Metal Ions and Neurodegenerative Disorders

Metal Ions and Neurodegenerative

Editor

Paolo Zatta

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Picture on cover Metallothionein-l-ll positive astrocytes in mouse cerebral cortex (Photo: Dr. Pamela Zambenedetti, Brain Bank, General Hospital, Dolo-Venice, Italy)

Metal Ions and Neurodegenerative Disorders

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Metal Ions and Neurodegenerative Disorders

Editor

Paolo Zatta CNR - National Research Council, Italy

'~world Scientific NEW JERSEY· LONDON· SINGAPORE· SHANGHAI· HONG KONG· TAIPEI· BANGALORE

Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: Suite 202, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

METAL IONS AND NEURODEGENERATIVE DISORDERS Copyright © 2003 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

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ISBN 981-238-398-0

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Preface Many metal ions are as essential to life (Williams and Fausto da Silva, 1996) as other constitutive elements within living matter, such as fats, carbohydrates, and proteins. Today, research into the role of metal ions is of particular interest to nutritional sciences, which have discovered that a wide range of common pathologies are caused by a lack of metalloions. Physiologists maintain that the constitutive and functional roles of some metal ions (such as Na, K, Ca, Mg, Fe, Co, Mn, Cu, and Zn) are vital to the smooth functioning of cells, as well as various organs and tissues. Toxicologists have performed indepth research into the harmful properties of some metal ions (such as Al, Cd, Hg, and Pb), and environmental disasters, such as Camelot and Minamata, have enabled them to be studied on a vast scale. However, until recently, little attention had been paid to the role of metal ions as etiopathogenic agents for some neurodegenerative diseases such as Alzheimer's, Parkinson's, and Lateral Amyotrophic Sclerosis, to name but three. Today, a new field, metalloneurochemistry, is breaking fresh ground on account of the burgeoning number of studies which show the importance of metal ions as etiopathogenic factors or co-factors (Bush, 2000; Zatta, 2001). From this point of view, the concept of metallochaperones is comparatively new in that molecules that could be used for this function were only discovered in the mid-1990s (Halloran and Culotta, 2000). Theories that suggest metal ions are vital etiological agents in neurodegenerative diseases are finding it hard to break into mainstream thought not only on account of the technical and conceptual difficulties, but also of the financial complexities of investing in both basic and applied research in order to discover more about the molecular mechanisms that account for the physiopathological action of metalloions. Chelation therapy is still in the early days of its research into new products that could revolutionize treatment. Two international conferences, Metals and the Brain: From Neurochemistry to Neurodegeneration (University of Padova, Italy, 2000 and University of Fez, Morocco, 2002;

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see also Zatta, 2001), were held on metals and their role in physiopathology as a sign of their importance. This book follows the same train of thought as these conferences in order to highlight the unquestionable importance of metal ions to research into the neurochemistry of neurodegenerative diseases. The excellent reputation of the scientists who have contributed to this project bears testimony to the quality of these studies, which we hope can be developed further with a whole host of new research within this field; one which is still in its infancy. PAOLO ZATTA

REFERENCES Bush AI. Metals and neuroscience. Curr Op Chem Biol 2000; 4:184-191. Halloran TV, Culotta VC. Metallochaperones: An intracellular shuttle service for metal ions. J Biol Chem 2000; 275:25047-25060. William RJP, Frausto da Silva JJR. The Natural Selection of the Chemical Elements. Oxford: Clarendon Press, 1996. Zatta P, editor. Metals and the brain. A special issue. Brain Res Bull 2001; 55:123-325.

Contents Preface

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List of Contributors

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Chapter 1. Metal-Catalyzed Redox Activity in Neurodegenerative Disease Marta A Taddeo, Mark A Smith, Quart Liu, Craigh S Atwood, Lawrence M Say re, George Perry Chapter 2. Metals Distribution and Regionalization in the Brain Margherita Speziali, Edoardo Orvini

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Chapter 3. The Olfactory Pathway as a Route of Entry of Metals into the Brain Hans Tjalve, Jonas Tallkvist

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Chapter 4. Aluminum in Neurological Disorders and Systemic Chelation Therapy Theo P Kruck, Walter J Lukiw

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Chapter 5. Alumium and Central Nervous System Morphology in Hemodialysis Erich Reusche

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Chapter 6. Zinc, Brain, and Aging Eugenio Mocchegiani, Mario Muzzioli, Robertina Giacconi, Tiziana Casoli, Giuseppina DiStefano, Patrizia Fattoretti

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Chapter 7. Transition Metals, Oxidation, Lipoproteins, and Amyloid-3: Major Players in Alzheimer's Disease Anatol Kontush

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Chapter 8. Molecular Basis of Copper Transport: Cellular and Physiological Functions of Menkes and Wilson Disease Proteins (ATP7A and ATP7B). David R Kramer, Roxana M Llanos, Julian F B Mercer

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Chapter 9.

Importance of Copper and Zinc in Alzheimer's Disease and the Biology of Amyloid-(3 Protein and Amyloid-(3 Protein Precursor Avi L Friedlich, Xudong Huang, Seiichi Nagano, Jack T Rogers, Lee E Goldstein, Ashley I Bush, Gerd Multhaup, Konrad Beyreuther, Wolfgang Stremmel, Thomas Bayer

Chapter 10. Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis Moon B Yim, P Boon Chock, Earl R Stadtman

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Chapter 11. Copper and Prion Disease Judyth Sasson, David R Brown

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Chapter 12. Metallothioneins in Neurodegeneration Michael Aschner, William F Silverman, Israel Sekler, Paolo Zatta

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Chapter 13. Iron and Neurodegeneration Stacey L Grab, James R Connor

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Chapter 14. Iron, Neuromelanin, and a-Synuclein in Neuropathogenesis of Parkinson's Disease Kay L Double, Kurt Jellinger, Luigi Zecca, Moussa B H Youdim, Peter Riederer, Manfred Gerlach

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Chapter 15. Iron and Epilepsy Wie-Yi Ong, Benjamin Kian-Chung Ong, Akhlaq A Farooqui, Chuang-Chin Chiueh, James R Connor

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Chapter 16. Role of Iron Metabolism in Multiple Sclerosis Maritha J Kotze, J Nico P De Villiers, Monique G Zaahl, Kathryn J H Robson

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Chapter 17. Manganese Toxicity: A Critical Reappraisal Patrizia Vernole, Maria Morello, Giuseppe Sancesario, Alessandro Martorana, Giorgio Bernardi, Antonella Canini, Palma Mattioli

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Chapter 18. Cupric and Mercuric Ions Affect the Structure and Functions of Cell Membranes Mario Suwalsky, Fernando Villena, Hernan Cardenas,Beryl Norris, Carlos Patricio Sotomayor, Paolo Zatta

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Chapter 19. Influence of Lead Exposure on Brainstem Functions Ombretta Mameli, Marcello Alessandro Caria

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Chapter 20. Neuroprotective Effects of Lithium Sophie Ermidiou-Pollet, Serge Pollet

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Chapter 21. Histopathological Changes in Brain of Uremic Patients on Chronic Hemodialysis Pamela Zambenedetti, Mario Andriani, Maurizio Nordio, Paolo Zatta

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Chapter 22. Clinical Neurotoxicity of Metals and Neurodegenerative Disorders Marcello Lotti

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Index

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List of Contributors Michael ASCHNER Departments of Physiology and Pharmacology Wake Forest University School of Medicine Winston-Salem, NC, USA Email: [email protected] Craigh S ATWOOD Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Thomas BAYER Department of Psychiatry University of Bonn Medical Center Bonn, Germany Giorgio BERNARDI Department of Neuroscience University of Rome Tor Vergata and IRCCS S Lucia 00100 Rome, Italy Konrad BEYREUTHER ZMBH-Center for Molecular Biology University of Heidelberg Heidelberg, Germany David BROWN Department of Biology and Biochemistry University of Bath Bath BA2 7AY, UK Email: [email protected] XI

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Ashley I BUSH Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Email: [email protected] Antonella CANINI Department of Biology University of Rome Tor Vergata 00100 Rome, Italy Hernan CARDENAS Faculty of Biological Sciences University of Concepcion Casilla 160-C Concepcion, Chile Marcello Alessandro CARIA Department of Biomedical Sciences Human Physiology Division University of Sassari Viale San Pietro 43/B 07100 Sassari, Italy Email: [email protected] Tiziana CASOLI Neurobiology Center Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 89 60121 Ancona, Italy Email: [email protected] Chuang-Chin CHIUEH National Institute of Mental Health LCS, NIH 10/3D-41 Bethesda, MD 20892-1264, USA

List of Contributors

List of Contributors

P Boon CHOCK Laboratory of Biochemistry National Heart, Lung and Blood Institute National Institutes of Health Building 50, Room 2152 50 South Drive, MSC-8012 Bethesda, MD 20892-8012, USA James R CONNOR Department of Neuroscience and Anatomy Pennsylvania State University Hershey, PA 17033-0850, USA Email: [email protected] J Nico P DE VILLIERS Division of Human Genetics Faculty of Health Sciences University of Stellenbosch Tygerberg 7500, South Africa Giuseppina DiSTEFANO Neurobiology Center Research Department Italian National Research Centers on Aging (INRCA) Via Birarelli 89 60121 Ancona, Italy Kay L DOUBLE Prince of Wales Medical Research Institute Sydney, Australia Email: [email protected] Sophie ERMIDIOU-POLLET Department of Biochemistry Medical School, University of Athens Athens, Greece Email: [email protected]

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Akhlaq A FAROOQUI Department of Molecular and Cellular Biochemistry The Ohio State University Columbus, Ohio OH 43210, USA Patrizia FATTORETTI Neurobiology Center Research Department Italian National Research Centers on Aging (INCRA) Via Birarelli 89 60121 Ancona, Italy Email: [email protected] Avi L FRIEDLICH Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Manfred GERLACH Clinical Neurochemistry Department of Child and Youth Psychiatry and Psychotherapy University of Wurzburg Wiirzburg, Germany Robertina GIACCONI Immunology Center Section Nutrition, Immunity and Aging Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 8 60121 Ancona, Italy Lee E GOLDSTEIN Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA

List of Contributors

Stacy L GRAB Department of Neuroscience Pennsylvania State University College of Medicine/Milton S. Hershey Medical Center Hershey, PA 17033, USA Email: [email protected] Xudong HUANG Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Kurt JELLINGER Institute of Clinical Neurobiology Vienna, Austria Email: [email protected] Anatol KONTUSH INSERM Unite 551 Papillon Benjamin Delessert Hospital de la Pitie, 83 Boulevard de l'Hopital 75651 Paris Cedex 13, France Email: [email protected] Maritha J KOTZE Division of Human Genetics Faculty of Health Sciences University of Stellenbosch Tygerberg 7500, South Africa Email: [email protected] David K KRAMER Center for Cellular and Molecular Biology School of Biological and Chemical Sciences Deakin University, 221 Burwood Hwy Melbourne 3125, Australia Email: [email protected]

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Theo P KRUCK Surrey Place Center and Department of Physiology University of Toronto Ontario M5S 2C2, Canada Email: [email protected] Quan LIU Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Roxana M LLANOS Center for Cellular and Molecular Biology School of Biological and Chemical Sciences Deakin University 221 Burwood Hwy Melbourne 3125, Australia Email: [email protected] Marcello LOTTI Dipartimento di Medicina Ambientale e Sanita Pubblica Universita di Padova, Azienda Ospedaliera di Padova Via Giustiniani 2 25128 Padova, Italy Email: [email protected] Walter J LUKIW LSU Neuroscience Center and Department of Ophthalmology Louisiana State University Health Science Center New Orleans, LA 70112, USA Email: [email protected] Ombretta MAMELI Department of Biomedical Sciences, Human Physiology Division University of Sassari Viale San Pietro 43/B 07100 Sassari, Italy Email: [email protected]

List of Contributors

Alessandro MARTORANA Department of Neuroscience University of Rome Tor Vergata and IRCCS S Lucia 00100 Rome, Italy Palma MATTIOLI Department of Biology University of Rome Tor Vergata 00100 Rome, Italy Julian F B MERCER Center for Cellular and Molecular Biology School of Biological and Chemical Sciences Deakin University 221 Burwood Hwy Melbourne 3125, Australia Email: [email protected] Eugenio MOCCHEGIANI Immunology Center Section Nutrition, Immunity and Aging Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 8 60121 Ancona, Italy Email: [email protected] Maria MORELLO Department of Neuroscience University of Rome Tor Vergata 00100 Rome, Italy Gerd MULTHAUP Institut fuer Chemie/Biochemie Freie Universitat Berlin Universitat Berlin Thielallee 63 14195 Berlin, Germany Email: multhaup @ chemie.fu-berlin.de

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Mario MUZZIOLI Immunology Center Section Nutrition, Immunity and Aging Research Department Italian National Research Center on Aging (INRCA) Via Birarelli 8 60121 Ancona, Italy Seiichi NAGANO Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Maurizio NORDIO Nephrology Division General Hospital Venice, Italy Email: [email protected] Beryl NORRIS Faculty of Biological Sciences University of Concepcion Casilla 160-C Concepcion, Chile Benjamin Kian-Chung ONG Department of Medicine National University of Singapore Lower Kent Ridge Road Singapore 119260 Wei-Yi ONG Department of Anatomy National University of Singapore Lower Kent Ridge Road, Singapore 119260 Email: [email protected]

List of Contributors

Edoardo ORVINI Department of General Chemistry University of Pavia, Viale Taramelli 12 27100 Padova, Italy Email: [email protected] George PERRY Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Email: [email protected] Serge POLLET Department of Biochemistry Medical School, University of Athens Athens, Greece Email: [email protected] Erich REUSCHE Institute of Pathology and Neuropathology University of Lubeck Ratzeburgerallee 160 23538 Lubeck, Germany Email: [email protected] Peter RIEDERER Clinical Neurochemistry Department of Psychiatry and Psychotherapy University of Wurzburg 97080 Wurzburg, Germany Email: [email protected] Kathryn J H ROBSON MRC Unit of Molecular Hematology Weatherall Institute of Molecular Medicine John Radcliffe Hospital, Headington Oxford OX3 9DS, UK

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Jack T ROGERS Laboratory of Oxidation Biology Genetics and Aging Research Unit Massachusetts General Hospital Building 114, 16th Street Charlestown, MA 02129, USA Giuseppe SANCESARIO Department of Neuroscience University of Rome Tor Vergata 00100 Rome, Italy Email: [email protected] Judyth SASSOON Department of Biology and Biochemistry University of Bath Bath BA2 7AY, UK. Email: [email protected] Lawrence M SAYRE Department of Chemistry Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Israel SEKLER Zlotowski Center for Neuroscience Faculty of Health Science Ben-Gurion University Beer Sheva, Israel William F SILVERMAN Zlotowski Center for Neuroscience Faculty of Health Science Ben-Gurion University Beer Sheva, Israel

List of Contributors

List of Contributors

Mark A SMITH Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Carlos Patricio SOTOMAYOR Faculty of Basic Sciences Catholic University of Valparaiso Casilla 4059 Valparaiso, Chile Margherita SPEZIALI CNR-Institute for Energetics and Interphases Department of General Chemistry, University of Pavia Via Taramelli 12 27100 Pavia, Italy Email: [email protected] Earl R STADTMAN Laboratory of Biochemistry National Heart, Lung and Blood Institute National Institutes of Health Building 50, Room 2152 50 South Drive, MSC-8012 Bethesda, MD 20892-8012, USA Wolfgang STREMMEL Department of Medicine Division of Gastroenterology University of Heidelberg, Germany Heidelberg, Germany Mario SUWALSKY Faculty of Chemical Sciences University of Concepcion Casilla 160-C, Concepcion, Chile Email: [email protected]

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Marta A TADDEO Institute of Pathology Case Western Reserve University 2085 Adelbert Road Cleveland, OH 44106, USA Jonas TALLKVIST Department of Pharmacology and Toxicology Faculty of Veterinary Medicine Swedish University of Agricultural Sciences Biomedicum, Box 573 Uppsala 751 23, Sweden Hans TJALVE Department of Pharmacology and Toxicology Faculty of Veterinary Medicine Swedish University of Agricultural Sciences Biomedicum, Box 573 Uppsala 751 23, Sweden Email: Hans .Tj alve @ farmatoxslu. se Patrizia VERNOLE Department of Public Health University of Rome Tor Vergata 00100 Rome, Italy Fernando VILLENA Faculty of Biological Sciences University of Concepcion Casilla 160-C Concepcion, Chile Moon B YIM Laboratory of Biochemistry National Heart, Lung, and Blood Institute National Institutes of Health Building 50, Room 2152, 50 South Drive, MSC-8012 Bethesda, MD 20892-8012, USA Email: [email protected]

List of Contributors

List of

Contributors

Moussa B H YOUDIM Department of Pharmacology B Rappaport Faculty of Medicine Eve Topf Neurodegenerative and National Parkinson Foundation Centers Technicon, Haifa, Israel Email: [email protected] Monique G ZAAHL MRC Unit of Molecular Hematology Weatherall Institute of Molecular Medicine John Radcliffe Hospital, Headington Oxford 0X3 9DS, UK Pamela ZAMBENEDETTI Anatomopathology Division and Brain Bank Dolo General Hospital Dolo-Venice, Italy Email: [email protected] Paolo ZATTA CNR-Institute for Biomedical Biotechnologies Padova Unit "Metalloproteins" Department of Biology, University of Padova Viale G. Colombo 3 35121 Padova, Italy Email: [email protected] Luigi ZECCA CNR-Institute for Biomedical Technologies Via Fratelli Cervi 93 20090 Segrate, Italy Email: [email protected]

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CHAPTER 1

Metal-Catalyzed Redox Activity in Neurodegenerative Disease Marta A Taddeo, Mark A Smith, Quan Liu, Craigh S Atwood, Lawrence M Sayre, George Perry

ABSTRACT Oxidative damage and response are major features of Alzheimer's disease and other neurodegenerative diseases. Metal-catalyzed oxidative events are at the center of radical formation and abnormalities in iron and copper have been noted. In this review, we consider the evidence implicating the central role of metals. Keywords: Amyloid-(3; copper; iron; metal-binding protein; oxidative stress; tau.

1. INTRODUCTION There has been considerable investigation into the roles of redox-active transition metals in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and prion diseases. Iron, copper, manganese, and other such metals are usually found in essential normal biological processes, and are also involved in enzymatic activities such as those related to respiration. In these functions, deficiency of these metals can be hazardous to normal organ function, including central nervous system activities. However, inappropriate accumulation of excessive metal deposits can also be cytotoxic. In short, disruption of the normal homeostatic metal balance in either direction can result in cellular disturbance. In the latter case, these changes are characterized by oxidative 1

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stress and increased production of free radicals. Oxidative stress results from excessive levels of reactive oxygen species (ROS) generated through imbalance of normal biochemical processes. These oxygen radicals can then damage all types of macromolecules, disrupting normal cellular functions and, ultimately, causing cell death. ROS can normally be found at some level in all aerobic organisms, usually arising from the secondary production of superoxide by mitochondrial reduction of molecular oxygen. Increased levels of ROS during oxidative stress point to increased oxygen processing by mitochondria as a possible source. In addition, greater oxidative stress susceptibility can be caused by production of H 2 0 2 by oxidases such as monoamine oxidase. Examination of nitric oxide (NO) has also gained recognition through the term "nitrosative stress", a form of oxidative stress characterized by the reaction of superoxide with NO to yield peroxynitrite, which is further capable of both oxidation chemistry and nitration reactions. The primary ROS implicated in oxidative stress is the hydroxyl radical, whose damage is dependent upon its diffusion capacity over nanometer distances from its generation site. Although peroxynitrite also appears capable of hydroxyl-like activity, most hydroxyl radicals reflect the Fenton reaction between reduced transition metals (usually iron[II] or copper[I]) and H 2 0 2 . Re-reduction of iron[III] and copper[II] can be performed by superoxide or other cellular reductants such as ascorbate. In addition to their role in redox processes, transition metals may contribute to neurodegeneration, along with redox-inactive metal ions, through their deleterious effects on protein and peptide structure, such as a pathological aggregation phenomenon. In such cases, transition metals can sometimes exert dual neurotoxic effects. In contrast, there are also a number of normal antioxidant defenses to combat the effects of ROS through both enzymatic and nonenzymatic pathways in mammalian cells. Cytosolic copper-zinc superoxide dismutase (CuZnSOD) and mitochondrial manganese superoxide dismutase (MnSOD) are two such important players in antioxidant defenses. These enzymes convert reactive superoxide to harmless 0 2 and H 2 0 2 . The latter is then removed by catalase and peroxidases, which are abundant throughout tissues. In addition, there are a number of proteins closely tied to metalinduced redox activity. These include proteins involved in metal transport

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and those which do not normally have a metal-binding function, but are able to bind metals for neuroprotection. This review explores the interplay between these various factors and evaluates recent work concerning the role of redox-active metals in neurodegenerative diseases.

1.1. Redox-Active Transition Metals in AD AD is pathologically diagnosed by the presence of neurofibrillary tangles (NFTs), senile plaques, neuropil threads, amyloid-beta (A(3) deposits, and selective loss of neurons (Perry and Smith, 1999). Though a number of hypotheses have been presented describing the origins of AD, the one receiving the most recent attention involves a role for oxidative stress (reviewed in Markesbery, 1999; Smith et al., 2000a; Perry et al., 2001). Past studies have already implicated a number of trace metals whose imbalances can contribute to AD, including aluminum, silicon, lead, mercury, zinc, copper, and iron (Lovell et al., 1998). Disruptions in iron and copper levels, in particular, have quite a substantial effect on levels of oxidative stress markers such as lipid peroxidation, as well as on damage related to NFT, senile plaque deposition, and nucleic acid oxidation (Smith et al., 2000a). It was recently found through microparticle-induced X-ray emission that Zn(II), Fe(III), and Cu(II) are significantly elevated in the AD neuropil, and that these metals are further concentrated in the core and periphery of senile plaques (Lovell et al., 1998). These results build on earlier studies reporting increased levels of iron, transferrin receptors and ferritin in AD. Using an in situ iron detection method, we found a marked association of redox-active iron with both NFT and senile plaques in AD (Smith et al., 1997). The association with NFT may partly involve iron binding to the primary NFT protein component, tau (Perez et al., 1998). At the same time, iron regulatory protein (IRP)-2 was found to co-localize with redox-active iron in NFT, senile plaque neurites, and neuropil threads (Smith et al., 1998). IRP-1, in contrast, was found to be present at similar levels in both AD and control brain tissues. These latter results suggest that alterations in IRP-2 may be linked to impaired iron homeostasis in AD (Smith et al., 1998). We have found that redox activity in AD lesions can be directly detected in tissue sections and homogenates using cytochemical methods

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(Smith et al., 1997; Sayre et al., 2000). This activity is inhibited by preexposure to copper- and iron-selective chelators, with incubation in metal solutions post-chelation restoring activity. These studies indicating the presence of iron, and probably also copper related to AD pathology, suggest that accumulations of these metals are major generators of ROS. This production is responsible for the numerous oxidative stress markers apparent in NFT and senile plaques, as well as the more global markers observed in AD (Sayre et al., 2001).

1.2. Aggregation of Amyloid-p and Amyloid |3 Protein Precursor/Amyloid-p-Linked ROS Production Evidence for a disruption of trace metal homeostasis in AD has sparked off efforts to identify possible associations between metal ions and A3 aggregation. Under neutral conditions, it was found that aluminum, iron, and zinc accelerated Ap aggregation, while calcium, cobalt, manganese, copper, magnesium, sodium, and potassium did not (Mantyh et al., 1993). More recent studies, however, suggest that this aggregation due to metals depends more critically on pH. For example, it was found that Cu(II) was able to induce A p ^ o aggregation when the pH was lowered from 7.4 to 6.8, and that this result was unique to this particular metal (Atwood et al., 1998). A mildly acidic environment, as well as increased levels of Zn(II) and Cu(II), is a common feature of inflammation associated with increased oxidative damage due to microglial-derived peroxynitrite. The association of Cu(II), Zn(II), and Fe(II) with amyloid-pi seen in vitro could explain the deposition of these metals in senile plaques in AD. It is interesting to note in these studies on A|3 aggregation that the effect of the transition metal does not involve a redox role. This contrasts with results which suggest a synergistic action of A(3 and copper or iron in mediating ROS production (Bondy et al., 1998). Also, Cu(II) binds to amyloid-|3 protein precursor (ApPP) and appears to be reduced to Cu(I) accompanied by production of a disulfide linkage (Multhaup et al., 1998). Subsequent exposure to H 2 0 2 results in reoxidation of Cu(I) along with site-specific cleavage of ApSPP. Redox chemistry associated with Ap- or ApPP-bound metals could, therefore, contribute to a perturbation of free radical homeostasis.

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Recent genetic studies have revealed a number of mutations in the genes encoding the A(3PP and the presenilin proteins (Perry and Smith, 1999). Alterations in these genes lead to overproduction and/or altered proteolytic processing of A(3PP, in both cases leading to increased levels of A(3. Transgenic mouse models expressing these mutations can develop senile plaques and some neurotoxicity, but the model is not complete for familial AD, and the mechanism of this demonstrated neurotoxicity may be unrelated to the neuronal loss present in AD (Perry et al., 2000; Smith et al., 2000b). Furthermore, even though genetic mutations in these genes account for the majority of familial AD cases, they only comprise a small percentage of total AD cases since the predominance of AD is sporadic.

1.3. Iron in Neurodegenerative Disease Free iron has long been implicated in neurodegenerative disease through its redox transitions in vivo. The consequential generation of oxygen free radicals can further induce oxidative stress in tissues. Abnormally high levels of iron and oxidative stress have been found in neurodegenerative disorders such as AD, PD, multiple system atrophy, and progressive supranuclear palsy (Smith et al., 1997; Sayre et al., 2000). Oxidative stress, under these conditions, has been associated with levels of free iron. An increased level of total iron does not necessarily signify increased oxidative stress if it is accompanied by a concomitant increase in iron storage proteins, which keep iron in a redox-inert state. One such iron storage protein, ferritin, contains a core of insoluble and unreactive ferrihydrate. The binding and release of iron to and from ferritin, however, occurs through the ferrous iron state, which is quite active in the generation of hydoxyl radicals by Fenton reactions. Microglia are the main locations of ferritin-bound iron and are thought to be somewhat responsible for oxidative damage in PD and other neurodegenerative diseases (Smith et al., 1994; Premkumar et al., 1995; Kakimura et al., 2002; Castellani et al., 1995; Schipper et al., 1995; Ham and Schipper, 2000). Microglia stimulated in vivo using phorbol ester demonstrate increased lipid peroxidation due to superoxide-dependent release of iron from ferritin (Yoshida et al., 1998). This release can also be induced by 6-hydroxydopamine, a neurotoxin already implicated in PD, and other easily oxidized catechols (Double et al., 1998).

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These studies support the hypothesis that release of iron from ferritin contributes to free radical-induced cellular damage in vivo by Fenton production of hydroxyl radicals. The interplay between oxidative stress and iron metabolism in the brain is further demonstrated by the finding of abnormal iron deposition accompanied by lipid peroxidation in a transgenic mouse model of progressive neurodegeneration (Castelnau et al., 1998). In addition to primary management by the transferrin receptor and ferritin, it has been shown more recently that the handling of iron at the cellular level can be regulated by the lactotransferrin receptor, melanotransferrin, ceruloplasmin (Castellani et al., 1999), and divalent cation transporter 1. Disruptions in the expression of these latter proteins in brain tissues probably contribute to altered iron metabolism in disorders such as AD and PD (Qian and Wang, 1998). Overall, regulation of cellular iron metabolism relies on the actions of two known iron regulatory proteins, IRP-1 and IRP-2. IRPs undergo significant alterations in AD patients, supporting a role for redox-active iron in this disease (Smith et al., 1998). Levels of extracellular H 2 0 2 rapidly activate IRP-1, suggesting a regulatory connection between iron regulation and oxidative stress. This activation has been recapitulated in vitro and shown to require a cellular membrane-associated component which is sensitive to temperature and alkaline phosphatase (Pantopoulos and Hentze, 1998). Future work in this area should result in a clearer understanding of the causative link between iron-induced oxidative stress and neuronal death.

1.4. Iron-Mediated Oxidative Stress in Parkinson's Disease Even though the etiology of PD remains undefined, several biochemical abnormalities in PD brain tissue have been identified, including a mitochondrial complex I deficiency, oxidative stress, and excess iron (Jenner and Olanow, 1996). In addition, the somewhat recent identification of a mutation in the a-synuclein gene in cases of familial Parkinsonism may ultimately lead to an understanding of the biochemical mechanisms of selective dopaminergic cell death in these cases. Iron accumulations have been found in astrocytes in the substantia nigra of old rats (Schipper, 1996; Schipper et al, 1998), along with an increase in

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the Fe(III), to Fe(II) ratio and a decrease in reduced glutathione (Riederer et al., 1989). One interpretation is that mitochondrial sequestration of redoxactive iron in aging nigral astroglia may predispose the senescent nervous system to Parkinsonism and other neurodegenerative disorders. In fact, a persistent condition of elevated oxidative stress associated with greatly perturbed intracellular redox equilibria is widely recognized as a pathogenic factor underlying neurodegeneration. Moreover, there is circumstantial evidence that the intracellular redox imbalance results in aberrant oxidation of dopamine to 6-hydroxydopamine, which in turn can undergo autoxidation to the corresponding quinone accompanied by the generation of superoxide. This reaction cascade, either by itself or amplified by redox cycling of the quinone, which leads to further generation of ROS while depleting cellular reductants, can serve to explain the ultimate demise of these neurons. Studies to clarify the mechanism of dopamine oxidation in vitro have demonstrated conversion to 6-hydroxydopamine in the presence of Fe(II) and either H 2 0 2 or alkyl peroxides (Pezzella et al., 1997).

1.5. Manganese and Parkinson's Disease Chronic exposure to manganese results in extrapyrimidal syndromes resembling PD. Manganese has, therefore, been labeled as an environmentally toxic factor that induces brain dysfunction. One hypothesis to explain these results suggests that manganese acts as a dopaminergic neurotoxin, in the same manner as iron, by promoting the generation of ROS and the subsequent nonenzymatic autoxidation of dopamine to the neurotoxin 6-hydroxydopamine. There is, however, no convincing in vivo evidence for a pro-oxidant role of manganese in the brain. Also, the clinical picture of manganese-induced Parkinsonism is unclear because there is conflicting evidence regarding whether it is selectively toxic to dopaminergic neurons (Calne et al., 1994). In addition, more recent findings suggest that manganese actually may act as an antioxidant rather than as a pro-oxidant (Sziraki et al., 1998). In this study using rats given Fe(II) intranigrally, Mn(II) was found to protect against the toxicity of Fe(II) in a dose-dependent fashion. This protection could result from the fact that Mn(II), while itself inactive in Fenton chemistry, competes with Fe(II) in oxidative cascades. Thus, it appears that the Parkinsonian-like condition induced by chronic

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manganese exposure has little connection to nigrostriatal damage occurring in idiopathic PD.

1.6. Antioxidant and Transition Metal Homeostasis Many studies of oxidative stress in AD have focused on the inducible mitochondrial MnSOD and the constitutive cytoplasmic CuZnSOD enzymes as possible targets for therapy. The CuZnSOD gene has been associated with AD neuropathology, and levels of both MnSOD mRNA and CuZnSOD were found to be increased in AD, whereas the total antioxidant status was decreased. However, since superoxide dismutase (SOD) enzymes are key cellular antioxidant components, any pro-oxidant mechanism linked to SOD must derive from the balance in the local concentrations of superoxide and H 2 0 2 , which together can produce hydroxyl radicals by the Haber-Weiss process. The development of transgenic mouse "knockout" and overexpression models have allowed a range of studies to critically evaluate the extent to which selected biological processes affect cell viability. For example, if one of the SOD enzymes serves a crucial antioxidant function, then knockout animals may exhibit increased oxidative stress parameters, either on a global level or localized to the specific cellular compartment normally protected by that SOD enzyme. In support of this notion, MnSOD knockout mice, which suffer a 50% drop in mitochondrial SOD activity with no reduction in CuZnSOD or glutathione peroxidase activity, were found to exhibit increased oxidative damage to mitochondria. This was shown by increased mitochondrial protein carbonyls and 8-hydroxydeoxyguanosine in mitochondrial DNA (Williams et al., 1998). In contrast, no damage to cytosolic proteins or to nuclear DNA was observed. Analysis of homozygote knockouts showed mitochondrial degeneration throughout the two weeks that the mice lived (Melov et al., 1998). These in vivo results suggest that decreases in MnSOD activity can account for increased oxidative damage in mitochondria and alterations in essential mitochondrial function. In other studies, a tenfold higher expression level of human CuZnSOD in both myocytes and endothelial cells of mice was able to quench a burst of superoxide (in electron paramagnetic resonance detection) and reduce functional damage following 30-minute global ischemia (Wang et al., 1998).

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These results suggest that superoxide is an important factor in protecting against post-ischemic injury, and it is apparent that decreases in CuZnSOD activity can lead to a perturbation of cellular antioxidant defense mechanisms, thereby promoting a pro-oxidant condition. It is surprising, however, that CuZnSOD knockout mice show little, if any, significant neurodegenerative phenotype (Bruijn et al., 1998). Hence, while absences of CuZnSOD have little effect, enzyme inactivation by metal-catalyzed oxidation can promote oxidative damage (Kwon et al., 1998). These differences, while seemingly paradoxical, may be particularly relevant to human diseases. Ceruloplasmin (CP) is an important copper storage protein, the major Fe(II)-oxidizing enzyme in the central nervous system, and one of the key proteins that responds to oxidative stress. A related inherited metabolic disorder, aceruloplasminemia, is associated with impairment in iron homeostasis and consequent neurodegeneration (Harris et al., 1998). Studies directed at clarifying the relationship between oxidative stress and tissue metal-ion levels indicate that both the ratio of copper to zinc and the levels of CP are significantly higher with increasing age, and higher yet in cases with neurodegeneration. Interestingly, while CP is increased in brain tissue and cerebrospinal fluid in AD, PD and Huntington's disease patients (Loeffler et al., 1996), neuronal levels of CP remain unchanged (Castellani et al., 1999). Therefore, while increased CP may indicate a compensatory response to increased oxidative stress in AD, its lack of increase in neurons may play an important role in metal-catalyzed damage. Since the copper to zinc ratio is significantly correlated with systemic oxidative stress (that is, lipid peroxidation), it is probable that an increased oxidative stress burden in aging and neurodegeneration may reflect, in large part, copper-mediated ROS production. In this case, redox-inert zinc may serve as an antioxidant by preventing binding of pro-oxidant copper at tissue sites. A novel glycolipid-anchored membrane-bound form of CP expressed by astrocytes in the mammalian CNS has been identified, and any possible role for this CP in the neurodegenerative process will be investigated. The escalating interest in prion diseases (such as Creutzfeldt-Jakob disease), characterized by infection with an altered prion protein conformation that is transmissible through endogenous protein, continues to question the role of this cell-surface glycoprotein in healthy individuals. Thus far,

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several lines of evidence indicate that the prion protein binds Cu(II) and, thus, as with CP, may serve a cytoprotective role (Brown et al., 1997, 1998). One possibility, consistent with the finding in cell cultures that copper stimulates endocytosis of prion protein from the cell surface, is that prion protein controls copper metabolism by serving as a recycling receptor for uptake of extracellular copper (Pauly and Harris, 1998).

1.7. CuZnSOD Mutations and Familial Amyotrophic Lateral Sclerosis A crucial breakthrough in our understanding of amyotrophic lateral sclerosis (ALS) comes from the finding that many cases of familial ALS (FALS) are associated with mutations in the CuZnSOD gene. The protein products of these mutations retain nearly identical SOD activity, but they take on altered properties related to oxidative stress, possibly involving a gain-of-function peroxidase activity (Wiedau-Pazus et al., 1996). Consistent with these findings, transgenic mice overproducing a human FALS CuZnSOD mutant display increases in protein carbonyls, suggestive of increased hydroxyl radical production or lipoxidation-derived radicals (Andrus et al., 1998). Also, using in vivo microdialysis, increased hydroxyl radical production in the striatum was seen for mice overexpressing mutant CuZnSOD relative to mice overexpressing the wildtype human enzyme (Bogdanov et al., 1998), as determined by conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid. The hypothesis that mutant SOD-induced neurodegeneration is associated with disturbances in neuronal free radical homeostasis is further supported by observations made on several neuronal cell cultures expressing the mutant SOD (Ghadge et al., 1998). The link between the SOD mutations and oxidative stress indicators appears not to be simply due to increased production of hydroxyl radicals, however, since there are no increases in hemeoxygenase. Adducts are seen in vitro for the Gly93 to Ala and Ala4 to Val mutant enzymes relative to the wild-type enzyme (Singh et al., 1998). Recent studies have shown that the mutant and wild-type SODs differ neither in their rates of superoxide dismutation nor in H202-mediated inactivation (Liochev et al., 1998; Goto et al., 1998). Structural analysis of the mutant enzymes did reveal, in some cases, alterations such as subunit asymmetry that suggest aberrant copper-mediated redox chemistry due to

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less tight folding and, thus, more open "active sites" (Corson et al., 1998; Hart et al., 1998). Therefore, the gain-of-function activity may reflect a "rechanneling" of the enzyme to oxidize biomolecules that would usually never gain access to the SOD oxidative half-reaction. Alternatively, the mutant SOD may possess weakened affinities for zinc or copper, leading to a fraction of enzyme with abnormal activity (Singh et al., 1998) or oxidative reactions associated with copper leakage (Goto et al., 1998). For the predominant, sporadic form of ALS, an imbalance of trace metal ions, possibly tied to increased oxidative stress, has been considered for some time. Recent studies provide evidence for decreases in copper in cerebrospinal fluid and serum, as well as increases in manganese in the serum of affected individuals versus age-matched controls.

2. CONCLUSIONS There has been an increasing awareness of the role that redox-active transition metals play in a variety of neurodegenerative diseases. The next step is to critically examine the importance of these basic research findings as they are translated into therapeutic modalities, such as antioxidants and chelating agents, to be used clinically. Hopefully, these treatments will yield promising results.

REFERENCES Andrus PK, Fleck TJ, Gumey ME, Hall ED. Protein oxidative damage in a transgenic mouse model of familial amytrophic lateral sclerosis. J Neurochem 1998; 71:2041-2048. Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NME, Romano DM, Hartshorn MA, Tanzi RE, Bush Al. Dramatic aggregation of Alzheimer Afi by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem 1998; 273:12817-12826. Bogdanov MB, Ramos LE, Xu Z, Beal MR Elevated "hydroxyl radical" generation in vivo in an animal model of amyotrophic lateral sclerosis. J Neurochem 1998; 71:1321-1324. Bondy SC, Guo-Ross SX, Truong AT. Promotion of transition metal-induced reactive oxygen species formation by P-amyloid. Brain Res 1998; 799:91-96. Brown DR, Qin K, Herms JW, Madlung A, Manson J, Strome R, Fraser PE, Kruck T, von Bohlen A, Schulz-Schaeffer W, Giese A, Westaway D, Kretzschmar H. The cellular prion protein binds copper in vivo. Nature 1997; 390:684-687. Brown DR, Schmidt B, Kretzschmar HA. Effects of copper on survival of prion protein knockout neurons and glia. J Neurochem 1998; 70:1686-1693.

