal cord injured patients failed to show any significant improvement with naloxone compared with placebo [15]. The noncompetitive NMDA antagonist dizocilpine was shown to improve multiple pathophysiological parameters following spinal cord injury and ischemia [24]. Serious side effects of competitive as well as noncompetitive NMDA blockers have hindered clinical trials of them so far. 15.3.2

Transition from Acute Care to Rehabilitation

Rehabilitation of a patient with spinal cord injury should initially be conducted together with acute care. Management must flow continuously from emphasis on acute care through the steps leading up to the ultimate rehabilitative efforts. The length of time a patient should be kept immobilized varies with the type of injury, its location, and whether surgery has been performed. Spine surgery is usually performed to facilitate early mobilization. After cervical injuries, early mobilization should be undertaken with caution. Patients who are ambulatory should use halo devices for 6 to 8 weeks. A body jacket is used by all patients with thoracolumbar or lumbar injuries. The degree of functional independence achieved after mobilization depends on the level and the severity of the lesion and the adequacy of training. Recovery of functions lost because of neurapraxic lesions of spinal nerve fibers can occur during the first 3–4 months after trauma. 15.3.3

Basic Aspects of Motor Rehabilitation: Neuronal Activity of the Isolated Spinal Cord

Early descriptions of involuntary stepping movements generated by the spinal cord in human subjects with complete paraplegia date back to the work of Lhermite [25] and Kuhn [26]. Later on, in a patient with a complete spinal cord lesion, rhythmic

15.4 Current Approaches in Motor Rehabilitation

contractions of the trunk and lower limb extensor muscles were described [27, 28]. This rhythmic activity could be stopped, induced and modulated by peripheral stimulation of flexor reflex afferents [29]. In line with this, in a patient with spinal cord injury to the cervical spinal cord, involuntary step-like movements were observed [30]. Furthermore, in persons with complete spinal lesion, spinal cord stimulation is followed by “stepping movements” with reciprocally organized EMG-activity of symmetric muscles of both sides [31]. Further indirect evidence for a spinal pattern generator for locomotion has been suggested from recordings of locomotor activity induced in patients with complete paraplegia standing on a moving treadmill with body weight support [32–34]; for review see [35, 35a]. According to such experiments, it appears that the “completeness” of the locomotor pattern depends on the level of lesion, i. e. the higher the lesion the more “normal” is the pattern [36]. This would imply that neuronal circuits up to a cervical level contribute to the locomotor activity, as it was suggested for the mudpuppy [37]. Recently, locomotor movements were induced by a driven gait orthosis (DGO) that allows to induce leg movements even with 100 % body unloading [38]. In such a condition, physiological locomotor-like movements alone do not lead to leg muscle activation. Only in combination with loading of the legs are stepping movements associated with patterned leg muscle activation in patients with complete para/tetraplegia as well as healthy subjects. This indicates that stretch reflexes play only a minor role in the generation of leg muscle activity in these patients [38]. According to these studies, the appearance of a locomotor pattern seems to depend on afferent input from “load receptors” in combination with hip joint positionrelated afferent input.

15.4

Current Approaches in Motor Rehabilitation

There is increasing evidence that a defective utilization of afferent input in central motor diseases, in combination with secondary compensatory processes is involved in typical movement disorders, such as occur after central motor lesions. Furthermore, it became evident from cat experiments that neuronal networks underlying the generation of motor patterns are quite flexible after central or peripheral neural lesions [39, 40]. This has implications for therapy. The aim of rehabilitation should be focused on the improvement of function by taking advantage of the plasticity of neuronal centers, rather than the correction of isolated clinical signs, such as reflex excitability. For the monitoring of outcome and for the assessment of the effectiveness of any interventional therapy, standardized functional tests should be established.

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15.4.1

Spinal Reflex Plasticity

There is increasing evidence that the isolated spinal cord exhibits some neuronal plasticity. Evidence for such plasticity at the spinal level has been obtained for the relatively simple monosynaptic reflex arc [41]. Monkeys could either be trained to voluntarily increase or decrease the amplitude of the monosynaptic stretch reflex in response to an imposed muscle lengthening [41], as well as of its analogue, the H-reflex [42]. The fact that the training effects persist after spinal cord transection [43] indicates that some kind of learning by neuronal circuits within the spinal cord is possible. Similarly, humans can be trained to change the gain of the monosynaptic stretch reflex [44]; for review see [45]. The idea that the spinal cord can learn is also supported by studies of spinal reflex conditioning. Simple hindlimb motor responses to cutaneous or electrical stimulation are enhanced in animals with transected spinal cords via classical reflex conditioning (i. e. pairing the stimulus with another stimulus that evokes a stronger motor response) [46]. These reflex responses are enhanced within minutes of conditioning, indicating that synaptic efficacy along the reflex arc has changed, perhaps through long-term potentiation [46]. 15.4.2

Use-Dependent Plasticity – Rehabilitative Approaches

There is convincing evidence in spinal animals that a use-dependent phenomenon exists that can be attributed to some form of learning [47, 48]. When stepping has been practiced in spinal cats, this task can be performed more successfully than when it has not been practiced [49, 50], indicating a use-dependent plasticity of the spinal cord. The training of any motor task provides sufficient stimulation to initiate reorganization of neural networks within the spinal cord and, for example, to generate locomotion. Consequently, the loss of motor capacity following neural injury can become more severe when locomotor networks are no longer used, for example following a spinal cord injury [47]. In contrast, a much greater level of functional recovery might be possible if the concept of use-dependence is applied in both clinical and rehabilitative settings [47]. A considerable degree of locomotor recovery in mammals with a spinal cord injury can be attributed to a reorganization of spared neural pathways [10, 11]; for review see [48]. It has been estimated that if as little as 10 % of the descending spinal tracts are spared, some locomotor function can recover [51, 52]. If the loss of supraspinal input to the spinal cord is complete, these neuronal networks that exist below the level of the lesion adapt to generate locomotor activity even in the absence of supraspinal input [53–55].

15.4 Current Approaches in Motor Rehabilitation

15.4.3

Task-specific Plasticity

It is becoming clear that the plasticity of the sensory-motor networks of the adult mammalian lumbo-sacral spinal cord is greater than has been previously assumed [56]; for review see [40, 47]. Subsequently, the most prominent aspect of research on CPGs has been the detailed assessment of the modifiability of neuronal network function [48, 57–59]. The lumbo-sacral spinal cord obviously can execute stepping or standing more successfully if that specific task is practiced. Observations in spinal cats indicate that if the training of a motor task is discontinued and no other task is subsequently trained, then the performance of the task previously trained is degraded [47]. Consequently, plasticity can be exploited for rehabilitative purposes, using specific training approaches following a neural injury. In the cat, recovery of locomotor function following spinal cord transection can be improved using regular training, even in adult animals [60, 61]. The provision of an adequate sensory input during such treadmill training is of great importance for achieving an optimal locomotor output of the spinal neuronal circuitry. Correspondingly, in association with hindlimb exercise, reflex activity becomes normalized in adult rats following spinal cord transection [62]. Exercise obviously helps to normalize the excitability of spinal reflexes. Several neurotransmitter systems within the spinal cord (glycinergic and GABAergic systems) are suggested to be involved in the adaptation to repetitive use [47]. In animals with a spinal cord transection, stepping can be induced by the administration of the nor-adrenergic agonist clonidine, which enhances the activity in the locomotor generative spinal circuits [63]; for review see [53, 54]. Furthermore, serotonin seems to be involved in the production of locomotor rhythm [64]. Training paradigms of stepping and standing can modify the efficacy of the inhibitory neurotransmitter, glycine [47]. For example, when strychnine is administered to a chronic spinal cat that has acquired the ability to step successfully, there is little change in its locomotor capability. If it is administered to a standtrained cat, it becomes able to step successfully with body support [47, 56]. These findings suggest that the effect of strychnine is in so far specific in its action as it enables spinal networks to integrate sensory input by reducing inhibition [53, 54]. 15.4.4

Effects of Locomotor Training in Spinal Cord Injury

As established elsewhere, the coordination of human gait seems to be controlled in much the same way as in other mammals (for reviews see [65–67]). Therefore, it is not surprising that in persons with a complete or incomplete paraplegia, due to a spinal cord injury, locomotor EMG activity and movements can be both elicited and trained in a similar way as in the cat. This is achieved by partially unloading (up to 80 %) patients who are standing on a moving treadmill [32, 68, 69]; for review see [66]. In severely affected patients, the leg movements usually have to be assisted

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externally, especially during the transmission from stance to swing. The approach is based on findings made in cats and rats and is directed to train functional movements. It is supposed that by moving the limbs through trajectories under close physiological conditions central spinal neuronal circuits become activated by appropriate sensory inputs. The timing of the pattern of leg muscle EMG activity recorded in such a condition is similar to that seen in healthy subjects. However, the amplitude of leg muscle EMG is considerably reduced and less well modulated. This makes body-unloading necessary for locomotor training. The analysis of the locomotor pattern in complete paraplegic patients indicates that it is unlikely to be due to rhythmic stretches of the leg muscles, because leg muscle EMG activity is, as in healthy subjects, equally distributed during muscle lengthening and shortening [70]. In addition, recent observations indicate that locomotor movements induced in patients who are completely unloaded do not lead to leg muscle activation [38]. This implies that the generation of leg muscle EMG patterns in these patients is programmed at a spinal level rather than generated by stretch reflexes. During the course of daily locomotor training, the amplitude of the EMG in the leg extensor muscles increases during the stance phase and inappropriate leg flexor activity decreases. Such training effects are seen both in complete and incomplete paraplegic patients [32, 68]. These training effects lead to a greater weight-bearing function of the extensors, i. e. body unloading during treadmill locomotion can be reduced during the course of training. This indicates that the isolated human spinal cord has the capacity not only to generate a locomotor pattern but also to show some plasticity. However, only patients with incomplete paraplegia benefit from the training program in so far as they can learn to perform unsupported stepping movements on solid ground [32, 68]. In complete paraplegic patients, the training effects on leg muscle activation becomes lost after training has been stopped [55]. There are several reports about the beneficial effect of locomotor training in incomplete paraplegic patients [71, 72]; for review see [35] and patients who undergo locomotor training have a greater mobility compared to a control group without training [73]. Afferent input from receptors signaling contact forces during the stance phase of gait is essential for the activation of spinal locomotor centers (cf. [34, 38] and for achieving training effects in paraplegic patients [32, 68]. Furthermore, hip joint-related afferent input seems to be essential to generate a locomotor pattern [38]. The improvement of locomotor activity can also be attributed to some spontaneous recovery of spinal cord function that can occur over several months following a spinal cord injury [11]. However, recent observations indicate in both incomplete and complete paraplegic patients, that the increase of leg extensor EMG activity also occurs independently of the spontaneous recovery of spinal cord function, as assessed by clinical and electrophysiological means [11, 69]. Therefore, one might conclude that in persons suffering a spinal cord injury specific training effects on spinal locomotor centers lead to a similar improvement of locomotor function, as described for the spinal cat [69]. However, part of the recovery in loco-

15.5 Search for Reliable Clinical Assessment

motion might also be attributed to changes that occur in the muscles during the training period [69] similar as observed in the rat [47]. Unfortunately, patients with complete or almost complete paraplegia do not, as yet, profit from locomotor training for their mobility. In the future, these patients may profit from a combination of regeneration and exploitation of neuronal plasticity, as the research in spinal cord regeneration appears to be quite encouraging (for review see [74]).