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Bruijn LI, Houseweart MK, Kato S, Anderson KL, Anderson SD, Ohama E, Reaume AG, Scott RW, Cleveland DW. Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science 1998; 281:1851-1854. Calne DB, Chu NS, Huang CC, Lu CS, Olanow W. Manganism and idiopathic Parkinsonism: similarities and differences. Neurology 1994; 44:1583-1586. Castellani R, Smith MA, Richey PL, Kalaria R, Gambetti P, Perry G. Evidence for oxidative stress in Pick disease and corticobasal degeneration. Brain Res 1995; 696:268-271. Castellani RJ, Smith MA, Nunomura A, Harris PLR, Perry G. Is increased redox-active iron in Alzheimer disease a failure of the copper-binding protein ceruloplasmin? Free Radic Biol Med 1999; 26:1508-1512. Castelnau PA, Garrett RS, Palinski W, Witztum JL, Campbell IL, Powell HC. Abnormal iron deposition associated with lipid peroxidation in transgenic mice expressing interleukin-6 in the brain. J Neuropathol Exp Neurol 1998; 57:268-282. Corson LB, Strain JJ, Culotta VC, Cleveland DW. Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants. Proc Natl Acad Sci USA 1998; 95:6361-6366. Double KL, Maywald M, Schmittel M, Riederer P, Gerlach M. In vitro studies of ferritin iron release and neurotoxicity. J Neurochem 1998; 70:2492-2499. Ghadge GD, Lee JP, Bindokas VP, Jordan J, Ma L, Miller RJ, Roos RP. Mutant superoxide dismutase-1-linked familial amyotrophic lateral sclerosis: Molecular mechanisms of neuronal death and protection. JNeuwsci 1998; 17:8756-8766. Goto JJ, Gralla EB, Valentine JS, Cabelli DE. Reactions of hydrogen peroxide with familial amyotrophic lateral sclerosis mutant human copper-zinc superoxide dismutases studies by pulse radiolysis. J Biol Chem 1998; 273:30104-30109. Ham D, Schipper HM. Heme oxygenase-1 induction and mitochondrial iron sequestration in astroglia exposed to amyloid peptides. Cell Mol Biol 2000; 46:587-596. Harris ZL, Klomp LW, Gitlin JD. Aceruloplasminemia: An inherited neurodegenerative disease with impairment of iron homeostasis. Am J Clin Nutr 1998; 67:S972-S977. Hart PJ, Liu H, Pellegrini M, Nersissian AM, Gralla EB, Valentine JS, Eisenberg D. Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis. Protein Sci 1998; 7:545-555. Jenner P, Olanow CW. Oxidative stress and the pathogenesis of Parkinson's disease. Neurology 1996; 47:S161-S170. Kakimura J-I, Kitamura Y, Takata K, Umeki M, Suzuki S, Shibagaki K, Taniguchi T, Nomura Y, Gebicke-Haerter PJ, Smith MA, Perry G, Shimohama S. Microglial activation and amyloid-|3 clearance induced by exogenous heat-shock proteins. FASEB J 2002; 10.1096/fj.01-0530fje. Kwon OJ, Lee SM, Floyd RA, Park JW. Thiol-dependent metal-catalyzed oxidation of copper, zinc superoxide dismutase. Biochim Biophys Acta 1998; 1387:249-256. Liochev SI, Chen LL, Hallewell RA, Fridovich I. The familial amyotrophic lateral sclerosisassociated amino acid substitutions E100G, G93A, and G93R do not influence the rate of inactivation of copper- and zinc-containing superoxide dismutase by H 2 0 2 . Arch Biochem Biophys 1998; 352:237-239.

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Loeffler DA, LeWitt PA, Juneau PL, Sima AA, Nguyen HU, DeMaggio AJ, Brickman CM, Brewer GJ, Dick RD, Troyer MD, Kanaley L. Increased regional brain concentrations of ceruloplasmin in neurodegenerative disorders. Brain Res 1996; 738:265-274. Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158:47-52. Mantyh PW, Ghilardi JR, Rogers S, DeMaster E, Allen CJ, Stimson ER, Maggio JE. Aluminum, iron, and zinc ions promote aggregation of physiological concentrations of beta-amyloid peptide. J Neurochem 1993; 61:1171-1174. Markesbery WR. The role of oxidative stress in Alzheimer's disease. Arch Neurol 1999; 56:1449-1452. Melov S, Schneider JA, Day BJ, Hinerfeld D, Coskun P, Mirra SS, Crapo JD, Wallace DC. A novel neurological phenotype in mice lacking mitochondrial manganese superoxide dismutase. Nature Genetics 1998; 18:159-163. Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Bill E, Pipkorn R, Masters CL, Beyreuther K. Copper-binding amyloid precursor undergoes a site-specific fragmentation in the reduction of hydrogen peroxide. Biochemistry 1998; 37:7224-7230. Pantopoulos K, Hentze MW. Activation of iron regulatory protein-1 by oxidative stress in vitro. Proc Natl Acad Sci USA 1998; 95:10559-10563. Pauly PC, Harris DA. Copper stimulates endocytosis of the prion protein. J Biol Chem 1998;273:33107-33110. Perez M, Valpuesta JM, de Garcini EM, Quintana C, Arrasate M, Lopez Carrascosa JL, Rabano A, Garcia de Yebenes J, Avila J. Ferritin is associated with the aberrant tau filaments present in progressive supranuclear palsy. Am J Pathol 1998; 152:1531-1539. Perry G, Smith MA. Alzheimer's disease. In: Adelman G, Smith BH, editors. Encyclopedia ofNeuroscience, 2nd edition. Amsterdam: Elsevier Science BV, 1999: 59-61. Perry G, Nunomura A, Avila J, Perez M, Rottkamp CA, Atwood CS, Zhu X, Aliev G, Cash AD, Smith MA. Oxidative damage and antioxidant responses in Alzheimer's disease. In: Iqbal K, Sisodia SS, Winblad B, editors. Alzheimer's Disease: Advances in Etiology, Pathogenesis and Therapeutics. Chichester: John Wiley & Sons, 2001: 371-378. Perry G, Nunomura A, Raina AK, Smith MA. Amyloid-P junkies. Lancet 2000; 355:757. Pezzella A, d'Ischia M, Napolitano A, Misuraca G, Prota G. Iron-mediated generation of the neurotoxin 6-hydroxydopamine quinone by reaction of fatty acid hydroperoxides with dopamine: A possible contributory mechanism for neuronal degeneration in Parkinson's disease. J Med Chem 1997; 40:2211-2216. Premkumar DRD, Smith MA, Richey PL, Petersen RB, Castellani R, Kutty RK, Wiggert B, Perry G, Kalaria RN. Induction of heme oxygenase-1 mRNA and protein in neocortex and cerebral vessels in Alzheimer's disease. J Neurochem 1995; 65: 1399-1402. Qian ZM, Wang Q. Expression of iron transport proteins and excessive iron accumulation in the brain in neurodegenerative disorders. Brain Res Rev 1998; 27:257-267. Riederer P, Sofic E, Rausch W-D, Schmidt B, Reynold GP, Jellinger K, Youdim MBH. Transition metals, ferritin, glutathione, and ascorbic acid in Parkinsonian brains. J Neurochem 1989; 52:515-520.

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Sayre LM, Perry G, Harris PLR, Liu Y, Schubert KA, Smith MA. In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals. J Neurochem 2000; 74:270-279. Sayre LM, Smith MA, Perry G. Chemistry and biochemistry of oxidative stress in neurodegenerative disease. Curr Med Chem 2001; 8:721-738. Schipper HM, Cisse S, Stopa EG. Expression of heme oxygenase-1 in the senescent and Alzheimer-diseased brain. Ann Neurol 1995; 37:758-768. Schipper HM. Astrocytes, brain aging, and neurodegeneration. Neurobiol Aging 1996; 17:467^180. Schipper HM, Vininsky R, Brull R, Small L, Brawer JR. Astroctye mitochondria: A substrate for iron deposition in the aging rat substantia nigra. Exp Neurol 1998; 152:188-196. Singh RJ, Karoui H, Gunther MR, Beckman JS, Mason RR Kalyanaraman B. Re-examination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated CuZn superoxide dismutase mutants and H 2 0 2 . Proc Natl Acad Sci USA 1998; 95:6675-6680. Smith MA, Taneda S, Richey PL, Miyata S, Yan S-D, Stern D, Sayre LM, Monnier VM, Perry G. Advanced Maillard reaction end products are associated with Alzheimer's disease pathology. Proc Natl Acad Sci USA 1994; 91:5710-5714. Smith MA, Harris PLR, Sayre LM, Perry G. Iron accumulation in Alzheimer's disease is a source of redox-generated free radicals. Proc Natl Acad Sci USA 1997; 94:9866-9868. Smith MA, Wehr K, Harris PLR, Siedlak SL, Connor JR, Perry G. Abnormal localization of iron regulatory protein (IRP) in Alzheimer's disease. Brain Res 1998; 788:232-236. Smith MA, Nunomura A, Zhu X, Takeda A, Perry G. Metabolic, metallic, and mitotic sources of oxidative stress in Alzheimer's disease. Antiox Redox Signal 2000a; 2:413^120. Smith MA, Joseph JA, Perry G. Arson: tracking the culprit in Alzheimer's disease. Ann NY Acad Sci 2000b; 924:35-38. Sziraki I, Mohanakumar KP, Rauhala P, Kim HG, Yeh KJ, Chiueh CC. Manganese: A transition metal protects nigrostriatal neurons from oxidative stress in the iron-induced animal model of Parkinsonism. Neuroscience 1998; 85:1101-1111. Wang P, Chen H, Qin H, Sankarapandi S, Becher MW, Wong PC, Zweier JL. Overexpression of human copper, zinc-superoxide dismutase (SOD1) prevents postishemic injury. Proc Natl Acad Sci USA 1998; 95:4556-4560. Wiedau-Pazus M, Goto JJ, Rabizadeh S, Gralla EB, Roe JA, Lee MK, Valentine JS, Bredesen DE. Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 1996; 271:515-518. Williams MD, Van Remmen H, Conrad CC, Huang TT, Epstein CJ, Richardon A. Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J Biol Chem 1998; 273: 28510-28515. Yoshida T, Tanaka M, Sotomatsu A, Hirai S, Okamoto K. Activated microglia cause irondependent lipid peroxidation in the presence of ferritin. Neuro Report 1998; 9:1929-1933.

CHAPTER 2

Metals Distribution and Regionalization in the Brain Margherita Speziali, Edoardo Orvini

ABSTRACT A review of the literature on trace element levels in normal and diseased human brain is undertaken in an attempt to recognize possible distribution patterns of aluminum, iron, copper, and zinc. Trace element concentration changes are known to occur in brain areas of subjects affected with neurodegenerative diseases. We have considered the levels of the four metals in Alzheimer's disease, Parkinson's disease, and Western Pacific Parkinsonism-dementia along with amyotrophic lateral sclerosis. For each disease, we selected articles that consider well-defined brain sites for which the element values observed in controls were found to be significantly different from those determined in patients. For a more complete picture statistically not significant differences of the same element concentrations in the same regions, whenever available, were also taken into consideration. Keywords: Trace elements; human brain; brain areas; brain regions; Alzheimer's disease; Parkinson's disease; amyotrophic lateral sclerosis.

1. TRACE ELEMENT DISTRIBUTION IN NORMAL HUMAN BRAIN Minor and trace element levels appear to be unevenly distributed throughout the human central nervous system. Several studies have reported on different metal concentrations in various areas of the brain (encephalon) in the same subjects; sometimes, they also focus on possible changes in relation to age. We have considered aluminum (Al), iron (Fe), copper (Cu), 15

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and zinc (Zn). Generally, the authors found the amounts of these four elements to be higher in gray than in white matter. When comparing the metal content of various sites, the relative composition of both matters could not be negligible.

1.1. Aluminum, Iron, Copper and Zinc Distribution in Normal Human Brain Sites in Relation to Age We summarize the data on autoptic samples taken from individuals with no signs of neurologically related diseases ("controls") in an attempt to identify possible distribution patterns of Al, Fe, Cu and Zn in "normal" human brain regions. We consider experiments in which the number of sites examined were not very low, as our purpose could be attained whether or not the areas taken into account in the different experiments were mostly the same. In trace element determination, every scientific team analyzed brain samples in different number, areas, and gray/white matter ratio. Most Authors published tables with all the values obtained, while others pulished selective information.

1.2. Aluminum McDermott et al. (1977) determined Al concentration in the frontal cortex/ hippocampus of three younger controls and six elderly controls (age not reported). They found a double Al level in the elderly compared to the younger subjects. McDermott et al. (1979) analyzed Al concentration in the lobe cortices, cerebellum and corpus callosum of three controls (25-65 y) and in another six controls (75-99 y). Considered together, the highest Al concentrations in the nine subjects were found in the hippocampus, the lowest in corpus callosum (white matter). In bulk brain, the mean Al content in the elder group was found to be considerably higher than in the younger one. Ehmann et al. (1980) analyzed Al levels in the brains of 20 controls in five different age groups. They observed a positive correlation between an abundance of Al and age. Markesbery et al. (1981), in their study of 28 controls (0-85 y) derived the same results. Ehmann et al. (1982) and Markesbery et al. (1984) published the results of a systematic study on minor and trace element concentrations over the

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whole human life span. Aluminum levels were determined in a very high number of brain regions in 29 adult controls (age 19-85) and seven infants (premature - 6 months). They found that the mean Al levels were highest in globus pallidus (GP), putamen, and middle temporal lobe, and lowest in the superior parietal lobule. No data on other areas were reported. Aluminum exhibited a significant increase in the brain with advancing age. Roider and Drasch (1999) evaluated Al concentration in brain gray matter and white matter (sulcus lateralis), nucleus lentiformis, brainstem (pons) and cerebellum (arbor vitae) of 140 adults in dependence of age. The highest Al concentrations were found in gray matter, the lowest in white matter, with a statistically high significance difference. The differences in Al contents of gray matter, nucleus lentiformis and cerebellum were also shown to be highly significant statistically. The dependence of Al values on age appeared biphasic: there was an increase with age up to a first maximum at approximately 40 years followed by a plateau or even a small decrease at up to 70 years. A second increase then followed in the eighth and ninth decades of life. No significant gender differences in Al concentrations in the brain were noticed. Taking into account papers not related to Al changes in the different periods of life, Yoshimasu et al. (1976 and 1980) published Al determination in 10 brain sites of three Japanese controls (28^-0 y); the highest value was in gyrus praecentralis, the lowest in capsula interna (CI). Yoshimasu et al. (1982) analyzed Al in 20 brain areas of three Chamorro (from Guam island) controls (57-87 y); gyri praecentralis and postcentralis showed the highest contents, followed by parietal and temporal cortices; CI, putamen and cerebellar white matter had the lowest levels. Rajput et al. (1985) and Uitti et al. (1989) determined Al in frontal cortex, nucleus caudatus (NC), substantia nigra (SN), and cerebellum in nine (no age given) and 12 individuals (average age = 70 y), respectively. The highest amounts were found in NC. Yasui (1991a and 1991b) evaluated Al contents in three areas of five control brains (65-75 y); gyrus praecentralis showed higher levels than CI and cms cerebri. Yasui et al. (1992) reported Al values in 10 areas of the same brains; the highest contents were found in SN, nucleus ruber (NR), pons and cerebellar cortex; the lowest was found in GR Xu et al. (1992) analyzed Al levels in the middle frontal gyrus, hippocampus, inferior parietal lobule, and superior and middle temporal gyri

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of 10 controls (57-93 y). Middle frontal gyrus and inferior parietal lobule contained higher values than the other two areas. Andrasi et al. (1989) studied Al in 10 regions of nine controls (65-75 y). The highest amounts were found in GP and cerebellar cortex, the lowest in putamen and pulvinar thalami. In another study (1994), they evaluated Al contents in 12 sites of 11 (no age given) subjects; they found the highest Al levels in NR and SN, the lowest in pulvinar thalami, putamen and NC. In a later study (1995), they determined Al concentrations in 20 (average age = 70 y) individuals. Samples from the cortices, thalamus and GP showed the maximum levels, while NC, Ammon's horn and other cortical specimens showed the minimum levels. Rajan et al. (1997) determined Al contents in 12 regions of eight individuals (50-60 y); they found the highest amounts in the temporal cerebrum and thalamus, the lowest in pons and cerebellum. In the case of aluminum, only GP, SN, and NR were analyzed and found to be the richest in Al by different teams. A positive correlation between Al amounts in the brain and age was observed.

1.3. Iron Hallgren and Sourander (1958) published the levels of nonhemin Fe in 16 brain sites of 37-59 individuals (30-100 y). The highest Fe values were found in the structures of the extrapyramidal system: the maximum amount occurred in the GP, while somewhat lower levels were obtained for NR and SN, followed by putamen, nucleus dentatus (ND) and NC. As for the cerebral cortex, the highest concentrations were observed in the motor cortex followed by the occipital, sensory and parietal cortices; the lowest levels were found for the prefrontal and temporal cortices. An increase in nonhamin Fe occurred with advancing age in all regions examined, except in the medulla oblongata. The rise in Fe values was rapid during the first two decades of life, and then became more gradual. Sundermann and Kempf (1961) analyzed Fe content in the brainstem ganglia of 100 human brains divided into 10 age groups. The Fe value was richest in GP followed by, in order of decreasing content, putamen, NC, thalamus, cerebral cortex, NR and cerebral medulla. While the increase in iron content with increasing age could be proven statistically for GP, putamen, and NC, the thalamus and cerebral cortex showed a clear increase in

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iron content only up to the fourth or fifth decade of life. In old age, however, a sharp tendency towards reduction appeares. Schicha et al. (1971) analyzed Fe in 16 brain sites of six subjects (newborn - 74 y). In four adults (34-74 y), the highest Fe concentration was found in GP and the lowest in NR. NC and putamen showed the same intermediate concentration. In cortical areas considerably lower Fe levels than in the basal ganglia were observed. The average Fe concentration in the brain of a newborn is only 20% that of an adult and about 60% that of a 4-year-old child. The Fe values in adults appeared to be reached between seven and nine years of age. The Fe concentration in Broca's center of speech and the motor cerebral cortex was, in all cases, higher in the left hemisphere than in the right. Volkl and Ule (1972 and 1974) determined the Fe concentration in 13 defined areas of the brain in 33 subjects (newborns - 80 y). The highest values were found in GP, putamen, and NC; the lowest in the oliva inferior and corpus callosum (CC), with intermediate values in the frontal and occipital cortices. The Fe content, in relation to age, seemed to differ considerably among the various areas examined. There was a sharp rise in the basal ganglia during the first year of life followed by a gradual leveling off. The increase in Fe content in the cerebral cortex was observed to be much less marked. Both the thalamus and hippocampus showed little change in their Fe content during life, while the olives tended to lose iron. Volkl and Ule (1974) analyzed Fe contents in 13 regions of six infant (two were born premature, three were born at full term, and one infant was 21 months old) and juvenile (3 children of 6-11 y) brains, and compared these levels to those of adult brains. Fe rose in the juvenile years in gray matter, exhibiting intermediate values; in white matter, Fe rose by approximately 50% compared to perinatal levels. Along with the increase in Fe, typical regional distributions developed until the juvenile years thereby resembling the distribution in an adult brain. Ehmann et al. (1982) and Markesbery et al. (1984) observed the highest mean Fe values in the GP and putamen; the cerebellar vermis and hippocampus contained the lowest concentrations. Iron appeared to peak between 40 and 79 years of age before declining, although this trend was not significant. Connor et al. (1992) studied Fe contents in the superior temporal gyrus, motor cortex, and occipital cortex (both white and gray matters in each site) of nine normal adults (mean age, 55 years; range, 44 to 64 years) and 11 elderly (mean age, 71.6 years; range, 65 to 80 years).

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In both groups, Fe was highest in the motor cortex. In the gray matter in the occipital cortex, there was a 130% increase in Fe with age; in the white matter, no statistically significant increase occurred with age. Loeffler et al. (1995) determined Fe concentration in eight young adult controls (80% with age in SN, NC, and putamen, but decreased by 46% in GP; these differences were not statistically significant. Hebbrecht et al. (1999) determined Fe levels in the cerebral and cerebellar cortex, white matter, basal ganglia, and brainstem of 18 individuals (7-79 y) to assess any changes related to age. They observed an age-related increase in Fe concentration. Zecca et al. (2001) measured the concentration of Fe in SN from normal subjects aged one to 90 years old. Fe levels in SN appeared to increase by the fourth decade of life and to remain stable until 90 years of age. Hill (1988) reviewed the distribution of iron in the brain in relation to age. Dealing with papers not related to Fe changes in aging, Harrison et al. (1968) analyzed Fe contents in 10 regions of 35 individuals (no age given). The highest Fe amounts were found in the GP, putamen, and NC; the lowest in the corpus callosum and cerebellar white matter. Hock et al. (1975) determined Fe amounts in 27 sites of seven brains (23-66 y). The maximum level of Fe was detected in the GP, followed by the putamen, NC, and SN; the minimum in the cms fornicis, corpus callosum, corpus geniculatus lateralis, and corpus semiovale (white matter). Goldberg and Allen (1979 and 1981) studied Fe amounts in six sites of three control subjects (no age given). They observed the maximum Fe level in SN and elevated contents in GP, NC, and putamen, while the minimum level was found in the hippocampus and cerebral cortex. Duflou et al. (1989) analyzed Fe contents in about 50 brain sites of 12 subjects (7-69 y). They found the highest Fe concentrations in the plexus choroideus, GP, SN, and putamen. The lowest concentrations were observed in the pyramid, tractus corticospinalis, and nucleus olivaris inferior. Kornhuber et al. (1994) measured Fe in seven brain regions of 12 individuals (41-91 y). The highest amounts were detected in NC and the corpus mamillare, the lowest in the hypothalamus and gyrus cinguli. Bush et al. (1995) analyzed gray and white matters of the lobes along with

Metals Distribution and Regionalization in the Brain

21

six other areas of 30 subjects (18-85 y). The highest Fe values were found in the putamen, SN, GP, and NC, while the lowest values were found in the white matter of the occipital and parietal lobes. Rajan et al. (1997) observed the highest Fe concentrations in the frontal, temporal, and parietal cerebrum, and the lowest in the medulla oblongata and pons. Andrasi et al. (1999) determined Fe amounts in 20 sites of five control subjects (>65 years). The highest concentrations were found in the putamen, followed by GP, NC, and SN; the lowest concentrations were found in NR and gyrus cinguli. Most authors found the highest Fe concentrations in GP, followed by the putamen, NC (caput), and SN. The metal showed a different increase with age in various areas.

1.4. Copper Ule et al. (1974) and Volkl and Ule (1974) determined Cu amounts in 13 brain areas of 29 and 33 subjects (age 0-80 y), respectively. In adults, the highest concentrations of Cu appeared to occur in SN, which was significantly different from the frontal cortex. High levels were also observed in the occipital cortex, neostriatum, and nucleus dentatus, while the lowest levels were seen in the thalamus and centrum semiovale. The Cu level of newborns was found to be somewhat lower than that of adults, while between the fourth and seventh decades of life these values remained independent of age. Volkl et al. (1974) observed that Cu rose during the juvenile years in the gray matter; in white matter, Cu changed little during maturation. Along with the increase in Cu, typical regional distributions developed until the juvenile years, thereby resembling the distribution in an adult brain. Saito et al. (1993 and 1994) analyzed Cu levels in the brains of 14 subjects (4-54 y) in five brain regions (lobes and cerebellum). The concentration of Cu in the cerebellum appeared to be significantly higher than that in the temporal lobe. No relation to age was observed. Tandon et al. (1994) examined 17 controls (age 33-85) and observed a significant positive correlation between Cu content in bulk brain and age. Considering experiments not related to age, Warren et al. (1960) and Thompson (1961) reported the results of analyses of Cu contents in 26 areas of nine brains (no age given). The highest amounts were found in the

22

Speziali M & Orvini E

locus coeruleus, followed by SN, ND, and parietal cortex (all samples were gray matter); the lowest amounts were found in the optic chiasma, base of pons, and corpus callosum (all made of white matter). SmeyersVerbeke et al. (1974) evaluated the distribution of Cu in 13 brain areas of 11 subjects (no age given). The maximum values were observed in the cerebellar cortex followed by the occipital cortex and basal ganglia, while the lowest values were found in the brainstem and corpus callosum. Harrison et al. (1968) detected the highest Cu amounts in the cerebellar gray matter, putamen, and NC; the lowest amounts were found in the corpus callosum, thalamus, and frontal and cerebellar white matter. Smeyers-Verbeke et al. (1974) verified the distribution of Cu in the brain. The highest values were observed in the cerebellar and occipital cortices and basal ganglia; the lowest values were found in the corpus callosum and brainstem. Goldberg and Allen (1981) observed maximum Cu level in SN and elevated contents in the putamen and NC, while the minimum levels were found in the hippocampus and cerebral cortex. Bonilla et al. (1984) determined Cu concentrations in 38 brain areas of seven subjects (11-75) y. The highest Cu levels were detected in the olfactory bulb, gyrus postcentralis superior, NC (tail), and calcarine cortex (cuneus); the lowest levels were found in the frontal, occipital, and parietal lobes' white matter along with the optic chiasma and thalamus. Duflou et al. (1989) found the highest Cu concentrations in SN, ND, lobulus flocculo-nodularis cerebelli, paraflocculus ventralis cerebelli, and putamen. The lowest concentrations were observed in chiasma opticum, tractus corticospinalis, and corpus fornicis. In Kornhuber et al. (1994), the maximum levels were detected in NC, followed by the hypothalamus and cortex; the lowest levels were found in the hippocampus and corpus mamillare. Deibel et al. (1997) analyzed Cu contents in 13 regions of five subjects (no age given). They found the highest levels in the amygdala and cerebellum, with the lowest levels in the cerebellum and hippocampus. In Rajan et al. (1997), the highest Cu concentrations were determined in the cerebellum and midbrain, with the lowest levels in the pons and thalamus. Andrasi et al. (1999) found the highest concentrations of Cu in SN, NR, and putamen, and the lowest in the genu corporis callosi. Most authors found the highest concentrations of Cu in SN, ND and other cerebellar regions, putamen, and NC. Copper seemed to increase at least until adult age, possibly by the fourth decade of life.

Metals Distribution and Regionalization in the Brain

23

1.5. Zinc Hu and Friede (1968) determined Zn in 24 regions of five adults (34-87 y). They found the maximum level in Amnion's horn, SN, ND, and amygdala, and the lowest level in the pyramids, optic chiasma, and cerebral peduncle. In 17 areas of two newborns (four to five days' old), Amnion's horn, SN, cerebellar white matter, and cortex showed the highest Zn concentrations; the frontal and occipital white matters showed the lowest concentrations. Ule et al. (1974) and Volkl and Ule (1974) found the highest zinc concentrations in adults in the hippocampus, followed by the frontal cortex, NC, oliva inferior, and SN; in the last three sites, the Zn levels sometimes appeared to increase with age. The lowest values were seen in the centrum semiovale. Between the fourth and seventh decades of life, the Zn values remained independent of age. Volkl et al. (1974) observed that Zn rose during the juvenile years in regions of gray matter; in white matter, Zn changed little during maturation. The perinatal distribution pattern of Zn was already similar to that found in adult brains. Ehmann et al. (1982), Markesbery et al. (1984), and Ehmann et al. (1984) reported on Zn levels in 29 adult controls (age 19-85) and seven infants (=S6 months of age), by considering many regions. For controls aged 20 and above, the highest mean Zn levels were found in the hippocampus, NC, amygdala, and cerebellar vermis, and the lowest in SN, middle frontal lobe, and GP. Among infants, Zn concentrations were observed to be highest in the posterior occipital lobe, middle frontal lobe, and putamen; the lowest concentrations were detected in the thalamus. The difference between the infants and the adults was significant. Zn concentrations in controls aged above one exhibited a highly significant positive correlation with age. However, if the 19-year-old patient was eliminated from the group, the Zn concentration showed no significant correlation with age. In the hippocampus, the Zn level reached a maximum in those aged between 40 and 59, but declined gradually afterwards. This was also true of the putamen and superior parietal lobe. No regular patterns, as a function of age, were observed for any of the other brain regions in the adult control group. Saito et al. (1994) found that Zn concentration in the frontal lobe was significantly related to age. Hebbrecht et al. (1999) observed an age-related increase in Zn concentration.

24

Speziali M & Orvini E

Regarding works in which Zn possible changes in aging are not considered, Kornhuber et al. (1994) found the highest Zn values in the hippocampus and cortex, and the lowest in the corpus mamillare and hypothalamus. Rajan et al. (1997) observed the highest levels in parietal cerebrum and medulla oblongata, and the lowest in the hippocampus, hypothalamus, and pons. In Smeyers-Verbeke et al. (1974), the highest Zn values were seen in the temporal, frontal, and cerebellar cortices, while the lowest levels were seen in the corpus callosum centrum ovale, and CI. Hock et al. (1975) found the highest Zn levels in the epiphysis, gyrus dentatus, hippocampus, and insula, and the lowest in the corpus callosum, crus fornicis, and centrum semiovale (white matter). In Harrison et al. (1968), the hippocampus and frontal gray matter showed the highest Zn amounts, while the frontal and cerebellar white matter, together with the corpus callosum, showed the lowest. Duflou et al. (1989) found the highest Zn concentrations in the epiphysis, uncus hippocampi, plexus choroideus, and amygdala. The lowest concentrations were observed in the radiatio optica, pedunculus cerebri, CI, and genu corporis callosi (all samples were of white matter). Andrasi et al. (1999) found the highest Zn concentrations in the gyrus frontalis medius, putamen, and NC (caput), and the lowest amount in the genu corporis callosi. From the papers examined, it appears that several authors found the highest concentrations of Zn in the hippocampus, amygdala, NC, and epiphysis. Zn seems to increase until adult age, depending on the areas being considered.

2. TRACE ELEMENT IMBALANCES IN NEURODEGENERATIVE DISEASES The biochemical role that the redox-active transition metals, especially Fe, Cu, and Zn, play as mediators of oxidative stress in a variety of neurodegenerative diseases has recently been reviewed by Sayre et al. (1999 and 2000), Bush (2000), and Campbell et al. (2001). The last paper also considers Al. The role of metal ions (mainly Fe and possibly Cu) in oxidative processes and aging was reviewed by Floyd and Carney (1993). In single elements, the role of Al in human brain disease was explained by Yase (1980), Crapper McLachlan and De Boni (1980a and

Metals Distribution and Regionalization in the Brain

25

1980b), Crapper McLachlan et al. (1983), Crapper McLachlan and Farnell (1985), Wisniewski et al. (1985), Wisniewski and Sturman (1989), Savory et al. (1991), Youdim (1994 and 2001), and Yokel (1997). The function of Cu in neurodegenerative diseases was described in Scheinberg (1988), Waggoner et al. (1999), Rotilio et al. (2000), Brown (2001), and Strausak et al. (2001). The implication of Fe in neurodegenerative diseases was elucidated by Beard et al. (1993), Gelman (1995), Qian et al. (1997), Pinero and Connor (2000), and Thompson et al. (2001). Fe and Al homeostases in neural disorders were treated by Joshi et al. (1994). A book on Fe metabolism in Central Nervous System disorders was published by Riederer and Youdim in 1993. Gerlach et al. (1994 and 2000) also reviewed, besides metabolic studies, papers on Fe evaluation in different brain regions in several neurodegenerative diseases. The role of Zn in the same disorders was discussed by Wallwork (1987), Cuajungco and Lees (1997a), and Yasui et al. (1997). Sandyk (1991) has examined the relationship between Zn deficiency and cerebellar diseases, and a review on Zn, human diseases, and aging was published by Fabris and Moccheghiani in 1995. Cu and Zn functions and levels in the brain in health and neurological disease were reviewed by Cumings (1965). Analytical studies on trace element imbalances in agerelated neurological diseases were published by Ehmann et al. in 1987 and 1993. Markesbery and Ehmann (1988) have reviewed the possible trace element (especially Al and Zn) concentration changes in dementing disorders; while Ehmann and Vance (1996) have reviewed studies of trace element involvement in neurodegenerative diseases using activation analysis. For this overview, we have taken into consideration literature data on the tissue level, with concentrations expressed as the amount of element per gram of tissue or protein. Concentrations as a ratio between a couple of elements were not considered, as well as observations at the cellular or subcellular level. For each neurological disease, original papers with analytical data on both control subjects and patients were retrieved from the literature. Of these, those reporting on Al, Fe, Cu, and Zn concentration values in bulk samples, in wide regions such as lobes, lobe cortices, cerebellum, or in not so clearly delimited sites were excluded. We selected only articles that consider well-defined brain sites in which the element

26

Speziali M & Orvini E

values were significantly or highly significantly different between the control and patient groups. To provide a more complete picture, statistically not significant (NS) differences of the same element concentrations in the same regions, whenever available, were reported in the tables. Captions valuable for all tables are shown in Fig. 1.

SD = standard deviation; M = male, F = female; S* = significant or highly significant difference; pS = possibly significant difference;

SEM = standard error of the mean; av = average; mS = marginally significant difference; NS = not significant difference.

w = concentration expressed on a wet weight basis. To allow a comparison among all the values, the data were converted to a dry weight basis; the values obtained are reported, in parentheses, beside. When possible (Thompson et al., 1988 and Cornett et al., 1998b) conversion was made using the FD/WET weight ratio provided by the authors; in the other cases, the data were calculated using the ratio 0.21. v subjects from Eastern Canada; * combined data for olfactory bulb, tract and olfactory trigone; A data converted from nmol/g;

A subjects from United Kingdom; • combined data for olfactory bulb, tract and nucleus; 0 data expressed as mg Fe/g of protein.

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Gly, Tyr —> Phe, and His —> Arg at positions 5, 10, and 13, respectively) is unaffected by Zn 2+ or Cu 2+ at low micromolar concentrations (Atwood et al., 1998), perhaps explaining why rodents do not deposit cerebral amyloid. The molecular risk factor, apoE, modulates the precipitation of A|3 by Cu 2+ and Zn 2+ (Moir et al., 1999). We found that that the apoE4 isoform, which is an associated risk factor for the development of AD, is the poorest solubility chaperone for A(3 when compared to the other forms, apoE2 and apoE3. This finding holds regardless of whether the precipitating stress is Cu 2+ or Zn 2+ . CuZn selective chelators markedly enhance the resolubilization of Afi deposits from post-mortem AD brain samples (Cherny et al., 1999), supporting the possibility that Cu and Zn ions play a significant role in assembling these deposits. The metallochemistry of A(3 links oxidative damage and amyloidosis in AD. In vitro precipitation of Ap by Cu 2+ or Zn 2+ can be completely reversed by chelation (Huang et al., 1997). In the post-mortem AD brain, selective Cu or Zn chelators can induce the resolubilization of A$ from plaques (Cherny et al., 1999). Oxidative damage may be the earliest pathologic event in AD (Nunomura et al., 2001) and a negative correlation exists between amyloid burden and levels of 8-hydroxyguanosine, a marker of hydroxyl radical activity (Cuajungco et al., 2000). This is possibly because Zn suppresses H 2 0 2 formation from Ap. Thus, the mature plaque in AD may form through a compensatory

Importance of Copper and Zinc in Alzheimer's Disease

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mechanism where Zn 2+ is sequestered into accumulations of A(3. However, the quenching of H 2 0 2 formation by Zn 2+ is inefficient and, therefore, if the mass burden of plaque amyloid becomes sufficient, oxidative damage predominates (Opazo et al., 2002).

5. COPPER AND ZINC METABOLISM IS A PROMISING THERAPEUTIC TARGET IN ALZHEIMER'S DISEASE Preclinical and clinical data now support Cu and Zn metabolism as a valid therapeutic target in AD. Clioquinol (CQ), a retired antibiotic with a 0.5 billion patient-day history, was evaluated by Cherny et al. (2001) for efficacy in 21-month-old A(3PP2576 transgenic mice. Administration by lavage of 30 mg/kg for nine weeks resulted in a 49% decrease in brain A(3 load and a decrease in serum A(3, with no evidence of drug-induced toxicity or systemic loss of metals. General health and body weight parameters were significantly improved in the treated animals after only 16 days of treatment. Treatment of A(3PP2576 mice with CQ induced a «15% increase in brain Cu and Zn, indicating that the therapeutic mechanism of CQ is not simply chelation and clearance. A phase II double-blinded clinical trial of CQ in Alzheimer patients has recently been completed. CQ decreased plasma Ap levels and, in more advanced AD patients, slowed the rate of cognitive decline (Masters et al., personal communication). Continued investigation into Cu and Zn metabolism, the metallobiology of A(3 and A|3PP, and the mechanism of action of CQ seems likely to increase the understanding of AD pathogenesis and lead to new treatments for the millions of patients suffering from the disorder.

REFERENCES Asahina M, Yoshiyama Y, Hattori T. Expression of matrix metalloproteinase-9 and urinarytype plasminogen activator in Alzheimer's disease brain. Clin Neuropathol 2001; 20:60-63. Assaf SY, Chung S-H. Release of endogenous Zn 2+ from brain tissue during activity. Nature 1984; 308:734-736. Atwood CS, Moir RD, Huang X, Bacarra NME, Scarpa RC, Romano DM, Hartshorn MA, Tanzi RE, Bush Al. Dramatic aggregation of Alzheimer Ap by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem 1998; 273:12817-12826.

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Atwood CS, Huang X, Moir RD, Tanzi RE, Bush AI. Role of free radicals and metal ions in the pathogenesis of Alzheimer's disease. Met Ions Biol Syst 1999; 36:309-364. Atwood CS, Scarpa, RC, Huang X, Moir RD, Jones WD, Fairlie DP, Tanzi RE, Bush AI. Characterization of copper interactions with Alzheimer AB peptides—identification of an attomolar affinity copper binding site on AB1-42. JNeurochem 2000; 75:1219-1233. Backstrom JR, Miller CA, Tokes ZA. Characterization of neutral proteinases from Alzheimer-affected and control brain specimens: Identification of calcium-dependent metalloproteinases from the hippocampus. J Neurochem 1992; 58:983-992. Basun H, Forssell LG, Wetterberg L, Winblad B. Metals and trace elements in plasma and cerebrospinal fluid in normal aging and Alzheimer's disease. J Neural Transm Park Dis Dement Sect 1991; 3:231-258. Bayer TA, Cappai R, Masters CL, Beyreuther K, Multhaup G. It all sticks together—the ABPP-related family of proteins and Alzheimer's disease. Mol Psychiatry 1999; 4:524-528. Blacker D, Wilcox MA, Laird NM, Rodes L, Horvath SM, Go RC, Perry R, Watson B Jr, Bassett SS, Mclnnis MG, Albert MS, Hyman BT, Tanzi RE. Alpha-2 macroglobulin is genetically associated with Alzheimer' disease. Nat Genet 1998; 19:357-360. Borchardt T, Camakaris J, Cappai R, Masters CL, Beyreuther K, Multhaup G. Copper inhibits B-amyloid production and stimulates the non-amyloidogenic pathway of amyloid precursor protein (ABPP) secretion. Biochem J 1999; 344:461-467. Borchardt T, Schmidt C, Camakaris J, Cappai R, Masters CL, Beyreuther K, Multhaup G. Differential effects of zinc on amyloid precursor protein (ABPP) processing in copperresistant variants of cultured Chinese hamster ovary cells. Cell Mol Biol 2000; 46:785-795. Bush AI, Martins RN, Rumble B, Moir R, Fuller S, Milward E, Currie J, Ames D, Weidemann A, Fischer P. The amyloid precursor protein of Alzheimer's disease is released by human platelets. J Biol Chem 1990; 265:15977-15983. Bush AI, Multhaup G, Moir RD, Williamson TG, Small DH, Rumble B, Pollwein P, Beyreuther K, Masters CL. A novel zinc(II) binding site modulates the function of the AB4 amyloid protein precursor of Alzheimer's disease. J Biol Chem 1993; 268:16109-16112. Bush AI, Pettingell WH, de Paradis M, Tanzi RE, Wasco W. The amyloid-B protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily. J Biol Chem 1994a; 269:26618-26621. Bush AI, Pettingell WH, Multhaup G, Paradis MD, Vonsattel JFG, Beyreuther K, Masters CL, Tanzi RE. Rapid induction of Alzheimer AB amyloid formation by zinc. Science 1994b; 265:1464-1467. Bush AI. Metals and neuroscience. Curr Opin Chem Biol 2000; 4:184-191. Castellani RJ, Smith MA, Nunomura A, Harris PL, Perry G. Is increased redox-active iron in Alzheimer's disease a failure of the copper-binding protein ceruloplasmin? Free Radic Biol Med 1999; 26:1508-1512. Chelly J, Turner Z, Tonnesen T, Petterson A, Ishikawa-Brush Y, Tommerup N, Horn N, Monaco AP. Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat Genet 1993; 3:14-19.

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Cherny RA, Legg JT, McLean CA, Fairlie D, Huang X, Atwood CS, Beyreuther K, Tanzi RE, Masters CL, Bush AI. Aqueous dissolution of Alzheimer's disease A(3 amyloid deposits by biometal depletion. J Biol Chem 1999; 274:23223-23228. Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron 2001; 30:665-676. Corrigan FM, Reynolds GP, Ward NI. Hippocampal tin, aluminum and zinc in Alzheimer's disease. Biometals 1993; 6:149-154. Cuajungco MP, Goldstein LE, Nunomura A, Smith MA, Lim JT, Atwood CS, Huang X, Farrag YW, Perry G, Bush AI. Evidence that the beta-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of abeta by zinc. J Biol Chem 2000;275:19439-19442. Curtain CC, Ali F, Volitakis I, Cherny RA, Norton RS, Beyreuther K, Barrow CJ, Masters CL, Bush AI, Barnham KJ. Alzheimer's disease amyloid binds Cu and Zn to generate an allosterically-ordered membrane-penetrating structure containing SOD-like subunits. J Biol Chem 2001; 276:20466-20473. Danscher G, Jensen KB, Frederickson CJ, Kemp K, Andreasen A, Juhl S, Stoltenberg M, Ravid R. Increased amount of zinc in the hippocampus and amygdala of Alzheimer's diseased brains: A proton-induced X-ray emission spectroscopic analysis of cryostat sections from autopsy material. J Neurosci Methods 1997; 76:53-59. Deibel MA, Ehmann WD, Markesbery WR. Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer's disease: Possible relation to oxidative stress. J Neurol Sci 1996; 143:137-142. Du Y, Ni B, Glinn M, Dodel RC, Bales KR, Zhang Z, Hyslop PA, Paul SM. Alpha2macroglobulin as AP-amyloid peptide-binding plasma protein. J Neurochem 1997; 69:299-305. Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 1989;31:145-328. Frederickson CJ, Suh SW, Silva D, Thompson RB. Importance of zinc in the central nervous system: The zinc-containing neuron. J Nutr 2000; 130:S1471-S1483. Glenner GG, Wong CW. Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984; 120:885-890. Gonzalez C, Martin T, Cacho J, Brenas MT, Arroyo T, Garcia Berrocal B, Navajo JA, Gonzales Buitrago JM. Serum zinc, copper, insulin and lipids in Alzheimer's disease epsilon 4 apolipoprotein E allele carriers. Eur J Clin Inv 1999; 29:637-642. Hambidge M, Krebs NF. Interrelationship of key variables of human zinc homeostasis: Relevance to dietary zinc requirements. Ann Rev Nutr 2001; 21:429^152. Hartter DE, Barnea A. Brain tissue accumulates 67copper by two ligand-dependent saturable processes. J Biol Chem 1988a; 263:799-805.

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Hartter DE, Bamea A. Evidence for release of copper in the brain: Depolarization-induced release of newly taken-up 67copper. Synapse 1988b; 2:412-415. Hershey CO, Hershey LA, Varnes A, Vibhakar SD, Lavin P, Strain WH. Cerebrospinal fluid trace element content in dementia: Clinical, radiologic, and pathologic correlations. Neurology 1983; 33:1350-1353. Hesse L, Beher D, Masters CL, Multhaup G. The beta A4 amyloid precursor protein binding to copper. FEBS Lett 1994; 349:109-116. Howell GA, Welch MG, Frederickson CJ. Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 1984; 308:736-738. Huang X, Atwood CS, Moir RD, Hartshorn MA, Vonsattel J-P, Tanzi RE, Bush AL Zincinduced Alzheimer's AfU-40 aggregation is mediated by conformational factors. J Biol Chem 1997; 272:26464-26470. Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, Scarpa RC, Cuajungco MP, Gray DN, Lim J, Moir RD, Tanzi RE, Bush, AL The AB peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 1999a; 38:7609-7616. Huang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall J, Hanson GR, Stokes KC, Leopold M, Multhaup G, Goldstein LE, Scarpa RC, Saunders AJ, Lim J, Moir RD, Glabe C, Bowden EF, Masters CL, Fairlie DP, Tanzi RE, Bush AL Cu(II) potentiation of Alzheimer AB neurotoxicity: Correlation with cell-free hydrogen peroxide production and metal reduction. / Biol Chem 1999b; 274:37111-37116. Jacob C, Maret W, Vallee BL. Control of zinc transfer between thionein, metallothionein, and zinc proteins. Proc Natl Acad Sci USA 1998; 95:3489-3494. Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Multhaup G, Beyreuther K, Muller-Hill B. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 1987; 325:733-736. Kapaki EN, Zournas CP, Segdistsa IT, Xenos DS, Papageorgiou CT. Cerebrospinal fluid aluminum levels in Alzheimer's disease. Biol Psychiatry 1993; 33:679-681. King JC, Shames DM, Woodhouse LR. Zinc homeostasis in humans. J Nutr 2000; 130:S1360-S1366. Krebs NF. Overview of zinc absorption and excretion in the human gastrointestinal tract. J Nutr 2000; 130:S1374-S1377. Leake A, Morris CM, Whateley J. Brain matrix metalloproteinase 1 levels are elevated in Alzheimer's disease. Neurosci Lett 2000; 291:201-203. Lee DY, Prasad AS, Hydrick-Adair C, Brewer G, Johnson PE. Homeostasis of zinc in marginal human zinc deficiency: Role of absorption and endogenous excretion of zinc. J Lab Clin Med 1993; 122:549-556. Lee J-Y, Mook-Jung I, Koh J-Y. Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice. J Neurosci 1999; 19:1-5. Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR. Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 1998; 158:47-52. Maret W, Vallee BL. Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci USA 1998; 95:3478-3482.

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Masters BA, Quaife CJ, Erickson JC, Kelly EJ, Froelick GJ, Zambrowicz BP, Brinster RL, Palmiter RD. Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles. J Neurosci 1994; 14:5844-5857. Mercer JF. The molecular basis of copper-transport diseases. Trends Mol Med 2001; 7:64—69. Miura T, Suzuki K, Kohata N, Takeuchi H. Metal binding modes of Alzheimer's amyloid B-peptide in insoluble aggregates and soluble complexes. Biochemistry 2000; 39:7024-7031. Moir RD, Atwood CS, Romano DM, Laurans MH, Huang X, Bush AI, Smith JD, Tanzi RE. Differential effects of apolipoprotein E isoforms on metal-induced aggregation of AB using physiological concentrations. Biochemistry 1999; 38:4595-4603. Molina JA, Jimenez-Jimenez FJ, Aguilar MV, Meseguer I, Mateos-Vega CJ, GonzalezMunoz MJ, de Bustos F, Porta J, Orti-Pareja M, Zurdo M, Barrios E, Martinez-Para MC. Cerebrospinal fluid levels of transition metals in patients with Alzheimer's disease. J Neural Transm 1998; 105:479^188. Morita H, Ikeda S, Yamamoto K, Morita S, Yoshida K, Nomoto S, Kato M, Yanagisawa N. Hereditary ceruloplasmin deficiency with hemosiderosis: A clinicopathological study of a Japanese family. Ann Neurol 1995; 37:646-656. Multhaup G, Schlicksupp A, Hesse L, Beher D, Ruppert T, Masters CL, Beyreuther K. The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I). Science 1996; 271:1406-1409. Multhaup G, Ruppert T, Schlicksupp A, Hesse L, Bill E, Pipkorn R, Masters CL, Beyreuther K. Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide. Biochemistry 1998; 37:7224—7230. Multhaup G, Scheuermann S, Schlicksupp A, Simons A, Strauss M, Kemmling A, Oehler C, Cappai R, Pipkorn R, Bayer T. Possible mechanisms of ABPP-mediated oxidative stress in Alzheimer's disease. Free Radic Biol Med 2000; 33:45-51. Nunomura A, Perry G, Aliev G, Hirai, K, Takeda A, Balraj EK, Jones PK, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood CS, Petersen RB, Smith MA. Oxidative damage is the earliest event in Alzheimer's disease. J Neuropathol Exp Neurol 2001; 60:759-767. Opazo C, Huang X, Cherny R, Moir R, Roher A, White A, Cappai R, Masters C, Tanzi R, Inestrosa N, Bush AI. Metalloenzyme-like activity of Alzheimer's disease B-amyloid: Cu-dependent catalytic conversion of dopamine, cholesterol and biological reducing agents to neurotoxic H 2 0 2 . J Biol Chem 2002; 277:40302^10308. Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J 1995; 14:639-649. Palmiter RD, Cole TB, Findley SD. ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J 1996; 15:1784—1791. Palmiter RD, Cole TB, Quaife CJ, Findley SD. ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc Natl Acad Sci USA 1996; 93:14934-14939. Panayi AE, Spyrou NM, Iversen BS, White MA, Part P. Determination of cadmium and zinc in Alzheimer's brain tissue using inductively coupled plasma mass spectrometry. J Neurol Sci 2002; 195:1-10.

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Van Nostrand WE. Zinc(II) selectively enhances the inhibition of coagulation factor XIa by protease nexin-2/amyloid-beta-protein precursor. Thromb Res 1995; 78:43-53. Wenstrup D, Ehmann WD, Markesbery WR. Trace element imbalances in isolated subcellular fractions of Alzheimer's disease brains. Brain Res 1990; 533:125-131. White AR, Multhaup G, Galatis D, McKinstry WJ, Parker MW, Pipkorn R, Beyreuther K, Masters CL, Cappai R. Contrasting, species-dependent modulation of copper-mediated neurotoxicity by the Alzheimer's disease amyloid precursor protein. J Neurosci 2002; 22:365-376. White AR, Reyes R, Mercer JFB, Camakaris J, Zheng H, Bush AI, Multhaup G, Beyreuther K, Masters CL, Cappai R. Copper levels are increased in the cerebral cortex and liver of ApPP and APLP2 knockout mice. Brain Res 1999; 842:439-444. Wong PC, Waggoner D, Subramaniam JR, Tessarollo L, Bartnikas TB, Culotta VC, Price DL, Rothstein J, Gitlin, JD. Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA 2000; 97:2886-2891. Yu WH, Lukiw WJ, Bergeron C, Niznik HB, Fraser PE. Metallothionein III is reduced in Alzheimer's disease. Brain Res 2001; 894:37-45.

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CHAPTER 10

Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis Moon B Yim, P Boon Chock, Earl R Stadtman

ABSTRACT More than 90 individual mutations in copper-zinc superoxide dismutase are associated with familial cases of amyotrophic lateral sclerosis and they are mostly fatal within one to five years of the onset of the disease. Results obtained from studies with transgenic mice, transfected cells, and patients suggest that familial amyotrophic lateral sclerosis copper-zinc superoxide dismutase mutants have gains or enhancements in one or more cytotoxic properties, which are not related to the superoxide dismutation activity. The proposed nature of the cytotoxic gain-of-function of the familial amyotrophic lateral sclerosis copperzinc superoxide dismutase mutants under debate includes enhanced free radicalgenerating (peroxidative) activity, enhanced activity of peroxynitrite-mediated tyrosine nitration, and familial amyotrophic lateral sclerosis mutant-associated aggregate formation due to failure of protein folding. We review these proposals and summarize our findings from the studies with purified familial amyotrophic lateral sclerosis copper-zinc superoxide dismutase mutants. Keywords: Copper-zinc superoxide dismutase; familial amyotrophic lateral sclerosis; free radical; protein oxidation; protein aggregation.

1. INTRODUCTION Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is an age-dependent, fatal degenerative disorder of motor neurons in the spinal cord, motor cortex, and brainstem. Up to 10% of all ALS patients 263

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are familial ALS (FALS) and about 20% of FALS are associated with dominantly inherited missense mutations in the coding regions of superoxide dismutase 1 (SOD1), the gene for copper-zinc SOD (Cu,ZnSOD) (Rosen et al., 1993; Deng et al., 1993), which catalyzes the dismutation of superoxide radical anions (0 2 ") to hydrogen peroxides (H 2 0 2 ) and oxygen molecules (0 2 ). Over 90 individual point mutations in Cu,ZnSOD are known to cause FALS, which is usually fatal within one to five years of onset of the disease, generally at the age of 45 years and older (Gaudette et al., 2000; Cleveland and Rothstein, 2001), when the antioxidant defenses start to deteriorate (Moskovitz et al., 2002). The mutation sites are scattered throughout the structure of the wild-type Cu,ZnSOD protein. Several mutated residues are positioned within the electrostatic active site channel and in the active site (H46R, H48Q, or H80R). Another group of mutations causes truncation of the final 20 to 33 amino acids. The most abundant sites of mutation are located in conserved interaction regions critical to the subunit fold and dimer contact. Initial studies suggested that erythrocytes and brain tissues of FALS patients exhibited a marked reduction in Cu,ZnSOD activity compared to that of normal individuals (Deng et al., 1993). This reduction in SOD activity may induce oxidative damage and cause FALS symptoms. However, several studies with transgenic mice (Gurney et al., 1994; Ripps et al., 1995; Bruijn et al., 1997; Wong et al., 1995), transfected cells (Borchelt et al., 1995; Rabizadeh et al., 1995), and lymphoblasts of patients (Deng et al., 1993; Borchelt et al., 1994) revealed that levels of total Cu,ZnSOD activities remain unchanged or higher than normal. These results imply that the FALS mutants have gained or enhanced one or more cytotoxic properties, which are not related to superoxide dismutation (SOD) activity. Although the nature of the cytotoxic gain-of-function caused by Cu,ZnSOD FALS mutants is debatable, several mechanisms have been proposed. They include enhanced free radical-generating (or peroxidative) activity with H 2 0 2 and small anions as substrates (Yim et al., 1996, 1997; Wiedau-Pazos et al., 1996), enhanced activity of peroxynitritemediated tyrosine nitration, especially with Zn-depleted enzymes (Crow et al., 1997a, 1997b; Estevez et al., 1999), and FALS mutant-associated aggregate formation due to failure of protein folding (Bruijn et al., 1998; Chou et al., 1998). For the first two mechanisms, oxidation reactions catalyzed by Cu ions at the active sites or other bound sites are required. In

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contrast, the aggregate formation for the last mechanism may not require Cu incorporation into Cu,ZnSOD, even though enhanced free radicalgenerating activity would lead to aggregation of macromolecules. We review these proposals and summarize our findings obtained from the studies with purified FALS Cu,ZnSOD mutants.

2. STRUCTURE OF Cu,ZnSOD Cu,ZnSOD is a homodimer with each subunit containing 153 amino acids and an active site that contains one Cu ion at the catalytic site (ligated by the four histidines (His) of 46, 48, 63, and 120) and one Zn ion at the structural site (ligated by the three His of 63, 71, and 80 and one Asp, 83) (Tainer et al., 1982; Getzoff et al., 1983). The Cu and Zn ions are bridged by His-63. The catalytic site can be reached by the substrate through a positively charged channel shaped like a funnel, which leads to the catalytically essential Arg 143 and a narrow hydrophobic access (< 4 A) to the Cu ion (see Fig. 1). One oxygen molecule of the superoxide anion binds to the Cu ion at the active site, while the other oxygen molecule forms a hydrogen bond with the positively charged guanidinum nitrogen on Arg 143, which is located 5 A away from the Cu ion. This positively charged channel, which consists the basic residues Arg 143, Lys 122, and Lys 136, is responsible for the favorable electrostatic guidance of the anionic substrate 0^" to the active site to yield an unusually rapid catalytic rate constant (2 X 109 M _ 1 s _1 ) for superoxide dismutation (Klug et al., 1977; Tainer et al., 1982; Getzoff et al., 1983). Other small anions, such as azide, cyanide, halides, and phosphate, are also known to have easy access to the channel and bind to either the active site or the positively charged guanidinum nitrogen on Arg 143 (Rigo et al., 1977; Mata de ( ^ ^

24A

> J

*Thr5§\

5A |

5A

("

L

10A

Argl43"-\

Glu 133

>J (Thrl37

WA^ Cu

Fig. 1. Shape and dimension of the active site channel. The Lys 136 and Lys 122 located front and back of the paper, respectively, at the top of the channel.

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Freitas and Valentine, 1984). Thus, these anions, as well as many other small anions, behave as inhibitors in the superoxide dismutation reaction and exert profund effects on the general reactions involving Cu,ZnSOD.

3. PRO-OXIDANT, FREE RADICAL-GENERATING (PEROXIDATIVE) ACTIVITY OF Cu,ZnSOD Besides its superoxide dismutation activity, Cu,ZnSOD also reacts with its own reaction product, H 2 0 2 , which leads to its inactivation (Hodgson and Fridovich, 1975a; Fuchs and Borders, 1983). The inactivation appears to be caused by the oxidation of one or more amino acid residues of the enzyme. This includes His, which is ligated to the Cu ion at the active site of the enzyme. A reaction mechanism has been proposed for the inactivation reaction (Hodgson and Fridovich, 1975a): Enz-Cu(II) + H 2 0 2 -> Enz-Cu(I) + 0 2 * + 2H + , Enz-Cu(I) + H 2 0 2 -> Enz-CuQIKOH + OH", Enz-Cu(II)-"OH + ImH -> Enz-Cu(II) + Im" + H 2 0.

(1) (2) (3)

The intermediate product, Enz-Cu(II)-"OH, is a highly oxidizing species, such as free, caged, or Cu-bound "OH radical. The reactive species can react with the enzyme itself, resulting in damage to amino acid residues, such as the imidazole moiety of a His residue at the active site. Analysis of the stable products of the inactivated SOD revealed that 2-oxohistidine (His-118 for bovine SOD) was produced during the inactivation reaction by H 2 0 2 (Uchida and Kawakishi, 1994). Recent spin-trapping electron paramagnetic resonance (EPR) spectroscopic experiments have shown that 2-oxohistidine was formed by the reaction of 0 2 with the 2-histidinyl radical intermediate. This was confirmed by comparing the EPR spectrum of the authentic histidinyl radical generated by the reaction of the free "OH radical with His (Gunther et al., 2002). Some small anions, such as azide and formate, which have easy access to the active channel, protect the enzyme against inactivation caused by H 2 0 2 by scavenging free or metal-bound "OH radicals generated in reaction 3 (Hodgson and Fridovich, 1975b). During this reaction, the scavenger anions were oxidized. Hence, Cu,ZnSOD exhibits peroxidative activity (Hodgson and Fridovich, 1975b). The scavengers that can be oxidized are not limited to small anions that protect the enzyme

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against inactivation, but also include proteins and relatively large molecules, such as ferrocytochrome-c and bilirubin, or neutral molecules, such as alcohols. These molecules or proteins do not protect the Cu,ZnSOD against inactivation by H 2 0 2 . These results may suggest that reactive species were released from the Cu ion to oxidize larger molecules or proteins. In the initial spin-trapping EPR experiments (Yim et al., 1990, 1993), it was demonstrated that during these reactions "OH radicals and other free radical species originating from scavengers are generated and some stable free radicals may diffuse away from the active site to exert oxidative damage on other macromolecules in the cell. Thus, Cu,Zn-SOD can function as a pro-oxidant to generate free radicals that may initiate harmful chain reactions in vivo (Yim et al., 1990, 1993, 1999).

4. FREE RADICAL SPECIES GENERATED BY Cu,ZnSOD In initial studies (Yim et al., 1990, 1993), two spin traps, 5,5'-dimethyl-1pyrroline-N-oxide (DMPO) and N-tert-butyl-a-phenylnitrone (PBN), were used to convert transient free radicals to stable free radical adducts according to the following reaction: DMPO (or PBN) + R' -» DMPO-R* (or PBN-R').

(4)

The nature of the trapped free radical was identified by EPR spectroscopy. When H 2 0 2 was added to the solution containing active Cu,Zn-SOD and DMPO, EPR resonance lines of the hydroxyl radical adduct of DMPO (DMPO-'OH) were observed (Yim et al., 1990). The formation of this radical adduct was greatly affected by various buffer systems: the signal amplitude of DMPO-'OH obtained in phosphate (25 mM, pH 7.3) or borate (25 mM, pH 8.1) buffers was increased dramatically in the NaHC0 3 (23.5 mM)/C0 2 buffer at pH 7.6 (Yim et al, 1990). A recent study (Sankarapandi and Zweier, 1999a) suggested that bicarbonate may bind to the Arg 141 of bovine Cu,ZnSOD and anchors H 2 0 2 at the active site Cu for easier redox cleavage. In the presence of "OH radical scavengers with anionic characteristics, such as HCO^, N J , or glutathione, DMPO adducts of the scavenger-derived radicals, DMPO-C0 2 \ DMPO-Nj, or DMPO-GS' were observed besides DMPO-'OH (Yim et al., 1990; Kwak et al., 1995). Surprisingly, with neutral 'OH radical scavengers, such as ethanol, DMPO-hydroxyethyl radical adducts were

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formed, but in very small quantities compared to those of anionic scavengers. However, with a different spin trap, PBN, in place of DMPO, PBN-hydroxyethyl radical adducts were formed in a quantity for a competition reaction for free *OH radicals. These seemingly contradictory results can be explained by differences in the affinities of the spin traps used to bind to the positively charged active channel of Cu,ZnSOD, where the "OH radicals are generated (Yim et al., 1990, 1993). For this purpose, we measured binding constants of the spin traps to the active site/channel using a negatively charged chromogen, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) (Yim et al., 1993). The ABTS is known to react with the "OH radical at a diffusion-controled rate (reaction 5) to produce a stable oxidized free radical, ABTS+", which has strong absorption at 415 nm (E415 - 3.6 X 104 JVT' cm - 1 ): ABTS + "OH -> ABTS + " + OH~

k = 1.2 X 1010 M _ 1 s"1.

(5)

Addition of active Cu,ZnSOD also produced ABTS+" in the presence of H 2 0 2 and ABTS. The formation of ABTS+* followed first-order kinetics with respect to Cu,ZnSOD and H 2 0 2 . However, it showed a binding isotherm with ABTS that yielded a dissociation constant for ABTSCu,ZnSOD of Kd = 7.1 ± 0.5. The formation of ABTS+" was inhibited by the presence of DMPO or PBN in the reaction mixture. By performing a competition study, Kd values for DMPO and PBN were obtained as 0.63 mM and 11 mM, respectively. A radical scavenger, formate anion, also inhibits the formation of ABTS+", whereas ethanol does not. These results indicate that DMPO and anionic scavengers have much more accessibility to the cavity of the active channel than PBN and neutral ethanol. Consequently, DMPO or anionic scavengers are in a position to intercept the newly formed "OH radicals and drastically reduce the probability for the reaction of "OH radicals with ethanol. Therefore, the concentrations of DMPO-hydroxyethyl radical adducts observed are much lower than that expected under equally competitive conditions. PBN and ethanol, however, are able to compete in the bulk solution for free "OH radicals released from the active site of Cu,ZnSOD. Thus, both PBN-hydroxyethyl radical and PBN-OH can be detected. Conversely, these results indicate that free 'OH radicals and scavenger-derived radicals can reach the bulk solution and may cause oxidative damage to the biological environments.

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Considering the short half-life of "OH radicals, the majority of the radicals released to biological environments will be secondary cascading radicals formed by scavenging the initial 'OH radicals within a short distance of their formation (Yim et al, 1990, 1993, 1999). The term "bulk solution" should not be misunderstood. It is defined as a few bonding distances away (5 A) from the active Cu ion site due to the unique topography of the channel. As shown in Fig. 1, the Cu ion is located about 5 A inside the narrow channel ( 'Enz-Cu(II) —> Inactivation, oxidized protein, (7) -> Enz-Cu(II) + 'OH > DMPO-'OH, B'. (8) DMPO orBH

Several groups (Sankarapandi and Zweier, 1999b; Singh et al, 1998; Goss et al., 1999; Zhang et al., 2000, 2002) carried out similar experiments and concluded against the release of free "OH radicals from metal ions for various reasons. Singh et al. (1998) and Goss et al. (1999) carried out DMPO spin-trapping experiments using O-17-enriched H 2 17 0 2 and H 2 17 0. Their experimental spectra clearly showed that a significant fraction (—65%) of DMPO-'OH (and also with a different spin trap, DEPMPO-'OH) formed during SOD reaction was derived from oxygen of H 2 0 2 , but DMPO cannot protect the enzyme against inactivation. This result supports the above mechanism, although they reached a different conclusion. A recent report (Zhang et al., 2000) by the same group, however, claimed that 100% of oxygen in DMPO-'17OH originated from H 2 17 0, and not from H 2 17 0 2 . They suggested that the DMPO-'OH adducts observed originated entirely from the hydrolysis of DMPO-carbonate radical adducts, which were formed by

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the scavenging reaction that occurred in the bicarbonate buffer. However, DMPO-'OH adducts can also be observed in phosphate and borate buffers (Yim et al., 1990). In free radical chemistry, propagating radicals and chain carriers frequently govern the final products, rather than initiating species of free radical reactions. Thus, a variety of secondary free radicals can be produced by the reaction involving Cu,Zn-SOD and H 2 0 2 in vivo, irregardless of the identity of the initiating species, be it free, caged, or metal-bound "OH. These secondary free radical species, including radicals originating from glutathione (Kwak, et al., 1995), formate (Yim et al., 1990, 1993), bicarbonate (Goss et al., 1999), neurotansmitters (Yim et al., 1993), and many other radicals that have yet to be identified, may diffuse away from the active channel. These diffused radicals could initiate damaging reactions to proteins and other macromolecules and/or behave as second messengers to induce apoptosis (Guegan et al., 2001).

5. ENHANCEMENT OF FREE RADICAL GENERATION BY FALS Cu,ZnSOD MUTANTS Yim et al. (1996, 1997) and Wiedau-Pazos et al. (1996) have investigated whether there are any differences in the free radical-generating activity between the wild-type and FALS mutant Cu,ZnSOD. For this purpose, we cloned the wild-type and FALS G93A and A4V cDNA of human Cu,ZnSOD, overexpressed them in insect cells (Sf9), purified proteins, and measured their enzymatic activities. Our results revealed that the G93A, A4V, and wild-type Cu,ZnSOD have similar dismutation activities. However, the free radical-generating function of the G93A and A4V mutants measured by the spin-trapping method is enhanced relative to that of the wild-type enzyme (wild-typeA4V (13 mM)]. Thus, A4V and G93A have higher affinities for H 2 0 2 than the wild type. This increased affinity for H 2 0 2 could favor its conversion to "OH radicals. This enhancement of free radical-generating activity may provide, in part, an explanation for the association of the FALS Cu,ZnSOD mutants to ALS. We have also investigated whether FALS mutants have enhanced capacity for tyrosine nitration mediated by peroxynitrite (unpublished

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results) by using two substrates, free tyrosine and a synthetic pentadecameric peptide, which correspond to the tyrosine phosphorylation site of p34cdc2 kinase used in our previous work (Kong et al., 1996). The results showed that the nitration activity of the FALS mutants is virtually identical to that of the wild-type enzyme. Several reports, however, suggested that FALS mutants have a lower affinity to Zn ion (Lyons et al., 1996; Crow et al., 1997a) and Zn-deficient enzyme, either wild-type or mutant, shows enhanced capacity for nitration involving peroxynitrite (Estevez et al., 1999). Considerable experimental results support the involvement of free radicals in FALS. Hall et al. (1998) and Andrus et al. (1998) demonstrated that lipid peroxidation and protein carbonyl levels, a marker of protein oxidative damage, in G93A transgenic mice were elevated in comparison to nontransgenic mice, respectively. Liu et al. (1998) carried out spin trapping by using azulenyl nitrone, which produces azulenyl aldehyde through reaction with free radicals. The results obtained from G93A transgenic mice showed significant enhancement of free radical generation in the spinal cord prior to motor neuron degeneration compared to wild-type transgenic control mice. Bogdanov et al. (1998) used a microdialysis method to measure oxidation of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid. They observed elevated hydroxyl radical generation in vivo in G93A transgenic mice, but not in wild-type transgenic mice. Aguirre et al. (1998) demonstrated that the fibroblasts obtained from FALS patients showed an increased sensitivity to H 2 0 2 stress, which was explained by the increased affinity of the mutants to H 2 0 2 . Liu et al. (2002) showed that infection of mouse NSC-34 motor neuron-like cells with the G93Acontaining vector increased cellular oxidative stress, indicating mitochondrial dysfunction. Pretreatment of cells containing the mutant with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and enhanced cell death, but not with cells containing wild-type Cu,ZnSOD. Pretreatment with DMPO prevents mutant-mediated mitochondrial dysfunction and cell death. Roe et al. (2002) trapped free radicals generated in the FALS mutants (G93A, G93C, L38V, and A4V) expressed in yeast cells using the a-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) spin trap. They found that mutant Cu,ZnSOD produced a greater concentration of trapped free radical adducts. Zhang et al. (2002) used the spinal cords of homogenized mice to detect the oxidation of dichlorodihydrofluoroscein

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(DCHF) to dichlorofluoroscein. Their results showed a significant increase in the oxidation of DCHF in spinal cord extracts from G93A transgenic mice (90 days' old) relative to that of wild type. This observation strongly suggests that G93A has enhanced free radical-generating activity, unlike their previous assertion that their EPR data obtained with G93A and wild-type Cu,Zn-SOD showed no difference in free radicalgenerating activity (Singh et al, 1998).

6. AGGREGATION INDUCED BY FALS MUTANTS Bruijn et al. (1998) suggested that poorly or unstably folded mutants mitigate Cu,ZnSOD-containing aggregates that are toxic to motor neurons. They suggested that this process is induced by an as yet unidentified chemical reaction, rather than the involvement of free radicals. Subramaniam et al. (2002) reported results recently obtained from FALS transgenic mice (G93A, G37R, or G85R), in which the gene encoding the Cu chaperone for SOD1 (CCS) has been disrupted. They found that the incorporation of Cu ion into mutants was very much diminished in the absence of CCS. However, it did not modify at all (neither accelerate nor delay) the onset and progression of motor neuron disease in transgenic mice, relative to the mice with intact CCS. They concluded that the CCS-dependent Cu loading to mutant Cu,ZnSOD plays no role in the pathogenesis of disease in these mouse models (Subramaniam et al., 2002). The results, however, are not easily understood by invoking the aggregation hypothesis alone. In general, the apo-forms of metalloproteins are less stable and aggregate more easily than their holo forms (Rodriguez et al., 2002). Thus, if mutant-mediated aggregation is the cause of Cu,ZnSOD-associated FALS disease, the onset and progression of the disease should be accelerated in the CCS-disrupted FALS mouse model. Protein aggregates containing Cu,ZnSOD are commonly observed in FALS patients and transgenic mice expressing human Cu,ZnSOD (Kaytor and Warren, 1999; Julien, 2001; Watanabe et al., 2001). Johnston et al. (2000) have studied the formation of FALS Cu,ZnSOD-associated aggregates and insoluble protein complexes (IPCs) in G93A and G85R transfected human embryonic kidney and G93A transgenic mice. They found that SOD IPCs were present in the spinal cords of transgenic mice expressing mutant, but not wild-type SOD, at least three months before either SOD inclusion bodies or motor neuron dysfunction were first

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manifested. They proposed that the accumulation of SOD IPC could cause neuronal death by disrupting the capacity of the ubiquitin-proteasome pathway (Johnston et al., 2000). Several groups have reported the involvement of glycation in the formation of the aggregates. These include the observation of advanced glycation endproducts in neurofilament-associated conglomeration of motor neurons (Chou et al., 1998), detection of glycated SODl-positive inclusions (Kato et al., 2000), and observation of protein glycoxidation in the spinal cords of sporadic ALS patients (Shibata et al., 2001). Advanced glycation endproducts are heterogeneous products that may include various crosslinked proteins. It has been shown that oxidative conditions enhance glycation reactions. In addition, we have shown previously that protein glycation creates catalytic sites for free radical generation in protein-crosslinked products (Yim et al., 1995, 1998; Lee et al., 1998). Thus, the aggregation hypothesis may not exclude the possibility that the aggregation is mediated by free radicals initially, and these crosslinked proteins will then function as free radical generators to create autocatalytic phenomena for the progression of the disease (Yim et al., 1999).

7. CONCLUSIONS It is clear that the mechanism(s) by which FALS Cu,ZnSOD mutants induce ALS is far from resolved. The available data suggest that the processes caused by FALS mutants will eventually lead to the formation of Cu,ZnSOD-containing aggregates, which are commonly found in FALS patients and are believed to be toxic to motor neurons. The formation of aggregates can be facilitated by free radical reactions, protein misfolding and demetallation, and/or protein glycation. All these processes may be mechanistically linked as causes (enhanced free radical-generating activity and/or protein glycation) and consequences (protein glycation and aggregates). Thus, effects of wild-type and mutant Cu,ZnSOD on the formation of aggregates in vitro and characterization of their associated proteins found in the aggregates of FALS patients deserve further investigation.

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CHAPTER 1 1

Copper and Prion Disease Judyth Sassoon, David R Brown

ABSTRACT The prion protein, PrP c , is a neuronal cell surface glycoprotein. In an abnormal isoform, termed PrPSc, it is associated with the family of neurodegenerative conditions called prion diseases. PrP c was recently shown to bind copper and there is strong evidence that it has a role in the regulation of brain copper metabolism. Its expression alters copper uptake into cells and enhances copper incorporation into superoxide dismutase enzymes. Also, PrP c has a superoxide dismutase-like function and may, therefore, protect neurons from the onslaught of reactive oxygen species. Furthermore, several lines of evidence have suggested that copper ions play a role in the biology of both PrP c and PrPSc and may influence the conversion of PrP c to PrPSc. This chapter provides an overview of the latest findings in this field and discusses structural and functional aspects of the prion protein. Keywords: Antioxidant; copper; neurodegeneration; prion; PrP.

1. INTRODUCTION Prion diseases are a group of fatal neurodegenerative conditions, such as Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy and scrapie in ruminant animals (Collinge, 2001). The infectious factors associated with these conditions are proteins, composed of PrPSc a post-translationally modified form of a normal cellular protein, designated PrP c (Prusiner, 1998) (Fig. 1). Spectroscopic studies have revealed that PrP c has a high a-helical content, whereas PrPSc is rich in P-sheets (Caughey et al., 1991; Pan et al., 1993; Liu et al., 1999; Zahn et al., 2000). These two isoforms differ biochemically, with PrPSc displaying reduced solubility in nondenaturing detergents and partial resistance to 279

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#

*

*

%

B Fig. 1. Detection of PrPSc in the brains of mice with experimental mouse scrapie. The histoblot technique was used to examine brain sections of mouse brain infected with the ME7 strain of scrapie at (A) 30 days and (B) 120 days post-inoculation. The sections were treated with proteinase K to destroy protease-sensitive PrR An antibody to PrP then allowed detection of PrPSc. This is detected heavily in the 120-day brain.

protease digestion (Oesch et al, 1985; Meyer et al, 1986). The "proteinonly hypothesis" proposed by Prusiner (Prusiner, 1982, 1991) implies that prpSc a i o n e c a n a c t a s t k e infectious agent. However, experiments with recombinant prion protein converted to the PrPSc-like form failed to induce disease in mice (Hill et al., 1999). It is, therefore, possible that some other element is necessary for infectivity or that PrP interacts with some other factor which catalyzes the conversion of PrP c to PrPSc. Although there is still no evidence for a virus or DNA element in prion diseases, a modified version of the protein-only hypothesis is now clearly required to explain the mechanism of infection. Although much data is available about PrPSc in relation to prion diseases, the physiological function of the normal cellular PrP c is still a matter of contention. There is clear evidence that the expression of PrP c and its conversion to PrPSc is connected in some way with disease-associated

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neurodegeneration. It has been shown that mice lacking expression of PrP c cannot be infected with the disease and suffer no neurodegeneration, or any of the symptoms associated with prion diseases (Biieler et al., 1992, 1993). Furthermore, cultured neurons lacking PrP c expression are resistant to the neurotoxicity of PrPSc or peptide homologs, both in vivo and in vitro (Brown et al., 1994; Brandner et al., 1996). In the course of research directed towards understanding the function of PrP c , several lines of evidence suggested that copper (Cu) might play a key role in prion biology (Brown et al., 1997a). Most importantly, through studies on synthetic peptides and recombinant protein, several laboratories have shown that Cu ions bind to the octapeptide repeat region in the N-terminal portion of the mammalian PrP c (Hornshaw et al., 1995a; Brown et al., 1997a; Stockel et al., 1998; Miura et al., 1999; Viles et al., 1999; Whittal et al., 2000). Binding of Cu is pH-dependent and apparently induces a conformational change in the normally unstructured N-terminus of the molecule. In addition, it was shown that Cu rapidly and reversibly stimulated endocytosis of PrP c from the surface, raising the possibility that PrP c may serve as a receptor for cellular uptake and efflux of Cu (Pauly and Harris, 1998; Brown et al., 1999a). An enzymatic function for PrP c has also been claimed based on the observation that Cu binding confers superoxide dismutase (SOD) activity on the protein (Brown et al., 1999b). Other connections between PrP c and Cu have been proposed, but have remained controversial. PrP c was postulated to be a major Cubinding protein in brain based on the observation that the content of Cu is 5% to 50% of normal in membrane fractions derived from the brains of mice which carry a disrupted PrP gene (Brown et al., 1997; Herms et al., 1999). The activity of SOD-1 was also reported to be 50% of normal in the brains of these mice, and neurons cultured from the animals were found to be more susceptible to oxidative stress, further implying a role for PrP c in protection from oxidative damage (Brown and Besinger, 1998; Brown et al., 1999b). There are also several other results suggesting interactions between Cu and the abnormal prion, PrPSc. A role for Cu in mediating the disease process has been proposed for the following reasons. First, Cu facilitated the restoration of protease resistance and infectivity during refolding of guanidine-denatured PrP Sc (McKenzie et al., 1998). Secondly, the protease cleavage pattern of PrPSc derived from the brains

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of patients with CJD was altered by addition or chelation of Cu and another trace metal zinc (Zn), suggesting a role for metal occupancy in determining prion strain properties (Wadsworth et al., 1999). In addition, Cu added at high concentration to the prion protein causes it to refold into a protease-resistant form. (Quaglio et al., 2001). Finally, it was reported almost 25 years ago that administration of the Cu- chelating agent cuprizone to mice caused spongiform degeneration of the brain similar to scrapie (Pattison and Jebbett, 1971a). In view of all this, much current research is now being directed towards defining and characterizing the function of Cu within the prion protein.

2. PRION PROTEIN AND COPPER BINDING A link between prion disease and Cu metabolism was first proposed in the early 1970s. Pattison and Jebbett (1971a) noticed that the histopathology of mouse scrapie resembled that induced by the chemical, cuprizone. Cuprizone (bis-cyclohexanone oxaldihydrazone) is a Cu chelator which interferes with oxidative phosphorylation and induces spongiform encephalopathy and gliosis following chronic exposure. Interestingly, this resembles the well-known phenomenon in Wilson's disease, a human disease related to the loss of activity of a Cu-transporting ATPase, which also demonstrates spongiform degeneration (Tanzi et al., 1993). When cuprizone is injected into animals, it depletes the brain of Cu and modifies the activity levels of Cu-dependent enzymes (Venturini, 1973). This effect is not observed if the chelator is first charged with Cu before injection. Many of the symptoms observed in cuprizone-treated hamsters are similar to the enzymatic changes seen in scrapie-infected hamsters (Kimberlin et al., 1974; Kimberlin et al., 1976). Further studies were made in which scrapie-infected mice were treated with cuprizone to see if this would alter incubation times. Although a number of effects were observed, no clear picture emerged (Kimberlin and Millson, 1976). Research on cuprizone, in relation to prion diseases, was not pursued with any great enthusiasm following the publication of studies on PrPSc in the early 1980s (Prusiner, 1982) and the proposal of the "protein-only hypothesis". At that time, the emphasis turned towards determining the proposed function of PrPSc in infectivity rather than to look at the range of other possible factors involved in the disease.