15.5

Search for Reliable Clinical Assessment

Neurorehabilitation is one of the most expanding fields in medicine leading to increasing costs for other forms of treatment. However, little is known about the effect of any rehabilitative program and, therefore, new standardized functional tests have to be established. 15.5.1

Physical Signs and Function

Movement disorders are usually the first and most pronounced symptoms indicating an impaired functioning of motor centers. The physical signs obtained during clinical examination, such as reflex excitability and muscle tone, can lead to diagnosis, but give little information about the pathophysiology, course and appropriate treatment of a movement disorder. For example, stretch reflex excitability and muscle tone are fundamentally different between passive (clinical examination) and active (movement) conditions. An appropriate treatment should not be cosmetic, i. e. the correction of an isolated clinical parameter which does not affect function. For an adequate therapy of a movement disorder, it is essential to understand and analyze the function of reflexes and motor centers involved in the particular motor task that is impaired. This requires an evaluation of the behavior and function of neuronal (EMG) and biomechanical (joint movement) measurements, as any changes within these systems may lead to a movement disorder. Nevertheless, one has to be aware that any change in a measurement may be secondary or compensatory to the primary dysfunction of the motor systems involved in the impaired movement. The effect of any treatment, either by drugs or physical therapy, has to be assessed on the basis of function. An appropriate evaluation of a therapeutical effect on function requires (1) that the spontaneous recovery of function be separated from the effect of any therapy; (2) that the intensity and duration of a particular physical therapy, which strongly influences its effect [75] be determined and, (3) that patientsl functional impairments become comparable by internationally standardized scores for classification.

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15.5.2

Appropriate Assessment of Function – Basis for New Interventional Therapies

At present it is a common, well-accepted approach to score isolated clinical measures, such as reflex excitability, muscle tone, or voluntary force of single muscles. For example, muscle tone and spasm frequency can be assessed by the Ashworth scale and Penn spasm frequency scale, respectively [76]. For patients with spinal cord injury (SCI), the American Spinal Injury Association (ASIA) has developed a standardized neurological assessment, i. e. the ASIA-classification of motor and sensory deficits [4]. The first question is whether such scoring systems can serve as a sensitive outcome measure for new interventional therapies, and secondly, whether they can reflect the functional impairment, which is the most important aspect in terms of a patients’ quality of life. Only recently has a score been developed which relates to function. Locomotor ability has been classified into 19 items [77]. A current study indicates that a close relationship between motor scores and locomotor ability exists only in patients with moderately impaired motor function. Patients with a low motor score undergoing an intensive locomotor training can achieve an improved locomotor function without a change in motor score (Fig. 15.2). In these cases, relatively little voluntary force in the leg muscles (reflected in the ASIA score) is required to achieve the ability to walk, as long as functional training is applied. For the future, the effectiveness of any new interventional therapy should be assessed by internationally accepted functional scores in combination with motor scores of selected limb muscles. Motor and sensory scores are most likely to reflect the spontaneous recovery of function, as they depend on the integrity of corticospinal connections. In contrast, improvement of locomotor function after SCI also reflects the plasticity of neuronal spinal centers below the level of lesion. With the combined assessment of voluntary force and automatic function, the superiority of any new interventional therapy on functional movements might reliably become assessed. In conclusion, effective rehabilitation after a central motor lesion critically depends on the following points: (1) Knowledge about the neuronal mechanisms involved in the normal movement and about the interactions between central program and afferent inputs; (2) taking into account that a movement disorder is the consequence not only of the primary motor lesion, but also of secondary processes that can be compensatory and should be supported during rehabilitation; (3) the aim of rehabilitation should be improvement of function by taking advantage of the plasticity of neuronal centers. It should be directed less to the correction of isolated clinical signs, such as the reflex excitability; (4) for the monitoring of outcome and for the assessment of the effectiveness of any new interventional therapy, standardized functional tests have to be established.

15.5 Search for Reliable Clinical Assessment

Figure 15.2 Examples of 3 patients (A–C) with incomplete paraplegia following spinal cord injury, undergoing locomotor training. There is a differential course of motor score (according to the ASIA standards) and locomotor function (WISCI score) from [78].

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15.6

Conclusion

Based on animal experiments, recent investigations have shown that isolated human spinal cord contains neuronal centers which are able to generate complex movements. However, these centers can only activated within certain limits in order to improve mobility of paraplegic patients. (1) After an acute spinal cord injury (SCI), basic changes in the spinal neuronal activity take place, partly associated with alterations of signs assessed in clinical examination. These changes seen in the clinical signs, ranging from spinal shock to spasticity, were shown to be only partially reflected in the neuronal correlates, as assessed by electrophysiological recordings, for example, when increased muscle tone becomes established and muscle spasms appear. The discrepancy between clinical signs of spasticity and the probable neuronal correlates indicates that non-neuronal changes may contribute to spasticity. However, one has to be aware that at present we are able to evaluate only part of spinal neuronal activity. (2) Based on observations of the locomotor capacity of the spinal cat, several studies have indicated that spinal locomotor centers can be activated and trained in patients with complete or incomplete paraplegia, when the body is partially unloaded. The level of electromyographic activity (EMG) in the leg extensor muscles, however, is considerably lower in patients with central motor lesions than in individuals without neurological impairment. During the course of a locomotor training program, the amplitude of the EMG increases, associated with an improvement of locomotor function in the patients. Only incomplete paraplegic patients profit from this training for their locomotor ability. Furthermore, there are also unspecific training effects on muscles and ligaments. (3) In complete para-/tetraplegic patients, the level of lesion determines locomotor activity in spinal man. The higher the level of spinal cord lesion, the more “normal” is the locomotor pattern. This suggests that neuronal circuits underlying locomotor pattern generation extend from thoraco-lumbal to cervical levels. This observation has important therapeutic implications in the future when it might become possible to induce some regeneration of spinal tract fibers. (4) In complete or almost complete paraplegic patients who at present do not yet profit from locomotor training might, in the near future, benefit from experimental therapies that facilitate regeneration of adult CNS axons. Potential therapeutic interventions include growth-promoting factors, such as bridging transplants and neutralization of myelin-associated factors that inhibit axonal outgrowth.

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Acknowledgement

The work included in this review was supported by the Swiss National Science Foundation.

16 Neurodegeneration Jrg B. Schulz, MD Although there is an increasing number of symptomatic treatments available for neurodegenerative brain diseases, for most diseases, no causal or protective treatment has been established. Depending on the underlying pathomechanism, diseases are characterized by progressive neuronal dysfunction and/or loss of vulnerable neurons. It is the aim of all protective approaches to interfere with the cascade of deleterious events that lead to neuronal dysfunction and cell death. Although neuroprotective effects for many substances haven been reported in cell culture and experimental animal models for neurodegenerative disorders, only very few studies have also proven efficacy in clinical studies in patients. In this chapter I will review the most important clinical studies aimed to show neuroprotective effects, as well as point out the reasons why so many of these studies failed.

16.1

What Is Neuroprotection?

The term neuroprotection refers to “pharmacological interventions that produce enduring benefits by favorably influencing underlying etiology or pathogenesis and thereby forestalling onset of disease or clinical decline [1]. It is the aim of neuroprotective strategies to interfere with cascades that lead to neuronal dysfunction or cell death. Although the initiating events are obviously different, the downstream mechanisms leading to cell death are similar, if not identical. Currently discussed, important cell death mechanisms include accumulation of intra- or extracellular protein aggregates, mitochondrial dysfunction, generation of reactive oxygen species, excitotoxicity, activation of MAP kinases, immunogenicity and apoptosis. Neuroprotection may be achieved by (1) blocking glutamate receptors, that are activated under pathological conditions, (2) blocking certain signaling mechanisms, e. g. the MAP kinase pathway, or (3) inhibiting certain enzymes that produce noxious substances, e. g. nitric oxide synthase, or are ultimately involved in the execution of cell death, e. g. caspases. In many instances, endogenous defense mechanisms are up-regulated following an initial insult, a phenomenon that also occurs during preconditioning. This includes the activation of antioxidative defense mechanisms and Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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the induction of neurotrophins. Therapeutically, neuroprotective agents may induce or support these endogenous defense mechanisms. Neuroprotection may also be achieved by preventive activities that enhance endogenous defense mechanisms or support the metabolic activities of neurons. Optimal therapeutic strategies include: (1) early detection of reversible or irreversible injury of neurons, (2) high sensitivity and specificity of diagnostic tests, (3) a repertoire of therapeutic options, (4) capacity to delay, stop, or improve neuronal deterioration, and (5) no risk of serious unwanted side effects. 16.2

Outcome Measures in Clinical Studies

Most important for the success of clinical studies are study design and – as part of the design – primary and secondary clinical outcome measures. To evaluate severity of the disease, natural history and disease progression over time, tools are needed that permit to assess neurological function and to quantify the course of a disease. To achieve this goal, all studies include repeated measures of clinical scales. These scales are part of validated and standardized protocols. Examples are the Unified Parkinson’s Disease Ranking Scale (UPDRS) for Parkinson’s disease, the Unified Huntington’s Disease Ranking Scale (UHDRS) for Huntington’s disease, the Barthel Index, the National Institutes of Health Stroke Scale (NIHSS) and the Modified Rankin Scale for stroke, and the Alzheimer’s Disease Assessment Scale (ADAS) for Alzheimer’s disease. Studies on the effects of riluzole in amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease) have demonstrated the advantage of robust end points. End points in the ALS studies on the efficacy of riluzole were time to death or tracheostomy [2, 3]. Validated scales allow to study and compare the course of diseases in patients treated with (neuroprotective) agents or placebo. However, in most neurodegenerative diseases, the natural course is highly variable, making direct comparison impossible, due to the low numbers of patients. Furthermore, in many diseases, it is unclear whether there is a linear relationship between disease symptoms and neuronal injury. In stroke, for example, there is no linear relationship between infarct size and functional deficit [4]. The size of the lesion significantly determinates the neurological deficit. Furthermore, age, co-morbidity, and individual mechanisms to compensate after occlusion of the main cerebral arteries (e. g. collateralisation) influence clinical symptoms, size and localization following a stroke. For some diseases, especially Parkinson’s disease and increasingly also for Alzheimer’s disease, the interference with established symptomatic treatments (dopamimetics and acetylcholinesterase-inhibitors, respectively) is an important co-variable. Even if the study design controls for these medications, the individual response to and side effects of these treatments have an influence on the study outcome. Finally, some substances that were thought to provide neuroprotection had symp-