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2.1. PrPc Structure There is now a considerable amount of biochemical information on the structure and amino acid sequence of PrP c . Translation of the original DNA sequences of the mouse and hamster prion genes revealed a 254amino acid protein (253 in humans) encoded by a single copy gene, Prnp (Locht et al., 1986) (see Fig. 2), The coding region of the prion protein is preceded by a 22-amino acid signal peptide at the N-terminus and followed by a 23-amino acid region at the C-terminus (Easier et al., 1986). Under normal physiological conditions, these N- and C-terminal signal peptides are proteolytically cleaved, leaving residues 23 to 231 in the mature mammalian protein. A glycosyl phosphatidylinositol (GPI) anchor is attached at serine 231. This marks the protein for expression at the cell surface and makes it conceivable that PrP c may be similar to other GPI-anchored proteins that are commonly associated with cell to cell signaling, adhesion, or cellular defense (Sendo et al., 1998). The C-terminal region of PrP c forms a globular structure comprising three a-helices and two short (3-strands Octamerie repeats (5) N-terminal cleavage

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Proposed fifth site Histidine at 111 Fig. 2. Schematic representation of the human prion protein primary sequence showing the main features. Indicated are the main Cu binding sites and two of the possible sites at which a fifth atom of Cu may bind.

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(Riek et al., 1996, 1997; Donne et al., 1997) and, during the process of prion infection, can refold into protease-resistant (3-sheet enriched aggregates (McKinley et al, 1983; Oesch et al, 1985; DeArmond et al., 1985; Caughey et al., 1991; Pan et al., 1993). In contrast, the N-terminal domain is highly flexible and typically includes at least four tandem copies of a conserved octapeptide repeat motif (Riek et al., 1997; Donne et al., 1997; Oesch et al., 1985). Evidence suggests that Cu binding occurs predominantly within this region. The N-terminal domain of human PrP c contains these "octarepeat" sequences (PHGGGWGQ: within residues 60 to 91) and one homologous sequence lacking the histidine residue (PQGGGWGQ: residues 51 to 59) (Kretzschmar et al., 1986) Residues 51 to 91 of mouse PrP c consists of four octarepeat sequences (PHGGSWGQ: residues 60-89) and one homologous sequence lacking histidine (PQGGTWGQ: residues 51 to 59) (Westaway, 1987).

2.2. Copper Binding in the Octameric Repeat Region Although the octarepeat motifs within PrP c have no sequence homology to classical Cu-binding proteins (Bazan et al., 1987), evidence suggests that these motifs bind Cu with a remarkable degree of selectivity; as such, they may comprise the prototype of a new class of Cu binding motif. Recombinant PrP c (rPrPc) can be expressed and purified in large amounts from inclusion bodies in an E.coli expression system (Wong et al., 1999). The protein is denatured in 8 M urea and subsequently refolded by gradual removal of urea. rPrP c can be refolded in the presence of Cu, which binds specifically in the octameric repeat region and was also demonstrated to have increased solubility (Brown et al., 1999a). Cu binding to the N-terminal octarepeat region of human recombinant PrP23-98 has now been demonstrated using equilibrium dialysis (Brown et al., 1997a) and in synthesized peptides using mass spectrometry (Hornshaw et al., 1995a; Whittal et al., 2000), fluorescence Spectroscopy (Whittal et al., 2000), Raman spectroscopy (Miura et al., 1999), circular dichroism, proton nuclear magnetic resonance (NMR) spectroscopy (Viles et al., 1999), electron paramagnetic resonance (EPR), and electron spin-echo envelope modulation spectroscopy (Aronoff-Spencer et al., 2000). Cu binding has also been reported for nearly full-length forms of Syrian hamster (residues 29 to 231) (Stockel et al., 1998) and human PrP

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(residues 91 to 231) (Jackson et al., 2001) and full-length mouse PrP (residues 23 to 231) (Kramer et al., 2001). Affinity chromatography using immobilized Cu ions has been used to purify mature, glycosylated PrP c isolated from hamster brain (Pan et al., 1993). With regard to stoichiometry, the number of Cu(II) binding sites in the N-terminal region of PrP c has been variously reported as between two and 5.6 and pH-dependent (Brown et al., 1997a; Whittal et al., 2000; Viles et al., 1999; AronoffSpencer et al., 2000; Stockel et al., 1998; Hornshaw et al., 1995b). At neutral pH, Cu binding to the N-terminal domain occured in the micromolar range with positive co-operativity. There was a remarkably close correlation of Hill coefficients calculated by different laboratories: 3.4 (PrP23 to 98) (Brown et al., 1997a), 3.3 (PrP58 to 91) (Viles et al., 1999), and 3.6 (PrP23 to 98) (Kramer et al., 2001). More recently, the suggestion was made that there are two independent high affinity Cu binding sites of 10" 14 M and 4 X 10 _14 M deduced from the analysis of PrP58 to 98 and PrP91 to 231, respectively (Jackson et al, 2001). Other authors provided different data regarding the binding of Cu at the octameric repeat region, using a mutant protein lacking this region, PrPA59-91. They found that four Cu atoms were missing from each molecule of the mutant protein compared to full-length rPrP c . This corresponded well with evidence showing that the histidines in the octameric repeat region co-ordinate with the four Cu atoms (Brown, 1999a). Other mutants lacking one, two, or three octarepeats were also studied and showed that the amount of Cu binding is directly proportional to the number of repeats, although affinity values were not calculated (Brown et al., 2000). Several other authors also confirmed that Cu can bind to PrP c along the more structured C-terminal domain of the protein (Cereghetti et al., 2001; van Doorslaer et al., 2001). Continuous wave EPR studies demonstrated that, on binding, Cu first fills the C-terminal binding sites before occupying the octarepeats at the N-terminus (Cereghetti et al., 2001).

2.3. Copper Co-Ordination in PrPc Cu in the form of Cu(II) can adopt a range of co-ordination geometries in proteins and peptides and can interact with nitrogen, oxygen, and sulfur atoms. Electron spin resonance spectra have shown that, in PrP peptides, the complexes co-ordinate with tetragonal symmetry (Aronoff-Spencer

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et al., 2000). The first study to determine the co-ordination of Cu in the octameric repeat region of PrP c was performed by Viles et al. (1999). Their model proposed that each Cu atom was co-ordinated by nitrogens of two histidine imidazole rings and a nitrogen from a proline residue, as well as with the oxygen of a water molecule. Aronoff-Spencer et al. (2000), however, disputed this model. Their EPR studies suggested that their spectra are composed of two components. The first apparently arose from three nitrogen atoms, and one oxygen atom, while the second consisted of two nitrogens and two oxygens. The first component came from studies of peptides that are equal to or longer than a single octarepeat, and the sequence HGGGW gave a pure spectrum of this kind. Thus, the fragment probably bound Cu(II) in a way that is nearly equivalent to that of peptides containing multiple repeats and, therefore, comprises the fundamental binding unit. Thus, each of the four metal ions bound to the octameric repeat region probably binds to a single HGGGW region. In the three nitrogen co-ordination component, Aronoff-Spencer et al. (2000) proposed that the first nitrogen is contributed by a histidine imidazole ring and the other two originate from deprotonated backbone amide groups. The idea that each Cu is bound by residues primarily within the octarepeat region is further supported by titration experiments on one, two, and four octarepeat-containing peptides (Aronoff-Spencer et al., 2000; Burns et al., 2002). Thus, the ratio of Cu bound within an octarepeat region is one to one and this conflicts with the findings of Viles et al. (1999), who proposed that the Cu interacted with two imidazole rings. An imidazole bridge between a single pair of Cu(II) ions leads to exchange interactions and the resulting EPR transitions take place solely among the two-spin triplet levels. If, in addition, the exchange interaction is large compared to the kT, the Boltzmann factor will favor the singlet ground state, thereby decreasing the integrated EPR absorption signal relative to that expected from uncoupled spins. The work of Aronoff-Spencer et al. (2000) found no evidence for this, suggesting that the exchange coupling does not occur in the binding of Cu to multirepeat peptides. The finding that one repeat binds one Cu ion also agrees with the findings of Miura et al. (1999), whose work was based on Raman spectroscopy studies. They suggested that HGGG is the fundamental binding unit. For a fully Cu(II) loaded protein, the model of Aronoff-Spencer et al. (2000) implies that the metal ion binding sites in the N-terminal PrP c

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domain are like beads on a string, where each bead is a Cu-HGGGW segment separated by intervening Gly-Gln-Pro links. Interestingly, glycine and proline often participate in p-turns. Thus, the intervening links may provide a mechanism that allows the Cu-binding segments to fold and, perhaps, collapse together. Co-ordination dominated by a single histidine would make a PrP c molecule bound to Cu pH sensitive, supporting the idea that it may function in vivo as a Cu transporter, taking Cu into the acidic environment of lysosomes and releasing it there. Although there is now little doubt that Cu binds in the octameric repeat region, the "fifth Cu binding site" (Brown et al., 1997a; Jackson et al., 2001) is still a matter of controversy. Some authors propose that Cu binds in the form of Cu(II) in the C-terminal domain of PrP c . A mutation in amino acid residue 198 abolishes this binding, suggesting that the histidine involved in binding might be at the amino acid residue 187. Other authors suggest that the site of Cu binding is in the region of the toxic peptide PrP106 to 126. If the latter is true, the histidine within the peptide would be the one involved in binding, with additional co-ordination from the appropriate nitrogen atoms in the vicinity. It would also require two peptides to create this site. Recent work by Qin et al. (2002) used mass spectroscopic-based footprinting techniques to attempt to position the histidine-dependent metal co-ordination sites in Cu bound to PrP c . This technique allowed the total number of histidines involved in metal co-ordination to be determined by measuring the mass differences between apo- and metal co-ordinated proteins or peptides. They confirmed the Cu co-ordination sites at the four octarepeat histidines PHGGG/SWGQ (residues 60,68,76, and 84 of mouse PrPc) and also suggested a second type of site involving histidine 95, in the related sequence GGGTHNQ, possibly in conjunction with histidine 110 (equivalent to histidine 111 in human PrP). (Jackson et al., 2001; Hasnain et al., 2001). They reported that these sites had estimated binding affinities ranging from 2.2 X 10 - 6 M to 10" 14 M for the octarepeats and 5 X 10~6 M to 4 X 10~14 M for the site involving histidine-95 (Qin et al., 2000).

2.4. Crystal Structure of Copper-Bound PrPc Following the work reported by Aronoff-Spencer et al. (2000), the group of Glenn Millhauser made further studies on the ability of the sequence

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HGGGW to bind Cu. The first crystal structure of Cu binding to this region of the protein was recently reported (Burns et al., 2002). The binding of Cu to this peptide segment is consistent with EPR and other spectroscopic studies. The HGGGW-Cu complex included six ordered water molecules (Fig. 3). Thus, Cu(II) is found in a penta-co-ordinate environment with equatorial ligation from the dl nitrogen of the imidazole ring and deprotonated amide nitrogens of the next glycine residues. The second glycine residue contributes its amide carbonyl oxygen. Except for the histidine backbone nitrogen and a-carbon, all atoms from the histidine through to the nitrogen of the third glycine lie approximately in the equatorial plane and the Cu lies just above this plane, which is consistent with a pentaco-ordinated complex. The tryptophan indole ring also participates in the co-ordination environment in a rather unusual fashion. The indole nitrogen from the tryptophan side-chain is 3.9 A from the oxygen of water bound axially to the Cu(II), suggesting the presence of hydrogen bond. This arrangement places the plane of the indole ring above the Cu, such that it is nearly parallel to the equatorial plane. Two additional water molecules make hydrogen bonds to the axial water to form a network extending from the backbone carbonyl, preceding the histidine, to the carbonyl of the third glycine. There are no equatorial water molecules involved in this model.

Fig. 3. Model of the crystal structure of Cu(II) binding to a fragment of a single octarepeat based on Burns et al. (2002).

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Examinations of the intermolecular contacts found in the crystal structure reveal a potentially important docking interaction between HGGGW-Cu units that may explain previously observed co-operative binding of Cu to PrP c . However, there is still some controversy about this co-operativity as this was not evident from previous studies (Jackson et al., 2001) and the suggestions remain to be confirmed.

3. FUNCTION OF COPPER-BOUND PrP The in vivo function of the prion protein has been investigated, but remains an enigma. Nevertheless, the interaction of PrP c with Cu may hold clues about its physiological role. Much of the evidence discussed above shows that PrP c sequesters Cu. Possible functions for the Cu-bound prion protein might be either in the distribution of Cu in the cellular environment or, alternatively, the Cu might be essential to a specific physiological activity that the protein displays.

3.1. Copper Transport Experiments designed to look at the possible function of PrP c in Cu transport employed the use of Cu67. The studies involved three strains of mice: those overexpressing PrP c , those deficient in PrP c , and wild-type controls. Transport of unchelated Cu67 by cerebellar cells was recorded in all three strains, but kinetic studies showed that histidine-chelated Cu67 was taken up at a rate proportional to PrP c expression (Brown, 1999a). Kinetic parameters for Cu67 transport were also determined, and the Vmax values increased with higher expression of PrP c while the Km values (in the nM range) were not greatly different. These results suggested that there might be an increase in the number of Cu binding sites within the cerebellar cells, which can be related to PrP c expression. Further experiments in which cytosolic enzyme SOD-1 was immunoprecipitated from cells loaded with Cu67 showed that the Cu could be incorporated into SOD-1 in proportion to the level of PrP c expression in the cells studied. Thus, PrP c may participate in the incorporation of Cu into other cellular proteins (Brown and Besinger, 1998). In the brain, the highest concentrations of PrP c are found at the synapses and synaptosomes of PrP0/0 knockout mice demonstrated a strong reduction in Cu concentration. Also, Cu binding in the synaptic

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cleft showed a significant influence on synaptic transmission. Cu is released from the synapse during quantal release and it has been proposed that PrP c may have a function in regulating Cu at levels at neuronal synapses. Several studies have been performed to study this phenomenon. In one set of experiments, neuronal cells were loaded with Cu67 and allowed to release Cu spontaneously. Subsequently, they were treated with the depolarizing agent, veratridine, which blocks Na + channels in neurons. Veratridine released Cu from cells at levels corresponding to the expression of PrP c in those cells. Cells which did not express PrP demonstrated almost no veratridine-induced Cu release, while cells which overexpressed PrP c released higher quantities of Cu compared to wild-type cells (Brown and Besinger, 1998). Furthermore, electrophysiological experiments indicated that Cu applied to cerebellar slices inhibited the amplitude and frequency of inhibitory currents measured on Purkinje cells of PrP c deficient cells, but not wild-type cells, suggesting that some protection against Cu is absent from the synapses lacking PrP c . One proposal has been that PrP c itself performs this protective function (Brown et al., 1997b).

3.2. Copper-Dependent Antioxidant Function of PrP It has been demonstrated that both recombinant and brain-derived PrP c have SOD-like activity when bound to Cu (Brown et al., 1999b, 2001). The depletion of PrP c from cell extracts results in a lower SOD activity in the extract (Wong et al., 2000). Also, when PrP c converts to PrPSc, its SOD function is abolished (Thackray et al., 2002). Thus, it is possible that the normal function of PrP c is to act as a SOD-like enzyme and control oxidative stress. It has, however, been demonstrated that PrP c is different from cellular Cu/ZnSOD, especially in the way that it forms its complex with Cu (van Doorslaer et al., 2001). In addition, a very current question concerns oxidative stress as a factor causing neuronal damage in prion diseases and it is, therefore, of considerable interest to consider the SODlike function of PrP in this context. Recombinant PrP c , purified from E.coli expression systems and refolded with Cu can be used in quantitative assays designed to measure SOD activity. Experiments showed that both chicken and mouse rPrP c could catalyze the dismutation of the superoxide radical at a rate equivalent to one-tenth of SOD-1. SOD-1 is a very potent enzyme which

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catalyzes the reaction at around 100,000 times the spontaneous rate of superoxide degradation (Fridovich, 1975). It was, therefore, concluded that PrP c had significant SOD activity. This was also confirmed for native protein immunoprecipitated from mouse brain (Brown et al., 1999b). Strict controls were used to ensure that the prion protein SOD activity was a real enzymatic activity, and not simply due to Fenton chemistry arising from Cu ligation within the protein. The deletion of the specific octameric repeats of the N-terminal region abolished the SOD activity, despite there still being Cu bound at the C-terminal domain (Brown et al., 1999b). In addition, a peptide based on the octarepeat region with Cu bound to it had no SOD activity. When rPrP c was refolded without Cu and the Cu subsequently added to the refolded protein, the mixture did not demonstrate SOD activity to the level of the co-ordinated Cu in the protein (Brown et al., 1999b). It is, therefore, clear that regions outside the N-terminal octarepeat domain are required for SOD activity of the PrP c . Further evidence for this comes form amino acid analysis of rPrP c after Cu binding. It was found that methionine residues were oxidized in rPrP c with Cu incorporated during refolding (Wong et al., 1999). This is characteristic of certain antioxidant Cu binding enzymes, such as SOD-1 (Chowdhury et al., 1995). Thus, it could be concluded that the observed catalytic SOD activity was not due to the presence of Cu alone, but was more likely due to a true enzymatic activity. It would appear that the dismutation of superoxide could, indeed, be one of the normal functions of the prion protein. At least two Cu atoms per molecule of PrP c were necessary to endow the protein with SOD activity. Binding of Cu also induced a more ordered •structure on the molecule. On binding of two more Cu atoms, a further increase in SOD activity was observed and an additional molecular ordering was found (Miura, 1996, 1999). There are currently three known SODs in mammals (Fridovich, 1997): the cytosolic Cu/ZnSOD-1, mitochondrial MnSOD or SOD-2, and extracellular SOD or SOD-3. The former two are found in all cells at varying concentrations and often show increased expression under conditions of oxidative stress. Extracellular SOD exists in three different isoforms, binds one atom of Cu per molecule, and is either released into the extracellular matrix or remains bound at the cell surface. In brain tissue, the expression of SOD-3 is very low (Ookawara et al., 1998), although it is elevated in PrP knockout mice (Brown et al., 2001). It is interesting to

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correlate these observations with the in vivo expression of PrP c , considering its SOD activity. The expression of PrP c is highest in the brain and particularly abundant at synapses. It is also present at neuromuscular junctions (Gohel et al., 1999). Thus, it has been proposed that PrP c may serve as a synaptic SOD and may be released during transmission at the synapse. Superoxide is known to inhibit synaptic transmission and the presence of SOD activity in these regions of the nervous system may have a protective role. The possible protective role of PrP c against oxidative stress was confirmed in cell culture using PC 12 rat tumor cells, which can be differentiated into neurons using nerve growth factor (NGF). It was also observed that PrP c expression increased in PC 12 cell cultures on exposure to oxidative stress (Brown et al., 1997b).

4. ALTERED METAL BINDING IN THE OCTAREPEAT REGION OF PrPc It was recently demonstrated that recombinant PrP c also had the capacity to bind metal ions other than Cu (Brown et al., 2000). Most significantly, the bivalent anion manganese was found to bind in both the octarepeats and C-terminal sites (Collinge, 2001). In vitro metal ion occupancy experiments showed that when manganese replaced the Cu ion in the prion protein, PrP c altered its structure and took on a more PrPSc-like conformation (Brown et al., 2000). Manganese-bound prion protein also lost its SODlike function (Brown et al., 2000). Several lines of investigation have been taken to see if the metal binding of PrP c is altered in TSEs, if metal imbalances also correlate with the loss of antioxidant function in PrP c , and whether these alterations correlate with the disease phenotype, such as PrPSc and the PrP genotype at codon 129, which influences the manifestation of the disease (Worrall et al., 1999). Investigations on alterations in metal ion concentrations were carried out using mouse scrapie models (Thackray et al., 2002) and in samples from sporadic CJD cases (Wong et al., 2001). Changes in the levels of Cu and manganese were detected in the brains of scrapie-infected mice early in the disease, prior to the onset of clinical symptoms. In addition, a major increase in blood manganese was also noted in the early stages of the disease. Analysis of purified PrP from the brains of scrapie-infected mice also showed a reduction in Cu binding to the protein and a proportional decrease in antioxidant activity between 30 to 60 days post-infection.

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A striking elevation of manganese and, to a lesser extent, Zn accompanied by a significant reduction in Cu binding to purified PrP were found in subtypes of sporadic CJD the most common type of human prion disease. Studies were made using brain tissues and affinity purified PrP preparations (that is, PrP c , PrPSc, and possibly other abnormal PrP species) obtained from four major subtypes of sporadic CJD. These were identified according to the genotype at codon 129 of the PrP gene and the PrPSc type established by Parchi et al. (1999). Both Zn and manganese were undetectable in PrP c preparations from control brain preparations. However, Cu and manganese changes were pronounced in sporadic CJD subjects homozygous for methionine at codon 129 and carrying PrPSc type 1. It was also found that a decrease of up to 50% of Cu and an approximately ten fold increase in manganese occurred in the brain tissues of sporadic CJD subjects. Antioxidant activity of purified PrP was dramatically reduced by up to 85% in the sporadic CJD variants, and correlated with an increase in oxidative stress markers in sporadic CJD brains. These results clearly point to the fact that metal ion occupancy alterations in PrP play a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sporadic CJD variant, they may contribute to the diversity of PrPSc and disease phenotypes in sporadic CJD (Wong et al., 2001). These fascinating and significant results could also have a bearing on potential approaches to the diagnosis of CJD. The increase in brain manganese associated with prion infection is potentially detectable by magnetic resonance imaging, and the binding of manganese by PrP in sporadic CJD might represent a novel diagnostic marker.

5. PLASMINOGEN ACTIVATION BY PrP Pericellular proteolytic activity plays an important role in many physiological situations in various organs, including the brain. This proteolytic activity can act to degrade components of the extracellular matrix or activate bioactive molecules, such as growth factors, making it a key regulator of cellular behavior (Werb, 1997). The broad specificity serine protease plasmin is one of the principal activities involved in these processes. Plasmin is generated from the abundant zymogen plasminogen by a single proteolytic cleavage catalyzed by either of the two plasminogen activators, uPA and tPA. At the functional level, the activity of the plasminogen activation system is largely regulated by mechanisms that enhance the generation of

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plasmin (Ellis and Whawell, 1997). Thus, uPA-catalyzed plasminogen activation is stimulated by the binding of uPA to its cell surface receptor, uPR (Ellis et al., 1991), and tPA-catalyzed plasminogen activation juxtaposed to the plasminogen activator, either on the same co-factor molecule (as with fibrin) or to discrete cellular binding sites. Interactions with these molecules are mediated by "lysine-binding sites" in the kringle modules of plasminogen, which can be antagonized by lysine, such as eACA (Marti et al., 1997). These kringle modules preferentially bind C-terminal lysine residues (that is, those with a free carboxylate group), which can either be present in the native proteins or generated by the proteolytic action of plasmin. It was reported that plasminogen, which can act as a pro-protease and is implicated in neuronal excitotoxicity, is bound to PrPSc, but not PrP c (Fischer et al., 2000). Therefore, plasminogen represents the first endogenous factor that can discriminate between normal and pathological prion proteins. Binding was abolished if the conformation of PrPSc was disrupted by 6M urea or guanidine, but the isolated lysine-binding site of plasminogen (kringle modules I to III) retained the binding activity. Ellis et al. (2002) tested the hypothesis that, due to its reported ability to bind plasminogen, PrPSc is a regulator of plasminogen activation. They observed the effect of recombinant PrP, either containing Cu (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA. PrP was found to have no effect on plasminogen activation by uPA, but the activity of tPA was stimulated by up to 280 times. This was observed only with the apo-PrP isoforms. Thus, they demonstrated that PrP can, indeed, regulate plasminogen activation, but that a critical determinant of the interaction between PrP and tPA was related to the absence of Cu in the PrP. Failure of Cu to bind PrP may be an early event in the conversion of PrP c to PrPSc and in the stimulation of plasminogen activation. The similarity in the interaction of PrPSc or Cu-free rPrP with plasminogen or plasminogen activators suggests that PrPSc may be a form of PrP devoid of Cu.

6. METAL ION BINDING AND NEURODEGENERATIVE DISEASES Research has suggested that Cu has an important role to play in the prevention or moderation of a number of neurodegenerative diseases, including

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the polyglutamine diseases such as Friedrich ataxia and Huntington's disease, Parkinson's disease (PD), Wilson's disease, Menkes' diseases, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD) (Strausak et al., 2001). Cu is an essential metal that serves as a co-factor for a number of proteins and enzymes. Many proteins associated with neurodegenerative diseases have metal-binding properties and/or metal-responsive expression (Bush, 2000). Besides PrP c , the ectodomain of the Alzheimer precurser protein (Multhaup et al., 1996), amyloid-f} peptide (Atwood etal., 2000), and SOD-1 (Fridovich, 1975) exist in Cu-bound forms. In these examples, metal binding relates to pathogenesis via an impact on aggregation or production of oxidative damage. Cells rely on a number of transition metals to regulate a wide range of metabolic activities and signaling functions. The diversity and efficiency of their physiological functions are derived from the specific atomic properties of transition metals, most notably an incomplete inner valence subshell. These properties enable the metals to fluctuate among a variety of positively charged ionic forms, allowing for a chemical flexibility that can ultimately impose conformational changes on the proteins to which they bind. By this means, transition metals can serve as catalytic centers of enzymes for redox reactions involving, for example, molecular oxygen and endogenous peroxides (Hamai et al., 2001). In general, neurodegenerative diseases display two commonly recognized metal-dependent reactions. First, there are the metal-protein associations causing abnormal aggregation of proteins. These can involve both redox-inert metal ions, such as Zn 2+ , or redox-active ions, such as Cu 2+ or Fe 3+ . Secondly, there are the metal-catalyzed protein oxidations leading to protein damage and denaturation. These reactions involve only the redox-active metal ions such as Cu 2+ , Fe 3+ , or Mn 2+ . Both reactions can lead to the functional demise of their target protein. It has been proposed that certain neurodegenerative diseases are caused by the abnormal interaction of specific, susceptible target proteins in neural tissues enriched in metals with metal ions to which they do not normally bind. The interaction can then cause aggregation and/or oxidation of neural tissue mediated by the abnormal redox-active metal ion associating with the target protein, and can lead to the loss of function of the protein. This important mechanism of oxidative damage can involve the following reactions of the bound redox-active transition metals: an

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initial reduction of the metal, such as Cu 2+ or Fe 3+ , by electron donors like the superoxide radical (0 2 ), catecholamines, L-ascorbate, and mercaptanes, as well as the subsequent generation of hydroxyl (OH*) radical through the reduction of H 2 0 2 by the reduced metals. This highly reactive free radical attacks neighboring amino acid residues, producing carbonylcontaining derivatives on the proteins. Carbonyls can also be introduced into the protein as a result of oxidative cleavage of the peptide backbone. Thus, the carbonyls produced are signs of oxidative damage in proteins (Berlett and Stadtman, 1997; Stadtman and Levine, 2000; Butterfield and Kanski, 2001). Cu is a transition metal with multiple valencies, Cu(I) and Cu(II). In a free, unbound state, it is a powerful catalyst of auto-oxidation reactions, such as oxidation of ascorbic acid, thiols, and catecholamines, that generate various reactive oxygen species (ROS) (Halliwell and Gutteridge, 1990). In view of this, Shiraishi et al. (2000) reasoned that PrP may sequester Cu(II) and may prevent oxidative damage induced by the coexistence of Cu(II) and auto-oxidizable compounds. For this to occur, the Cu(II) bound to PrP c should preferably be maintained in the redox-inactive state. Otherwise, the protein itself would be subjected to oxidative degradation. Shiraishi et al. (2000) tested the octarepeat region for inhibitory effects on Cu catalyzed oxidations of L-ascorbic acid or glutathione and generation of ROS, such as OH*. Their results showed that the catalytic activity of the first Cu(II) ion bound to the octarepeat region was completely suppressed. The valence state of the Cu under reducing conditions was Cu(II). Thus, they suggested that the in vivo function of the prion protein might be to sequester Cu ions in the redox-inactive state, rendering Cu-induced generation of ROS impossible. From the preceding discussion, it can be seen that some of the symptoms of prion diseases may be caused by an abnormality in the active Cubinding site on the PrP protein. A similar phenomenon can also occur within other proteins, such as amyloid-fS in AD or the Cu/Zn SOD in familial ALS, and can be a potential factor in other neurological diseases. Under normal conditions, Cu binding to appropriate proteins is essential to oxidative stress homeostasis. Such Cu-active sites are very likely exposed to constitutively high concentrations of ROS, such as 0 2 and H 2 0 2 . H 2 0 2 can react with Cu + , which is produced transiently at the active site of these proteins, and generate the highly reactive and detrimental ion,

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OH*. In the normally folded proteins (PrP c in TSEs, Alzheimer precusor protein in AD, and SOD-1 in Familial ALS), the Cu 2+ active site is probably shielded and, therefore, does not undergo this abnormal reaction. However, changes in the conformation of these proteins may expose the active site and make it more prone to react to produce the OH* radical.

7. COPPER AND THE ENVIRONMENT Transition metal ion pollution in the environment has been an issue now for some years. Recently, it has become a question in epidemiological studies of prion disease distribution (Purdey, 2000). For example, chronic wasting disease (CWD) is a sporadic prion disease in deer and elk and is found in specific regions of the United States. The pathology is similar to spongiform degeneration induced by Cu deficiency, as seen in cuprizonetreated mice (Pattison and Jebbett, 1971a, 1971b). The soil in regions where CWD is high apparently have a deficiency in Cu and this disease was proposed to be related to environmental Cu deficiency (Purdey, 2000). CWD was first described as a spongiform encephalopathy when plaques were detected in the brains of a large percentage of afflicted mule deer (Bahmanyar et al., 1985). Later, this was confirmed to contain PrPSc by immunodetection (Guiroy et al, 1991), thus confirming CWD as a prion disease, though some authors still regard it as a Cu deficiency disease (Yashikawa et al., 1996). Thus, it has been proposed that environmental Cu deficiency may exacerbate or even cause prion diseases (Purdey, 2000). However, despite certain coincidental observations, there is still no direct evidence linking Cu deficiency to prion diseases. It is worth noting that when metal ions are implicated in neurological syndromes associated with abnormally folded proteins, the source of the metal ions is thought to be environmental exposure. Ingestion, or some other method of internalizing metal ions, is often thought to be the original cause of the abnormal interaction between metal ions and proteins. This, however, may be a misconception because even the brain, under normal conditions, has more than enough metal ions to cause abnormal metal binding, damage, and a general malfunctioning of metabolism. Take Zn as an example. Zn 2+ is released during neurotransmission at concentrations of about 300 pJVL This concentration is sufficient to be lethal to neuronal cell cultures (Frederickson, 1989). It is, therefore, necessary for

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the brain to have efficient homeostatic mechanisms and buffers that prevent the decompartmentalization of these metal ions. The blood-brain barrier is relatively impermeable to fluctuating levels of plasma metal ions. Some metal regulatory transport systems are energy-dependent, one of these being the Cu-ATPase, which functions in Wilson's disease (Tanzi et al., 1993). It is possible that damage to the blood-brain barrier or energy compromise in the brain perturbs metal levels and lead to abnormal binding in proteins. Thus, it is not necessary to hypothesize that neurological conditions, such as prion diseases, are caused by environmental exposure to metals alone. Clearly, other factors may play a primary role in these diseases.

8. CONCLUSION There is now a steadily increasing body of evidence confirming that the function of the normal prion protein, PrP c , is that of an antioxidant and/or a Cu-sequestering protein active in the prevention of Cu toxicity in neurons. The implication for prion diseases is that the loss of function of PrP c through conversion to, or interaction with, PrPSc may be responsible for some of the pathological changes seen in this family of diseases. Though PrP c unquestioningly binds Cu ions, it is still unclear whether PrPSc can associate with Cu in the same way. There is evidence that Cu, possibly binding at an inappropriate site, can convert PrP to a protease-resistant form (Fig. 4). In the course of the disease, the amount of PrP c generated by cells increases to a very high level and much of the protein is converted to the protease-resistant form, which then probably remains in the brain until the death of the individual. If all the generated PrP c initially bound Cu then, on conversion, the accumulated PrPSc would represent a huge sink of Cu. It is also possible that Cu might become trapped nonspecifically within the PrPSc aggregates. However, initial experiments with scrapie-infected mice showed no large increases in brain Cu suggesting that the conversion of PrP c to PrPSc actually results in a protein that either does not bind Cu or that there is a reduction in Cu within the cells. The evidence suggesting that alternative metal ions bind in the place of Cu in prion diseases seems to support this idea. In the coming years, the changes in Cu metabolism during prion diseases will become clearer. Indeed, if it is consistently shown that Cu is

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Cu fails to bind during synthesis

Extracellular movement

Normal Cu binding induces protective change in conformation

Cu binds to exposed "wrong" sites in conformationally immature protein

Cu causes conformational corruption

(A)

Fig. 4. Alternative interactions of Cu with prion protein that cause either (A) protective activity or (B) conversion to a toxic species.

reduced during prion diseases, this may lead to treatments designed to enhance Cu uptake by cells in affected individuals. Alternatively, if Cu bound to PrPSc is acting as an oxidant generating H 2 0 2 or hydroxyl radicals, it might be possible to reverse the entombment of Cu or other cations in PrPSc and, thus, alleviate the disease. Whatever advances may come in the future, one thing remains clear: the pathogenesis of prion diseases is tightly linked to the function and distribution of metal ions, particularly Cu, in the brain.

ACKNOWLEDGMENTS We acknowledge the support of the BBSRC and DEFRA towards our research fund.

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CHAPTER

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Metallothioneins in Neurodegeneration Michael Aschner, William F Silverman, Israel Sekler, Paolo Zatta

ABSTRACT Metallothioneins are ubiquitous low molecular weight proteins characterized by an abundance of the thiol-containing amino acid, cysteine. Metallothionein I and metallothionein II, the most widely expressed isoforms, are co-ordinately regulated in all mammalian tissues, while a third variant, metallothionein III, is predominantly expressed in zinc-containing neurons and absent from non-neural tissue. Metallothionein proteins have been implicated as regulators of gene expression in homeostatic control of cellular metabolism of metals and in cellular adaptation to stress, including oxidative stress. They regulate transcription, replication, protein synthesis, metabolism, and other zinc-dependent biological processes. Because the intracellular concentration of zinc is buffered by complexing with apothionein to form metallothionein, and disordered metallothionein homeostasis results in changes in brain zinc levels, there has been great interest in the potential role of metallothionein regulation in the etiology of neurodegenerative disorders. Abnormalities in metallothionein and/or zinc homeostasis have been reported in a wide variety of neuropathologies, including Alzheimer's disease, epilepsy, Friedrich's ataxia, Pick's disease, amyotrophic lateral sclerosis, schizophrenia, hepatic encephalopathy, multiple sclerosis, Guillaine-Barre syndrome, Parkinson's disease, retinitis pigmentosa, retinal dystrophy, Wernicke-Korsakoff syndrome, and alcoholism, though a direct association with the etiology of neurodegenerative diseases has yet to be established. This chapter commences with a brief discussion of the various brain metallothionein isoforms, followed by a survey of the evidence that metallothioneins are involved in neurodegeneration. Keywords: Metallothionein; neurodegenerative disorders; Alzheimer's disease; prion diseases.

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1. METALLOTHIONEIN ISOFORMS AND ABUNDANCE IN THE CENTRAL NERVOUS SYSTEM The metallothionein (MT) family belongs to a class of low molecular weight, intracellular, cysteine-rich proteins devoid of aromatic amino acids with a high affinity to metal ions (Oz et al., 1999). MTs are multifunctional proteins and their roles remain elusive after more than 40 years of intense investigation. They are known to regulate the release of gaseous mediators, such as hydroxyl radical and nitric oxide, apoptotic signaling, and the binding and exchange of heavy metals (such as copper (Cu), zinc (Zn), cadmium, and silver) (Simpkis, 2000). A relationship between MT and several diseases, including cancer, cardiovascular diseases, septic shock, and immunological alterations, has been widely investigated. The involvement of MTs in the physiopathology of disorders of the central nervous system (CNS) has been recently reviewed by Hidalgo et al. (2001). MTs are referred to as "housekeeping" proteins. Their basal mRNA expression level in the CNS is low and induction is not always apparent even with potent peripheral MT inducers, most likely because the brain is protected by the restrictive properties of the blood-brain barrier (Saijoh et al., 1994). Four major isoforms of MTs have been described: MT-I, MT-II, MT-HI, and MT-IV. MT-I and MT-II are found in all vertebrate tissues, differing at neutral pH by a single negative charge. Each represents a number of different isoproteins designated as MT-Ia, MT-Ib, MT-Ic, and so forth. MT-I and MT-II are single-chain proteins containing between 61 and 68 amino acids (species-dependent), about 20 of which are cysteines. Noncysteine amino acids serve as flexible spacers connecting the metalchelating cysteine residues. The formation of stable clusters is accomplished by the folding of the peptide backbone in a way that minimizes the strain on the conformation of the protein (Kille et al, 1994; Vasak et al., 1999). MT-III, a brain-specific MT isomer, was originally described as a growth inhibitory factor (GIF) (Uchida et al., 1991). It contains 68 amino acids; thirty-eight of the amino acids are identical both in alignment and type to the human MT-I and MT-II isoforms. MT-III possesses close structural homology to other mammalian MTs. Indeed, when aligned with MT-I and MT-II, the position of all 20 constituent cysteine residues is completely conserved (Kille et al., 1994; Aschner, 1996).

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The most abundant MT mRNA in the CNS is MT-I, followed by MT-III (Aschner, 1996). Both MT-I and MT-II mRNA are highly expressed in glia in the cerebellum, olfactory bulb, and eye. MT-III transcripts are particularly abundant in neurons in regions with high concentrations of vesicular Zn, such as the hippocampus, piriform cortex, and amygdala, though strong expression is also observed in the cerebellum, an area with little synaptic Zn (Masters et al., 1994).

2. THE ROLE OF METALLOTHIONEIN IN NEURODEGENERATIVE DISORDERS 2.1. Amyotrophic Lateral Sclerosis Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by loss of motor neurons in the spinal cord, brainstem, and motor cortex. Only 10% of cases are of a familial form that is linked to point mutations in the gene encoding for cytosolic Cu, Zn, and superoxide dismutase (SOD) (Sillevis-Smitt et al., 1994). In earlier studies of sporadic ALS, the amount of MTs appeared higher in the liver, kidney, and spinal cord of ALS patients than in control subjects (Blaauwgeers et al., 1996). In ALS, MTs are highly expressed in the gray matter of protoplasmatic astrocytes from the spinal cord. This likely reflects an increase in metal exposure or enhanced oxidative stress (Sillevis-Smitt et al., 1994). In wildtype mouse spinal cord, expression of MT-I-II has been observed in ependymal cells and in a subset of astrocytes of the white matter. MT-III is limited to neurons within the gray matter (Gong and Elliott, 2000). Compared to wild-type mice, transgenic mice (G93A) carrying the SOD1 mutation show a significant increase in expression of MT-I-II within astrocytes, both in the white and gray matter. Furthermore, MT-III in the neurons of G93A mice is also significantly upregulated (Gong and Elliott, 2000).

2.2. Multiple Sclerosis Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS. Experimental autoimmune encephalitis (EAE) has been extensively used to study the human autoimmune disease MS. EAE is characterized by significant macrophage activation, T-lymphocyte infiltration, astrogliosis

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in the spinal cord, brainstem, and cerebellum with maximum manifestation at two weeks post-immunization (Penkowa and Hidalgo, 2000). Exogenous administration of Zn-MT-II significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE (Penkowa and Hidalgo, 2001). The same authors also observed a significant decrease in the expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-a in the CNS of EAE animals, which might have also played a causal role in the decrease in clinical manifestations and reduced rate of neuronal apoptosis. None of these effects could be ascribed to Zn content, suggesting that MT-I-II might be used as a pharmacotherapy in EAE/MS.

2.3. CNS Immune Responses and Seizures A recent study examined the role of MT-I and MT-II during experimental EAE (Penkowa et al., 2001a). In genetically altered mice that are MTI- and MT-II-deficient (MT knockout; MT-I/II-KO), a significantly higher susceptibility to the development of EAE relative to wild-type mice was noted. Furthermore, the inflammatory responses elicited by EAE were significantly altered in these mice. This was characterized by diminished rates of astrocytosis and increased macrophage and T-lymphocytes infiltration. Additional alterations in MT-I/II-KO mice included increased expression of proinflammatory cytokines (IL-lbeta, IL-6, and TNF-a) and increased oxidative stress and apoptosis in response to EAE. An earlier study by the same authors established that MT-I/II-KO mice were dramatically deficient in their ability to induce IL-6-induced angiogenesis (Penkowa et al., 2000a). Increased epileptiform activity and hippocampal degeneration following kainic acid-induced seizures have also been reported in MT-I/II-KO mice (Carrasco et al., 2000). These results support the notion that MT-I and MT-II modulate inflammatory responses in the CNS, thereby playing a neuroprotective role during EAE ontogeny and inflammation (Penkowa et al., 2001a), as well as during kainic acidinduced seizures (Carrasco et al., 2000). MT-I and MT-II have also been suggested to enhance neuronal survival during kainic acid-induced seizures, either via impaired Zn regulation or compromised antioxidant activity. Significantly, compared to wild-type controls, IL-6 deficient mice also demonstrate increased hippocampal neuronal injury and impairment in inflammatory responses following kainic acid-induced seizures,

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an effect that is also associated with reduced MT-I and MT-II protein levels (Penkowa et al., 2001b). The changes in neuronal tissue damage and brain regeneration of IL-6-KO mice were associated with an IL-6-dependent decrease in MT-I and MT-II expressions (Penkowa et al., 2000b). Another study, conducted on aged animals under constant stress conditions, observed MT-induced depletion of Zn from plasma and tissues. This mechanism was invoked to explain increased MT levels and a concomitant decrease in free Zn ion bioavailability, thereby perturbing immune responses known to be Zn-dependent (Mocchegiani et al, 2000, 2001). Recently, a chimeric fusion protein consisting of MT flanked by the cyan and yellow forms of the green fluorescent protein has been generated and expressed in endothelial cells. This paradigm has been used to explore metal interactions with MTs (Pearce et al., 2000). By measuring changes in fluorescent resonance energy transfer (FRET), changes in protein conformation resulting from release of metal may be followed, such as Zn and MT. These experiments indicate that interactions of Zn and Cu with MTs are dynamic and are regulated by cell signal transduction, such as the nitric oxide pathway.

2.4. Parkinson's Disease Parkinson's disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta. The etiology of PD is unknown, though oxidative stress and mitochondrial inhibition have been suggested to be involved (see review by Jenner, 2001). Oxidative stress results either from excess generation or reduced scavenging of reactive oxygen species. Thus, in rodents and primates the systemic or intracranial application of the synthetic heroin analog, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), leads to the rapid loss of nigrostriatal dopaminergic neurons and the development of a syndrome virtually indistinguishable from idiopathic PD (Davis et al., 1979). Several studies have examined MT-I and MT-II in the context of oxidative stress. Rojas et al. (1996), for example, examined the effect of 6-hydroxydopamine (6-OHDA), an oxygen-radical generator and dopaminergic neurotoxin, on levels of Zn and MTs in the brain. They reported that 6-OHDA, introduced at a concentration producing a nearly

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complete lesion of the dopaminergic innervation of the striatum, reduced Zn and MT-I levels there. A lower concentration, sufficient to cause oxidative stress but not lethal to dopaminergic neurons, resulted in a robust upregulation of MT-I mRNA in various brain regions, though not in the striatum. The authors suggest that Zn or MT are altered in conditions where oxidative stress has occurred, and propose that areas of the brain, such as the striatum, which contain high iron levels but low levels of inducible MT, are particularly vulnerable to oxidative stress. Nevertheless, the idea that MT is neuroprotective against free radicals should be viewed with caution, since the study did not take into account key mediators of cell death, presumably involved in the degeneration of nigral dopaminergic neurons and their striatal projections. Furthermore, little data exist which directly link Zn or MT with mechanisms believed to underlie the development of PD. Interestingly, these same authors have demonstrated that another free radical generator, MPTP, reduces MT-I mRNA and protein and MT-II protein content in the striatum of mice (Rojas et al., 2000), and does not protect against MPTP toxicity (Rojas et al., 1996), further weakening the contention that MT is a principal antioxidant in the context of PD. Little information is available regarding MT-III and PD. MT-III has been shown to possess free radical scavenging ability (Uchida et al., 1991). Indeed, a study on a glial cell line suggested that oxidative stress might be a key factor in modulating MT-III mRNA expression (Sogawa et al., 2000). However, another study which looked at MT-III in the context of free radicals and dopaminergic neurotoxicity (Miyazaki et al., 2000) found that MT-III transcripts decreased in the striatum after exposure to 6-OHDA. More data is needed before the question of a role for MT-III as an antioxidant in the CNS can be answered. Another important question that has not, to our knowledge, been addressed is the role of Zn and MT in Lewy body formation. These are eosinophilic inclusion bodies present in the cytoplasm of nigral dopaminergic neurons in PD (Forno, 1969). They are formed as a result of disordered metabolism and/or transport of neurofilaments, and contain both amyloid-|3 (A3) and the non A(3 components of Alzheimer's disease (AD) amyloid-P protein precursor (A(3PP) or alpha-synuclein. Because of recent insights into the role of Zn in Alzheimer's type inclusion bodies (Cherny et al., 2001), it seems likely that regulators of intracellular

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Zn-like MTs will influence, or be influenced by, cellular changes that result in Lewy body formation. Indeed, alpha-synuclein has been shown to undergo precipitation and conformational change by heating in the presence of Zn (but not calcium or manganese) in vitro (Kim et al., 2000). Such a change could be involved in the abnormal aggregation of the protein observed in PD.

2.5. Alzheimer's Disease Alzheimer's disease (AD) is a chronic neurodegenerative disorder whose pathogenic mechanisms are poorly understood. In normal human brain, MT-I and MT-II are expressed in astrocytes, but not in neurons. Using antibodies to detect cells expressing MT-I-II in AD brain sections, a high level of expression of these proteins has been noted in the cortex, cerebral white matter, and cerebellum. In particular, MT-I-II have been observed in astrocytes and microcapillaries, as well as in the granular, but not in the molecular, layer of the cerebellum in AD brains (Zambenedetti et al., 1998). A role for the MT-III isoform in the etiology of AD has been proposed (Richarz and Bratter, 2002). Initially purified as a component of normal human brain extract, MT-III was originally postulated to be secreted by astrocytes and to suppress neurotrophic activity within the CNS (Uchida et al., 1991). These authors demonstrated that extracts prepared from AD brains increased the survival of rat cortical neurons in vitro. Additional studies indicated that this neurotrophic activity of AD brain extracts was due to a reduction of GIF, which was subsequently shown to be a new member of the MT gene family, and designated MT-III. Subsequent studies established that MT-III is predominantly expressed in the hippocampus and, specifically, within neuronal populations that sequester Zn in synaptic vesicles (Masters et al., 1994; Erickson et al., 1995). Additional analyses of MT-III protein and its mRNA revealed that its expression was decreased up to tenfold in AD brains (Yu et al., 2001), leading to speculation that downregulation of MT-III and aberrations in Zn homeostasis could lead to excessive neuritic sprouting and the formation of neurofibrillary tangles characteristic of the disease. In addition, MT-III was shown to protect cortical neurons in culture from the toxic effect of A3 peptides (Irie and Keung, 2001).

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An important pool of Zn in the CNS is present in synaptic vesicles in a subgroup of glutamatergic neurons in the hippocampus. "Free" or synaptic Zn is readily released by electrical stimulation, serving to modulate responses at receptors for a number of different neurotransmitters, including the excitatory and inhibitory receptors, N-methyl-D-aspartate and 7-aminobutyric acid, respectively (Cuajungco and Lees, 1997). Although this pool of Zn is the only one that is readily stained by histochemistry, it represents only about 5% of the total Zn content in the CNS (Frederickson et al., 2000). Given that Zn is readily released from MT by disulfides, which occurs under conditions of oxidative stress, consideration should be given to the possibility that changes in MT protein expression can alter intracellular Zn homeostasis. This would be very significant since even small increases in intracellular Zn, such as those which follow the massive release of extracellular Zn accompanying some neuropathological events such as stroke (Cuajungco and Lees, 1997), could result in neuronal death. The pathophysiological role of Zn has been highlighted recently by the demonstration that oral treatment of Tg2576 transgenic mice (overexpressing human mutant AfSPP) with clioquinol, an antibiotic and Cu/Zn chelator, reduced A(3 deposition by almost 50% (Cherny et al., 2001). Reduction in deposition of Ap protein in the clioquinol-treated animals was also accompanied by a significant improvement in the health of the mice. There was no monitoring, however, of MT expression in the antibiotic-treated subjects. Another recent study tying Zn to amyloid plaques showed that synaptic Zn is an important component of amyloid deposition in hApPP(+) mice (Lee et al., 2002). Indeed, female mice exhibited higher levels of synaptic Zn in soluble A(3 and plaques than males, suggesting a mechanism for the predominance of AD in females (Katzman et al., 1989). This difference disappeared in mice lacking synaptic Zn, supporting a direct role for this pool of Zn in promoting the formation of amyloid plaques. Evidence for altered Zn metabolism in AD includes the following (Bush et al., 1994; Cuajungco and Lees, 1997): decreased levels of Zn in the temporal lobe; increased cerebrospinal fluid concentration of Zn; increased hepatic Zn with reduced Zn bound to MT; increased extracellular Zn2+-metalloproteinase activity in the hippocampus; hippocampal cholinergic differentiation, a neurochemical deficit associated with AD which leads to elevation of Zn concentrations within this

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region; a pervasive abnormality of Zn metabolism is a common complication of Down's syndrome, a condition that is frequently associated with the development of premature AD; desferoxamine, a drug which chelates iron and aluminum has been reported to arrest the clinical progression of AD; Zn has been shown to aggregate A(3 amyloid, a form which is potentially neurotoxic, the principal component of the cerebral amyloid-containing plaques characterizing AD, specifically and saturably binds Zn; Zn increases the peptide's adhesive properties and resistance to proteolytic digestion, potentially representing the putative upstream lesion in the pathogenic cascade of amyloid formation; the Zn-dependent transcription factors NF-kappa B and Spl bind to the promoter region of the ApPP gene; and Zn inhibits enzymes that degrade A(3PP to nonamyloidogenic peptides which, in turn, degrade the soluble form of Ap (Cuajungco and Lees, 1997). A number of studies have examined but failed to find an association of MT-III with AD. For example, Erikson et al. (1994) examined the association between neurotrophic activity and MT-III expression in the frontal cortices of eight AD and five control brains, focusing on the proposed role of MT-III (that is, GIF) in inhibiting cell proliferation. They reported that extracts from the cortex of AD brains stimulated the survival of approximately twofold more rat cortical neurons than control extracts, demonstrating that the AD brain possesses elevated neurotrophic activity. Furthermore, when recombinant MTs were added to cultures grown in the presence of brain extract, MT-III, but not MT-I, had a negative effect on neuron survival, further supporting the idea that MT-III is a specific inhibitory factor in this assay. However, in contrast to previous reports, neither MT-III mRNA nor MT-III protein levels were significantly decreased in the AD group. Therefore, the authors concluded that the difference in neurotrophic activity between the AD and control brain samples examined was not directly mediated by MT-III, suggesting that MTIII downregulation is not a key pathogenic event in the etiology of AD. Similarly, the fate and significance of MT-III in the AD brain was evaluated in both the temporal and frontal cortices, hippocampus, and cerebellum of 11 AD patients and two groups of five and six control subjects, respectively (Amoureux et al., 1997). Reverse transcription-polymerase chain reaction was used to quantify the levels of MT-III mRNA in these brains. The distribution of MT-III was found to be similar to that of

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control, constitutive RNAs, that is, beta-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and ribosomal RNA 18S (rRNA 18S), measured simultaneously. This shows that downregulation of MT-III mRNA in the frontal and temporal cortices and hippocampus is not associated with AD. The content of MT-III mRNA in the brain of AD patients is also not detectably altered (Amoureux et al., 1997) compared to neurologically unimpaired individuals. It is noteworthy that the contradictory results obtained on MT-III expression in AD may derive from the utilization of antibodies that face problems in targeting small epitopes due to steric hindrance by their surface peptide chains (Yanagitani et al., 1999). Mice lacking MT-III mRNA (MT-III-KO), following targeted gene inactivation, have decreased concentrations of Zn in several brain regions, including the hippocampus (Erickson et al., 1997). The pool of histochemically reactive Zn, however, does not appear to be altered in these animals. Indeed, MT-III-KO displayed normal spatial learning in the Morris water maze and were insensitive to systemic Zn or cadmium exposure. Furthermore, neuropathology or behavioral deficits were undetected in two-year-old MT-III-KO mice. However, the MT-III-deficient mice were more susceptible to kainic acid-induced seizures and exhibited an increase in neuronal injury in the CA3 field of the hippocampus. Conversely, mice overexpressing MT-III were more resistant to seizureinduced neuron injury in CA3, leading Erickson et al. (1997) to propose a role for MT-III in Zn regulation during neural stimulation.

2.6. Prion Diseases Prion protein (PrP) is a plasma membrane glycosylphosphatidylinositol (GPI)-anchored protein prevalently expressed in neurons. Its conservative presence among species suggests its relevant physiological role(s). Prion diseases are genetic or sporadic neurodegenerative and transmissible disorders characterized by an overdeposition of an abnormal isoform of PrP. These diseases include the sporadic form of Creutzfeld-Jacob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSSS), and fatal insomnia. Analogous to other Cu-binding proteins, PrP has high a affinity to Cu (Km = nM) (Brown, 2001; Wong et al., 2001) and Cu binding to the PrP modifies its biochemical properties (Lehmann, 2002). However, PrP also binds other metal ions, such as manganese and Zn, albeit at a lower affinity (Brown, 2001).

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Changes in the concentration of Cu and manganese in the brain of scrapie-infected mice before the onset of clinical symptoms have been observed and, interestingly, a major increase in manganese in the blood in the early stage of the disease has been reported (Thackray et al, 2002). The Cu content of the PrP in the brains of scrapie-infected mice has been seen to reduce with a reduction in the antioxidant properties at between 30 and 60 days post-inoculation (Thackray et al., 2002). Several properties have been ascribed to PrP, such as Cu uptake, protection against oxidative stress, cell adhesion, differentiation, signaling, and cellular survival (Martins et al., 2002). Cu binding and the ensuing changes in the tertiary structure of PrP are pH-dependent (Gustiananda et al, 2002). The possible link between prion disease and Cu dismetabolism has been reviewed by Brown (2001). Few papers report data concerning the expression of MT in prion disease. These are summarized in Table 1. Expression of MT in the brain of human prion disease patients, with and without PrP gene mutation and polymorphisms, has been studied by Kawashima et al. (2000). MT immunoreactivity was positive in the cytoplasm and processes of astrocytes from the cerebral cortex and white matter, both in normal and prion diseased brains. However, in CJD brains, immunoreactivity varies from case to case and apparently depends on the duration of the disease. In CJD with a long disease course, MT-I-II-III immunoreactivity was significantly reduced in astrocytes, which is

Table 1. MTs and prion diseases. Disorder

MT I and MT-II

CJD

Induced in reactive astrocytes in the cerebral cortex Depends on the duration of the disease Varies from case to case mRNA increased Preserved

Uchida, 1991

Reduced

Kawashima et al, 2000

GSSS Kuru

MT-III

Preserved

Reduced in the vicinity of plaques

CJD = Creutzfeld-Jakob MT = metallothionein.

disease,

Reference

Kawashima et al, 2000 Kawashima et al., 2000

Kawashima et al., 2000

GSSS = Gerstmann-Straussler-Scheinker

syndrome,

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analogous to observations of MT-I-II in GSSS, while MT-III was unmodified. Conversely, in the vicinity of the Kuru plaques, astrocytes showed a weak or negative immunoreactivity for MT. Increased expression of MT-II in scrapie has been reported (Duguid et al., 1988), where a high level of MT-II mRNA has been observed only at the terminal stage of the disease (Dandoy-Dromn et al., 1998). Finally, to the best of our knowledge only one paper deals with the high expression of MT in Pick's disease (Duguid et al., 1989).

3. SUMMARY Given the prominent role played by MTs in modulating genetic processes through redistribution of Zn and regulation of reactive oxygen species, it is not surprising that a number of different MT genes are expressed in mammalian cells. This may also represent a type of redundancy that may serve as a "safety valve," providing a reserve capacity of MTs under stressful conditions. Functionally identical MTs might be regulated independently, thus permitting cell-specific MT expression. Alternatively, the distinct MT isoforms may have distinct functions, such as that of MT-III in regulating glutamatergic neurotransmission in the hippocampus. Though MT knockout and overexpressing animal models show permutations in normal immune responses, direct evidence for the role of MTs in neurodegenerative disorders is lacking. Nevertheless, given the cardinal role played by MTs in buffering intracellular Zn, additional studies of MT gene and protein expression are necessary to assess their contribution to both neuroprotection and neurodegeneration.

REFERENCES Amoureux MC, Van Gool D, Herrero MT, Dom R, Colpaert FC, Pauwels PJ. Regulation of metallothionein-III (GIF) mRNA in the brain of patients with Alzheimer's disease is not impaired. Mol Chem Neuropathol 1997; 32:101-121. Aschner M. The functional significance of brain metallothioneins. FASEB J 1996; 10:1129-1136. Blaauwgeers HG, Anwar-Chand M, van den Berg FM, Vianney de Jong JM, Troost D. Expression of different metallothionein messenger ribonucleic acids in motor cortex, spinal cord and liver from patients with amyotrophic lateral sclerosis. J Neurol Sci 1996; 142:39^4. Brown DR. Copper and prion disease. Brain Res Bull 2001; 55:165-173.

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Bush AI, Pettingell WH, Multhaup G, Paradis MD, Vonsattel J-P, Gusella JF, Beyreuther K, Masters CL, Tanzi RE. Rapid induction of Alzheimer's A(3 amyloid formation by zinc. Science 1994; 265:1464-1467. Carrasco J, Penkowa M, Hadberg H, Molinero A, Hidalgo J. Enhanced seizures and hippocampal neurodegeneration following kainic acid-induced seizures in metallothioneinI + II-deficient mice. Eur J Neurosci 2000; 12:2311-2322. Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron 2001; 30:665-676. Cuajungco MP, Lees GJ. Zinc metabolism in the brain: Relevance to human neurodegenerative disorders. Neurobiol Dis 1997; 4:137-169. Dandoy-Dromn F, Guillo F, Bendoudjema L, Deslys JP, Lasmezas C, Dormont D, Tovey MG, Dron M. Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts. J Biol Chem 1998; 273:7691-7697. Davis GC, Williams AC, Markey SP, Ebert MH, Caine ED, Reichert CM, Kopin IJ, et al. Chronic Parkinsonism secondary to intravenous injection of meperidine analogues. PsychiatrRes 1979; 1:249-254. Duguid JR, Bohmont CW, Liu NG, Tourtellotte WW. Changes in brain gene expression shared by scrapie and Alzheimer's disease. Proc Natl Acad Sci USA 1989; 86: 7260-7264. Duguid JR, Rohwer RG, Seed B. Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library. Proc Natl Acad Sci USA 1988; 85:5738-5742. Erickson JC, Hollopeter G, Thomas SA, Froelick GJ, Palmiter RD. Disruption of the metallothionein-III gene in mice: Analysis of brain zinc, behavior, and neuron vulnerability to metals, aging, and seizures. J Neurosci 1997; 17:1271-1281. Erickson JC, Masters BA, Kelly EJ, Brinster RL, Palmiter RD. Expression of metallothionein-III in transgenic mice. Neurochem Int 1995; 27:35^-1. Erickson JC, Sewell AK, Jensen LT, Winge DR, Palmiter RD. Enhanced neurotrophic activity in Alzheimer's disease cortex is not associated with downregulation of metallothionein-III (GIF). Brain Res 1994; 649:297-304. Forno LS. Concentric hyalin intraneuronal inclusions of Lewy type in the brains of elderly persons (50 incidental cases): Relationship to Parkinsonism. J Am Geriatr Soc 1969; 17:557-575. Frederickson CJ, Suh SW, Silva D, Frederickson CJ, Thompson RB. Importance of zinc in the central nervous system: The zinc-containing neuron. J Nutr 2000; 130(5s suppl):1471s-1483s Gong YH, Elliott JL. Metallothionein expression is altered in a transgenic murine model of familial amyotrophic lateral sclerosis. Exp Neuro 2000; 162:27-36. Gustiananda M, Harris PI, Milburn PJ, Gready JE. Copper-induced conformational changes in a marsupial prion protein repeat peptide probed using FTIR spectroscopy. FEBS Lett 2002; 512:38^12.

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Hidalgo J, Aschner M, Zatta P, Varsak M. Roles of the metallothionein family of proteins in the central nervous system. Brain Res Bull 2001; 55:133-146. Irie Y, Keung WM. Metallothionien-III antagonizes the neurotoxic and neurotrophic effects of amyloid beta peptides. Biochem Biophys Res Commun 2001; 282(2):416^420. Jenner P. Parkinson's disease, pesticides and mitochondrial dysfunction. Trends Neurosci 2001;24:245-247. Katzman R, Aronson M, Fuld P, Kawas C, Brown T, Morgenstern H, Frishman W, Gidez L, Eder H, Ooi WL. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant A(3PP transgenic mice. Ann Neurol 1989; 25: 317-324. Kawashima T, Doh-ura K, Torisu M, Uchida Y, Furuta A, Iwaki T. Differential expression of metallothioneins in human prion diseases. Dement Geriatr Cogn Disord 2000; 11:251-262. Kille P, Hemmings A, Lunney EA. Memories of metallothionein. Biochim Biophys Acta 1994;1205:151-161. Kim TD, Paik SR, Yang C, Kim J. Structural charges in a-synuclein affect its chaperonelike activity in vitro. Protein Sci 2000; 9(12):2489-2496. Lee JY, Cole TB, Palmiter RD, Suh SW, Koh JY. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant AfJPP transgenic mice. Proc Natl Acad Sci USA 2002; 99:7705-7710. Lehmann S. Metal ions and prion diseases. Curr Opin Chem Biol 2002; 6:187-192. Martins VR, Linden R, Prado MAM, Walz R, Sakamoto AC, Izquierdo I, Brentani R. Cellular prion protein: On the road of functions. FEBS Lett 2002; 512:25-28. Masters BA, Quaife CJ, Erickson JC, Kelly EJ. Froelick GJ, Zambrowicz BP, Brinster RL, Palmiter RD. Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles. J Neurosci 1994; 14:5844-5857. Miyazaki I, Sagawa CA, Asanuma M, Higashi Y, Tanaka KI, Nakanishi T, Ogawa N. Expression of metallothionien-III mRNA and its regulation by levodopa in the basal ganglia of hemi-parkinsonian rats. Neurosci Lett 2000; 293(l):65-68. Mocchegiani E, Giacconi R, Cipriano C, Muzzioli M, Fattoretti P, Bertoni-Freddari C, Isani G, Zambenedetti P, Zatta P. Zinc-bound metallothioneins as potential biological markers of aging. Brain Res Bull 2001; 55:147-153. Mocchegiani E, Muzzioli M, Giacconi R. Zinc, metallothioneins, immune responses, survival and aging. Bio gerontology 2000; 1:133-143. Oz G, Pountney DL, Armitage IA. Metallothionein structure uptake. In Klaasen CD, editor. Metallothionein IV. Basel: Birkhauser Verlag, 1999:37^13. Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt H, Kanai AJ, McLaughlin MK, Pitt BR, Levitan ES. Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Math Acad Sci USA 2000; 3:477^4-82. Penkowa M, Espejo C, Martinez-Caceres EM, Poulsen CB, Montalban X, Hidalgo J. Altered inflammatory response and increased neurodegeneration in metallothionein I+11 deficient mice during experimental autoimmune encephalomyelitis. J Neuroimmunol 2001a; 119:248-260.

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Penkowa M, Carrasco J, Giralt M, Molinero A, Hernandez J, Campbell IL, Hidalgo J. Altered central nervous system cytokine-growth factor expression profiles and angiogenesis in metallothionein-I+II deficient mice. J Cerebral Blood Flow Metabolism 2000a; 20:1174-1189. Penkowa M, Giralt M, Carrasco J, Hadberg H, Hidalgo J. Impaired inflammatory response and increased oxidative stress and neurodegeneration after brain injury in interleukin-6deficient mice. Glia 2000b; 32:271-285. Penkowa M, Hidalgo J. Metallothionein-I-II expression and their role in experimental autoimmune encephalomyelitis. Glia 2000; 32:247-263. Penkowa M, Hidalgo J. Metallothionein treatment reduces proinflammatory cytokines IL-6 and TNF-alpha and apoptotic cell death during experimental autoimmune encephalomyelitis (EAE). Exp Neurol 2001; 170:1-14. Penkowa M, Molinero A, Carrasco J, Hidalgo J. Interleukin-6 deficiency reduces the brain inflammatory response and increases oxidative stress and neurodegeneration after kainic acid-induced seizures. Neuroscience 2001b; 102:805-818. Richarz AN, Bratter P. Speciation analysis of trace elements in the brains of individuals with Alzheimer's disease with special emphasis on metallothioneins. Anal Bioanal Chem 2002; 372:412^117. Rojas P, Cerutis DR, Happe HK, Murrin LC, Hao R, Pfeiffer RF, Ebadi M. 6-hydroxydopamine-mediated induction of rat brain metallothionein I mRNA. Neurotoxicology 1996; 17:323-334. Rojas P, Hidalgo J, Ebadi M, Rios C. Changes of metallothionein I + II proteins in the brain after l-mefhyl-4-phenylpyridinium administration in mice. Progress NeuroPsychopharmacol Biol Psychiatr 2000; 24:143-154. Saijoh K, Katsuyama H, Sumino K. Brain metallothionein gene expression and regulation. Biol Signals 1994; 3:150-156. Sillvetis-Smitt PA, Mulder TP, Verspaget HW, Blaauwgeers HG, Troost D, de Jong JM. Metallothionein in amyotrophic lateral sclerosis. Biol Signals 1994; 3(4): 193-197. Simpkis CO. Metallothionein in human disease. Cell Mol Biol 2000; 46:465-488. Sogawa CA, Miyazaki I, Sogawa N, Asanuma M, Ogawa N, Furuta H. Antioxidants protect against dopamine-induced metallothionein-III (GIF) mRNA expression in mouse glial cell line (VR-2g). Brain Res 2000; 853(2):310-316. Thackray AM, Knight R, Haswell SJ, Bhjdoso R, Brown DR. Metal imbalance and compromised antioxidant function are early changes in prion disease. Biochem J 2002; 362:253-258. Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M. The growth inhibitory factor that is deficient in Alzheimer's disease is a 68 amino acid metallothionein-like protein. Neuron 1991;7:337-347. Vasak M, Bogumil R, Faller P, et al. Structural and biological studies on native bovine Cu, Zn-metallothionein-3. In: Klaasen CD, editors. Metallothionein IV. Basel: Birkhauser Verlag, 1999: 15-22. Wong BS, Brown DR, Sy MS. A ying-yang role for metals in prion disease. Panminerva Med 2001; 43:283-287.

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Yanagitani S, Miyazaki H, Nakahashi Y, Kuno K, Ueno Y, Matsushita M, Naitoh Y, Taketani S, Inoue K. Ischemia induces metallothionein III expression in neurons of rat brain. Life Sci 1999; 64(8):707-715. Yu WH, Lukiw WI, Bergeron C, Niznik HB, Fraser PE. Metallothionein III is reduced in Alzheimer's disease. Brain Res 2001; 894(1):37^15. Zambenedetti P, Giordano R, Zatta P. Metallothioneins are highly expressed in astrocytes and microcapillaries in Alzheimer's disease. J Chem Neuroanat 1998; 15:21-26.

CHAPTER 13

Iron and Neurodegeneration Stacey L Grab, James R Connor

ABSTRACT Iron-related pathology is present in many neurodegenerative diseases, and the effects of iron mismanagement can serve as either primary or secondary causes of neurodegeneration. There are many mechanisms by which iron mismanagement can precipitate neurodegeneration, including misregulation of iron import and export, iron deficiency or accumulation, and oxidative damage resulting from loss of iron homeostasis. While the crucial role of iron in neurodegeneration is, in general, beginning to be appreciated, the mechanisms by which loss of iron homeostasis in the brain occurs are still unclear and questions regarding opportunities for therapeutic intervention involving iron chelation remain unanswered. Keywords: Iron transport; brain iron accumulation; neurodegeneration; oxidative stress.

1. INTRODUCTION Many neurodegenerative disorders share several general pathogenic processes, including oxidative stress and free radical activity, accumulation of intracellular aggregates, and mitochondrial dysfunction. An imbalance of iron (Fe) concentration is perhaps a common mediator of these effects, thus making an understanding of its regulation essential to attaining a complete picture of neurodegeneration. Fe is physiologically essential, a fact readily demonstrated by the syndromes associated with Fe-deficient states, including Fe deficiency anemia and cognitive deficits induced by developmental dietary Fe deficiency. The capacity of Fe to interconvert between ferric and ferrous states by readily accepting and donating electrons makes it quite useful to 323

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cytochromes and hemoglobin, and as a co-factor for enzymes. This property of Fe, however, illustrates the need for its tight regulation, as it can also catalyze free radical formation (Andrews, 1999). In the brain, Fe is the most abundant trace metal (Beard et al., 1993), with its concentration varying by region. Systemically, Fe circulates bound to transferrin (Tf). Tf is largely produced in the liver and has a half-life of eight days (Finch and Huebers, 1982). Within cells, Fe is stored as ferritin (Ft), which is a large, 24-subunit protein consisting of light and heavy chains that can store up to 4,500 atoms of Fe (Harrison and Arosio, 1996). The amount of Fe uptake into a cell is largely dependent upon the amount of transferrin receptor (TfR) on the membrane, with each TfR molecule having the ability to bind two diferric Tf molecules (four Fe 3+ atoms). TfR is regulated by cellular Fe and cytoplasmic ribonucleic acid (RNA) iron regulatory proteins (IRPs) (Rouault, 2001). IRPs interact with specific elements in messenger RNA (mRNA) transcripts referred to as iron response elements (IREs). Ft mRNA contains an IRE in the 5' untranslated region (5'UTR) and Tf contains five IREs in the 3' untranslated region (3'UTR) (Mullner and Kuhn, 1988; Leibold and Munro, 1988). There exist two distinct IRPs, IRP1 and IRP2. The presence or absence of Fe-sulfur clusters determines the functionality of IRP1 (Emerit et al., 2001). When the cluster is present, which occurs in conditions of sufficient Fe supply, IRP1 acts as an aconitase, interconverting citrate, and isocitrate. In states of Fe depletion, the cluster is absent and IRP1 takes on the role of a high-affinity binding site for IREs. IRP2 does not contain an Fe-sulfur cluster, and does not have aconitase activity. IRP2 is rapidly degraded under conditions of adequate Fe supply (Kuhn, 1998). Under low Fe conditions, IRPs bind to the 5'UTR of Ft and prevent its translation. Concurrently, IRPs bind to the 3'UTR of TfR and act to stabilize its RNA for receptor translation. In the presence of sufficient Fe, IRPs are released from both Ft and TfR, allowing for ribosomal attachment and subsequent translation of Ft and degradation of TfR mRNA (Rouault and Klausner, 1997). This elegant system of IRP-IRE Fe regulation allows for close control of cellular Fe homeostasis by the Fe molecule itself. The factors governing certain aspects of this system, such as Fe-sulfur cluster synthesis, are not fully understood, but may be affected by oxidative stress. It is also possible that dysfunction of any aspect of the system by various mechanisms may occur or result in

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neurodegenerative disease. IRP-IRE interaction in the brain is disrupted in some individuals with Alzheimer's disease (AD) (Connor et al., 1992), and targeted deletion of the IRP2 gene in mice may cause misregulation of Fe metabolism and a neurodegenerative disease characterized by neuronal Fe accumulation (LaVaute et al., 2001).

2. BRAIN IRON TRANSPORT AND NEURODEGENERATION Much is known about the regulation of Fe uptake and transport within the systemic circulation (Andrews, 2000), but less is understood about those compartments within organs that are physically separate from the systemic circulation and, therefore, cannot acquire Fe directly from serum Tf. Among these compartments is the central nervous system (CNS). Unlike endothelial cells of the systemic organs, the cells of the brain blood vessels are joined by tight junctions that present a physical barrier to the passage of proteins and molecules. Little is definitively known about the uptake and export of Fe within the brain, or about its carrier state and method of distribution within the CNS, but it seems clear that the behavior of Fe within the CNS is quite different from that within the systemic organs. These differences will be presented in this section. The role of Tf in CNS Fe regulation is not as straightforward as Tf in the systemic circulation. Fe and Tf distribution in the brain are seemingly paradoxical, in that the areas of highest Fe concentration — the globus pallidus, substantia nigra, red nucleus, dentate gyrus, thalamus, and putamen — have relatively low Tf concentrations. The highest Tf concentrations are found in the hippocampus and cortical region, and these areas demonstrate relatively low Fe levels. Notably, the areas of highest Fe concentration are associated with several neurodegenerative diseases, including Parkinson's disease (PD), AD, Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Recent data from Zerpa et al. (2000) may help in understanding this apparent paradox. They note that while Tf is synthesized in oligodendrocytes, it does not appear to be secreted by oligodendrocytes. Furthermore, Tf was localized in the cytosol and not in the secretory compartment, as would be expected for a secreted protein. Zerpa et al. (2000) propose that such findings suggest that Tf synthesized in the brain is functionally different than Tf elsewhere in the body,

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and that Tf plays a role other than Fe transport in oligodendrocytes and myelination. The role of additional Fe transport proteins in the CNS is one of growing importance, as it appears that proteins such as ceruloplasmin (CP), lactotransferrin (Lf), melanotransferrin (MTf), and divalent metal transporter 1 (DMT1) exert a substantial influence on Fe trafficking. The uneven distribution of these proteins among brain regions implies that Fe transport is specific and regional within the brain. Hence, increased or decreased production of some of these proteins is not surprisingly implicated in some neurodegenerative diseases. It has long been observed that Fe accumulations are often present in the brains of sufferers of neurodegenerative disease, yet only recently have the mechanisms of these accumulations begun to be understood and, with this knowledge, the realization of the primary importance of errors in Fe metabolism and transport.

2.1. Dysfunctional Iron Import 2.7.7.

Lactotransferrin

Lactotransferrin (Lf) is found normally in breast milk and saliva, where it serves as a potent antibacterial agent by binding free Fe 3+ and denying it to invading bacteria. Lf is also found in the lesions of some neurodegenerative disorders, most dramatically in Guamian ALS-Parkinsonismdementia complex (Levuegle et al., 1994). This complex refers to the specific geographic isolate of motor neuron disease localized to Guam. Betz cells are immunoreactive for Lf in the affected areas of the ALS brain (Levuegle et al., 1994), suggesting the importance of increased Fe deposition. Nevertheless no evidence has, to date, convincingly suggested a purpose for Lf in the brain, nor a mechanism by which Lf functions in the brain or gains access to the brain. The overproduction of Lf and its receptor has also been associated with the degeneration of dopaminergic neurons in the substantia nigra in PD (Faucheux et al., 1995). Lactotransferrin receptor (LfR) immunoreactivity in the mesencephalon of PD brains was most pronounced in those areas with severe loss of dopaminergic neurons, and immunoreactivity intensity in the substantia nigra was greatest in those brains with the greatest dopaminergic loss. Expression of Lf-positive neurons was found to be decreased in PD, but

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of those neurons surviving, immunolabeling demonstrated higher Lf levels compared to control cases. Similarly, increased Lf expression has been found in AD, but LfR expression has not been studied, again suggesting the importance of further investigation focusing on Lf and LfR functions, mechanisms, and transport in both normal and diseased brains.

2.7.2.

Melanotransferrin

Melanotransferrin (MTf) is also associated with AD via overexpression and may contribute to excess Fe accumulation (Jeffries et al., 1996). MTf is an Fe-binding protein that was first identified on melanoma cells. The protein is encoded on chromosome 3 in humans. While it is structurally similar to Tf and Lf, its function has not been well determined. MTf has been shown to be expressed on reactive microglial cells in AD patients and on associated amyloid plaques in post-mortem tissue. MTf was not expressed on those microglia not associated with plaques, and was not found in tissue from PD, HD, or ALS brains, indicating that MTf is a marker for AD. In addition, a correlation has been shown between increasing serum MTf concentration and disease progression, suggesting the role of MTf as a provider of the excess Fe found in senile plaques (Kennard et al., 1996). The distribution and expression of MTf and its physiological role warrant further investigation.

2.2. Dysfunctional Iron Export 2.2.7. Ceruloplasmin Ceruloplasmin (CP) is serum glycoprotein belonging to a family of blue copper oxidases. Its primary role appears to be as a ferroxidase, aiding in Fe efflux from cells before binding to apotransferrin, but it also functions as an amine oxidase, an antioxidant, and a copper transporter (Qian and Wang, 1998). CP is mainly synthesized in hepatocytes, with a smaller amount being produced in the CNS most likely by astrocytes (Klomp et al., 1996). Mutations in the CP gene cause an autosomal recessive disease known as aceruloplasminemia. The disease is characterized by Fe accumulation, and is often clinically manifested by diabetes and retinal and basal ganglia degeneration. The cerebrospinal fluid contains not only increased Fe levels, but also increased peroxidation products and

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superoxide dismutase (SOD) activity. Though it is widely accepted that CP functions in Fe efflux, there is also evidence to support a dual role for CP, with functions both in Fe efflux and influx (Qian and Shen, 2001). Alteration of Fe efflux has been described in aceruloplasminemia, and it is likely that defects in other Fe transport mechanisms are responsible for other diseases that have not yet been elucidated.