16.3 Specific Diseases

tomatic effects that did not allow to finally draw conclusions on the neuroprotective potencies of these drugs [5]. Because so many factors have an influence on clinical scales, the use of surrogate markers is becoming increasingly important. In developing valid and reliable biological markers of neurodegeneration lies an important challenge. Surrogate markers that detect the progression of brain disease in the living patient have enhanced the experimental therapeutics of demyelinating disorders. In this process, magnetic resonance imaging (MRI) has helped to monitor the distribution and load of demyelination [6]. Other neuroimaging tools have already shown promise as markers of neurodegenerative activity, helping to put into a biological perspective the impact on disability observed in a clinical trial. [7]. But biological markers should be viewed as an enhancement of neuroprotective investigation rather than a substitute for relevant and enduring clinical outcomes.

16.3

Specific Diseases 16.3.1

Wilson’s Disease

Although in general, neuroprotection remains a largely unrealized therapeutic goal, decoppering therapy for Wilson’s disease can be considered a prototype of neuroprotective therapy. Wilson’s disease is an autosomal recessively inherited condition of abnormal copper metabolism. As a result of decreased copper excretion by the hepatocytes into the biliary system, copper is deposited in liver, brain and other tissues. The estimated prevalence of Wilson’s disease is 3–4:100.000, the frequency of heterozygous mutations carriers is thought to be 1:180 [8]. WD typically manifests between 10 and 25 years of age, but occasionally as early as 5 years or as late as 50 years. In 40 % of all cases, neurologycal features are the first presenting signs. Untreated, homozygous gene carriers eventually develop hepatitis, cirrhosis, aminoaciduria, and progressive neurologic deterioration characterized by movement disorder, as well as speech and swallowing problems. The causative gene for Wilson’s disease encodes a copper binding ATPase (ATP7B) on chromosome 13q14.3. More than 100 mutations of ATP7B have now been described [9]. The copper-binding ATPase encoded by ATP7B is localized intracellularly and imports copper into the Golgi apparatus. Normal function of this enzyme is a prerequisite for the synthesis of copper-containing enzymes such as cytochrome C oxidase or superoxide dismutase 1 (SOD1) in the Golgi apparatus. Intracellular elevation of copper leads to a translocation of this ATPase to the cytoplasmic vesicular component, presumably providing intracellular copper homeostasis by copper sequestration [10]. Copper toxicity in the brain and other tissues is predominantly due to the formation of oxyradicals following its reactions with molecular oxygen [11]. Early diagnosis of Wilson’s disease is of particular importance because a combination of low-copper diet and chelate-forming agents such as D-penicillamine leads to a negative copper balance. In asymptomatic patients, this combined therapeutic

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strategy can prevent the disease from manifesting itself. However, once the disease has manifested itself, clinical improvement can only be reached in 60–70 % of all patients. Wilson’s disease is also a prototype for prevention strategies whereby decoppering therapies can prevent the clinical manifestations of illness in healthy individuals who are known to be homozygotic gene carriers. Notably, penicillamine decoppering therapy was developed more than three decades before the mutant gene for Wilson’s disease was eventually identified on chromosome 13. 16.3.2

Parkinson’s Disease DATATOP Study All currently established medical and surgical therapies of Parkinson’s disease have symptomatic effects. Reactive oxygen species probably play an important role in the pathogenesis and the progresssion of dopaminergic cell death in the substantia nigra. The first comprehensive clinical trial using antioxidants was the DATATOP (deprenyl and tocopherol antioxidative therapy of Parkinsonism) study. This study has been one of the most comprehensive clinical trials aimed at slowing down disability resulting from neurodegeneration. Initial findings from this trial in patients with early Parkinson’s disease indicated that deprenyl (selegiline), a type B monoamine oxidase inhibitor, significantly delayed disability associated with early Parkinson’s disease; however, therapeutic effects were not sustained or translated into enduring benefits such as reduced levodopa-related adverse effects or extended lifespan. It is likely that the slowing of disability has been due to weak dopaminergic effects of deprenyl. The antioxidant a-tocopherol had no benefit in slowing disability or extending life. Notably, research participants in DATATOP, regardless of treatment assignment, lived as long as age-matched controls without Parkinson’s disease [5, 12–14]. Patients who continuously received deprenyl for years were more likely to develop dyskinesias but less likely to develop freezing of gait than those who discontinued medication with deprenyl [15]. Currently, treatment with deprenyl is recommended to achieve symptomatic but not neuroprotective effects. 16.3.2.1

Dopamine Agonists It is controversially discussed whether or not L-Dopa and dopamine contribute to dopaminergic cell death through the generation of reactive oxygen species following autooxidation of dopamine. Although L-Dopa is toxic to dopaminergic neurons in culture at low concentrations, L-Dopa treatment of rats at high concentrations after partial denervation of the nigrostriatal pathway does not promote cell death [16]. For symptomatic treatment, L-Dopa is combined with dopamine agonists. Monotherapy with a dopamine agonist or the combination of a dopamine agonist with L-Dopa for the first 3 to 5 years delays the occurrence of L-Dopa associated dyskinesia. Such data have been published for ropinirole, pramipexole and cabergoline [17–20]. Similar data have been reported for pergolide, although these data have not been published yet. Dopamine agonists do not only save L-Dopa in the symp16.3.2.2


tomatic therapy, but provide own neuroprotective effects in vitro and in experimental animal studies. Direct free radical scavenging effects of dopamine agonists and the stimulation of autoreceptors, which leads to a reduction of dopamine metabolism and the dopamine-mediated generation of reactive oxygen species, are discussed as potential underlying mechanisms. PET and SPECT studies have provided evidence that treatment with dopamine agonists slows the reduction of striatal dopaminergic markers, suggesting protective effects of dopamine agonists against the progressive degeneration of the nigrostriatal system [21, 22]. However, the significance of SPECT and PET imaging studies to monitor disease progression and effects of drugs on disease progression has been debated [23–25].

Anti-Excitotoxic Treatment Both excitotoxicity and apoptosis are considered important cell death mechanisms in idiopathic Parkinson’s disease. At least in Europe, low affinity agonists of glutamatergic N-methyl-D-aspartate receptors (amantadine, memantine) have been used for years as symptomatic and potentially neuroprotective treatments of Parkinsonian syndromes. However, there is no clinical evidence forthcoming to draw final conclusions on their putative protective effects on dopaminergic neurons and the natural course of the disease. Based on the positive data of riluzole in the treatment amyotrophic lateral sclerosis [3] an international randomized, placebo-controlled clinical trial to evaluate the effects of riluzole on the progression of Parkinson’s disease was initiated (RP 54274X-320). After randomization, newly diagnosed patients with idiopathic Parkinson’s disease were treated either with riluzole or placebo. The primary endpoint was the time delay until a symptomatic treatment with L-Dopa or dopamine agonists became available. Repeated evaluation of the UPDRS and dopaminergic function measured with 18F-Dopa-PET over a period of 2 years were used as secondary endpoints. After the primary endpoint did not show any significant difference between riluzole- and placebo-treated patients, the study was terminated. There is a major drawback in this particular study design that unfortunately applies to most studies looking at neuroprotective effects in newly diagnosed patients with idiopathic Parkinson’s disease – most patients seeing their physician for the first time already need symptomatic treatment. The symptoms interfere with their normal quality of life or their ability to work. Patients should be treated symptomatically already at this time point. To include patients in a study the initiation of this treatment is delayed but often is initiated in accordance to the study protocol at the earliest time point possible. In my view it is unrealistic to expect a difference in this time delay between patients treated with a placebo and those treated with a putative protective drug that does not provide symptomatic treatment. 16.3.2.3

Neurotrophins Neurotrophins, neuroimmunophilins, gangliosides, anti-apoptotic and anti-inflammatory agents provide neuroprotective effects in animal models of Parkinson’s 16.3.2.4

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disease. First clinical studies employing intrathecal application of glial cell linederived neurotrophic factor (GDNF) were performed that initially did not provide positive effects, but demonstrated a limited diffusion [26]. In contrast, lentivirusmediated expression of GDNF in the striatum or the substantia nigra provided functional benefit against toxicity induced by 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [27]. Recently, it was reported in 5 patients that continuous infusion of GDNF into the putamen improved the off-medication motor sub-score of the UPDRS and the activities of daily living sub-score. Medication-induced dyskinesia was reduced and could not be observed off medication during chronic GDNF delivery [28]. However, it is debated whether treatment with GDNF really provides protection for neurons by preventing death of dopaminergic neurons or whether its action is limited to a neurotrophic effect, enabling sprouting and metabolic enhancement, e. g. higher dopamine production, in the remaining dopaminergic neurons [29]. In a model of cerebral ischemia, at least, long-term expression of GDNF may actually result in toxicity [30]. Before GDNF is applied to humans in large numbers, it needs to be shown that long-term metabolic enhancement of dopaminergic neurons does not result in toxicity.