2.2.2. Secondary iron accumulation Other diseases of Fe accumulation do not directly affect Fe storage and transport molecules, but are the result of mutations in molecules that interact closely with Fe and can affect its homeostasis. Hallervorden-Spatz syndrome (HSS) is a rare neurodegenerative disease of childhood in which extrapyramidal dysfunction is characterized by rigidity, dystonia, and choreathetosis. The globus pallidus and substantia nigra of these patients have substantial Fe accumulation. The underlying mutation in a small number of patients with this disease appears to affect the PANK2 pantothenate kinase, an enzyme essential to co-enzyme A synthesis and catalysis of the phosphorylation of vitamin B5 (pantothenate) in a cysteine-consuming reaction (Zhou et al., 2001). PANK2 is specifically expressed in the brain, and its absence in these patients likely results in the observed cysteine accumulation in degenerating areas of the brain. A possible mechanism for Fe-mediated damage implicates initial cysteine accumulation in the brain and resultant Fe chelation by cysteine. It is notable that the causative mutation is a loss-of-function mutation. Thus, possible therapeutic strategies for this subset of HSS sufferers may target delivery of phosphopantothenate or co-enzyme A. Beyond those diseases for which a specific means of Fe mishandling has been described, there exist several other potential mechanisms for production of Fe-induced pathology. The condition known as neuroferritinopathy provides an example of a defect not in Fe transport, but rather in Fe storage (Curtis et al., 2001). In this dominant condition, the insertion of an adenine within the gene that encodes for the Ft light polypeptide results in Fe accumulation in the basal ganglia. Identification of this gene mutation provides a suggestive therapy of Fe chelation with desferrioxamines. Accumulation of the pigment neuromelanin is known to occur in the brain with normal aging, and it is particularly abundant in the substantia

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nigra. Initially termed neuromelanin because of its similarity in appearance to cutaneous melanin, neuromelanin has recently been established as a true melanin by electron paramagnetic resonance and metal analysis studies (Enochs et al., 1993; Zecca et al., 1994). Neuromelanin can interact with several heavy metals, but binds Fe particularly strongly. Neuromelanin has been implicated in the development of PD, as an increased rate of degeneration of highly pigmented neurons has been observed in the substantia nigra. The concentration of Fe in the substantia nigra in PD increases by 30% to 35%. This accumulation seems to occur within neuromelanin granules, as the concentration of Fe in these granules is higher in PD patients than in normal subjects (Sofic et al., 1991; Good et al., 1992). In normal subjects, neuromelanin may play a protective role by sequestering redox-active Fe atoms, thus preventing oxidative stress and neuronal damage. When free Fe increases such that neuromelanin is saturated, however, neuromelanin may become cytotoxic, thereby catalyzing the production of free radicals. Hydrogen peroxide could potentially be produced in excess in such a situation and, because hydrogen peroxide can degrade neuromelanin, neuromelanin could be lost altogether, releasing more Fe and accelerating neuronal death (Zareba et al., 1995). As more is discovered about each of the Fe transport molecules and mechanisms of Fe storage, it is likely that their role in neurodegenerative diseases will become better understood. Two new animal models, the Belgrade rat in which a defect in the DMT protein is associated with decreased brain Fe acquisition (Burdo et al., 2001; Zywicke et al., 2002) and a Ft knockdown mouse that has normal Fe concentrations but a decrease in Ft concentration (Thompson et al., 2002), have considerable promise in elucidating the contribution of Fe mismanagement to neurodegenerative processes.

3. IRON AND INFLAMMATION The effect of inflammation on iron metabolism is widely accepted, especially in the example of anemia of chronic disease, but the processes underlying the relationship are poorly understood and represent an area in need of further investigation. One proposed mechanism of the role of iron in system-wide inflammation suggests that the TfR located on monocytes facilitates iron entry into the cell in early inflammation, while H ferritin-specific

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binding sites have a prolonged iron sequestration effect in macrophages (Scaccabarozzi, 2000). Inflammation can cause alterations in the bloodbrain barrier (BBB) that could result in increased iron influx. The mechanisms governing iron and inflammation in the CNS are not understood. It is known that inflammation increases serum ferritin, and that CSF ferritin is elevated in Multiple Sclerosis (MS) patients. Such observations suggest that CSF ferritin could be a marker of CNS inflammation. At the cellular level, cytokines secreted during inflammation may modify IRP function, altering cellular iron homeostasis and possibly catalyzing further iron accumulation. Inflammation also appears central to plaque formation in AD. AD is characterized by the formation of plaques throughout the brain. The principle component of these plaques is amyloid-beta (AfS), a protein derived by cleavage of amyloid-beta precursor protein (ApPP). ApPP is a transmembrane glycoprotein expressed in several types of mammalian cells that, when cleaved according to its major pathway, does not produce A(3. Evidence suggests that Fe availability, which itself appears to be affected by inflammation, may affect processing of ApJPP, as A(3PP mRNA contains a putative IRE based on sequence homology (Bodovitz et al., 1995). Neuritic plaques in the AD hippocampus demonstrate strong immunoreactivity for Ft, with the Ft accumulation being associated largely with reactive microglia (Grundke-Iqbal et al., 1990; Connor et al., 1992a, 1992b). The increased expression of IRP2, in association with neurofibrillary tangles, senile plaque neuritis, and neuropil threads, supports the role of altered Fe metabolism in the pathogenesis of AD (Smith et al, 1998). In addition, IRP-IRE interaction is altered in some AD brains. In the latter case, IRP is more difficult to dissociate from the IRE in AD brain tissue. The result of this abnormally tight association would be increased Tf receptor expression, decreased Ft expression, and increased cellular Fe accumulation. All of these are consistent observations in AD brain tissue (Pinero and Connor, 2000). Further related evidence for the role of Fe in AD is provided by magnetic resonance imaging of AD brains, which demonstrate increased Fe levels in the basal ganglia (Bartzokis et al., 2000). Alzheimer's plaques also contain oxidized and nitrated proteins of neuroinflammation, as well as a surrounding layer of activated microglia and astrocytes. Inflammatory response markers, including cytokines, acute phase reactants, and proteases, are also present in the AD brain. In addition, epidemiologic studies have demonstrated that anti-inflammatory

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agents and corticosteroids are associated with slowing the progression or delaying the onset of AD (Markesbery and Carney, 1999). Microglia cells, which serve as immunological scavengers in the brain, may be central to the inflammatory response in AD. Activated microglia cells are significantly increased in the AD brain, and are capable of releasing several interleukins (IL), including IL-1, IL-6, and the widely implicated tumor necrosis factor alpha (TNF-a) (Carpenter et al., 1993). Another related mechanism of damage in AD focuses on the ability of IL-1 to upregulate the expression of A|3PP. Such an upregulation could increase A@ and, in turn, cause hydrogen peroxide accumulation, thereby resulting in an IL-1-induced self-propagation of free radicals and neuron degeneration secondary to inflammation (Goldgaber et al., 1989). Recently, two studies have considered the possibility that carrying a mutation in the protein that causes hemochromatosis, an Fe overload disease, may place individuals at greater risk for AD or at least influence the age of onset of AD (Moalem et al., 2000; Sampietro et al., 2001). Although the brain has historically been considered protected from Fe accumulation in hemochromatosis, this theory has not been systematically studied (Sheldon, 1935) and the hemochromatosis (Hfe) protein that is associated with hemochromatosis is expressed on the brain microvasculature (Connor et al., 2001). Thus, there is reason to suspect that the presence of the Hfe mutation, the most common genetic mutation in Caucasians, could influence Fe accumulation in the brain (Connor et al., 2001).

4. BRAIN IRON DEFICIENCY Many of the neurodegenerative diseases, including AD, PD, and HD, share Fe accumulation as a common pathology. The converse situation of Fe deficiency can also have dire consequences. Systemic Fe deficiency adversely affects over half a billion people worldwide, with consequences ranging from anemia to cognitive deficits (Andrews, 1999). Fe is especially crucial during development, with the effects of early dietary Fe deficiency on the brain being largely irreversible. Given that Fe is required for proper myelination of the spinal cord and white matter of cerebellar folds, and is also a co-factor for enzymes in neurotransmitter synthesis (Larkin and Rao, 1990), it is not surprising that early Fe deficiency can have longterm effects. The oligodendrocyte is the predominant Fe-containing cell in

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the brain and is also the cell responsible for myelination (Hill, 1988; Connor and Menzies, 1996). In Fe deficiency, oligodendrocytes appear immature (Erikson et al., 1997). Disruption of oligodendrocyte maturation (as caused by some gene mutations) results in Fe accumulation that is only 50% of normal levels (Connor and Menzies, 1990). Restless legs syndrome (RLS) is an example of a disease of Fe deficiency. It is characterized by an irresistible urge to move the legs and, occasionally, the arms (Earley et al., 2000a). Symptoms are worst at night and when the sufferer is at rest, with the sensations being lessened by voluntary movement of the extremity. The majority of RLS sufferers demonstrate periodic limb movements of sleep and, consequently, suffer from sleep deprivation. A magnetic resonance imaging analysis has suggested that brain Fe levels in the substantia nigra are below normal in individuals with RLS (Allen et al., 2001). Both the substantia nigra and the putamen showed Fe losses in proportion to RLS severity. Cerebrospinal fluid levels of Ft, Fe, and Tf further support the theory that brain Fe is deficient in RLS. A 65% decrease in cerebrospinal fluid ferritin, as well as a threefold increase in cerebrospinal fluid Tf, was reported in RLS patients when compared to controls, despite normal serum levels of Ft and Tf in both populations (Earley et al., 2000b). A recent histopathological analysis also provides support that the RLS brain is Fe-deficient (Connor et al, under review). Finally, pregnancy, as an example of a secondary cause of RLS, supports the causative role of Fe deficiency in RLS, as there is an increased incidence of RLS in pregnancy attributable to Fe deficiency (Ekbom, 1960).

5. OXIDATIVE STRESS AND NEURODEGENERATION Beyond misregulation of Fe trafficking as a primary cause of neurodegeneration, consideration must also be given to defects in antioxidant defense mechanisms. The etiology of neurodegenerative diseases remains elusive, yet the body of evidence supporting the crucial role of oxidative damage is increasingly convincing. As the knowledge of the various mechanisms of oxidative stress grows, so too does the list of diseases in which oxidative stress has implications. Oxidative stress has gained recognition as a powerful mechanism of both primary and secondary pathology in a growing list of neurodegenerative diseases.

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Oxidative stress refers to the damage that can ensue in the presence of excess free radical production, either endogenous or exogenous, or from a reduced capacity to neutralize free radicals. A free radical, by definition, can be any independent molecule containing an unpaired electron. This unpaired electron makes such molecules highly reactive with macromolecular structures, potentiating tissue injury and homeostatic disruption. The most important mechanism of radical production in vivo is likely the decomposition of superoxide and hydrogen peroxide as catalyzed by transition metals (Young and Woodside, 2001). Normal cellular function involves the production of free radicals, yet these free radicals represent a precarious balance between health and disease. Any failure in the decomposition or scavenging of these radicals can result in a wide array of consequences, with neurodegenerative diseases being perhaps the most devastating of these. HD is a neurodegenerative disorder related to defective free radical detoxification. It is caused by a trinucleotide (CAG) repeat expansion, and is characterized by degeneration of the striatum and disturbances in motor and cognitive functions (Huntington's Disease Collaborative Research Group, 1993). While the generation of a toxic N-terminus fragment of unknown function known as huntingtin is likely the primary mechanism of pathogenesis, striatal Fe accumulation has been noted in presymptomatic Huntington's sufferers. It is thought that generation of free radicals, via this mechanism, plays a role in the neurodegeneration (Dexter et al., 1991). Further evidence supporting the crucial role of oxidative damage in neurodegeneration is demonstrated by both AD and PD. Alzheimer brains have an increase in Fe without an accompanying increase in Ft in certain regions of the brain, including the superior temporal gyrus and the frontal cortex. Such an abnormal Fe accumulation could put cells at an increased risk for oxidative stress; perhaps not coincidentally, these areas of the brain often demonstrate severe histopathology in AD. A recently developed animal model, in which the Ft protein is only 20% of normal despite normal Fe concentrations in the brain, is likely to provide insight into the contribution of Fe and oxidative stress to the neurodegeneration seen in AD brains (Thompson et al., 2002). Similarly, in PD, the primary site of neurodegeneration and the death of dopaminergic neurons in the substantia nigra pars compacta, a site that also often demonstrates increased Fe in individuals with severe PD. As in AD, Ft expression is also decreased in

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PD. Of the surviving neurons in PD, some may contain Lewy bodies, which are intracytoplasmic inclusions of neurofilaments, ubiquitin, and alpha-synuclein. It has been shown that alpha-synuclein aggregation can be induced in vitro by Fe-related oxidative stress, providing further evidence for the linkage between impaired Fe metabolism and pathologic intracellular aggregates in PD (see Chapter 14). ALS is a type of motor neuron disease characterized by progressive muscle weakness and wasting. Its prevalence is about five in 100,000, with approximately 10% of cases being familial autosomal dominant ALS. Mutations in the copper-zinc SOD1 gene are responsible for some forms of familial ALS, with greater than 50 mutations having been described in the SOD1 gene on chromosome 21. SOD1 normally converts superoxide anion to hydrogen peroxide, but can also form toxic hydroxyl radicals. The SOD1 mutations support an excitotoxicity and free radical damage basis of disease in familial ALS. The presence of Fe in spinal motor neurons may make them especially susceptible to ALS-type degeneration by facilitating the generation of free radicals (Kasarskis et al., 1995). Familial ALS represents only a fraction of all ALS cases, however, and a causal relationship for sporadic ALS is less clear.

5.1. Mitochondrial Dysfunction In considering Fe metabolism and oxidative stress in the context of neurodegeneration, the intermediary role of mitochondria is a crucial consideration. Mitochondria are the major intracellular source of free radicals, and thus any damage to mitochondrial deoxyribonucleic acid (DNA) or to nuclear DNA coding for mitochondrial proteins often has neurodegenerative implications. The primary role of mitochondria is to provide adenosine triphosphate (ATP) by aerobic metabolism. Thus, these organelles are found in particularly high concentrations in tissues with high aerobic activity, including the skeletal and cardiac muscles and the brain. In addition to ATP production, mitochondria play a key role in the regulation of apoptosis. Mitochondria have their own circular, doublestranded DNA, with a given mammalian mitochondria containing between two to 10 molecules of mitochondrial DNA (mtDNA). Of this DNA, 13 proteins form components of the respiratory chain and oxidative phosphorylation system (OXPHOS), with the remaining 70 proteins being

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supplied by nuclear genes. Neurodegenerative disorders involving mitochondria can accordingly be divided into those caused by OXPHOS abnormalities of either mtDNA or nuclear proteins (pure myopathies, complex I and II deficiencies), those resulting in OXPHOS abnormalities caused by nuclear mutations' coding for nonmitochondrial proteins (HD), and those caused by nuclear genes encoding mitochondrial non-OXPHOS proteins (Wilson's disease, Freidreich's ataxia (FA), and hereditary spastic paraplegia) (Orth and Schapira, 2001). Still, other diseases have unassigned mitochondrial involvement, including PD, AD, and ALS, as discussed in the previous section. FA is a dramatic example of the consequence of oxidative stress in the context of mitochondrial dysfunction. It is an autosomal recessive disorder with a prevalence of one in 30,000 live births. It is characterized by progressive ataxia, neuropathy, and cardiomyopathy resulting from the loss of the mitochondrial protein frataxin with resultant mitochondrial Fe overload. An expanded GAA trinucleotide repeat on chromosome 9 is responsible for the decreased frataxin levels (Campuzano et al., 1996). Neurons and cardiac muscle are affected to the greatest degree, a finding that correlates with the strong dependence of these tissues on a continuous demand for Fe uptake and a high level of mitochondrial energy production. Mouse and yeast models of FA demonstrate decreased aconitase activity, suggesting a role for frataxin in mitochondrial-cytosolic Fe cycling. Antioxidant co-enzyme Q and free radical scavenger idebenone represent potential cardiac therapeutics for FA patients (Rustin et al., 1999). In a cellular model to determine the intracellular events associated with oxidative stress and Fe, mitochondrial membrane potential and ATP production were both decreased when Fe-loaded astrocytes were exposed to a pro-oxidant, and were conserved in the presence of an Fe chelators (Robb et al., 1999).

6. POTENTIAL THERAPEUTICS There remain a great number of unanswered questions regarding the mechanisms of neurodegenerative diseases and, accordingly, there are few truly effective treatments for these diseases, but many potential therapeutic targets. Little is known about the impact of dietary antioxidants on the development and progression of neurodegenerative diseases, and past studies have been insufficient in design and methodology. The natural

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antioxidants, including vitamin E, carotenoids, and flavonoids, do not readily cross the blood-brain barrier in adults. Thus, other antioxidants, such as spin traps and low molecular mass oxygen scavengers, are being investigated (Halliwell, 2001). Another possibility to be investigated is that some drugs already in therapeutic use, such as selegiline, apomorphine, and nitecapone, may owe some of their effects to antioxidant action. Current treatment of Wilson's disease represents a prototype of neuroprotective therapy. It is a rare autosomal recessive disturbance of copper incorporation and resultant accumulation of copper in the brain, liver, kidney, and eyes. Decoppering therapy in such patients can slow or reverse the neurologic deterioration associated with copper accumulation. Such success suggests the importance of developing protective and preventive therapies for other neurodegenerative diseases associated with elemental Fe accumulation, including AD, PD, and HD. One obstacle to overcome involves a primary difference between copper and Fe excretion: there is an active, regulated mechanism of excretion for excess copper, but no such mechanism has been identified for Fe (Andrews, 2002). The delivery of nonlipophilic compounds to the brain is limited by the blood-brain barrier. The TfR itself possesses a potential route of therapy in neurodegenerative and other diseases, as antibodies that bind the TfR have been shown to selectively target blood-brain barrier endothelium. Specifically, the OX26 antibody against rat TfR has shown potential for brain drug and gene delivery when conjugated to immunoliposomes (Shi and Pardridge, 2000). In addition, conjugates between OX26 and a variety of therapeutic agents, including neuropeptides, polyamide nucleic acids, and nerve growth factor, have shown markedly increased delivery to the brain compared to intravenous administration alone (Kordower etal., 1994; Park et al., 1998). To significantly improve the clinical management of neurodegenerative diseases, genetic risk, susceptibility factors, and prodromal symptoms must be better characterized in order that preventive strategies can be targeted toward healthy subjects to postpone illness onset. In addition, it is plausible that effective treatment of many neurodegenerative diseases would involve a bimodal approach, with both a reduction in brain Fe levels and inhibition of free radical formation. Such a method could involve, for example, combination therapy with an antioxidant and an Fe chelator. Both the antioxidants and the chelators must have high penetrability of the blood-brain barrier.

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Qian ZM, Shen X. Brain iron transport and neurodegeneration. Trends Mol Med 2001; 7:103-108. Qian ZM, Wang Q. Expression of iron transport proteins and excessive iron accumulationin the brain in neurodegenerative disorders. Brain Res Rev 1998; 27:257-267'. Robb SJ, Robb-Gaspers LD, Scaduto RC, Thomas AP, Connor JR. Influence of calcium and iron on cell death and mitochondrial function in oxidatively stressed astrocytes. JNeurosciRes 1999; 55:674-686. Rouault TA. Systemic iron metabolism: A review and implications for brain iron metabolism. Pediatr Neurol 2001; 25:130-137. Rouault T, Klausner R. Regulation of iron metabolism in eukaryotes. Curr Topics Cell Regulation 1997;35:1-19. Rustin P, von Kleist-Retzow JC, Chantrel-Groussard K, Sidi D, Munnich A, Rotig A. Effect of idebenone on cardiomyopathy in Friedreich's ataxia: A preliminary study. Lancet 1999; 354:477-479. Sampietro M, Caputo L, Casatta A, Meregalli M, Pellagatti A, Tagliabue J, Annoni G, Vergani C. The hemochromatosis gene affects the age of onset of sporadic Alzheimer's disease. Neurobiol Aging 2001; 22:563-568. Scaccabarozzi A, Arosio P, Weiss G, Valenti L, Dongiovanni P, Fracanzani AL, Mattioli M, Levi S, Fiorelli G, Fargion S. Relationship between TNF-[alpha] and iron metabolism in differentiating human monocytic THP-I cells. Bri J Haematol 2000; 110:978-984. Sheldon JH. Hemochromatosis. London: Oxford University Press, 1935: 155-159. Shi N, Pardridge WN. Noninvasive gene targeting to the brain. Proc Natl Acad Sci USA 2000; 97:7567-7572. Smith MA, Wehr K, Harris PLR, Siedlak SL, Connor JR, Perry G. Abnormal localization of iron regulatory protein in Alzheimer's disease. Brain Res 1998; 788:232-236. Sofic E, Paulus W, Jellinger K, et al. Selective increase of iron in substantia nigra zone compacta of Parkinsonian brain. J Neurochem 1991; 56:978-982. Thompson K, Menzies SL, Muchkenthaler M, Torti FM, Wood T, Torti SV, Hentze M, Beard J, Connor JR. Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress. J Neurosci Res 2002, in press. Thompson KJ, Shoham S, Connor JR. Iron and neurodegenerative disorders. Brain Res Bull 2001; 55:155-164. Young IS, Woodside JV. Antioxidants in health and disease. / Clin Pathol 2001; 54:176-186. Zareba M, Bober A, Korytowski W, et al. The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems. Biochim Biophys Acta 1995; 1271:343-348. Zecca L, Pietra R, Goj C, et al. Iron and other metals in neuromelanin, substantia nigra, and putamen of human brain. J Neurochem 1994; 62:1097-1101.

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Zerpa GAA, Saleh M, Fernandez PM, Guillou F, Monteros AE, Vellis J, Zakin MM, Baron B. Alternative splicing prevents transferrin secretion during differentiation of a human oligodendrocyte cell line. J Neurosci Res 2000; 61:388-395. Zhou B, Westaway SK, Levinson B, Johnson MA, Gitschier J, Hayflick SJ. A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome. Nat Genet 2001;28:345-349. Zywicke HA, Van Gelderen P, Connor JR, Burdo JR, Garrick MD, Dolan KG, Frank JA, Bulte JWM. Microscopic R2 mapping of reduced brain iron in the Belgrade rat. Ann Neurol 2002; 52:102-105.

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CHAPTER

14

Iron, Neuromelanin, and ot-Synuclein in Neuropathogenesis of Parkinson's Disease Kay L Double, Kurt Jellinger, Luigi Zecca, Moussa BH Youdim, Peter Riederer, Manfred Gerlach

ABSTRACT Parkinson's disease is a common neurodegenerative disorder characterized clinically by motor dysfunction. The primary pathological changes in the Parkinsonian brain are the degeneration of pigmented dopaminergic neurons of the substantia nigra and the development of pathological inclusions called Lewy bodies. Progressive cell loss in Parkinson's disease is suggested to result from self-sustaining mechanisms related to oxidative mechanisms and mitochondrial dysfunction. A common factor linking oxidative damage, mitochondrial dysfunction, and the development of Lewy bodies in Parkinson's disease is the presence of a significant and pathological increase in the amount of iron in the degenerating substantia nigra. The role of iron in biochemical pathways proposed to mediate these mechanisms and their association with the etiology of Parkinson's disease is discussed. Keywords: Iron; Parkinson's disease; neuromelanin; alpha-synuclein; neurodegeneration; oxidative stress.

1. INTRODUCTION In 1817, James Parkinson (Parkinson, 1918) first described the disease which came to be known as Parkinson's disease (PD). The most common form of this disease is the idiopathic form, which is clinically diagnosed 343

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as distinct from the rarely encountered genetic forms of Parkinsonian syndromes accompanied by multisystem degeneration on the basis of its clinical presentation and progression. The primary pathological change in the Parkinsonian brain is the progressive degeneration of the neuromelanin-containing neurons of the substantia nigra pars compacta in the ventral midbrain (Jellinger, 1989). The loss of these neurons, and the consequent loss of dopamine in the striatum (putamen and caudate nucleus), disrupts the normal control of motor activity (Gerlach and Riederer, 1993). The pathological changes characterizing this disease are well documented, but the cellular mechanisms responsible for the progressive death of these neurons have yet to be established. The greatest risk factor for PD is age (Le Couteur et al., 2002). The reason for this association is not well understood, although a range of subtle changes in cellular systems are common to both normal aging and neurodegenerative diseases. As for other neurodegenerative conditions, it is thought that multiple triggering events may initiate degeneration in PD. The disease trigger, or triggers, in any one individual may combine a variety of genetic and environmental influences. The initiation of neurodegeneration may occur many years prior to the development of symptomology (Le Couteur et al., 2002). Once initiated, however, the evidence suggests that a small number of self-sustaining mechanisms result in ongoing cell loss. Two primary, and interrelated, mechanisms implicated in the etiology of PD are oxidative pathways and mitochondrial dysfunction. Increments in indices of oxidative damage to proteins, lipids, and deoxyribonucleic acid (DNA) and correlated changes in the endogenous antioxidant systems are associated with cell damage in the Parkinsonian brain (reviewed in Double et al., 2000). Interestingly, age-related changes include alteration that favor increases in oxidative damage, such as pro-oxidative changes in mitochondrial pathways, decreases in antioxidant protective systems such as the glutathione system, and changes in cytoplasmic calcium concentrations and membrane potential (Nicholls, 2002). Changes in mitochondrial function, in particular inhibition of complex I activity, have also been proposed to be causally related to cell death in PD (Jenner and Olanow, 1998). Complex I activity is readily restricted by oxidative damage, and the resulting changes in respiratory function further stimulate oxidative mechanisms (Nicholls, 2002). Oxidative mechanisms are also believed to underlie the

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development of Lewy bodies, pathological inclusions characterizing the Parkinsonian substantia nigra. A common factor linking oxidative damage, mitochondrial dysfunction, and the development of Lewy bodies is the presence of a significant and pathological increase in the amount of iron (Fe) in the degenerating substantia nigra in PD (Fig. 1). This article briefly reviews the role of Fe in the biochemical pathways proposed to mediate these mechanisms and their association with the etiology of PD.

2. REGIONAL AND SELECTIVE INCREASE IN BRAIN IRON IN PARKINSON'S DISEASE Fe is unevenly distributed within the brain, with the highest levels found in the basal ganglia (substantia nigra, putamen, caudate nucleus, and globus pallidus), red nucleus, and dentate nucleus (Sourander and Hallgren, 1958; Youdim, 1985; Riederer et al., 1989). Brain Fe is deposited early in life. Subsequent entry of Fe into the mature brain is tightly regulated and, unlike other tissues, the turnover of brain Fe is extremely slow and serum Fe has little access to this organ (Youdim, 1985). Once inside the brain, Fe is highly sequestered within organic storage forms, such as ferritin (Octave et al., 1983), with relatively little in a free and reactive form. Despite the highly developed regulatory systems for the control of Fe in the mature brain, increases in brain Fe are associated with a variety of neurodegenerative diseases and with neurotoxin-induced neurodegeneration, suggesting an association with the neurodegenerative process (reviewed in Gerlach et al., 1994). Increased regional brain Fe has been identified in Parkinsonian syndromes, such as PD, progressive supranuclear palsy, and multisystem atrophy, in trinucleotide repeat disorders, such as Huntington's disease and dentatorubral pallidoluysian atrophy, and in dementia disorders, such as Alzheimer's disease (AD) and dementia with Lewy bodies. In all of these disorders, the striking feature of this change is the close association between the increased tissue Fe and the degenerating brain region. In movement disorders, Fe levels are increased in the basal ganglia, the brain regions controling movement (Table 1), while in AD increased Fe is associated with the pathological hallmarks of this disease in the vulnerable cortical regions (Connor et al., 1992; Smith et al., 1997). Furthermore, the number of regions affected also parallel the pattern of degeneration seen in each disease. Thus, in PD significantly

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Changes to blood brain barrier

Disease triggers (unknown, but probably includes both genetic and environmental factors) Dysregulation of Fe buffering systems (ferritin, neuromelanin, and /Lewy bodies)

Changes in buffering/ antioxidant systems (TSOD/MAO-B activity \> glutathione peroxidase activity 4 catalase activity iGSH)

Increased nigral Fe (source/timepoint?)

Oxidative pathways Toxic metabolic products

(1) Fe 2+ +H 2 0 2 ->'OH+T)H+H 2 0+Fe 3+ (Fenton reaction) (2) 20 2 -»20 2 ->H 2 0 2 -?°OH+~OH+2H 2 0 (Mitochondrial respiration)

Neuronal death in substantia nigra

Parkinsons's disease

Fig. 1. Interacting molecular mechanisms underlying neurodegeneration in Parkinson's disease.

increased Fe levels are only found in the degenerating substantia nigra (Berg et al., 2001), while multisystem atrophy and progressive supranuclear palsy are characterized by increased Fe not only in the substantia nigra, but also in the degenerating caudate nucleus and putamen (Dexter et al., 1991, 1993). Hence, the topographical distribution of the increased Fe suggests a direct relationship with the disease state.

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Total Fe content in most brain areas do not differ between control and Parkinsonian brains (Riederer et al., 1989; Sofic et al., 1988; Dexter et al., 1989). However, an increase in total Fe levels of approximately 77% was measured in the substantia nigra of patients with PD compared to control subjects (Sofic et al., 1988). Furthermore, this increase was shown to be confined to the substantia nigra pars compacta, the tissue region containing the vulnerable dopaminergic neurons, but does not occur in the pars reticulata (Sofic et al., 1991). The presence of increased nigral Fe levels in PD has been confirmed by many research groups (Gerlach et al., 1994; Jellinger, 1999) using different quantitative methods (such as metal ion detection by inductively coupled plasma mass spectroscopy, X-ray microanalysis) and histochemical techniques. Magnetic resonance imaging studies using specialized sequences are also often quoted as supporting the accumulation of Fe in the nigrostriatal system in patients with PD (Gorell et al., 1995; Bartzokis et al., 2000; Graham et al, 2000). The topographic association between hypointensity on T2-weighted magnetic resonance images (bulk water proton spin-spin relaxation time) and localization of Fe has led many investigators to conclude that Fe is the factor primarily responsible for the reduced signal intensity observed on such images in PD.

3. POTENTIAL SOURCE OF INCREASED IRON The source of the localized increase Fe in PD is unknown, but several possibilities present themselves. First, the increase might result from increased entry of peripheral Fe into the substantia nigra via an alteration in the blood-brain barrier. In the adult brain, the entry of Fe into brain is highly regulated. However, circumstances such as local inflammation may disrupt this control and allow the access of Fe. Alternatively, rather than being the result of some pathological process increased Fe might also enter the substantia nigra via normal Fe regulatory systems, such as an increase in Fe transport sites. Studies have shown that the major Fe transport site, transferrin, is not upregulated in the substantia nigra in the PD brain (Morris et al., 1994; Faucheux et al., 1995a), although specific polymorphisms in the transferrin gene have recently been associated with PD (Borie et al., 2002). Another Fe-binding protein, lactoferrin, however, is reported to be upregulated in surviving neurons in the Parkinsonian

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substantia nigra (Leveugle et al., 1996) and increased numbers of receptors for this protein are present on neurons in the Parkinsonian nigra (Faucheux et al., 1995b). The translation of many proteins involved in Fe regulation is controled by two cytoplasmic proteins called iron regulatory protein 1 (IRP1) and iron regulatory protein 2 (IRP2). These proteins bind to stem loop structures called Fe-responsive elements of the protein's mRNA. If Fe is depleted, IRP binding results in decreased synthesis of proteins involved in Fe storage and mitochondrial metabolism, such as ferritin and mitochondrial aconitase, and increased synthesis of proteins, such as the transferrin receptor, involved in Fe uptake. While IRPmediated control systems are currently an area of intensive research and changes in these systems have been associated with Fe accumulation in animal models (LaVaute et al, 2001), recent data suggest that these systems remain unchanged in the substantia nigra in PD (Faucheux et al., 2002). A body of evidence indicates that disturbances in Fe metabolism occur at multiple levels PD (reviewed in Berg et al., 2001) and may reflect genetic differences in Fe regulatory systems in these individuals (Borie et al., 2002). Nevertheless, to date no convincing explanation for the selective increase in Fe in the Parkinsonian substantia nigra is available. Furthermore, while no clear environmental cause of PD has been recognized, epidemiological studies have implicated several environmental factors associated with an increased risk of PD. Of these, the association between long-term exposure to heavy metals, such Fe, copper, and manganese, and an increased risk of PD is one of the best described, suggesting that exposure to external sources of metals may be involved in the etiology of PD (reviewed in Gorell et al., 1999). Alternatively, rather than entering from outside the brain, increased levels of nigral Fe might be derived from within the brain itself. The substantia nigra is directly connected to the globus pallidus, one of the most Fe-rich regions of the brain, via afferent 7-aminobutyric acid pathways systems. To date, however, no know mechanisms exist to explain the movement of Fe from one brain region to another. A third possibility is that the measured increases in Fe reflect not an increase in total Fe content, but rather a redistribution of Fe from other cellular sources. One possible source of the measured increased in Fe is an increase in nigral glial cells. Glial cells, predominantly oligodendroglia, but also microglia and astrocytes, are the major source of the Fe storage protein

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ferritin. An increase in the number of Fe-positive glial cells is associated with nigral degeneration in PD (Jellinger et al., 1990, 1992). Despite increased numbers of ferritin-positive glial cells, total nigral ferritin concentrations appear to be unchanged in PD (Dexter et al., 1990; Mann et al., 1994; Connor et al., 1995). More recently, it has been demonstrated that expression of ferritin mRNA is increased in glial cells adjacent to neurons in the Parkinsonian substantia nigra. Despite increased nigral Fe content, no increased expression of ferritin mRNA nor changes in binding activity of associated IRPs were detected within the Parkinsonian dopaminergic neurons (Faucheux et al., 2002). The co-localization of Fe and ferritin in the Parkinsonian substantia nigra is supported by histochemical studies. Perl's staining demonstrated increased Fe(III) in Parkinsonian substantia nigra pars compacta localized in macrophages, astrocytes, and reactive microglia (Perl and Good, 1992), as well as in the cytoplasm of occasional nonpigmented neurons. Histochemically, Fe could not be demonstrated within the neuromelanin-containing neurons of the substantia nigra pars compacta, intracytoplasmic melanin, or extracellular melanin granules in the neuropil (Perl and Good, 1992), although Lewy bodies contain Fe (Hirsch et al., 1991; Perl and Good, 1992; Castellani et al., 2000; Jellinger et al., 1990). Ferritin-bound Fe can be released by a variety of physiological (Biemond et al., 1984; Monteiro and Winterbourn, 1989; Rief and Simmons, 1990; Yoshida et al., 1995; Double et al., 1997) and nonphysiological stimuli (Double et al., 1997), resulting in oxidative, mediated membrane damage (Double et al., 1997). Moreover, a dysregulation of ferritin homeostatic systems appears to occur in PD (Connor et al., 1995). A neurodegenerative syndrome exhibiting neuropathological and symptomatic similarities with PD, associated with a mutation in the ferritin gene, has also been described (Curtis et al., 2001). The question of the temporal association between the reported increase in Fe and neurodegeneration in PD is also nuclear. Early reports suggest that the increase in Fe might be a late or secondary event in the disease process. In 1994, Dexter et al. reported unchanged nigral Fe levels in cases of so-called incidental Lewy body disease characterized by mild neuronal loss and Lewy bodies in nigral neurons. The authors suggested that these pathological findings may represent an early preclinical state of PD. They concluded that increased Fe only occurs in advanced stages of neurodegeneration. This is supported by another report that nigral Fe concentrations are

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unchanged in "mild" cases PD, assessed semi-quantitatively as a mild nigral neuronal loss (Riederer et al., 1989). In contrast, more recent work using transcranial ultrasound indicates that increased Fe can be demonstrated in the substantia nigra of patients with PD even prior to the onset of clinical symptomology (Berg et al., 2002), suggesting the increase in Fe might occur earlier than previously thought. While both the source and timing of the apparent increase in Fe substantia nigra is still controversial, the presence of increased quantities of this metal in this brain region appears to be involved in the two primary cellular pathways associated with neurodegenerative changes in PD: oxidative stress and formation of Lewy bodies.

4. ROLE OF IRON IN OXIDATIVE PATHWAYS IN PARKINSON'S DISEASE The "oxidative stress" hypothesis of PD specifically proposes an imbalance between the formation of cellular oxidants and the antioxidative processes. Decreased activity of the endogenous antioxidant molecules, glutathione peroxidase and catalase, and reduced concentrations of reduced glutathione (GSH) are reported in the Parkinsonian substantia nigra. The significant and apparently early loss of systems designed to protect the brain, together with site-specific increases of superoxide dismutase and monoamine oxidase activity, may play an important role in priming the substantia nigra for oxidative damage in PD (reviewed in Beard et al., 1994; Gotz et al., 1994; Gerlach et al., 1995a; Bharath et al., 2002). Oxidative stress, resulting from increased formation of hydrogen peroxide and oxygen-derived free radicals, can damage biological molecules and initiate a cascade of events, including dysfunction of mitochondrial respiration, excitotoxicity, and a fatal rise in cytosolic calcium (Gerlach et al., 1995a; Nicholls, 2002). Hydrogen peroxide is produced in human tissues by several enzymes, such as superoxide dismutase, 1-amino acid oxidase, glycollate oxidase, xanthine oxidase, and monoamine oxidase. In dopaminergic nerve cells, it is mainly generated by monoamine oxidase via deamination of dopamine, and nonenzymatically by autoxidation of dopamine. Hydrogen peroxide is relatively inert and is not toxic to cells. The interaction of hydrogen peroxide with the reduced forms of transitional metal ions, such as Fe(II) or copper(I), however, decomposes

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hydrogen peroxide to the highly reactive hydroxyl free radical via the Fenton reaction (Eq. [1] in Fig. 1). In addition, hydroxyl radicals are produced in the mitochondria of nerve cells during oxidative phosphorylation (Eq. [2] in Fig. 1). Hydroxyl radicals have a strong reactivity with almost every molecular species found in living cells. Such reactions include breakage of single- and double-stranded DNA, chemical alterations of the deoxyribose purine and pyrimidine bases, membrane lipids, and carbohydrates, leading to a cascade of events with subsequent damage to the mitochondial electron transport system, disturbance of intracellular calcium homeostasis, induction of proteolysis by proteases, increased membrane lipid peroxidation, release of excitotoxic amino acids (glutamate and aspartate), and finally cell death (reviewed in Gotz et al., 1994; Gerlach et al., 1995a). While measurements of oxidative load within the degenerating human Parkinsonian substantia nigra are impractical, a considerable body of indirect evidence from experimental models and post-mortem studies support the hypothesis that oxidative stress contributes to the loss of dopaminergic neurons in patients with PD (reviewed in Beard et al., 1994; Gotz et al., 1994; Gerlach et al., 1995a). Not only is total nigral Fe content increased in PD, but the ratio of Fe(II) to Fe(III) shifts from two to one in control brains to one to two in the brains of PD patients (Sofic et al., 1988). This indicates an increased rate of synthesis of hydroxyl radicals. Post-mortem studies also report increased basal levels of thiobarbituric acid-reactive substances in the substantia nigra of PD patients (a measure of secondary products of lipid peroxidation), coupled with a decrease in the levels of polyunsaturated fatty acids, the substrates for lipid peroxidation (reviewed in Beard et al., 1994; Gotz et al., 1994; Gerlach et al, 1995a). In addition, there may be DNA damage as indicated by raised 8-hydroxydeoxyguanosine (Sanchez-Ramos et al., 1994), a product of free radical attack on guanine in DNA. Further evidence for the occurrence of oxidative stress in PD comes from studies on experimental models of this disease. For example, the Fe chelator desferrioxamine (desferal) and vitamin E protect rats against the 6-hydroxydopamine (6-OHDA)-induced reduction in striatal dopamine content and decrease of dopamine-related spontaneous locomotor activity (Ben-Shachar et al., 1991a). These findings indicate the prevention of 6-OHDA-induced degeneration of nigrostriatal dopaminergic neurons. 6-OHDA is thought to induce nigrostriatal dopaminergic lesions via

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generation of hydrogen peroxide and hydroxyl radicals derived from it, presumably initiated by a transition metal such as Fe. In fact, it has been shown by magnetic resonance imaging (Hall et al., 1992) and neurochemical and histochemical studies (such as Oestreicher et al., 1994) that Fe is increased in the striatum of 6-OHDA-lesioned rats. Furthermore, it has been shown that 6-OHDA releases Fe from ferritin in vitro (Monteiro and Winterbourn, 1989). Finally, intranigral injections of Fe(III) produce neurotoxic effects similar to those observed with 6-OHDA (Ben-Shachar and Youdim, 1991).

5. ROLE OF IRON IN FORMATION OF LEWY BODIES One of the primary neuropathological criteria for a confirmed postmortem diagnosis of PD is the presence of cytosolic filamentous inclusions known as Lewy bodies and Lewy neurites in some surviving dopaminergic nigral neurons. The major fibrillar material of these inclusion bodies is a presynaptic protein of unknown function, a-synuclein (Spillantini et al., 1997, 1998). While no prominent genetic cause of sporadic or idiopathic PD has been identified, specific mutations in the a-Synuclein gene have been correlated to the onset of Parkinsonism in certain forms of autosomal dominant early onset PD in humans (Polymeropoulos et al., 1997; Kriiger et al., 1998) and animal models of the disorder (Masliah et al., 2000; Feany and Bender, 2000). a-synuclein naturally aggregates (Conway et al., 2000) and in PD, as well as in other disorders such as dementia with Lewy bodies, the protein aggregates into the filamentous structures present in Lewy bodies. The reason for this aggregation is unknown, but it has been shown that even small amounts of di- and tri-valent metals, including Fe, increase the rate of a-synuclein fibrillation (Uversky et al., 2001). An analysis of Lewy bodies in the Parkinsonian nigra demonstrated that these pathological inclusions contain redox-active Fe (Castellani et al., 2000), suggesting that this mechanism may also occur in vivo. Recently, it was shown that, in the presence of Fe, a-synuclein stimulated the production of hydrogen peroxide in vitro via Fenton chemistry (Turnbull et al., 2001), possibly via the binding of Fe(II) to the protein (Golts et al., 2002). This effect was not seen for P- and -y-synuclein, which are not associated with neurodegenerative disease (Turnbull et al., 2001). While the relationship between the aggregation

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state of the protein and free radical production is unclear, this may represent an Fe-mediated mechanism by which hydroxyl radicals may be produced inside the nigral neurons. Oxidative stress is reported to induce the aggregation of a-synuclein (Hashimoto et al., 1999), leading to fibril formation (Jellinger, 2002). Aggregated a-synuclein in diseased brains displays evidence of oxidative damage (Giasson et al., 2000), suggesting a mechanistic link between Fe, oxidative stress, protein aggregation, and cell death in PD and other synucleionopathies (Fig. 1). An unresolved question is whether the formation of Lewy bodies within a neuron exerts a protective or toxic influence on the cell (Goldberg and Lansbury, 2000). The concentration and aggregation of proteins have been demonstrated in model systems to be detrimental to the cell (for example, see Ostrerova-Golts et al., 2000; Feany and Bender, 2000). On the other hand, there appears to be no correlation between the density of Lewy body formation and cell loss (Kremer and Bots, 1993; Gomez-Isla et al., 1999; Gomez-Tortosa et al., 1999; Henderson et al., 2000), and the low number of cells containing Lewy bodies in any brain region (less than 5% of the total neuronal number) would not be expected to result in a significantly altered synaptic function. Indeed nucleolar size, an indicator of RNA synthesis, does not vary in substantia nigra cells containing Lewy bodies compared to those who do not (Gertz et al., 1994), suggesting that Lewy bodies do not disturb cell metabolism. Lewy bodies have also been proposed to represent a protective reflex within the cell. Fibrillar neuronal inclusions may sequester toxic species, diverting asynuclein from toxic assembly pathways (Goldberg and Lansbury, 2000), thereby protecting the cell (Saha et al., 2000). Nevertheless, it is clear that significant intracellular protein aggregation and Lewy body formation are pathological processes, reflecting changes in the cellular environment.

6. PUTATIVE ROLE OF NEUROMELANIN IN PARKINSON'S DISEASE Histochemical studies ascribe the localization of Fe in the substantia nigra primarily to the glial compartment and changes in glial-associated ferritin have been described in PD. Fe-mediated oxidative pathways are difficult to attribute primarily to glial-associated Fe, however, because highly active free radicals produced in glial cells are unlikely to diffuse across the

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extracellular space to neurons prior to being inactivated. A more plausible source of free radicals is, thus, likely to come from a physically less remote source, for example, free radicals produced by an intraneuronal source. While some Fe sequestration appears to occur in a-synuclein in Lewy bodies, the importance of this Fe per se for neurodegeneration is unknown. A more plausible source of intraneuronal Fe that may be involved in oxidative pathways is the neuromelanin, which characterizes the dopaminergic neurons of the human substantia nigra (reviewed in Gerlach et al., 1994, 1995a). Histological studies have demonstrated that neuromelanin occurs as granules, possibly inactivated lyosomes, within the neuronal perikaryon (Gerlach et al., 1995a). Neuromelanin appears to be an efficient binder of a variety of transition metals, of which its interaction with Fe is of particular physiological interest. Studies using synthetic dopamine-melanin have demonstrated that the binding of Fe to neuromelanin is pH- and concentration-dependent and appears to be relatively specific, as flunitrazepam did not bind to dopamine-melanin and spiperone demonstrated low affinity. Moreover, Fe(III) could only be displaced from dopamine-melanin by compounds with Fe-chelating capacity, such as the 21-aminosteroid U74500A (lazaroid) and desferoxamine, but not by dopamine, spiperone, MPTP, MPP + , and apomorphine (Ben-Shachar et al., 1991b). Also, a high affinity (KD = 13 nM) and low affinity binding site for Fe(III) (KD = 200 nM) were demonstrated on synthetic dopamine-melanin (Ben-Shachar et al., 1991b). These data, obtained from a melanin model, concurs with data from purified human neuromelanin isolated from the substantia nigra of control individuals, which exhibit Mossbauer spectra corresponding to high-spin Fe(III), but not Fe(II) (Gerlach et al., 1995b). The capacity of neuromelanin to bind Fe appears to be considerable, and is estimated to represent up to 20% of the total Fe content of the substantial nigra (estimated to be up to 12 |xg/mg) (Zecca et al., 1996, 2001; Shima et al., 1997). Energy-dispersive X-ray analysis in the scanning transmission electron microscopy mode also demonstrated weak, but significant, Fe peaks in intraneuronal neuromelanin granules of dopaminergic neurons in the substantia nigra pars compacta of Parkinsonian patients post-mortem. No such peaks were evident in neuromelanin granules from the brains of matched control substantia nigra (Jellinger et al., 1992). No peaks were seen in nonmelaninized cytoplasm or adjacent neuropil of nigral neurons

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in both Parkinsonian patients and controls, in Lewy bodies within these neurons, or in synthetic dopamine-melanin uncharged with Fe (Jellinger et al., 1992). Similar results were also reported by Good et al. (1992). In substantia nigra tissue, neuromelanin is only about 50% saturated with Fe (III), suggesting that it maintains an important residual chelating capacity which can protect against Fe toxicity (Zecca et al, 1996; Shima et al., 1997). It appears that Fe is bound to neuromelanin via catecholic groups. Electron paramagnetic resonance studies indicate that ferric Fe in the substantia nigra is bound to neuromelanin as a high spin complex with an octahedral configuration (Zecca and Swartz, 1993; Zecca et al., 1996; Shima et al., 1997). Mossbauer spectroscopy also demonstrates the chelation of ferric Fe by the neuromelanin polymer and suggests that the Fe sites are arranged in a ferritin-like ironoxyhydroxyde cluster form (Gerlach et al, 1995b; GalazkaFriedman et al., 1996; Lopiano et al., 2000; Zecca et al., 2001). X-ray absorption fine structure spectroscopy (Kropf et al., 1998) and infrared spectroscopy (Bridelli et al., 1999) studies confirm that Fe in neuromelanin is bound by oxygen-derived phenolic groups in a octahedral configuration. The data to date suggests that Fe bound to neuromelanin represents a significant pool of intraneuronal Fe inside the vulnerable dopaminergic neurons (Zecca et al., 2001). While the physiological role of neuromelanin is unclear, it is thought to participate in scavenging metal-induced free radicals within the healthy brain (Gerlach et al., 1994). Thus, neuromelanin would play a role analogous to that played by melanin in the skin, where epidermal melanin acts as a protection against ultraviolet light-induced damage to the skin, probably by involvement of hydroxyl radical-induced melanin degradation. Indeed, both native and synthetic dopamine-melanin have been shown to diminish basal lipid peroxidation in rat brain tissue, as suggested by lower levels of thiobarbituric acid reactive substances (Ben-Shachar et al., 1991b; Double et al., 1999). Under pathological conditions, however, such as increased availability of Fe(III) occurring in PD, neuromelanin is suggested to potentiate the formation of oxygen-derived radicals (Ben-Shachar and Youdim, 1990). The production of hydroxyl radicals in the presence of melanin is significantly greater when Fe(III) is predominant (Pilas et al., 1988; Zareba et al., 1995), and significantly enhanced cerebral membrane damage can be demonstrated in vitro in the presence of a neuromelanin Fe(III) complex (Double et al., 1999). Accordingly, neuromelanin appears to be able to potentiate or

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inhibit the formation of oxygen-derived free radicals, thereby playing a dichotomous role, depending on the cellular environment. The selective increase of Fe(III) in the substantia nigra pars compacta (Sofic et al., 1988, 1991) and the susceptibility of the melaninized dopaminergic neurons in the nigra to degeneration in PD (Hirsch et al., 1988) have led Youdim et al. (1989) to suggest that this disorder could be a progressive siderosis of the substantia nigra. The increased formation of a neuromelanin/Fe(III) complex (Jellinger et al., 1992) is consistent with the hypothesis that neuromelanin acts as a intraneuronal pool of Fe, which subsequently contributes to free radical-producing or other pathways. The absence of significant quantities of alternative Fe-binding molecules, such as ferritin, within pigmented neurons, together with a decreased antioxidant capacity in the Parkinsonian substantia nigra, suggests that an increase in the amount of Fe available to interact with neuromelanin may result in such interactions exerting a substantial influence upon oxidative-mediated pathways and, thus, cell survival. Thus, although Fe levels are increased in areas of pathological change in a variety of neurodegenerative disorders, the presence of neuromelanin in the human substantia nigra may contribute to the vulnerability of these neurons in the Parkinsonian brain. While the cause of the increased Fe concentration in the Parkinsonian substantia nigra is unclear, the interaction of this metal with cellular constituents, such as a-synuclein and neuromelanin, appears to be important for the development of the characteristic neuropathology characterizing the disease and, possibly, oxidative-mediated neurodegeneration. These pathways may not represent the initial trigger of the disease processs, as suggested by recent work investigating the time course of dopaminergic cell death and Fe accumulation in animals models of PD (He et al., 2003). They may, however, reflect as yet unidentified alterations in Fe homeostasis and represent secondary, but important, mechanisms involved in the progressive nature of the disease. Recent unpublished data from one of the current authors and colleagues demonstrated that the novel brain permeable Fe chelator, VK-28, protects against striatal dopamine depletions induced by 6-OHDA toxicity in the rat (Youdim, unpublished observations). These data suggest that the development of Fe-chelating substances suitable for use in the central nervous system may provide novel points for therapeutic intervention in PD and other neurodegenerative disorders associated with increases in central Fe levels.

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15

Iron and Epilepsy Wei-Yi Ong*, Benjamin Kian-Chung Ong, Akhlaq A Farooqui, Chuang-Chin Chiueh, James R Connor

ABSTRACT Injections of iron salts into the sensorimotor cortex, hippocampus, and amygdala of experimental animals results in chronic recurrent focal paroxysmal electroencephalographic discharges, behavioral convulsions, and electrical seizures. The induction of epilepsy may be related to generation of free radicals, lipid peroxidation of neuronal membranes, increased intracellular calcium concentrations through reverse action of sodium-calcium exchanger/reduced activity of plasma membrane or endoplasmic reticulum calcium ATPases, increased release of excitatory neurotransmitters, including aspartate and glutamate, and increased influx of ions through glutamate receptors. Some of the above effects of iron can be abrogated by inhibitors of phospholipase A2 (PLA2) indicating that the damaging effects of iron may be due to perturbation of the lipid environment essential to normal functioning of membrane proteins. Iron in hemoglobin, or by itself, is also likely to be the cause of human epilepsy, in instances where there is increased iron load in the brain. These include subarachnoid hemorrhage, intraparenchymal hemorrhages due to head injury and stroke, malaria, human immunodeficiency virus encephalitis, and possibly, neuroleptic drug use. A reduced level of haptoglobin, a hemoglobin-binding protein, has also been observed in select kindred relatives affected with familial idiopathic epilepsy. An accumulation of iron has been observed in the motor cortex with age, and it is possible that this might contribute to the increased incidence of epilepsy among the elderly. Iron accumulates with time in rat hippocampus after kainateinduced epilepsy. The accumulation occurs in oligodendrocytes, and is likely to be a reflection of the high levels of iron in the extracellular space. The accumulation of iron is correlated with increased expression of the divalent metal transporter-1 in astrocytes in the glial scar and increased expression of heme

"•"Corresponding author. 365

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Ong W-Yetal. oxygenase-1 in reactive astrocytes and microglia, as well as degenerating neurons at the edge of the scar. The increased divalent metal transporter-1 expression could lead to increased uptake of iron, followed by redistribution to the extracellular space. In this model, iron is the consequence of epilepsy, although it is possible that it can also be a cause of epilepsy. Further work is necessary to elucidate the effects of lipid peroxidation of the cellular membranes on function of membrane proteins and the role of phospholipases, including PLA2, in perturbing the lipid environment. The possible presence of iron in the human brain after epilepsy also needs to be elucidated. The causes of dysregulation of iron in the glial scar after neuronal injury need to be studied. In addition, possible beneficial effects of iron chelators, antioxidants that cross the blood-brain barrier, or neuroprotective gene induction on epilepsy, need to be evaluated. Keywords: Iron; epilepsy; free radicals; glutamate; GABA; phospholipase A2; kainate; excitotoxicity.

1. INTRODUCTION A seizure is a transient disturbance of the cerebral function caused by an abnormal, sudden, excessive, and disorderly discharge of cerebral neurons. Seizures can result from either primary central nervous system dysfunction or an underlying metabolic derangement or systemic disease (Simon et al., 1999). They can be classified into two types: partial (in which a focal or localized onset can be discerned) and generalized (in which the seizures appear to begin bilaterally). Partial seizures are classified as simple when consciousness is undisturbed, and complex when consciousness is impaired. Generalized seizures are of two types: convulsive and nonconvulsive. The common convulsive type is the tonic clonic (grand mal) seizure. The classic nonconvulsive, generalized seizure is the brief lapse of consciousness or absence (petit mal) (Adams et al., 1997). Epilepsy is a group of disorders in which seizures recur, usually spontaneously. Cerebral contusion, cortical laceration, intracerebral hematoma formation, and hemorrhagic cortical infarction cause extravasation of red blood cells, followed by hemolysis, decompartmentalization of iron (Fe), and increased incidence of epilepsy (Triggs and Willmore, 1984; Willmore et al., 1990). Fe is a key component of cytochromes a, b, and c cytochrome oxidase and the Fe sulfur complexes of the oxidative chain. It is, therefore,

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important for the production of ATP. Fe is also a co-factor for tyrosine hydroxylase and tryptophan hydroxylase, which are enzymes involved in the synthesis of neurotransmitters (Chen et al., 1995), ribonucleoside reductase, the rate-limiting enzyme of the first metabolic reaction committed to DNA synthesis, and succinate dehydrogenase and aconitase of the TCA cycle (reviewed in Connor et al., 2001). The highest levels of Fe are found in the motor system, and the globus pallidus, substantia nigra zona reticulata, red nucleus, and myelinated fibers of the putamen show the highest rate of staining reactivity. Fe is predominantly accumulated in glial cells (Connor et al., 1992; Morris et al, 1992). Fe can cross the blood-brain barrier in at least two ways. In the first, Fe transferrin complex from the blood is transported intact across the capillary wall by receptor-mediated transcytosis. In the second, Fe transport is the result of receptor-mediated endocytosis of Fe-transferrin from the blood by capillary endothelial cells. This is followed by release of Fe from transferrin within the cell, recycling of transferrin to the blood, and transport of Fe into the brain (reviewed in Moos and Morgan, 2000). Metal transporters, such as the divalent metal transporter 1 (DMT1) and metal transport protein 1 (MTP-1), also play a role in brain Fe transport. DMT1 has been localized to astrocytic processes and end feet around blood vessels in the rat and monkey cerebral cortex (Wang et al., 2001, 2002a), as well as glial cells and neurons in the striatum, cerebellum, and thalamus in the rat brain (Burdo et al., 2001). The presence of DMT1 in astrocytic end feet in contact with blood vessels suggests that DMT1 may be important in the uptake of ferrous Fe presented on the abluminal membrane of endothelial cells of brain capillaries. The importance of DMT1 in Fe transport into the brain is shown in Belgrade rats, in which a defect in DMT1 is associated with lower levels of Fe in the brain. MTP-1 expression is robust in pyramidal neurons of the cerebral cortex, but is not detected in vascular endothelial cells and ependymal cells (Burdo et al., 2001). An increase in Fe without proper sequestration could increase the vulnerability of cells to oxidative stress. An important form of antioxidant defense is the sequestration of Fe in organic storage forms, such as ferritin (Hallgren and Sourander, 1958; Octave et al, 1983). Ferritin is highly expressed in oligodendrocyte lineage cells, including oligodendrocytes

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and oligodendrocyte precursors cells. The latter have the ability to internalize ferritin by receptor-mediated endocytosis (Hulet et al., 2000). It has been suggested that ferritin may form part of the cellular antioxidative stress proteins (Lin and Girotti, 1997; Garner et al., 1998; Oberle et al., 1998). Intracellular Fe concentration regulates the gene expression of ferritin, transferrin receptor, and heme, thus controlling Fe metabolism in brain cells. Fe regulates these proteins post-transcriptionally, through the regulation of the binding of the iron regulatory protein (IRP) to iron response elements (IRE) in the mRNA of proteins (Rouault et al., 1992; Crichton and Ward, 1995). A concordant regulation of ferritin and transferrin receptors exists, such that a cell can obtain Fe when it is needed, and sequester Fe when it is in excess (Pinero et al., 2000). In the presence of Fe and copper, hydrogen peroxide (H 2 0 2 ) is converted into reactive hydroxyl radicals (OH*) via the Fenton reaction: Fe 2+ + H 2 0 2 -> intermediate complexes -> Fe(III) + OH* + OH". The rate of generation of free radicals by Fe can be accelerated by ascorbate since ascorbate promotes the redox cycling of Fe complexes (Rauhala et al., 1998). Ferrous citrate complexes can generate reactive radicals through either the Haber Weiss reaction or Fenton reaction (Mohanakumar et al., 1994, 1998; Rauhala et al., 1996). Brain tissue contains a large amount of polyunsaturated fatty acids, including arachidonic acid, which are sensitive to Fe-induced oxidative stress and readily undergo lipid peroxidation, a free radical-induced chain reaction: - C H 2 - + OH*-* -C*H- + H 2 0 -C*H- + 0 2 -» -COO*H-. Lipid peroxidation of neural membranes and subsequent decomposition of lipid peroxides lead to increased membrane fluidity, generation of reactive lipid products (such as malondialdehyde and 4-hydroxynonenal), disturbance of calcium homeostasis, and finally cell death (Youdim et al., 1989; Halliwell and Gutteridge, 1999).

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2. IRON-INDUCED EPILEPSY (Fig. 1) 2.1. Animal Model Studies Most studies on Fe and epilepsy have focused on examining whether Fe could be a cause of epilepsy. A single injection of 5 |JLL to 10 |JLL of ferrous or ferric chloride into rat or cat sensorimotor cortex resulted in chronic recurrent focal paroxysmal electroencephalographic discharges, as well as behavioral convulsions and electrical seizures. Fe-filled macrophages, ferruginated neurons, and astroglial cells surrounded the focus of epileptic discharge (Willmore et al., 1978a, 1978b). Intracortical implantation of blood and blood products, including whole blood, hemolyzed erythrocytes, methemoglobin, ferritin, ferrous chloride, and ferric chloride, were also observed to result in the development of focal paroxysmal discharges which became more prominent and more frequent after several months (Hammond et al., 1980). As in the sensorimotor cortex, microinjection of ferric chloride solution in the frontal cortex induced epileptic discharges recorded by electrocorticography. The seizures lasted up to 60 days after the injection (Hattori et al., 1983). The features of the epileptic discharges in electrocorticograms were studied, and were found to include isolated spikes and spike and wave complexes. They lasted for more than 12 months after the injection. The isolated spike activity appeared unilaterally, while the spike and wave complexes appeared bilaterally (Moriwaki et al., 1990,1992). The amount of neuronal damage produced by Fe deposition increased with time. One day after injection, the mean number of fluoro-Jade-labeled degenerating neurons in the center of ferric ammonium citrate injection sites was five-fold higher than at ammonium citrate injection sites. This difference increased to 56-fold by day three. At five days post-injection, few dying neurons were observed at the control sites, but neurodegeneration continued beyond a week at the Fe-injected sites. Thus, Fe released during a brief episode of hypoxiaischemia or during a stroke may be neurotoxic for a protracted period (Bishop and Robinson, 2001). Fe deposition in the hippocampus also resulted in increased epileptic activity. Injection of 3 |JL1 of 100 mM ferrous chloride into the dorsal hippocampus resulted in convulsive seizures in rats (Willmore et al., 1986). Electrolytic deposition of Fe ions in the hilus of the dentate gyrus of rats

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(and not merely hilus tissue destruction) produced electrographic seizure activity within one to two hours of current passage. Seizures recurred most intensely for two to three hours, and sporadic epileptiform activity was detected for up to 12 hours. Motor seizures were observed in all seizing rats and always coincided with hippocampal seizure discharges. The dentate gyrus appeared to be the most epileptogenic site tested, since hippocampal Fe deposition that did not include the dentate gyrus, or Fe deposition in the entorhinal cortex, was significantly less epileptogenic (Pico and Gall, 1994). Injection of Fe into the amygdala were also epileptogenic. Rats microinjected with a 100 mM aqueous solution of ferric chloride into the amygdala showed epileptiform discharges from the ipsilateral amygdala soon after injection. Within five days, epileptiform discharges were also detected in the contralateral amygdala, and behavioral seizures were observed. These spontaneous seizures occurred with rearing and bilateral forelimb clonus. Seizures persisted during the 30 days of the experiment (Ueda et al., 1998). Free radicals generated by the action of Fe are important causes of damage, resulting in epilepsy. Free radicals could be formed as a result of the Fenton reaction, and could attack cell membranes, leading to lipid peroxidation and generation of lipid peroxidation products. An increase in levels of the lipid peroxidation product, malondialdehyde, was observed in the cortex of rats treated with Fe. The increase was reduced in rats treated with Fe and the Fe chelator desferrioxamine (Suzer et al., 2000). The effect of free radicals appears to be dependent on their ability to produce lipid peroxidation, and a correlation between lipid peroxidation and epileptiform discharges after intracortical injections of ferric chloride was demonstrated (Singh and Pathak, 1990). In contrast to injections of ferrous chloride, which caused an increase in fluorescent products of lipid peroxidation and permanent seizures, injection of cobalt chloride had no effect on fluorescent products, and caused only transient seizures. Since cobalt chloride injections failed to cause formation of lipid peroxidation products, whereas injections of Fe caused significant increase in fluorescent products, it was suggested that the occurrence of Fe-induced lipid peroxidation is of importance in the initiation of recurrent seizures (Triggs and Willmore, 1984). The link between free radicals, lipid peroxidation, and

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epileptic discharges was demonstrated by experiments, in which treatment with alpha tocopherol or 2 ppm selenium prevented the development of Feinduced epileptiform activity (Willmore and Rubin, 1981). Rats pretreated with 500 mg/kg DL-alpha-tocopherol acetate prior to the injection of 3 |JLL of 100 mM ferrous chloride into the dorsal hippocampus showed significantly less lipid peroxidation and fewer convulsive seizures than those not treated with tocopherol (Willmore et al., 1986). The free radical scavenger, melatonin, also decreased the concentration of thiobarbituric acid reactive substances, an index of oxidative damage, and suppressed or delayed the development of ferrous chloride-induced epileptic discharges (Kabuto et al., 1998). The species of radicals formed after Fe injections were examined using electron spin resonance, and found to include the superoxide radical (Willmore et al., 1983). The hydroxyl radical is an even more reactive free radical species than the superoxide radical, and scavengers of hydroxyl radicals, in particular, were shown to be effective in suppressing or delaying the occurrence of the ferrous Fe-induced epileptic discharges. Treatment of rats with epigallocatechin or a phosphate diester of vitamins E and C, which are potent hydroxyl radical scavengers, significantly inhibited the formation of malondialdehyde and epileptic discharges in the Fe-induced epileptic focus (Mori et al, 1990). Intraperitoneal injections of the hydroxyl radical scavengers, adenosine and 2-chloroadenosine, also suppressed or delayed the occurrence of epileptic discharges induced by ferric chloride injections into the sensorimotor cortex of rats (Yokoi et al., 1995). Oral administration of the hydroxyl radical scavanger, alpha-tocopheryl-L-ascorbate-2-O-phosphate diester, was recently shown to protect against oxidation of neural membranes and prevented the induction of epileptiform activities after ferric chloride injection into the rat motor cortex (Yamamoto et al., 2002). In addition to superoxide and hydroxyl radicals, other free radical species, such as the l,l-diphenyl-2-picrylhydrazyl radical, may also be involved in Fe-induced epilepsy (Hiramatsu et al., 1994). In contrast, the nitric oxide radical does not seem to be a prominent feature of Fe-induced damage. Intracortical injections of Fe have instead been reported to result in a significantly decreased nitric oxide synthase activity at the injection site (Kabuto et al., 1996). Nitric oxide has been shown to scavenge

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short-lived, highly reactive free radicals, such as superoxide anion, hydroxyl radical, peroxyl lipid radicals, and thiyl radicals (Chiueh, 1999). Although destructive at high concentrations, the presence of nitric oxide at the time of hydroxyl radical production can lead to HN0 2 , thus inhibiting the damaging effects of hydroxyl radicals. The hydroxyl radical scavenging actions of nitric oxide donors have been shown to provide protection against ferrous citrate-induced neurotoxicity (Rauhala et al., 1996, 1998; Mohanakumar et al., 1998). When added with 1 mM ferrous chloride, nitric oxide donors including NOC-18, SNAP, and NOR-4 (0.1-1 mM) have been shown to inhibit lipid peroxidation and suppress cell death in a concentration-dependent manner (Nara et al., 1999). In view of these observations, it is possible that the decreased nitric oxide synthase activity after Fe treatment might result in reduced level of nitric oxide, and could further contribute to hydroxyl ion-induced damage. The free radicals catalyzed by Fe could affect neuronal membranes and result in increased excitation or decreased inhibition of neurons and epilepsy. These possible effects could be direct effects of free radicals on receptor or transporter proteins, or indirect effects of Fe on lipid components of the cell membrane, changes in its composition and structure, and consequent interference with the function of membrane proteins. The functions of excitatory amino acids, including glutamate and aspartate have been reported to be affected by Fe-induced free radical formation. An increase in both luminol and lucigenin enhanced chemiluminescence was observed in the cortex, hypothalamus and hippocampus of rats that had received Fe injection into the cortex. In contrast, no increases in chemiluminescence was observed in Fe-injected rats pretreated with MK-801, an antagonist of the N-methyl-D-aspartate (NMDA) class of glutamate receptors, suggesting that these receptors play important roles in the development of post-traumatic epilepsy (Kucukkaya et al., 1998). Fe treatment may result in increased activity of ionotropic glutamate receptors. The maximal release of [3H] y-aminobutyric acid (GABA) from cultured retinal cells, evoked by NMDA under potassium-induced depolarization, was significantly higher in peroxidized cells compared with control cells. The change in the intracellular sodium concentration evoked by saturating concentrations of NMDA under depolarizing conditions was also significantly higher in peroxidized cells than in controls (Agostinho et al., 1996).

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Kainate, used at a subsaturating concentration, also evoked significantly greater increases of the intracellular sodium concentration in peroxidized cells, than in control cells. These results suggest that the activity of ionotropic glutamate receptors is increased during conditions of oxidative stress and lipid peroxidation (Agostinho et al., 1996). Interestingly, the effect of oxidative stress on the NMDA receptor was significantly reduced by a phospholipase A2 (PLA2) inhibitor (Agostinho et al., 1996). This suggests that the lipid environment of the NMDA receptor could be crucial to its function. The peroxidized cell membrane is more susceptible to hydrolysis by PLA2 (McLean et al., 1993). In contrast to hypoxia alone (which generates arachidonic acid leading to feedback inhibition of PLA2 and minimal losses of phospholipids), treatment of kidney tubule cells with Fe and peroxidation of cell membranes resulted in early phospholipid losses (Zager et al., 1999). The breakdown of phospholipids could produce a "packing defect" in the membrane, which could then affect the function of membrane proteins (Farooqui et al., 2000). There is evidence that the channel region of the NMDA receptor may be affected by this form of Fe-induced damage. Treatment with ferrous chloride led to marked inhibition of MK-801 binding to the channel of the NMDA receptor, in a concentration-dependent manner in rat brain synaptic membranes (Ogita et al., 1999). Of the various saturated and unsaturated free fatty acids, both oleic and arachidonic acids exclusively decreased the potency of ferrous chloride to inhibit MK-801 binding when rat brain membranes were first treated with fatty acids, followed by treatment with ferrous chloride. These results suggest that changes in membrane phospholipids may be responsible for the effect of ferrous ions on the NMDA channel (Ogita et al., 1999). The observation that fatty acids inhibited the ferrous chloride induced-changes in the NMDA receptor argues against a direct effect of fatty acids or their metabolites in effecting these changes. An accumulation of fatty acids could, on the other hand, result in the inhibition of PLA2 (Sevanian and Rashba-Step, 1997) and prevent the formation of "packing defects" in the membrane. The above observations support the notion that perturbation of the physical structure of the cell membrane may be critical in effecting changes to the NMDA receptor. The importance of an intact cell membrane in NMDA function is further supported by the

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observation that pretreatment with the detergent Triton X-100 resulted in potentiation of the ability of ferrous chloride to interfere with MK-801 binding (Shuto et al., 1997). Fe may also produce changes in glutamate uptake and glutamate transporters. The uptake velocities of glutamate (and GAB A) from synaptosomal fractions was severely reduced in the presence of Fe and other metals (Gabrielsson et al., 1986; Keller et al., 1997). Injection of ferric cations into rat hippocampus resulted in bilateral elevations in the expression levels of the neuronal glutamate transporter EAAC-1, but a decrease in levels of the glial glutamate transporter GLAST at 30 days after injection, at a time when experimental animals were experiencing spontaneous limbic behavioral seizures. The decreased expression of GLAST may result in increases in extracellular glutamate, which could lead to increased excitation of neurons and epilepsy (Ueda and Willmore, 2000). Fe may also cause an increase in glutamate or aspartate release. Fe and peroxide treatment increased the basal release of D-[3H] aspartate from cerebral cortical synaptosomes (Gilman et al., 1994). A significant increase in potassium-stimulated aspartate release, but no significant differences in the release of glutamate or in the uptake of glutamate and aspartate between Fe-injected and saline-injected cortex was also described, in the rat cerebral cortex (Janjua et al., 1990). Injection of ferrous chloride was reported to result in increased levels of both aspartate and glutamate in epileptic brain tissue in another study (Engstrom et al., 2001). Fe-induced free radical damage may also affect the function of the inhibitory neurotransmitter, GABA. As in excitatory amino acid transmission, GABA receptors, transporters, and release may be affected. Addition of H 2 0 2 to rat hippocampal slices resulted in decreased function of the GABAA receptor, as evidenced by decreased intracellular chloride concentration and reduced ability of a GABA agonist, muscimol, to increase intracellular chloride concentrations in CA1 hippocampal neurons (Sah et al., 2002). A superoxide radical-generating system consisting of xanthine plus xanthine oxidase also decreased muscimol-induced chloride uptake in rat cerebral cortical synapsosomes (Schwartz et al., 1988). This effect of free radical damage on decreasing the function of an inhibitory transmitter receptor contrasts with the facilitatory effect of radicals on the NMDA receptor described above, and may result in an imbalance in excitation/inhibition, and epilepsy.

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In addition to the GABAA receptor, a decrease in GABA transaminase activity has been reported after ferric chloride-induced epilepsy (Pathak et al., 1984). GABA transaminase is both a key synthetic enzyme for GABA and a degrading enzyme. A decrease in GABA uptake by rat brain synaptosomes was shown after lipid peroxidation, induced with xanthine and xanthine oxidase (Braughler et al., 1985) or Fe/ascorbate-induced treatment (Gabrielsson et al., 1986; Rafalowska et al., 1989; Palmeira et al., 1993). The effect of decreased uptake would be to increase GABA concentration at GABAergic synapses. It is not known whether this could result in increased inhibition of neurons, since oxidative injury also concomitantly reduced GABAA receptor function (Sah et al, 2002). As mentioned above, lipid peroxidation of neuronal cell bodies produced an increase in GABA release in response to NMDA (Agostinho et al., 1996). In contrast, peroxidation appeared to have the opposite effect on synaptosomes, and inhibited GABA release. Fe/ascorbate-induced lipid peroxidation produced a decrease in calcium-dependent and calcium-independent efflux of accumulated [3H] GABA in response to potassium pulses (Palmeira et al., 1993). Peroxidation also caused a significant inhibition of veratridine-dependent release of GABA from synaptosomes, although potassium-dependent release of these neurotransmitters was not affected (Rafalowska et al., 1989). Comparison of GABA uptake and release after peroxidation suggested that GABA release was more severely affected than GABA uptake. The difference between release and uptake may result in a reduction of GABAergic transmission, leading to decreased inhibition of neurons and seizures (Zhang et al., 1989). An increase in excitatory neurotransmitter release following Fe-induced oxidative stress could be due to an effect of Fe in increasing intracellular calcium concentrations. This could occur via increased influx of calcium ions or reduced efflux of calcium. Peroxidation of rat brain synaptosomes by ferrous Fe and H 2 0 2 was coupled with a rapid and large (two- to seven-fold) uptake of calcium by synaptosomes (Braughler et al., 1985). Fe-induced calcium uptake was blocked by high concentrations of either desferrioxamine or methylprednisolone. In contrast, calcium channel blockers did not affect calcium uptake induced by Fe (Braughler et al., 1985). The effect of Fe on calcium influx, therefore, does not appear to occur via increased calcium influx through voltage-dependent calcium channels. Instead, Fe appeared to inhibit the function of these channels.

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Fe and H 2 0 2 significantly reduced potassium (depolarization) induced calcium influx, and this reduction was abolished by hydroxyl radical scavengers. Hydroxyl radicals were also found to suppress calcium influx induced by BAY K8644, an activator of L-type voltage-dependent calcium channels (Shirotani et al., 2002). An alternative way in which Fe could increase intracellular calcium concentration is by affecting the sodium-calcium exchanger (Amoroso et al., 2000). The exchanger usually functions to remove calcium from the cell but, when working in reverse, can lead to calcium accumulation in the cell. An increase in sodium efflux and intracellular calcium concentration was observed in C6 glioma cells treated with sodium nitroprusside, a nitric oxide donor and an Fe-containing molecule. The effect was blocked by bepridil, a specific inhibitor of the sodium-calcium exchanger, indicating that the calcium influx occurred through reverse action of the exchanger (Amoroso et al., 2000). A hydroxyl radical-induced calcium influx in rabbit and rat myocardium due to reverse mode of the sodium-calcium exchanger has also been described (Zeitz et al., 2002). Fe may also reduce the activity of the sodium-potassium ATPase and the plasma membrane or endoplasmic reticulum sodium/calcium ATPases (Kaplan et al., 1997). This could lead to depolarization (and possibly calcium influx through voltage-dependent calcium channels, though these may be damaged as well) and/or reduced extrusion or sequestration of calcium. The calcium-dependent ATPase activity of synaptosomal plasma membranes was significantly depressed following peroxidation of membrane lipids, and the calcium ATPase activity of endoplasmic reticulum was also affected during ascorbate/Fe-induced oxidative stress (Pereira et al., 1996). As in the Fe-induced increase in NMD A receptor activity, the lipid environment of the membrane protein appears important, and the effect of Fe on ATPase activity was abrogated by PLA2 inhibitors. These observations support the concept that increased synaptosomal calcium concentration due to oxidative stress may result from inhibition of plasma membrane and endoplasmic reticulum membrane calcium ATPase activities, as a result of the alteration of the lipid environment required for activity of these proteins (Pereira et al., 1996). A calcium-independent, nonvesicular, release of neurotransmitters after Fe treatment has also been described, but is controversial. Sodiumdependent amino acid transporters normally function to remove amino

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acids from the synaptic cleft, but when working in reverse, can lead to the release of neurotransmitters. Treatment with peroxide, Fe alone, or peroxide with Fe was reported to significantly increase the calciumindependent basal release of D-[3H] aspartate (Gilman et al., 1994). However, another study reported that release of aspartate, glutamate, and GABA, which occurs through reversal of the sodium-dependent amino acid transporters was reduced following ascorbate/Fe-induced oxidative stress (Rego et al., 1996).

Ca2*

Membrane

Native phospholipid

•

4

Peroxidized phospholipid

cPLA, •*-

Depolarization

Accumulation of arachidonicacid Increase in eicosanoids VitC ROS OH"

•

Dama 9 e t 0 Phospholipids, proteins,and nucleic acids

Neuroinflammation

Neural cell injury Surface transport system and Fe-binding proteins

Membrane

Fe3*

Fig. 1. Hypothetical diagram showing the relationship among Fe, phospholipids, and phospholipase A2 during kainic acid (KA)-induced neurotoxicity. Membrane phospholipids are hydrolyzed by cytosolic phospholipase A2 (cPLA2). Peroxidized phospholipids are a better substrate for cPLA2 than the native phospholipids. In KA-induced neurotoxicity, increased cPLA2 activity after epileptic seizure results in loss of essential phospholipids and accumulation of arachidonic acid. This triggers off an uncontrolled arachidonic acid cascade with alterations in ion homeostasis and cellular redox (Farooqui et al., 2000). The reduced iron (Fe2+) catalyzes the formation of hydroxyl radicals in the presence of H 2 0 2 through the Fenton reaction, leading to more oxidative damage to native membrane phospholipids, proteins, and nucleic acids. ROS = reactive oxygen species, SOD = superoxide dismutase.

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It has also been suggested that Fe can affect ion channels by virtue of its magnetic properties (Dobson and St Pierre, 1996). Such interactions may lead to changes in channel properties that might underlie some forms of epilepsy.