MAP Kinase Inhibitors In humans [31] and the MPTP model [29], the activation of caspases, leading to apoptosis, appears to be an important cell death mechanism. The cascade leading to apoptosis may be activated through the MAP kinase pathway [32]. Treatment with CEP-1347, a small molecule MAP kinase inhibitor that crosses the blood brain barrier, is protective against MPTP in mice [33]. Recently, a safety and tolerability study of CEP-1347 in patients with Parkinson’s disease was completed, showing good tolerability, no interference with L-Dopa pharmacokinetics and no symptomatic effects over a treatment period of 4 weeks [34]. A large multicenter phase II/III treatment trial has been initiated. 16.3.2.5

Transplantation Several studies have been performed in the last decade – not with the aim of neuroprotection, but neurorestoration – to prove that transplantation of embryonic mesencephalic stem cells offers an alternative treatment for patients with Parkinson’s disease. Good functional, morphological and biochemical results have been reported in smaller cases series, PET and a few post mortem studies. However, two recent studies that questioned whether transplantation of fetal dopaminergic cells would be more effective than sham surgery in a double-blind randomized controlled trial showed that only patients younger than 60 years benefit from some effects. In contrast, 15–56 % of patients developed dyskinesia or dystonia that persisted after overnight withdrawal of dopaminergic medication [35, 36]. However, due to methodological weaknesses, these results were questioned [37, 38]. These data show that transplantation of fetal dopaminergic cells in Parkinson’s disease is currently not only limited by an inadequate availability of donor tissue 16.3.2.6


and ethical issues, but also by a lack of functional improvement, especially when compared with high frequency deep brain stimulation [39, 40]. As long as transplantation of stem cells does not result in full functional integration into the basal ganglia, these results are likely to remain unchanged. It has to be questioned whether transplantation of fetal dopaminergic cells into the striatum instead of the substantia nigra is a promising approach. The generation, ex vivo proliferation and differentiation of embryonic stem cells into dopaminergic neurons may solve the problem of limited availability of donor tissue [41] but will probably not result in better functional integration and a reduction in unwanted effects (dyskinesia, dystonia).

Deep Brain Stimulation The subthalamic nucleus is a key node in the functional control of motor activity in the basal ganglia. Its inhibition suppresses symptoms of Parkinson’s disease in animal models, and high frequency chronic stimulation does the same in human patients. Acute and long-term results after deep brain stimulation show a dramatic and stable improvement of a patient’s clinical condition [39, 40], which mimics the effects of levodopa treatment. The stimulation-induced disruption of overactivity of the subthalamic nucleus may also result in neuroprotection and a slowing of disease progression [42]. 16.3.2.7

Coenzyme Q10 Recently, a first study using oral treatment with coenzyme Q10, an antioxidant and electron acceptor of complex I and II of the mitochondrial electron transport chain, was completed [43]. Over a period of 16 months, treatment with coenzyme Q10 was safe and well tolerated at dosages of up to 1200 mg/d. Less disability developed in subjects assigned to coenzyme Q10 than in those assigned to placebo, and the benefit was greatest in subjects receiving the highest dosage. Coenzyme Q10 appears to slow down the progressive deterioration of function in Parkinson’s disease, but these results need to be confirmed in a larger study and over a longer period of time. Because differences between groups were already observed at early time points, potential symptomatic effects of coenzyme Q10 have to be taken into consideration [44]. 16.3.2.8

16.3.3

Multiple System Atrophy and Progressive Supranuclear Palsy

Multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) are akinetic-rigid movement disorder of unknown origin [45]. In contrast to Parkinson’s disease, patients with MSA or PSP only partially respond to L-Dopa treatment. Furthermore, MSA and PSP progress much more rapidly than idiopathic Parkinson’s disease, leading to a reduced life expectancy. Currently, a European multicenter treatment trial (NNIPPS; Neuroprotection and natural history in Parkinson

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Plus syndromes: A clinical trial of efficacy and safety of riluzole in Parkinson Plus syndromes) has been initiated to evaluate the effects of treatment with the antiglutamatergic substance riluzole on disease progression in PSP and MSA. Results are to be expected in 2005. 16.3.4

Alzheimer’s Disease

Incidence and prevalence of Alzheimer’s disease progressively increase with age. Including mild stages of the disease, the prevalence is 10 % in humans older than 65 years and 40 % in humans older than 85 years. The high prevalence in old age together with the increase in life expectancy leads to a shift in the age structure and increased demands on social and healthcare systems. Therefore, the development of effective symptomatic and protective treatments has high priority. The neuropathological characteristics of Alzheimer’s disease are neurofibrillary tangles and amyloid plaques. Their distribution in the brain of patients with Alzheimer’s disease is regionally specific. They are mainly located in hippocampus, entorhinal cortex, and in isocortical association areas. They lead to neuronal dysfunction, cell death, decline of cognitive function, neurological symptoms and finally death. New studies utilizing transgenic animal models show that the formation of amyloid plaques precedes the buildup of neurofibrillary tangles, thus suggesting that in addition to convincing genetic data, amyloid plaque formation is a prerequisite for the pathogenesis of Alzheimer’s disease [46]. Currently, the exact mechanisms leading to the formation of plaques and tangles are intensively studied. Factors suspected to induce or modify the pathological cascade include (1) mutations that lead to autosomal-dominant inherited disease, (2) genetic polymorphisms, (3) oxidative stress, (4) abnormal Tau phosphorylation that leads to intracellular accumulation of Tau and (5) unrecognized environmental factors. Amyloid plaques mainly contain the Ab peptide. Ab is derived from a much larger precursor, the amyloid precursor protein (APP) through proteolytical cleavage by b-secretase and g-secretase resulting in fragments of 40 (Ab40) and 42 (Ab42) amino acids (Figure 16.1). Because Ab42 is less soluble than Ab40, its tendency to aggregate and thus contribute to plaque formation is higher. Causal treatment should directly interfere with this cascade to stop or delay disease progression.

Symptomatic Treatment Until today only symptomatic treatments, consisting of four acetylcholine-esterase inhibitors, are available for the treatment of mild and moderate Alzheimer’s disease. These are tacrine, which, due to unwanted side effects, is not longer used, donepezil, rivastigmine, and galantamine [47, 48]. All four drugs lead in 30– 40 % of patients to a moderate improvement of cognitive function. Data from long-term treatment observations indicate that patients that interrupt the intake of acetylcholine-esterase inhibitors but then continue the intake do not again reach the same functional level as those patients that continuously received 16.3.4.1


Figure 16.1 Processing of the amyloid precursor protein and potential treatment opportunities.

medication. The conclusion that acetylcholine-esterase inhibitors also may provide protective effects needs further evaluation in prospective studies.

Antioxidants Reactive oxygen species are considered important mediators of neuronal death in Alzheimer’s disease. Patients receiving 2000 IU/day Vitamin E showed a significant delay in the primary outcome parameters death, institutionalization, and loss of the ability to perform basic activities of daily living [49]. In the same study, the use of selegiline did not show any significant effects. However, this study is methodologically questionable because patient groups were different at baseline, and Vitamin E led to more side effects (unexplained falls). 16.3.4.2

Anti-inflammatory Approaches Basic research studies provide substantial evidence that inflammatory processes present in the brains of patients with AD are destructive, and that anti-inflammatory drugs can provide protection. Although epidemiological studies suggest that anti-inflammatory drugs reduce the risk of AD, there is not yet any strong evidence from completed randomized controlled trials that anti-inflammatory treatment is beneficial [50]. The use of low dose glucocorticoids [51], hydroxychloroquine [52], indomethacin [53], and new cyclo-oxygenase-2 inhibitors [54, 55] resulted in no significant benefit. 16.3.4.3

Estrogen Replacement The situation is similar for estrogen. There is convincing basic research evidence that estrogen provides protective effects in models of Alzheimer’s disease. Observational studies have associated estrogen-containing hormone therapy with reduced AD risk. Limited clinical trial evidence suggests that estrogen therapy, begun after the onset of AD symptoms, is without substantial benefit or harm [56]. In the Women’s Health Initiative Memory Study – a randomized, placebo-controlled 16.3.4.4

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trial of women 65–79 years of age – oral estrogen plus progestin doubled the rate of dementia, with a heightened risk of rapid onset after initiation of treatment [56]. Based on current evidence, hormone therapy is thus not indicated for the prevention of AD.

NMDA Receptor Antagonists Pathological overactivation of glutamate receptors, particularly NMDA receptors, results in excitotoxicity, which is discussed as one of the major cell death mechanisms in neurodegenerative diseases. Memantine is a competitive NMDA receptor antagonist. Memantine binds to and blocks the receptor only after its activation and opening of the ion channel. This is the reason why memantine does not induce typical side effects of other NMDA receptor blockers, e. g. hallucinations. After several reports of positive effects in patients with Alzheimer’s disease and vascular dementia a recently published, randomized, double-blind study showed that treatment of patients with Alzheimer’s disease with 20 mg memantine per day for 28 weeks resulted in a significant slowing of disease progression [57]. Memantine was approved for the treatment of moderate to severe dementias in the US and in Europe. 16.3.4.5

16.3.4.6

Experimental Therapies

As shown in Figure 16.1, there are several ways of interfering with the production of b-amyloid: (1) blocking g-secretase activity, (2) blocking b-secretase activity, (3) immunization, and (4) treatment with statins. Several g-secretase inhibitors“,4> have been identified and may be used in clinical studies. However, toxic consequences may occur because g-secretase is essential for notch signal transduction, for the processing of epidermal growth factors (ErbB4) and possibly other substrates. b-secretases may be a better therapeutic target than g-secretases. The development of b-secretase inhibitors has proved to be difficult. In the mean time the crystal structure of b-secretase was published. This will certainly facilitate the development of specific inhibitors [58]. However, the binding pouch is quite large, making it difficult to develop small molecules that also cross the blood brain barrier. Furthermore, such inhibitors may have toxic properties and inhibit other proteases, or b-secretases may have other substrates as well. 16.3.4.7

Immunization

One of the most promising strategies in Alzheimer’s disease is immunization against Ab [59]. Both active [60] and passive [61] immunization reduce the load of amyloid plaques in transgenic mouse models of Alzheimer’s disease and improve cognitive function. Two broad theories about the mechanisms by which


Ab antibodies work in mice have emerged. First, evidence for an Fc-mediated uptake and clearance of Ab-antibody complexes by local activated microglia has been obtained [61]. Second, evidence of a net movement of Ab peptide out of the brain as a result of its binding and mobilization by Ab antibodies, in the serum and in the cerebrospinal fluid (CSF), has been provided [62]. These two proposed mechanisms are not mutually exclusive, and there may be additional ways in which antibodies decrease Ab-mediated synaptic and neuronal dysfunction. It should be noted, however, that passive immunization using monoclonal antibodies to Ab resulted in brain hemorrhage [63]. The finding that active vaccination with Ab had profound Ab-lowering effects in an animal model led to clinical trials in which an Ab1–42 synthetic peptide was administered parenterally with a previously tested adjuvant (QS21) to patients with mild to moderate Alzheimer’s disease. Although a Phase I safety study with a small number of patients failed to reveal significant side effects, a subsequent Phase II trial was discontinued shortly after its initiation when approximately 5 % of the treated patients developed what appeared to be a CNS inflammatory reaction. A recent post mortem case report has shown some macrophages and T cells infiltrate the CNS after Ab immunization [64]. Such cellular reactions were not detected in mice and other mammals exposed to the vaccine during preclinical safety and efficacy testing. According to recent findings that some elderly subjects and patients with Alzheimer’s disease have remarkably increased T cell reactivity to Ab [65], it is possible that Alzheimer’s disease patients with high Ab T cell reactivity develop severe T cell reactions in the CNS when immunized and boosted with Ab emulsified in the TH1 adjuvant QS21. Nonetheless, titers of Ab antibodies did not correlate with the occurrence of severity of symptoms or relapses [66], suggesting that the meningoencephalitis was not necessarily directly linked to Ab-reactive T cells. Ab-reactive T cells presumably contributed to effective production of Ab antibodies, but it is not clear yet whether they contributed to severe brain inflammation. Therefore, it is possible that the Ab-reactive T cells induced in the context of TH1 adjuvant mediated the first wave of T cell infiltration to the CNS, which was then followed by T cell responses to myelin antigens caused by the inflammatory response in the brain. It is also possible that the prolonged exposure to the adjuvant QS21 induced the stimulation of pathogenic myelin-specific T cells, independent of Ab-reactive T cells. The possibility that pathogenic myelin reactive T cells were involved is supported by magnetic resonance imaging (MRI) analysis which showed lesions in white matter of the brain where Ab is not normally expressed. There are several reasons why Ab immunization of transgenic mice did not cause severe inflammatory responses in the CNS: (1) Ab is not an encephalitogenic antigen; (2) animals were boosted with IFA, which could result in increased antibody production in the context of TH2 type T cell responses; (3) increased production of Ab in transgenic mice could induce central and peripheral T cell tolerance [67]; (4) Ab was not sufficiently immunogenic for T cell responses in the strains tested; and (5) NO-mediated T cell apoptosis was enhanced in the CNS of transgenic Alzheimer disease mice [65].

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Clinical evaluation after Ab immunization showed that patients who develop effective titers of plaque-binding Ab antibodies showed slower rates of decline of cognitive functions [68]. The data observed so far in this cohort of subjects suggest that high titers of plaque-binding Ab antibodies are important to achieve immunemediated beneficial effects in AD. In the post mortem case, there was clear evidence of decreased amounts of Ab plaques in neocortex regions as compared with nonimmunized patients with Alzheimer’s disease [64]. In some regions that were devoid of Ab plaques, Ab immunoreactivity was associated with T cell infiltrates in the CNS, activated microglia, and low titer of Ab antibodies [64]. Despite the adverse effects that occurred after Ab vaccination in humans, the results help define the path for future immunotherapy approaches.

Cholesterol-lowering Therapy Two recent retrospective clinical studies indicate that there is a decreased prevalence of Alzheimer’s disease associated with the use of statins to treat hypercholesterolemia [69, 70]. Those drugs cross the blood-brain barrier efficiently and reduce de novo cholesterol synthesis by inhibition of the ubiquitously expressed enzyme HMG-CoA reductase. In one study, Wolozin and colleagues compared the prevalence of probable AD in groups of patients receiving HMG-CoA reductase inhibitors with patients receiving medication to treat hypertension or cardiovascular disease [70]. They found that the likelihood to develop Alzheimer’s disease was 60–73 % lower in the cohort taking statins. In another study, Jick and colleagues [69] showed in a case-control analysis that the risk of dementia was up to 70 % lower in patients using statins compared to patients with untreated hyperlipidemia or patients receiving other lipid-lowering drugs (fibrates, cholestyramine or nicotinic acid). In addition, animal studies have revealed an association of amyloid production and cholesterol. While rabbits and rats fed with a cholesterol-rich diet have a tendency to accumulate Ab in the brain [71, 72], guinea pigs treated with statins have lower levels of Ab in the CSF [73]. How are these effects to be explained? Previous studies have shown that cholesterol modulates the processing of amyloid precursor protein in cultures of rat hippocampal neurons [74]. Ab production and secretion was dramatically reduced when cellular cholesterol levels were reduced by inhibiting de novo synthesis with statins alone or in combination with the cholesterol-extracting agent, methyl-b-cyclodextrin [73–75]. Cholesterol depletion also led to a marked reduction of the C-terminal b-stub, suggesting that b-secretase cleavage depended on cholesterol [73–75]. In contrast, secretion of the APP ectodomain generated by the non-amyloidogenic a-secretase pathway was shown to increase [75]. Thus, cholesterol depletion seems to inhibit the amyloidogenic (band g-secretase) pathway, while stimulating the non-amyloidogenic (a-secretase) pathway (Figure 16.2). In a randomized, placebo-controlled, double-blind study, we investigated whether statins alter cholesterol metabolites and reduce Ab levels in the cerebrospinal fluid of 44 patients with Alzheimer’s disease [76]. Individuals were given up to 80 mg simvastatin daily or placebo for 26 weeks. Simvastatin significantly decreased 16.3.4.8


Figure 16.2 Processing of the amyloid precursor protein is dependent on the cholesterol concentration in the cellular membrane.

Ab40 levels in the cerebrospinal fluid (CSF) of patients with mild Alzheimer’s disease. The reduction of Ab40 correlated with the reduction of 24S-hydroxycholesterol. These changes were not observed in more severely affected patients. Our study indicated that treatment of normocholesterolemic Alzheimer’s disease patients is well tolerated and leads to lower Ab concentrations in the CSF. Longterm trials in a larger population will have to clarify if statins slow the progression of cognitive symptoms in AD. Such studies have been initiated. 16.3.5

Huntington’s Disease

The cloning of the Huntington’s disease gene in 1993 [77] led to an explosion in our understanding of the early molecular events that underlie its pathogenesis. Many of these were unknown only 5–6 years ago and now promise the development of new and crucial therapeutic targets. Exploitation of this research for the identification of novel therapeutic compounds and assessment of efficacy utilizes a wide variety of disease models ranging from yeast (Saccharomyces cerevisiae) to the invertebrates Caenorhabditis elegans and Drosophila melanogaster and to mammalian species, in most cases, the mouse. The majority of Huntington’s disease preclinical mouse trials have assessed the efficacy of compounds that are of interest as neuroprotective agents – compounds that target free radical damage, excitotoxicity or membrane integrity. Those reported to be beneficial include creatine [78, 79], coenzyme Q10 [80, 81], remacemide [80, 81], riluzole [82], a-lipoic acid [83] and essential fatty acids [84]. The rationale for using these agents predates the cloning of the Huntington’s disease gene. Recent data, however, continue to support the role of these mechanisms in the disease process, and whilst these strategies are

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unlikely to be curative, there is every reason to believe that they might be beneficial. Therapeutic interventions targeted against specific molecular events include minocycline [85], a caspase 1 and 3 inhibitor, cystamine [86, 87], an inhibitor of tissue transglutaminase, Congo red [88], an inhibitor of polyglutamine aggregation and suberoylanilide hydroxamic acid, a histone deacetylase (HDAC) inhibitor [89]. In all cases, it is currently unknown if the compounds are acting on the target for which they were selected, for example riluzole [90] and minocycline [91] have both been found to be polyglutamine aggregation inhibitors, and cystamine also acts as an antioxidant [86]. The models that are being used to identify and develop novel therapeutic compounds will only be validated once they have delivered a therapy that shows efficacy in the clinic. Efficacy trials for Huntington’s disease, however, are very expensive, they require a major commitment from both patients and health professionals, and they are likely to take at least 5 years to complete. It will only be possible to pursue a small number of potential therapeutic compounds through phase III trials and, therefore, by necessity, the selection of these compounds will depend on the beneficial effects that have been detected in the disease models. To date, only one phase III clinical trial has been conducted for Huntington’s disease [92], which was designed to test the potential benefits of coenzyme Q10 and remacemide hydrochloride. Participants with early Huntington’s disease (n ¼ 347) were randomized to receive coenzyme Q10, remacemide, both or neither treatment, and were evaluated every 4–5 months for 30 months [92]. This study was powered to detect a 40 % attenuation of total functional capacity decline and whilst neither drug achieved this at the doses studied, patients receiving coenzyme Q10 showed a non-significant trend toward a slowing in total functional capacity decline (13 %). Given that both coenzyme Q10 and remacemide have been assessed in mouse models, does this shed any light on the ability of these models to predict efficacy in the clinic? Whilst it is tempting to speculate that the 15–20 % improvement in survival observed when coenzyme Q10 was administered to two mouse models might suggest comparable results in the preclinical and clinical trials, in reality the outcome measures are very different, and far more studies are needed.

16.4

Why Do So Many Clinical Studies Fail? What Do We Need?

There are quite a number of reasons why it has been so difficult to translate results from preclinical research into successful clinical trials. Major reasons are: x

The experimental conditions in culture and in animal models are very homogenous. This is not the case in human diseases, in which different genetic and environmental factors have an influence on age of disease onset and progression. Even in the case of monogenetic disorders (e. g. Huntington’s disease) the length of CAG expansion varies between the patients, whereas transgenic animals in a particular study are all derived from one strain.

References x

x

x

x

x

Age is an important factor for disease onset and progression. The endogenous defense mechanisms may decline with age. However, in most experiments only young animals are used. In many animal studies, very high concentrations of drugs are empirically used that may not be tolerated by humans, especially when used as a long-term treatment. In almost all clinical studies, monotherapies are used. However, it may not be sufficient to just block one cell death pathway. Putative neuroprotective therapies are initiated at a time point at which patients become symptomatic. At this time point many – if not the majority – if not the majority of neurons are dysfunctional or already degenerated. In experimental studies lesion volume, loss of neurons or survival are used as outcome measures. In most clinical trials, functional scales are used.

For the future we should consider x x x x

x

Better design and outcome measures of clinical studies, Better definition of the at-risk population, enabling an early onset of treatment, Better screening of promising interventions, Better knowledge and studies on drug delivery, drug targeting, and penetrance of blood-brain-barrier, Design studies that use combinational therapies. As shown in oncological studies, this will also make it possible to use lower concentrations and improve tolerability of drugs.

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Parkinson’s disease: results of the first year of treatment in a doubleblind comparison of cabergoline and levodopa. The PKDS009 Collaborative Study Group. Neurology 1997; 48:363–368. Rinne UK, Bracco F, Chouza C, Dupont E, Gershanik O, Marti Masso JF, Montastruc JL, Marsden CD. Early treatment of Parkinson’s disease with cabergoline delays the onset of motor complications. Results of a doubleblind levodopa controlled trial. The PKDS009 Study Group. Drugs 1998; 55:23–30. Rascol O, Brooks DJ, Korczyn AD, De Deyn PP, Clarke CE, Lang AE. A five-year study of the incidence of dyskinesia in patients with early Parkinson’s disease who were treated with ropinirole or levodopa. 056 Study Group. N Engl J Med 2000; 342:1484–1491. Group PS. Pramipexole vs levodopa as initial treatment for Parkinson disease: A randomized controlled trial. JAMA 2000; 284:1931–1938. Marek K, Seibyl J, Shoulson I, Holloway R, Kieburtz K, McDermott M, Kamp C, Shinaman A, Fahn S, Lang A, Weiner W, Welsh M. Dopamine transporter brain imaging to assess the effects of pramipexole vs levodopa on Parkinson disease progression. Jama 2002; 287:1653–1661. Whone AL, Watts RL, Stoessl AJ, Davis M, Reske S, Nahmias C, Lang AE, Rascol O, Ribeiro MJ, Remy P, Poewe WH, Hauser RA, Brooks DJ. Slower progression of Parkinson’s disease with ropinirole versus levodopa: The REAL-PET study. Ann Neurol 2003; 54:93–101. Marek K, Jennings D, Seibyl J. Do dopamine agonists or levodopa modify Parkinson’s disease progression? Eur J Neurol 2002; 9 Suppl 3:15–22. Morrish PK. REAL and CALM: what have we learned? Mov Disord 2003; 18:839–840. Morrish P. Is it time to abandon functional imaging in the study of neuroprotection? Mov Disord 2002; 17:229–232.

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17 Perspectives for Neuroprotective Therapies in Basic Research and Clinical Application Mathias Bhr and Pawel Kermer The final chapters of this book have outlined in detail the pathophysiological similarities and differences between animal models and human patients in regard to relevant neurological disorders. Furthermore, some of the problems of translating basic research data from the lab to clinical applications have been exemplified for several substances and procedures. At last, it is time to consider how the current approach to scientific and clinical handling of neurological disorders can be optimized and focused, thereby facilitating the transfer from what we learn at the bench to what we do at the bedside. Without doubt, interactions between and across disciplines need to be substantially improved. Building up research teams consisting of basic scientists, clinicians and other experts with broad knowledge in trial conception could be a solid basis to work out strategies for better study designs. In addition, new developments in the imaging field involving PET and SPECT scan as well as new MR techniques are necessary for the visualization of affected brain areas and cell populations with regard to their metabolism, thereby acquiring more information about the dimensions of structurally and/or only functionally damaged regions as well as the mechanisms resulting in cellular degeneration in the individual patient. The definition of markers and surrogate-markers like the expression patterns of pro- and anti-apoptotic proteins (e. g. Bcl-family proteins) for the prognosis of the respective neurodegenerative disorder already at the level of clinical testing may lead to a substantial improvement in the choice of patients for which a given neuroprotective strategy could be effective at all. At the same time, such knowledge must also be used to exclude patients when no significant rescue with a given therapy can be anticipated. One example of a successful small randomized double-blind trial with a potentially neuroprotective agent, is the recently published Erythropoetin Stroke Study [1]. In this study, only such stroke patients with a confirmed large territorial ischemia in the territory of the middle cerebral artery were recruited for testing the effects of erythropoetin (Epo) within a restricted time window. Patients with small lacunar types of stroke or patients with complete infarctions at the time of evaluation were excluded. Both, MRI parameters, CSF parameters and clinical scales were used to monitor these patients and a detailed statistical analysis provided first evidence, not only of good tolerance of Epo but also of efficacy showing Neuroprotection. Models, Mechanisms and Therapies. Edited by Mathias Bhr Copyright c 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN 3-527-30816-4

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a significantly improved outcome in the Epo-treated group [1]. Of course, this result has to be reproduced in a larger multi-center trial, which is currently under way, but it highlights some of our future avenues of clinical research: Selection of safe and easy-to-administer medications, strict patient definition and recruitment, application of several markers and surrogate-markers for a given pathology, realistic time frames of application and adjusted dosage of potentially protective agents in rather small trials with highly qualified and motivated research teams to test efficacy and applicability before larger trials are initiated. Regarding work at the bench, several strategies have been mentioned how the situation can be optimized. Animal and lesion models have to be improved to get closer to the situation in human disorders, where our therapeutic goal is the preservation of the remaining neurons rather than the rescue of neurons where the death process has already been initiated. For one given neurodegenerative disease, it may even be desirable to establish several different animal models reflecting the various pathophysiological mechanisms causing neuronal degeneration. For example, among the diverse forms of Parkinson’s disease, we can distinguish hereditary forms, idiopathic forms where the cause is not known, toxic forms resulting from poisoning, and a form of the disease that is embedded in syndromes where neuronal degeneration occurs not only in the substantia nigra, but in other independent brain regions at the same time (multiple system atrophies). It seems reasonable to speculate that, while sharing the same or at least similar symptoms, all these forms of Parkinson’s disease represent different entities, following different pathophysiological pathways and consequently requiring a differential therapeutic approach. On the other hand, lesion models and neuroprotective strategies should be adjusted to realistic time frames, drug application routes and procedures reflecting the situation in the patient and the possibilities in the hospital. Instead of testing yet another drug that acts on a protective pathway that has already been analyzed (and possibly already unsuccessfully tested in clinical trials), one might want to combine neuroprotective substances either acting on different pathways or at different levels of the same signal transduction pathway. Ultimately, results obtained by such studies might enable us to reduce adverse effects in clinical applications by decreasing the dosage of a single drug, while still benefiting from additive effects of the drug combination. Conceptually, however, it is still a problem to start a clinical trial with a combination of potentially protective substances, since most drug registration legislations require demonstration of efficacy of one compound first before it can be combined with another drug of yet unknown efficacy. Reduction of unwanted drug properties can also be achieved by modulation of binding characteristics of a protein, by deleting or adding domains, mutating binding sites etc. The perfection of protein alteration is the generation of small synthetic drugs that can mimic protective effects of natural proteins, but avoid the negative ones entirely. As mentioned before, some drugs already exist like Epo, which now serve as a template for the generation of new derivatives that cross the blood-brain-barrier more effectively, have fewer systemic effects and higher binding capacities e. g. to trophic factor receptors etc. Engineering other new molecules may also be helpful in that regard. An intact blood brain


barrier prevents proteins from entering the brain. Obviously, surgical approaches to circumvent this barrier are only feasible in a small number of patients. Thus, the design of compounds bearing moieties facilitating the transport into the central nervous system (for example TAT-proteins; [2–5]) represents a promising alternative. Moreover, genetically modified viral vectors appear as useful tools to specifically express therapeutic factors in certain regions or cell types where they are needed [6]. Antisense strategies, lowering the expression levels of disease-driving genes, if specific enough, represent another approach for the treatment of hereditary neurodegenerative diseases where mutated proteins are held responsible for pathophysiology [7]). A new alternative treatment option is the vaccination against pathological proteins. In Alzheimer patients, for example, specific antibodies against amyloid protein were generated [8]. However, this therapeutic approach has to be optimized since it was accompanied by severe side effects in those patients. Furthermore, attempts to transplant neuronal precursors into the brain have been promising in individuals suffering from Parkinson’s disease, restoring synaptic and terminal function of the dopaminergic system [9]. With regard to stem cell research, new data indicate the possibility to induce neurogenesis even in regions of the adult mammalian brain, like the neocortex, where it does not normally occur, via manipulation of endogenous multipotent precursors in situ. This suggests that elucidation of the relevant molecular controls over adult neurogenesis from endogenous neural precursors/stem cells may permit the development of neuronal replacement therapies for neurodegenerative diseases and other central nervous system injuries that may not require transplantation of exogenous cells [10]. As clinicians, we are in demand of – and have to come up with – new surrogatemarkers, not only for the prognosis of disease, as mentioned before, but also for disease progression and therapy monitoring, since up to now studies involving patients mostly use clinical parameters (scales, mortality) as outcome measures, which are far from being as accurate as the parameters used in animal model studies. Defining such markers on the basis of the different processes that lead to neuronal degeneration and applying new imaging techniques demonstrating cellular and axonal integrity/lesions in vivo would also be helpful in revising and maybe renewing current classifications of neurodegenerative diseases. Intensifying research on genetic mapping and potential marker proteins, we might be able to uncover new or at least alternative entities regrouping diseases according to their pathophysiological mechanism in addition to histology or symptoms. Hopefully, we will be able in the future to provide every patient suffering from neurodegenerative disease with a more specified and diversified therapy, taking into account individual symptoms as well as pathophysiology. Thus, the apparent gap between bench and bedside might not stay as wide as it is today.

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Index A acetyl CoA 193 action kinetics 303 active cell death (ACD) 155, see also apoptosis adult brain 263 ff – adult-generated neurons 266 – functional relevance of neurogenesis 276 – maturation 266 – neurogenesis 269 adult neurogenesis 269 – enriched environment 272 – learning 271 – physical activity 272 – regulation 270 ALS see amyotrophic lateral sclerosis Alzheimer patients 359 – vaccination against pathological proteins 359 Alzheimer’s Disease 71 ff, 158, 173, 176, 213, 221, 291 ff, 296, 304, 342 ff – active vaccination 345 – amyloid plaque formation 342 – anti-inflammatory approaches 343 – antioxidants 343 – cholesterol-lowering therapy 346 – estrogen replacement 343 – experimental therapies 344 – immunization 344 – neuropathological characteristics 342 – NMDA receptor antagonists 344 – symptomatic treatment 342 Alzheimer’s Disease Assessment Scale (ADAS) 336 American Spinal Injury Association (ASIA) 328 – interventional therapy 328 4-aminopyridine 141 AMPA 292 Amphiphysin 185 b-amyloid peptide (Ab) 296

amyloid polyneuropathy 213 amyloid precursor protein (APP) 87 amyotrophic lateral sclerosis (ALS) 51 f, 153, 173, 176, 221, 293 – progressive bulbar palsy 51 anabolism 191 anaerobic glycolysis 8 – lactate-acidosis-hypothesis 8 anemia 192 anti-excitotoxic therapy 298 anti-inflammatory cytokines 179 apomorphine 43 apoptosis 13, 153ff – active cell death (ACD) 155 – anti-apoptotic proteins 159 – caspase 157, 164 – caspase-3 155 – caspase-9 155 – cellular mechanisms 164 – cowpox virus 158 – death receptors 164 – developmental cell death 153 – DNA-laddering 201 – genetics 155 ff – human diseases 153 – mitochondria 164 – oligodendrocyte 86 – programmed cell death 13, 154 – proteins involved 155 ff – regulation of 164 apoptosis-inducing factor (AIF) 14, 154 ASK1 54 astrocyte swelling 235 astrocytes – associated inhibitory molecules 236 – chondroitin sulfate proteoglycans (CSPGs) 237 astrocytic edema 235 autoimmune brain diseases 173 axon growth


362

Index – inhibitors 229 ff, 236 – inhibitors transduction of signals 242 axonal transport 87 axons – re-growth 247 – regeneration 246 axotomy 113

B B lymphocytes 178 bacterial meningitis 178 baculovirus IAP repeat (BIR) 162 Bcl-2 family – Bid 14 – mitochondrial permeability transition pore 15 Bcl-family – Bad 15 – Bcl-2 15 – Bcl-xL 15 – Bcl-xL fusion protein 15 BEN 54 BH-domains 159 biological markers 337 blood-brain-barrier (BBB) 12 ff, 173 – astrocytes 12 – basal lamina 12 – endothelial cells 12 bone-marrow-derived cells (BMDC) 20 bovine spongiform encephalopathy (BSE) 214 brain 192 – adulthood 261 – blood flow and functional activity 194 – disturbances of protein synthesis 202 ff – disturbances of the energy metabolism 192 ff – interruption of the blood oxygen supply 197 – loss of adenosine 198 – mitochondrial dysfunction 195 ff – oxygen supply 192 – regulation of blood flow 194 – restoration of blood flow 198 – therapy of metabolic disturbances 208 ff – transplantation of stem- or precursor cells 278 brain ischemia 203, 279 – focal 198, 205 – global 204 – secondary energy failure 199 – transient global 196 – transplantation of restoring cells 279 brain trauma 153

brevican 134, 237 bromo-deoxy-uridine (BrdU) 261

C c-jun 138 CADASIL, notch3-gene 4 Caenorhabditis elegans 72, 155 calcineurin 161 calpain 88 cAMP 245 carboxy-terminal hydrolase L1 (UCHL1) 38 caspase recruitment domain (CARD) 157 caspases 14, 220 – caspase 1 14 – caspase 3 14 – caspases 8 and 9 14 – caspase 12 221 catabolism 191 caveolin 219 CD200 ligand 179 Cdr2 185 ced-1 156 ced-2 156 ced-3 156 ced-4 156 ced-5 156 ced-6 156 ced-7 156 ced-9 156 ced-10 156 ced-12 156 cell death 166 f, see also apoptosis – endoplasmic reticulum 167 – lysosomes 167 – mitochondria 167 cell replacement 19 f. cellular prion protein (PrPC) 214 ff central nervous system 229 – acute phase of injury 233 – adaptive immune responses 177 ff – autoimmune brain diseases 173 – axonal injury 229 – cell-mediated immunity 235 – glial cell loss 235 – glial scarring 233 – immune response 173, 175 – inflammation 235 – injured neurons 229 – maturation 230 – non-conducive growth environment 233 ff – promotion of repair 245 ff – regeneration failure 233 – secondary injury 235 ff – T cell activation 180

Index – T lymphocytes 180 – Wallerian degeneration 230 central nervous system (CNS) 173 cerebral energy metabolism 104 – ATP 104 – GTP 104 – N-acetylaspartate 104 – nicotinic enzymes (NAD, NADP) 104 ces-1 156 ces-2 156 chaperone 14-3-3 35 Charcot disease 51 cholesterol 346 choline acetyltransferase (ChAT) 53 chondroitin sulfate proteoglycans (CSPGs) 134, 237 chondroitinase ABC 134 chromatin 14 chromatolysis 230 clinical assessment 327 ff clinical parameters 359 coenzyme Q10 341 complex I 33 f controlled cortical impact (CCI) models, TBI 99 contusions 100 cortical neurogenesis – stroke 276 cortical plaques 88 corticosteroids 273 cowpox virus 158 creatine 60 creatine phosphate (CrP) 196 Creutzfeldt-Jacob disease (CJD) 214, 219 cryolesion 100 cyclo-oxygenase-2 inhibitor 55 cyclophilin D 165 cyclosporin A (CsA) 209 cytochrome C 14, 165 cytokine production 176 cytokines, inflammatory 236

Diabetes type 2 213 DIABLO 15 diffuse axonal injury 101 diphtheria toxin 161 dizocilpine 322 DJ-1 33 DNA dependent protein kinase (DNA-PK) DNA repair 15 ff, 17 – AP-endonucleases 16 – base excision repair (BER) 16 – DNA glycosylases 16 – DNA polymerase b 16 – mismatch repair 16 – nucleotide excision repair (NER) 16 – uracil-DNA-glycosylase (UDG) 16 DNA ligase 16 18 F-Dopa-PET 339 dopamine, synthesis 37 dopamine (DA) 31 dorsal root ganglia (DRG) 232 doublecortin (DCX) 265 Drosophila melanogaster 72 drug administration 299 – memantine 299 DSPA-a1 13

E

EEG 194 egl-1 156 eIF2alpha 203 EMG 326 encephalitis 178 endoplasmic reticulum stress response (ERS) 203, 220 enriched environment 272 EP 194 epidermal growth factor (EGF) 263, 276 epigenetic mechanisms 17 – DNA methylation 17 – histone deacetylases (HDAC) 17 – histone modification 17 epilepsy 274 – temporal lobe epilepsy (TLE) 274 D epileptic seizures 200, 207 DAT (DA transporter) 37 erythropoietin 12 DATATOP (deprenyl and tocopherol antioxiErythropoietin Stroke Study 357 dative therapy of Parkinsonism) 338 excitotoxicity 3, 291, 294 ff, 295 Daxx 54 – Alzheimer’s disease 296 death effector domain (DED) 157 death inducing signaling complex (DISC) 14 – anti-excitotoxic therapy 298 – clinical relevance 294 ff deep brain stimulation 341 – definition 294 f dendritic cells 218 – glutamate 294 deprenyl (selegiline) 338 – memantine 299 depression 273 – nitric oxide synthase (NOS) 295 developmental cell death 153

14

363

364

Index – pathophysiology 296 – stroke 295 – uncompetitive antagonist 299 – vascular dementia 295 experimental autoimmune encephalomyelitis (EAE) 89, 178, 184 ff

F FALS 51 familial PD 33 fampridine 141 Fas antigen 86 Fas-receptor 14 fatal familial insomnia (FFI) 214, 219 fibroblast growth factor-2 (FGF-2) 276 fluid-percussion injury (FPI) 98 fluoxetine 274 focal brain ischemia 198, 205 – recovery of protein synthesis 206 fragile-X-syndrome 18 free radicals 7 f., 235 – nitric oxide 7 – superoxide 7

G GABA antagonists 200 GAD 185 gangliosides 322 GAP-43 230 GDNF 55 gene expression 18 f Gephyrin 185 Gertmann-Straussler-Sheinker (GSS) 214 glaucoma 293 glial cell line-derived neurotrophic factor (GDNF) 340 glial cells 76 glial scar 133 – fibroblasts 133 – microglia 133 – oligodendrocytes 133 – reactive astrocytes 133 global brain ischemia 196, 204 glucocorticoid hypothesis 9 glucose regulated family proteins (Grps) 221 GluR3 185 glutamate 294 f – glutamate receptor hyperactivity 295 glycolysis 192 growth factors 20 – BDNF 20 – EGF 20 – FGF-2 20 – SCF 20

– VEGF 20 GSH 34 GTPases 243

H head trauma 201, 207 – post-traumatic apoptosis 208 hemodialysis-related amyloidosis 213 HIF-1 (hypoxia-inducible factor-1) 21 hippocampus 20 – CA1 region 20 HMG-CoA reductase 346 Homer1A 271 hPDGFb promoter 44 hsp-70 54 hu 185 Huntington’s disease 153, 173, 176, 213, 221, 279, 293, 347 ff, 348 – coenzyme Q10 348 – disease gene 347 – remacemide hydrochloride 348 – therapy 348 hydrogen peroxide 7 hydroxyl radicals 7 hyperphosphorylated tau 296 hypoxia 192

I IAP proteins 162 ICF 18 IFN-g 176 IGF-1 55 imaging techniques 359 inflammation 10 ff – adhesion molecules 10 – anti-inflammatory cytokines 10 – cyclo-oxygenase 2 (COX-2) 11 – cytokines 10 – ICAM-1 10 – IL-1 11 – IL-6 11 – IL-10 11 – inducible nitric oxide synthase (iNOS) 11 – b2 -integrins CD11b/CD18 10 – leucocytes 10 – MHCI and MHCII 10 – microglial cells 10 – monocyte-attractant protein 1 (MCP-1) 12 – neutrophil granulocytes 10 – TGF-1b 11 – TNFa 11 – TNFR1 and TNFR2 11 – VCAM-1 10 – VLA-4(a4 b1) 10

Index – NitroMemantines 306 – pharmacology 300 ff – uncompetitive antagonist 299 – uncompetitive open-channel block 300 ff – voltage-dependence 303 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 33, 291 ff, 339 – receptor 296 ff – receptor antagonists 291 ff – receptor model 298 ff K – receptors 293 ff kainate 292 methylazoxymethanol acetate (MAM) 277 kainic acid (KA) 274 methylprednisolone 321 mGluR1 185 L middle cerebral artery (MCA) 199 L-Dopa 31, 73, 338 middle cerebral artery occlusion (MCAO) 275 lactacidosis 193 Minocyclin 60 learning 277 missile injury 101 lecticans 237 mitochondrial dysfunction 195 ff Lewy bodies 31 mitochondrial stress 220 – alpha-synuclein 35 ff monocytes chemotactic protein 1 Lewy neurites 35 (MCP-1) 181 lipid peroxidation 201 g-motoneurons 51 lipid rafts 215 motoneuron degeneration, cell culture – detergent resistant-membranes models 53 ff (DRMs) 215 motoneuron disease, animal models 55 ff lipopolysaccharide (LPS) 175 – MS 89 long-term potentiation (LTP) 277 a-motoneurons 51 Lou Gehrig’s disease 51 – subpial demyelination 89 lymphocytes multiple sclerosis (MS) 85 ff, 173, 178, 293 – effector mechanisms 182 – axonal pathology 87 – cortical plaques 88 M – experimental autoimmune M-cells 218 encephalomyelitis 186 mAb IN-1 134 – MS progressive primary 85 macrophage 86 – MS progressive secondary 85 macrophage inflammatory protein 1a – MS relapsing-remitting 85 (MIP-1a) 181 – neuronal pathology 88 macrophages 177 multiple sclerosis plaque, MAG 240 pathologic characteristics 86 f magnetic resonance imaging (MRI) 337 major histocompatibility complex (MHC) 173 multiple system atrophy (MSA) 35, 72 multipotent adult precursor cell (MAPC) 279 mammalian brain trauma 108 Munc-18 185 MAO 34 mutant SOD1 mice 56 ff MAO B (monoamineoxidase) 37 myelin 86, 230, 234 matrix metalloproteinases (MMP) 12 – associated protein 239, 242 MELAS 4 – Nogo 240 Memantine 291 ff, 299 ff, 304, 308 myelin-associated glycoprotein (MAG) 86 – action kinetics 303 myelin-oligodendrocyte glycoprotein – blockade of NMDA current 302 (MOG) 186 – chemical structure 301 – human clinical trials 304 ff – macroscopic blocking and unblocking N Naþ-Kþ-ATPases 200 actions 308 NADþ 193 – neuroprotective efficacy 304

inhibitor of caspase-activated DNase (ICAD) 14 interferon-g 176 interleukin-1b-converting enzyme (ICE) 157 ischemic infarct 194 ischemic tolerance 6, 21 f – preconditioning 21 – Subliminal ischemia 21

365

366

Index NADH 193 naloxone 322 National Institutes of Health Stroke Scale (NIHSS) 336 necrosis 154 nestin 264 neural cell adhesion molecule (PSA-NCAM) 266 neural cell replacement 278 ff – therapeutic strategies 278 ff neural stem cells (NSCs) 262 – adult brain 263 ff – application and use 262 neurocan 134, 237 neurodegeneration 213, 335 ff, 337 – biological markers 337 – clinical studies 335 ff – validated scales 336 neurodegenerative dementias 71 ff – a-synuclein (aSYN) 71 ff neurodegenerative diseases 71 ff, 166, 292 neurogenesis 261 ff, 270 – cortical trauma 275 – endogenous 278 – epilepsy 274 – ischemia 275 – learning 277 – stress and depression 273 neuroimmunophilins 339 neurological disease 166 – cell death 166 neuronal apoptosis 219 neuronal inhibitor of apoptosis protein (NAIP) 162 neurons 296 – dorsal root ganglia (DRG) 232 – energy failure 296 – growth potential 231 – migration 267 – overcome growth inhibition 245 – persistence of adult-generated 269 ff – regeneration 230 – regenerative sprouting 231 – sprouting 230 neuroprotection 335 ff – biological markers 337 – definition 335 – excitotoxicity 293 – magnetic resonance imaging (MRI) 337 – therapeutic perspectives 357 ff – therapeutic strategies 336 – validated scales 336 – Wilson’s disease 337

neurospheres 263 neurotrauma 95 ff neurotrophins 339 NG2 DSD-1/phosphacan 134 NitroMemantines 306 f NMDA receptor 20, 296 ff – NR1 subunit of 13 NO synthase 54 Nogo 240 ff Nogo-66 135 Nogo-A 134 ff NOVA1 185 nuc-1 156 nuclear magnetic resonance (NMR) – spectroscopy 162

O 6-OHDA 42 oligodendrocyte-myelin glycoprotein (OMgp) 134, 242 oligodendrocytes 86, 234 organotypic slice cultures 107 oxidative stress 34 f, 98

P p38 54 p75 neurotrophin receptor 54 paraneoplastic neurological diseases 185 paraptosis 154 paraquat 33 parkin 33 Parkinson’s disease 153, 173, 176, 213, 221, 293, 338-339, 341, 358 – 18F-Dopa-PET 339 – antioxidants 338 – coenzyme Q10 341 – deep brain stimulation 341 – deprenyl (selegiline) 338 – environmental factors 33 – epidemiology 32 – genetic contribution 32 f – genetic models 43 ff – MAP kinase inhibitors 340 – neuroimmunophilins 339 – oxidative stress 34 f – parkin involvement 39 ff – progressive supranuclear palsy (PSP) 341 – proteosome 38 f – riluzole 339 – transplantation of fetal brain tissue 279 – transplantation of stem cells 340 Parkinson’s Disease (PD) 31 ff pathogen-associated molecular patterns (PAMP) 175

Index Pentylenetetrazole (PTZ) 274 penumbra 6 f penumbra concept 6 f peri-infarct depolarization 6, 9 f permeability transition pore 8, 165 Peyer’s patches 218 pH 104 physical activity 272 PI3K/Akt-pathway 15 pilocarpine 274 PKA 245 PKR-like ER kinases (PERK) 203 plasminogen activators (PA) 12 poly-(ADP-ribose)-polymerase (PARP) 14 positron emission tomography 200 preconditioning 21 – effector phase 21 – induction phase 21 – transduction phase 21 precursor cells – adult brain 263 ff primary lateral 51 prion diseases 214 – apoptosis 220 ff – associated with different mutations 214 – bovine spongiform encephalopathy (BSE) 214 – ER-stress 220 ff – fatal familial insomnia (FFI) 214 – Gertmann-Straussler-Sheinker (GSS) 214 – new variant CJD (vCJD) 214 – prion biology 215 ff – prion pathogenesis 218 ff – prion pathology 215 ff – protein misfolding 213 ff pro-inflammatory cytokines 176, 178, 235 programmed cell death (PCD) 13, see also apoptosis progressive motor neuronopathy (pmn) 61 progressive muscular atrophy 51 progressive supranuclear palsy (PSP) 341 proteasome 43 protein misfolding 213 ff, 223 protein synthesis 202 ff prox-1 265 PrP scrapie (PrPSc) 214, 218 – blood-brain barrier (BBB) 218 Purkinje cells 138

R R-Ras p23 161 Raf-1 161 Rasmussen encephalitis 185 reactive oxygen species (ROS) 195

recombinant adeno-associated virus (rAAV2) 45 recombinant tissue plasminogen activator (rtPA) 5 regeneration 230 regenerative sprouting 231 regional cerebral blood flow (rCBF) 5 rehabilitation 129 respiration 319 reticulon family proteins (RTNs) 240 – Nogo 240 – Nogo A 240 – Nogo B 240 – Nogo C 240 – RTN1, -2, -3 240 retina 230 Rett syndrome 18 Rho 136 Rho GTPases 244 – Cdc42 244 – Rac 244 – Rho 244 riluzole 56, 339 RING motif 163 RNA interference (RNAi) technique 63 ROCK 244 rotenone 33, 43

S SCG10 231 SCI 131 – axonal 131 – cyst formation 131 – demyelination 131 – edema 131 – excitotoxity 131 – hemorrhage 131 – infarction 131 – inflammation 131 – metabolic perturbation 131 – National Acute Spinal Cord Injury Study II (NASCIS II) 131 – neuronal necrosis 131 – oxidative damage 131 – paralysis 131 scrapie 214 ff, 219 b-secretase (BACE) 343 f semaphorins 234, 239 sensory-motor networks 325 – locomotor function 325 – plasticity 325 serotonin 274 serpin-deficiency disorder 213 SOD1 51

367

368

Index spinal cord 51 spinal cord injury (SCI) 129 ff, 317f, 320 f – acute neuroprotection 131 f – acute stage 318 – bladder 319 – body temperature 319 – bridging cysts 138 – cardiovascular function 319 – cellular replacement 138 – clinical aspects 318 ff – degree of disability 129 – dizocilpine 322 – enhancing plasticity 141 – epidemiology 318 – gangliosides 322 – glial scar 133 – locomotor training 317, 325 ff – methylprednisolone 321 – movement disorders 327 – naloxone 322 – neural transplantation 139 – peristalsis 320 – processes 130 – regeneration of nerve fiber tracts 132 ff – rehabilitation 129, 322 ff – replacement of neurons 139 ff – respiration 319 – scars 138 – secondary compression 319 – sensory-motor networks 325 – sexual functions 320 – signs and symptoms 320 – spastic movement disorder 320 – spinal shock 320 – strychnine 325 – therapy 317, 321 ff – treating demyelination 140 – treatments 130 spinal cord trauma 317 ff – flaccid paraplegia 317 – paraplegia is 317 – spastic, para- or tetraplegia 317 spinal muscular atrophy 153 – Spinal Muscular Atrophy type 1 (SMA1) 163 spinal shock 320 spongiform encephalopathies 213 sprouting 230 stem cells 261 ff – levels of potency 261 – neural stem cells (NSCs) 262 Stiff-man syndrome 185 stress 273 stroke 3 ff, 173, 292, 336

– cerebral bleeding 4 – cerebral ischemia 4 – focal cerebral ischemia 5 – infarct 6 – infarction 3 stroke induced immune depression (SIDS) 22 f substantia nigra pars compacta (SNc) 31 subventricular zone (SVZ) 261 survivin 163 synaptophysin 219 a-synuclein (a-syn) 33, 71 ff synucleopathies 36

T T lymphocytes 86, 180, 183 – activation 180 – CD4þ T cells 183 – CD8þ T cells 183 TAT-proteins 359 temporal lobe epilepsy (TLE) 274 tenascins 238 – tenascin-R 134 thrombolytic therapy 12 Thy1 promoter 75 tissue acidosis 8 f toll like receptor (TLR) 175 – TLR2 175 – TLR3 176 – TLR4 175 – TLR5 176 TRAIL 86 transcription factors 18 – AP-1 18 – HIF-1 18 – NF-kB 18 transgenic invertebrates 72 f transgenic mice 73 transient brain ischemia 196 transmissible spongiform encephalopathies (TSE) 213 ff traumatic brain injury – acceleration models 112 – compression injury models 110 – controlled cortical impact models 99 – dynamic brain injury models 97 – fluid-percussion injury (FPI) 98 – fluid-percussion models 111 – hydrodynamic model 112 – hydrostatic pressure models 111 – in vitro models 106 – in vivo models 96 – mechanical force 96 – pH 104

Index – shear injury models 112 – static brain injury models 97 – stretch injury models 109 ff – weight-drop model 111 traumatic brain injury (TBI) 95 – missile injury 101 – these techniques are 101 tricarbonic acid cycle (TCA) 192 b-III-tubulin 266 tumor necrosis factor receptor 1 (TNF-R1) 157 tyrosine hydroxylase (TH) 37

U U. S. Food and Drug Administration (FDA) 291 UCHL-1 33 Unified Huntington’s Disease Ranking Scale (UHDRS) 336

Unified Parkinson’s Disease Ranking Scale (UPDRS) 336 unstable energy equilibrium 198

V vascular dementia 294 VASE 137 ventral tegmental area (VTA) 31 versican 134, 237 VMAT (vesicular monoamine transporter) 37 voltage dependent channel (VDAC) 165

W Wallerian degeneration 62, 230 f, 234 Wilson’s disease 337 ff

X X-chromosome-linked IAP (XIAP) 15

369

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