2.2. Human Studies The presence of an intracranial hemorrhage, especially those occurring in the brain tissue itself, has a robust association with the development of epilepsy. When hemoglobin or myoglobin is exposed to low levels of H 2 0 2 (such as a one-to-one molar ratio of H 2 0 2 to protein), they are converted into Fe(IV) (heme ferry 1) species: Heme Fe(III) protein + H 2 0 2 —> heme [Fe(IV) = O] protein* + H+ + H20. The ferryl species can stimulate peroxidation of lipids and oxidize other molecules. When myoglobin or hemoglobin is exposed to an excess (more than a 10-to-one molar ratio) of H 2 0 2 in vitro, they are degraded, releasing both heme and Fe ions (from heme ring breakdown). The heme so released can stimulate lipid peroxidation. When added to lipids, heme proteins, in the absence of added H 2 0 2 , could stimulate peroxidation by a mechanism that probably involves the decomposition of pre-existing traces of lipid peroxides in the lipids to alkoxyl and peroxyl radicals (Hogg et al, 1994; Rice-Evans et al, 1993; reviewed in Halliwell and Gutteridge, 1999): Heme Fe(III) + LipOOH -» heme Fe 2+ + LOO* + H + , Heme Fe 2+ + LipOOH -* heme Fe(III) + LipO* + OH". As is evident from the above reactions, Fe in the heme molecule plays a key role in radical generation even before heme breakdown. Heme is broken down by the action of heme oxygenase (HO). This enzyme is found in the endoplasmic reticulum. It catalyzes the breakdown of heme to biliverdin, with the release of carbon monoxide and Fe ions (Maines, 1997, 2000). Two isoforms of HO have been characterized. A constitutive isoform (HO-2; predominant under physiological conditions) and a stress-induced (Keyse and Tyrrell, 1989) isoform (HO-1; identical to heat shock protein 32). The biliverdin produced by the action of HO is converted into bilirubin by the enzyme biliverdin reductase in the

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cytosol (Tenhunen et al., 1968; reviewed in Elbirt and Bonkovsky, 1999). Bilirubin is an antioxidant, whereas the Fe formed is a pro-oxidant (reviewed in Halliwell and Gutteridge, 1999). Intracranial hemorrhages can be classified into extradural, subdural, subarachnoid (based on their relationship with the dura and arachnoid layers of the meninges covering the brain), and intraparenchymal (within the brain tissue). The potential for epileptogenesis appears related to the location of the extravasated blood. Extradural hemorrhages occur between the dura mater and the skull. They commonly occur as a result of a temporal or parietal fracture with laceration of the middle meningeal artery or vein. Less often, there is a tear in a dural venous sinus. Though the brain tissue may be compressed as a result of the hematoma, extravasted blood does not come into direct contact with the brain tissues. There is no increased incidence of epilepsy following hemorrhage in this location. Subdural hemorrhages occur between the dura mater and the arachnoid mater. They commonly occur as a result of acceleration/deceleration injury following a blow to the head, and tearing of the cerebral veins before the point of entry into the dura mater. In this situation, blood may compress the brain tissue, but is separated from the brain tissue itself by the arachnoid mater and the subarachnoid space. Although convulsions are occasionally seen in patients with associated cerebral contusions, they cannot be regarded as a cardinal sign of subdural hematoma (Adams et al., 1997). Subarachnoid hemorrhages occur between the arachnoid mater and pia mater covering the brain tissue. There are many causes of subarachnoid hemorrhages, but the most common cause is a ruptured aneurysm in one of the constituent vessels forming the arterial circle of Willis at the base of the brain. The characteristic of this condition is the tendency for the hemorrhage to recur from the same site. In this location, blood is separated from the brain tissue by only a thin layer of pia mater. Convulsive seizures, usually brief and generalized, are rare and occur in fewer than 10% of cases of subarachnoid hemorrhage. They are not recurrent and occur in relation to acute bleeding or rebleeding from an aneurysm. These early seizures do not correlate with the location of the aneurysm (Adams et al., 1997). Seizures are, however, observed if subarachnoid hemorrhage is associated with intraparenchymal hemorrhage, such as that following rupture of an arteriovenous malformation (AVM) which partly involves the

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brain parenchyma, and bleeding into the subarachnoid space (see below). Fe released by action of HO-1 in the arterial walls could catalyze the formation of free radicals, which lead to sustained arterial contraction and vasospasm following subarachnoid hemorrhage (Findlay et al., 1991; Ono et al., 2000). The vasospasm was strongly associated with fixed ischemic deficits, but not epilepsy (Ildan et al., 2002). Intraparenchymal hemorrhages occur in the brain tissue itself. They can be the result of trauma to the head and contusion of the brain tissue, chronic hypertension and degenerative changes in cerebral arteries (primary/hypertensive intracerebral hemorrhage), or rupture of AVMs in the brain. The presence of parenchymal blood appears to be an important element in the pathogenesis of seizures (Mori et al., 1991; Ueda et al., 1998). Post-traumatic seizures are either focal in type or generalized with loss of consciousness (grand mal). Petit mal is, rarely if ever, due to trauma. The risk for seizures is directly related to the severity of the injury. For instance, it has been observed that seizures occur within one year in about 7% of civilian and 34% of military head injuries. This difference has been correlated with the much higher proportion of penetrating wounds in military cases which, in turn, may be related to the amount of bleeding and Fe released into the brain tissues (Evans, 1962). In the Vietnam Head Injury Study, increased risk for epilepsy was associated with retained metal fragments, intracranial hematoma, persistent neurologic deficits, and degree of brain volume loss (Salazar et al., 1985). The extravasated blood clots within hours and the hematoma is surrounded by petechial hemorrhages from torn arterioles and venules. The red blood cells are phagocytosed by macrophages within 24 hours and hemosiderin is first observed around the margins of the clot in five to six days. In two to three months, the clot is slowly absorbed, leaving a smooth-walled cavity or a yellow brown scar. The Fe pigment in macrophages becomes dispersed and stud adjacent astrocytes and neurons, and may persist well beyond the border of the hemorrhage for years (Adams et al., 1997). Following traumatic brain injury, accumulation of HO-1 positive microglia/macrophages was detected as early as six hours post-trauma, and was still pronounced at six months. HO-1 was also weakly expressed in astrocytes in the perifocal penumbra (Beschorner et al., 2000). The Fe released by the HO could have an epileptogenic effect. Electrocorticograms of the brain in regions adjacent to the old traumatic foci reveal a number of electrically

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active zones adjacent to scars. The microscopic pathology of these foci shows neuronal loss and an increase in astrocytes (Adams et al., 1997). Primary (hypertensive) intracerebral hemorrhage is due predominantly to chronic hypertension and degenerative changes in cerebral arteries. Hemorrhages are described as massive, moderate, small, slit, and petechial. Slit refers to an old collapsed hypertensive or traumatic hemorrhage that lies just beneath the cortex. The bleeding can occur in various places in the brain. The most common sites are the putamen and adjacent internal capsule, central white matter of the temporal parietal or frontal lobes, thalamus, cerebellar hemisphere, and pons. Seizures, usually focal, occur in the first few days in some 10% of supratentoral hemorrhage. Seizures have been reported especially in association with subcortical slit hemorrhages (Adams et al., 1997). A recent large-scale survey of seizures after stroke has shown that seizures occurred in 8.9% of patients with stroke, and the only risk factor for seizures after hemorrhagic stroke was cortical location (Bladin et al., 2000). A greater incidence of seizures in hemorrhagic stroke (4.3%) compared to ischemic stroke (2%), as well as an increased incidence of seizures in strokes with cortical involvement, was also found in a separate study (Arbiox et al., 1997). Thus, hemorrhage in or near the cerebral cortex appears to be most epileptogenic among the various sites of intracerebral hemorrhages. As in hemorrhagic stroke, cortical location was found to be a risk factor for epilepsy after ischemic stroke (Bladin et al., 2000). An AVM consists of a tangle of dilated vessels that form an abnormal communication between the arterial and venous systems. It is a developmental abnormality, representing persistence of an embryonic pattern of blood vessels. The tangled blood vessels interposed between arteries and veins are abnormally thin and do not have the structure of normal arteries or veins. Most AVMs are clinically silent for a long time, but sooner or later they bleed. When hemorrhage occurs, blood may enter the subarachnoid space, but since the AVM lies within cerebral tissue, bleeding is likely to be partly intracerebral. Seizures are commonly associated with AVMs and, in 30% of cases, it is the first and only manifestation (Adams et al, 1997). An increase in brain Fe accumulation can also result from infectious agents. These include cerebral malaria and human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) encephalopathy.

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Both diseases are associated with increased incidence of epilepsy. In cerebral malaria, capillaries and venules are packed with parasitized erythrocytes and the brain is dotted with many foci of necrosis surrounded by glia (Durck nodes). Cerebral malaria is fatal in 20% to 50% of affected patients. It is characterized by headache, seizures, and coma. A survey of Nigerian children with fevers and malaria parasitemia showed that approximately two-thirds had seizures. Of these, 69% had generalized seizures, 21% had partial and generalized seizures, and 10% had partial seizures (Akpede et al., 1993). An induction of HO-1 has also been observed in the brain in lesions of human cerebral malaria. A close association was observed between HO-1 expression with areas of bleeding, suggesting that released hemoglobin and heme, known inducers of HO-1, are mainly responsible for induction of monocytic HO-1 expression (Schluesener et al., 2001). It is possible that the Fe released by action of HO-1 on parasitized erythrocytes may at least be partly responsible for the seizures in cerebral malaria. The progression of HIV infection towards its more advanced stages is accompanied by increasing body Fe stores. Fe accumulates in macrophages, microglia, endothelial cells, and monocytes. The Fe burden is especially heavy in bone marrow, brain white matter, muscle, and liver (reviewed in Boelaert et al., 1996). The caudate nucleus and globus pallidus appeared to be particularly affected, based on magnetic resonance imaging studies (Miszkiel et al., 1997). At autopsy, the number of hemosiderin-laden macrophages was found to be significantly increased in the brains of patients with AIDS compared with age-matched control subjects (Gelman et al., 1992). Seizures have been reported in 4% to 11% of HIVinfected patients (Wong et al., 1990; Van Paesschen et al., 1995). The seizures may occur at any stage of the disease, and may even be the presenting symptom of HIV infection (Holtzman et al., 1989). An increased Fe uptake across the blood-brain barrier might also occur in patients undergoing neuroleptic drug treatment, resulting in epilepsy. Antipsychotic drugs, such as haloperidol and chlorpromazine, can alter the blood-brain barrier and enhance the normally restricted Fe transport into the brain in rats (Ben-Shachar et al., 1993). A common side effect of these drugs is epilepsy, and the increased brain Fe content has been suggested to relate to the toxic effects of these drugs (Ben-Shachar et al., 1993). Other neuroleptics, including chlorpromazine, thioridazine,

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and fluphenazine, showed a stimulatory effect on Fe uptake from Fe donors in synaptosomal preparations of rat cerebral cortex (Penatti et al., 1998). It is possible that the increased Fe might interfere with ionic homeostasis in nerve terminals, resulting in epilepsy. An increased amount of Fe/transferrin saturation in the blood does not appear to be the cause of epilepsy in the majority of patients. In a study of 130 patients with epilepsy, abnormally high transferrin saturations were observed in only 10 patients (Ikeda, 2001). In contrast, in select kindred relatives affected with familial idiopathic epilepsy, most individuals suffering seizures also have low levels of the plasma hemoglobin-binding protein, haptoglobin (Panter et al., 1984). Binding of hemoglobin to haptoglobin decreases the effectiveness of hemoglobin in stimulating lipid peroxidation. The hemoglobin-haptoglobin complexes are cleared from the circulation by the liver, and at least part of the complex is excreted intact into the bile (Halliwell and Gutteridge, 1999). It has been suggested that hypohaptoglobinemia, either inherited or acquired through traumatic processes, may prevent efficient clearing of interstitial hemoglobin from the central nervous system (Panter et al., 1985). There are several inborn errors of metabolism that could result in abnormal brain accumulation of Fe. These include Hallervorden-Spatz syndrome, aceruloplasminemia characterized by Fe overload in basal ganglia (Gitlin, 1998), and Friedreich's ataxia characterized by neuronal and myocardial mitochondrial Fe accumulation (Puccio and Koenig, 2000). In Hallervorden-Spatz syndrome, imaging and pathologic studies demonstrated that excess Fe accumulates in the globus pallidus and substantia nigra, the exact nuclei in which neurons are lost (Swaiman, 1991). Although the symptoms of Hallervorden-Spatz syndrome, such as dystonia, rigidity, and choreoathetosis, are mostly associated with damage to the basal ganglia, some patients have hard-to-control seizures (Elejalde et al., 1979; reviewed in Chiueh, 2001). Whether Fe might be a factor in childhood epilepsies is not clear. The immature nervous system is especially enriched in Fe, particularly around the time of myelination (Connor et al., 1995; Cao et al., 2001). The increased Fe is associated with increased amounts of the lipid peroxidation product, 4hydroxynonenal, in the supraventricular corpus callosum and overlying deep layers of the rat motor cortex during this time (Cao et al., 2001). It has been suggested that free Fe and the presence of lipid peroxidation products could

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set up the immature nervous system for increased cytotoxicity (Ferriero, 2001). This idea is supported by observations that children with febrile illness and seizures were less frequently Fe-deficient than those with febrile illness without seizures (Kobrinsky et al., 1995). It is possible that Fe could play a role in increased incidence of epilepsy among the elderly. There is an increase in Fe in the human brain with age (Connor et al., 1990; Samson and Nelson, 2000). The increase appeared particularly in the basal ganglia (Drayer, 1988; Pujol et al., 1992; Floyd and Carney, 1993; Schenker et al, 1993), but was also observed in the cerebral cortex (Imon et al., 1995; Hirai et al., 1996; Korogi et al., 1997). Fe is detected as low intensity areas by T2 weighted magnetic resonance imaging studies. In the cortex, low intensity areas were most frequently observed in the aged motor cortex, followed by the visual and sensory cortices (Imon et al., 1995; Hirai et al., 1996; Korogi et al, 1997). The amount of Fe in the cortex has been reported to be higher than that in the basal ganglia, based on results of studies on Tl relaxation rate (Ogg and Steen, 1998). The increase of Fe in the aging brain coincides with an increased incidence of epilepsy in the elderly. Seizures are the third most frequently encountered neurological problem in the elderly population, and the incidence of recurrent unprovoked seizures peaks in older patients (reviewed in van Cott, 2002). It is possible that Fe accumulation, particularly in the motor cortex, could be one of the causes of increased incidence of epilepsy among the elderly.

3. IRON AS A CONSEQUENCE OF EPILEPSY Unlike studies on increase of Fe as a cause of epilepsy, fewer studies have been carried out on the increase of Fe as a consequence of epilepsy. These have mostly been carried out on laboratory animals, and little published information is available on Fe as a consequence of human epilepsy. In animals, focal epilepsy is induced by the topical application of aluminum hydroxide or systemic or intracerebroventricular application of tetanus toxin, kainate, and pilocarpine. Most of these models share the characteristic of producing acute seizures followed, often after a latent period, by the development of chronic epilepsy with seizures. These behaviors are similar to human complex partial seizures. While the early seizures may arise from different mechanisms, the chronic models

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seem to depend largely on resultant neuropathological change. Another method of inducing epilepsy in animals is by kindling. In this model, repeated subconvulsive electrical stimulation to the amygdala leads to increasing after-discharge and, ultimately, behavioral seizures (Goddard et al., 1969). A study to elucidate the distribution of ferric and ferrous Fe in the dorsal hippocampus after epilepsy induced by intracerebroventricular injection of kainate in rats showed that very light staining for Fe was observed in the hippocampus in normal, saline injected, or 1-day postkainate injected rats. At one week post-injection, a number of ferric Fepositive cells, but very few ferrous Fe-positive cells, were present in the degenerating CA fields. At one-month post-injection, large numbers of ferric Fe-positive glial cells (Figs. 2A and 2B) and some ferrous Fepositive blood vessels were observed. At two months post-injection, large numbers of ferric and ferrous Fe-positive glial cells were present. The labeled cells had light and electron microscopic features of oligodendrocytes and were double-labeled with CNPase, a marker for oligodendrocytes. In addition, the study showed a shift in the oxidation state of the accumulated Fe, with more cells becoming positive for ferrous Fe at a late stage (Wang et al, 2002a). The observation of an increasing number of ferric and ferrous Fepositive cells in the degenerating hippocampus with time is consistent with the results of a nuclear microscopic study. This technique utilizes a variety of high energy (MeV) ion beam techniques at submicron spatial resolutions to provide structural and quantitative elemental analysis of biological tissue, down to the parts per million level of analytical sensitivity (Watt, 1995). An increased amount of Fe was detected in the degenerating hippocampus after intracerebroventricular kainate injection (Ong et al., 1999). The increase occurred continuously throughout the four-week post-lesion interval (the longest post-lesion time interval in which samples were scanned in the experiment). The majority of the increased Fe was distributed at the edge of the glial scar (Ong et al., 1999). A likely consequence of the high amounts of Fe in the hippocampus after kainate injection is that it could promote free radical damage in the adjacent neurons. Any Fe present is also likely to be more harmful to neurons than glial cells. This is due to the increased antioxidant defenses in glial cells, such as elevated levels of the antioxidant glutathione, observed

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Fig. 2. Light micrographs of normal and kainate-injected rats. The normal hippocampus is shown in A,C, and E. B and D show the hippocampus from rats that had been injected with kainate at one month, and F at 3 weeks, previously. (A and B) Perls' stained sections show light staining in the normal hippocampus (asterisk in A), but an increase in staining in oligodendrocytes (arrows) in the lesioned CA field (asterisk in B). (C and D) DMT1 stained sections show light staining of astrocytic end feet in the normal hippocampus (arrow in C), but dense staining of reactive astrocytes in the lesioned CA field (arrows in D). (E and F) HO-1 stained sections show only occasional labeled nonpyramidal neurons in the stratum oriens in the normal CA fields (arrow in E), but dense staining of reactive astrocytes in the lesioned CA field (arrows in F). Scale= 100 Jim.

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in astrocytes after kainate treatment (Ong et al., 2000). A chemiluminescence analysis study has also shown that despite greater accumulation of Fe and aluminum in glial cells than neurons, reactive oxygen species were twice as high in neurons than glial cells, indicating that the antioxidant defenses are greater in glial cells than neurons (Oshiro et al., 2000). A casual role for Fe in hippocampal damage after kainate treatment was shown by the observation that Fe deprivation resulted in decreased damage, whereas Fe supplementation resulted in increased damage and microgliosis in rats (Shoham and Youdim, 2000). It is therefore possible that although Fe may not be the initiator of epilepsy in the kainate model, it is accumulated consequent to neuronal injury and might contribute to neuronal injury and epilepsy. Besides kainate-induced injury, Fe deposition has also been observed after transient forebrain ischemia in the rat brain (Danielisova, 2002). An increase in Fe in the brain after epilepsy could be the result of increased transport of Fe across the blood-brain barrier. A recent study on DMT1 expression in the hippocampus of rats after kainate-induced epilepsy has shown a significant increase in the density ratios of DMT1/ 3-actin bands observed in Western blots in the one week, one month, and two months post-kainate injected hippocampus compared to uninjected and one-day post-kainate injected hippocampus (Wang et al., 2002b). The increase in DMT1 protein was paralleled by an increase in DMT1 immunoreactivity in astrocytes. Light staining for DMT1 was observed in the uninjected, saline-injected, and one-day post-kainate injected rat hippocampus. In contrast, an upregulation of DMT1 was observed in reactive glial cells at one week, one month, and two months post-kainate injection (Figs. 2C and 2D). Electron microscopy confirmed that the glial cells had morphological features of astrocytes. DMT1 is a cellular Fe transporter responsible for transport of metal ions from the plasma membrane to endosomes, and the observation that DMT1 is present on astrocytic end feet in contact with blood vessels suggests that astrocytes may be involved in uptake of Fe from endothelial cells (Wang et al., 2002b). In addition to increased Fe transport, it is also possible that focal deposits of Fe could result from a breakdown of Fe containing heme proteins, followed by Fe efflux and redistribution of the Fe. These activities could occur through HO-1. Transfection of HO-1 into mammalian cells stimulates Fe efflux, whereas Fe efflux is greatly diminished in fibroblasts from HO-1

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knockout mice (Ferris et al., 1999). An essential role for HO-1 in Fe efflux is supported by evidence of low serum Fe and accumulation of Fe in the tissues of HO-1 knockout mice (Poss and Tonegawa, 1997a, 1997b). The distribution of HO-1 mRNA and protein has been studied in the hippocampus of rats after intracerebroventricular injections of kainate (Matsuoka et al., 1998; Nakaso et al., 1999; Lu and Ong, 2001). At post-injection weeks one to three, HO-1 was observed in glial cells in the center of the lesion (Figs. 2E and 2F) and in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, while the neurons contained discontinuous cell membranes and nuclear outlines and had features of degenerating neurons. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than fifteen fold) were associated with reactive Fe and toxicity (Suttner and Dennery, 1999). It is therefore possible that the high levels of expression of the enzyme could lead to excessive Fe accumulation and toxicity to neurons. Little is known about the source of heme proteins that form the substrate of HO. One possibility is that heme in prosthetic moiety of hemoproteins, such as hemoglobin, catalase, soluble guanalylate cyclase, cytochrome b5, cytochromes P450, and nitric oxide synthase, are released or made available in degenerating neurons and form the substrate of HO. Although it remains to be demonstrated whether the increased expression of HO-1 protein expressed after epilepsy is accompanied by increased activity, it has been shown in Alzheimer's disease patients that levels of cerebrospinal fluid bilirubin derivatives are increased significantly compared with those of controls. This increase is not due to the increased permeability of the blood-brain barrier, and is presumably due to increased HO activity (Kimpara et al., 2000). Heme itself crosses the blood-brain barrier poorly and negligible amount of radioactivity was recovered from the brain, following intravenous or intramuscular injections of 59Fe labeled heme arginate (Linden et al., 1987).

4. CONCLUSION AND DIRECTIONS FOR FUTURE RESEARCH Injections of Fe salts into the sensorimotor cortex, hippocampus, and amygdala of experimental animals have been shown to result in chronic

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recurrent focal paroxysmal electroencephalographic discharges and behavioral convulsions and electrical seizures. The induction of epilepsy is related to the generation of free radicals, lipid peroxidation of neuronal membranes, increased intracellular calcium concentrations through reverse action of sodium calcium exchanger/reduced activity of plasma membrane or endoplasmic reticulum calcium ATPases, increased release of excitatory neurotransmitters including aspartate and glutamate, and increased influx of ions through glutamate receptors. Some of the above effects of Fe can be abrogated by inhibitors of PLA2, but not arachidonic acid, indicating that the damaging effects of Fe may be due to perturbation of the lipid environment essential to normal functioning of membrane proteins. Fe in hemoglobin, or by itself, is also likely to be the cause of human epilepsy in instances where there is increased Fe load in the brain. These include subarachnoid hemorrhage, intraparenchymal hemorrhages due to head injury and stroke, malaria, HIV encephalitis, and possibly neuroleptic drug use. A reduced level of haptoglobin, a hemoglobin-binding protein, has also been observed in select kindred affected with familial idiopathic epilepsy. An accumulation of Fe has been observed in the motor cortex with age. It is possible that this might contribute to the increased incidence of epilepsy among the elderly. Fe accumulates with time in the rat hippocampus after kainate-induced epilepsy. The accumulation occurs in oligodendrocytes and is likely to be a reflection of the high levels of Fe in the extracellular space. The accumulation of Fe is correlated with an increased expression of DMT1 in astrocytes in the glial scar, and an increased expression of HO-1 in reactive astrocytes and microglia, as well as degenerating neurons at the edge of the scar. The increased DMT1 and HO-1 expression in astrocytes could lead to increased uptake of Fe followed by efflux of Fe and redistribution to the extracellular space. In this model, Fe is the consequence of epilepsy, although it is likely that it can also be a cause of epilepsy. Further work is necessary to elucidate the effects of lipid peroxidation of the cellular membranes on the function of membrane proteins and the role of phospholipases, including PLA2 in perturbing the lipid environment. The possible presence of Fe in the human brain after epilepsy also needs to be elucidated. The causes of dysregulation of Fe in the glial scar after neuronal injury need to be studied, and the possible beneficial effects of Fe chelators or antioxidants that cross the blood-brain barrier or neuroprotective gene induction on epilepsy need to be evaluated.

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Puccio H, Koenig M. Recent advances in the molecular pathogenesis of Friedreich ataxia. Hum Mol Genet 2000; 9:887-892. Pujol J, Junque C, Vendrell P, Grau JM, Marti-Vilalta JL, Olive C, Gili J. Biological significance of iron-related magnetic resonance imaging changes in the brain. Arch Neurol 1992;49:711-717. Rafalowska U, Liu GJ, Floyd RA. Peroxidation induced changes in synaptosomal transport of dopamine and gamma-aminobutyric acid. Free Rad Biol Med 1989; 6:485^-92. Rauhala P, Khaldi A, Mohanankumar KP, Chiueh CC. Apparent role of hydroxyl radicals in oxidative brain injury induced by sodium nitroprusside. Free Rad Biol Med 1998; 24:1065-1073. Rauhala P, Mohanakumar KP, Sziraki I, Lin AM, Chiueh CC. S-nitrosothiols and nitric oxide, but not sodium nitroprusside, protect nigrostriatal dopamine neurons against iron-induced oxidative stress in vivo. Synapse 1996; 23:58-60. Rego AC, Santos MS, Oliveira CR. Oxidative stress, hypoxia, and ischemia-like conditions increase the release of endogenous amino acids by distinct mechanisms in cultured retinal cells. J Neurochem 1996; 66:2506-2516. Rice-Evans C, Green E, Paganga G, Cooper C, Wrigglesworth J. Oxidized LDL induce iron release from activated myoglobin. FEBS Lett 1993; 326:177-182. Rouault TA, Haile DJ, Downey WE, Philpott CC, Tang C, Samaniego F, Chin J, Paul I, Orloff D, Harford JN, Klausner RD. An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein. Biometals 1992; 5:131-140. Sah R, Galeffi F, Ahrens R, Jordan G, Schwartz-Bloom RD. Modulation of the GABA(A)-gated chloride channel by reactive oxygen species. J Neurochem 2002; 80:383-391. Salazar AM, Jabbari B, Vance SC, Grafman J, Amin D, Dillon JD. Epilepsy after penetrating head injury. I. Clinical correlates: A report of Vietnam Head Injury Study. Neurology 1985; 35:1406-1414. Samson FE, Nelson SR. The aging brain, metals and oxygen free radicals. Cell Mol Biol 2000; 46:699-707. Schenker C, Meier D, Wichmann W, Boesiger P, Valavanis A. Age distribution and iron dependency of the T2 relaxation time in the globus palldius and putamen. Neuroradiology 1993; 35:119-124. Schluesener HJ, Kremsner PG, Meyermann R. Heme oxygenase-1 in lesions of human cerebral malaria. Acta Neuropathol 2001; 10:65-68. Schwartz RD, Skolnick P, Paul SM. Regulation of gamma aminobutyric acid/barbiturate receptor-gated chloride ion flux in brain vesicles by phospholipase A2: Possible role of oxygen radicals. J Neurochem 1988; 50:565-571. Sevanian A, Rashba-Step J. Phospholipase A2 activation: An early manifestation of oxidative stress. In: Forman HJ, Cadenas E, editors. Oxidative Stress and Signal Transduction. New York: Chapman and Hall, 1997: 77-107. Shirotani K, Katsura M, Higo A, Takesue M, Mohri Y, Shuto K, Tarumi C, Ohkuma S. Suppression of Ca 2+ influx through L-type voltage-dependent calcium channels by hydroxyl radical in mouse cerebral cortical neurons. Brain Res Mol Brain Res 2002; 92:12-18.

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Shoham S,Youdim MB. Iron involvement in neural damage and microgliosis in models of neurodegenerative diseases. Cell Mol Biol 2000; 46:743-760. Shuto M, Ogita K, Minami T, Maeda H, Yoneda Y. Inhibition of [3H]MK-801 binding by ferrous (II) but not ferric (III) ions in a manner different from that by sodium nitroprusside (II) in rat brain synaptic membranes. J Neurochem 1997; 69:744-752. Simon RP, Aminoff MJ, Greenberg DA. Clinical Neurology, 4th ed. Comecticut: Appleton and Lange, 1999. Singh R, Pathak DN. Lipid peroxidation and glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities in FeCl3-induced epileptogenic foci in the rat brain. Epilepsia 1990; 31: 15-26. Suttner DM, Dennery PA. Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. FASEB J 1999; 13:1800-1809. Suzer T, Coskun E, Demir S, Tahta K. Lipid peroxidation and glutathione levels after cortical injection of ferric chloride in rats: Effect of trimetazidine and desferoxamine. Res Exp Med 2000; 199:223-229. Swaiman KF. Hallervorden-Spatz syndrome and brain iron metabolism. Arch Neurol 1991;48:1285-1293. Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA 1968; 61:748-755. Triggs WJ, Willmore LJ. In vivo lipid peroxidation in rat brain following intracortical Fe 2+ injection. / Neurochem 1984; 42:976-980. Ueda Y, Willmore LJ. Sequential changes in glutamate transporter protein levels during Fe3+-induced epileptogenesis. Epilepsy Res 2000; 39:201-209. Ueda Y, Willmore LJ, Triggs WJ. Amygdalar injection of FeCl3 causes spontaneous recurrent seizures. Exp Neurol 1998; 153:123-127. Van Cott AC. Epilepsy and EEG in the elderly. Epilepsia 2002; 3:94-102. Van Paesschen W, Bodian C, Maker H. Metabolic abnormalities and new-onset seizures in HIV-seropositive patients. Epilepsia 1995; 36:146-150. Wang XS, Ong WY, Connor JR. A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus. J Neurocytol 2001;30:353-360. Wang XS, Ong WY, Connor JR. Increase in ferric and ferrous iron in the rat hippocampus with time after kainate-induced excitotoxic injury. Exp Brain Res 2002a; 143:137-148. Wang XS, Ong WY, Connor JR. A light and electron microscopic study of divalent metal transporter-1 distribution in the rat hippocampus after kainate-induced neuronal injury. Exp Neurol 2002b; in press. Watt F. Nuclear microscopy in the life sciences. Nucl Instr Meth B 1995; 104:276-284. Willmore LJ. Post-traumatic epilepsy: Cellular mechanisms and implications for treatment. Epilepsia 1990; 31:S67-S73. Willmore LJ, Hiramatsu M, Kochi H, Mori A. Formation of superoxide radicals after FeCl3 injection into rat isocortex. Brain Res 1983; 277:393-396. Willmore LJ, Rubin JJ. Antiperoxidant pretreatment and iron-induced epileptiform discharges in the rat: EEG and histopathologic studies. Neurology 1981; 31:63-69.

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Role of Iron Metabolism in Multiple Sclerosis Maritha J Kotze, J Nico P De Villiers, Monique G Zaahl, Kathryn J H Robson

ABSTRACT Multiple sclerosis is a chronic inflammatory disease of the central nervous system caused by a complex interaction between genetic and environmental factors. Support for a role of iron metabolism in multiple sclerosis was obtained by the analysis of the gene encoding the solute carrier family 11 (proton-coupled divalent metal ion transporters) member 1 (SLC11A1), formerly known as NRAMP1, in the genetically homogenous Afrikaner population of South Africa. Under-representation of allele 2 of the functional Z-DNA forming promoter polymorphism in patients compared with population-matched controls largely excluded the hypothesis that multiple sclerosis is primarily caused by a virus infection, since this allele has previously been linked to various infectious diseases and appears to protect against autoimmune disease. Differentially increased expression of alleles 3 and 5 upon stimulation with ferric ammonium citrate (p < 0.05) provided a direct link between the regulation of iron and susceptibility to autoimmune disease, especially since co-existence of these alleles is associated with multiple sclerosis in the South African population. Keywords: Iron metabolism; multiple sclerosis; SLCllAlgene; transcriptional activity.

1. INTRODUCTION Elucidation of the etiology of neurological diseases where iron (Fe) has been implicated would require a better understanding of the role of genes involved in Fe metabolism and their involvement in oxidative damage and heme biosynthesis. Although a melange of immunologic abnormalities have been documented in patients with Fe deficiency and in Fe overload 399

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states, the clinical importance of such abnormalities remains largely undefined (Bryan, 1991). Fe is essential to many metabolic processes, including DNA, RNA, and protein synthesis, as a co-factor of many heme and nonhaem enzymes, the formation and maintenance of myelin, and the development of the neuronal dendritric tree (Gerlach et al., 1994; Connor and Menzies, 1996). Since hemproteins are involved in electron transport, their optimal functioning is critical to axonal and myelin integrity. The complexity of Fe metabolism pathways is much greater than is presently appreciated and, although the transport of Fe into mitochondria represents an important step in Fe metabolism, this is often overlooked. Mitochondria utilize most of the cellular Fe to produce heme- and Fe-sulphur cluster-containing proteins, such as the cytochromes. An imbalance in the compartmentalization of Fe, rather than a decrease or increase in the absolute amount of body Fe, may result in neurological deficit. This is evident in patients with aceruloplasminemia where, unlike other Fe overload syndromes, neurological manifestations appear to dominate (Harris et al., 1998). Reduction in Fe binding to transferrin, as a consequence of defective oxidation of ferrous Fe to ferric Fe, results in impaired transport of Fe from intracellular stores to plasma. This leads to decreased serum Fe and microcytic anemia. Affected patients eventually die from effects of Fe accumulation in the basal ganglia, while the initial problem lies with the supply of Fe for key synthesis processes required for cell growth. These may include optimal expression of the ceruloplasmin gene implicated in neuronal survival in the retina and basal ganglia (Gitlin, 1998) and, although not investigated, interference of Fe delivery for heme biosynthesis involved in energy production and neurological integrity (Meyer et al., 1998). Although Fe deficiency-induced anemia is the most common Fe metabolism-related disease globally, much is still to be learned about the potential consequences of low availability of Fe for essential cellular processes, particularly in genetically predisposed individuals. Fe deficiency can be caused by inadequate dietary Fe absorption, loss or dysfunction of absorbing enterocytes, increased blood losses associated with malignant and inflammatory diseases, and abnormally high gastric pH. Fe withholding of the host upon infection with pathogens requiring Fe for proliferation might also result in hypoferremia and/or anemia of inflammation. It is generally assumed that these conditions are a consequence of

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the blockage of the macrophage release of senescent red blood cellsderived Fe (Roesser, 1980; Lee, 1983). Since Fe modulates cytokine activities, nitric oxide production, and immune cell proliferation (Weiss, 2002), this metal has to be immediately available for mounting a successful immunological defense and, at the same time, be effectively unavailable to the invading pathogen. The continuous battle for Fe between the host and invading pathogens may result in cytokine-mediated responses, which are involved in various infectious and autoimmune diseases. In this overview, the role of the solute carrier family 11 (protoncoupled divalent metal ion transporters) member 1 (SLC11A1), formerly known as the natural resistance-associated macrophage protein 1 (NRAMP1), will be discussed in relation to Fe homeostasis and disease susceptibility. This gene has been linked to various autoimmune and infectious diseases, which led to speculation that regulation of Fe by SLC11A1 may be of major importance in this context (Blackwell et al., 2000). Support for a role of Fe homeostasis in the pathogenesis of multiple sclerosis (MS), known to involve an autoimmune process, was obtained from recent detection of an association with the SLC11A1 gene (Kotze et al., 2001).

2. IRON AND MULTIPLE SCLEROSIS Defining the cause of MS represents a major challenge because little is certain about the pathogenesis of this disease. The varied clinical picture raises the possibility that MS is not a well-defined etiology, but consists of genetically different subtypes in addition to subtypes with no genetic contribution (phenocopies) (Rasmussen and Clausen, 2000). Different genes and environmental factors may be involved in the induction and progression of MS, with external factors affecting the population risk and genetic susceptibility accounting for familial risk. The proposed role of Fe in the etiology of MS could explain this complexity. Fe represents the most abundant metal in the human body and maintenance of Fe homeostasis involves a complex interplay between Fe receptor cells (erythrocyte precursors, duplicating and growing cells, and hepatocytes) and Fe donor cells (macrophages, intestinal mucosal cells, and hepatocytes). The regulatory process occurs within the context of different Fe transport proteins that are responsible for influx or efflux of Fe, and reductases with extracellular or intracellular activity.

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The high demand of Fe in the brain and the central nervous system, as well as their sensitivity to Fe-induced peroxidative damage, suggests the need for stringent regulation of Fe availability in these organs. Hulet et al. (1999) have demonstrated that the normal pattern of transferrin and ferritin binding is disrupted in the brain tissue of MS patients. This finding provided evidence of ferritin binding in human brain and suggests that loss of ferritin binding is involved in, or is a consequence of, demyelination associated with MS. Differences in Fe parameters measured in the cerebrospinal fluid (CSF) of MS patients have been reported by several groups (LeVine et al., 1999; Weller et al., 1999; Zeman et al., 2000). Zeman et al. (2000) have highlighted the potential role of transferrin determination in CSF as a means to distinguish between relapsing-remitting, secondary progressive, and primary progressive MS subtypes. They noted that transferrin is also a growth factor of importance in the proliferation of activated T lymphocytes. It has been hypothesized that cytokines from T cells play a functional role in regulating the expression of transferrin and in modulating Fe status through intestinal Fe absorption and transport (Lieu et al., 2001). The functional linkage between immune function and Fe absorption became evident when the major histocompatibility complex (MHC)encoded hemochromatosis (HFE) protein was identified. This finding provided evidence that MHC-encoded class I molecules might play a role in Fe metabolism (Salter-Cid et al., 2000). Although increased mean serum ferritin levels were reported by Valberg et al. (1989) in MS patients, no subjects with clinically manifested hereditary hemochromatosis (HH) were recognized by these authors among 1,700 patients with MS. Since MS and HH affect the same ethnic group, these findings may indicate interaction of the HFE gene with Fe-related genetic and/or environmental factors involved in the MS phenotype. Identification of a South African family where two sisters with MS were found to be homozygous for the common HH mutation C282Y has excluded the likelihood of a linkage disequilibrium effect with a putative MS-related gene on chromosome 6. Lack of clinical manifestation of HH in the MS patients, despite the presence of high transferrin saturation and ferritin levels (Kotze et al., 1999), raises the possibility that MS and clinically manifested HH may be mutually exclusive.

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2.1. Association Between Multiple Sclerosis and SLC11A1 Gene The first molecular study performed in MS patients to investigate the potential role of Fe metabolism, in association with infectious and/or autoimmune disease susceptibility linked to the SLCllAlgene, was performed in the genetically homogenous Afrikaner population of South Africa (Kotze et al., 2001). The unique genetic background of this population representing Dutch, German, French, and British origins provides the advantage to study the genetics of complex diseases on the basis of an expected limited number of disease-associated genes/mutations introduced from Europe. The SLC11A1 gene has been linked to various infectious and autoimmune diseases (Hofmeister et al., 1997; Marquet et al., 1999; Graham et al., 2000), which may relate largely to the functional activity of the Z-DNA forming GT-repeat polymorphism in the promoter region of the gene (Searle and Blackwell, 1998). The different alleles of the GT-repeat polymorphism detected in the South African Afrikaner population is shown in Table 1 (modified from Kotze et al., 2001), together with the allelic distribution observed in patients and controls. The allelic distribution differed significantly between the MS patients and population-matched controls, with underrepresentation of allele 2 previously implicated in susceptibility to infectious diseases. Analysis of a second age- and population-matched control group of 336 South African Caucasians in comparison with the MS patients confirmed this association (p < 0.03) (data not shown).

Table 1. Comparison of allelic distribution of the SLC11A1 GT-repeat polymorphism between South African MS patients and controls.

Alleles 2 [t(gt)5ac(gt)5ac(gt)10g] 3 [t(gt)5ac(gt)5ac(gt)9g] 5 [t(gt)4ac(gt)5ac(gt)10ggcaga(g)]

Multiple sclerosis (n=208)*

Controls (n= 1,076)*

41 (20%) 160 (77%) 7 (3%)

320 (30%) 752 (70%) 4 (0.4%)t

*From Kotze et al., 2001. tMS versus control group: p < 0.01, 2 df, %2 = 35.2.

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Samples were obtained from the parents and siblings of one of the MS patients with SLC11A1 genotype 3/5, whose aunt on the paternal side has also been diagnosed with definite MS. The pedigree is illustrated in Fig. 1. The finding that allele 5 was inherited from the unaffected mother of the index case, while her aunt with MS is from the paternal side of the family, highlights the complexity of the disease mechanism, which appears to be dependant on the co-existence of appropriate environmental and genetic factors. Allele 5 was associated with different haplotypes as defined by intragenic SLC11A1 polymorphisms in the two MS patients, probably due to a crossover or recurrent mutational event (data not shown). The presence of genotype 3/5 in several family members without MS in all three generations is in accordance with a polygenic genetic basis for this disease. Recently, Vitale et al. (2002) have demonstrated that even in a MS family with an autosomal dominant inheritance pattern, co-inheritance of two loci are required for the development of the disease. It seems likely that different combinations of gene-environment interactions may cause MS in diverse population groups. SLC11A1, which regulates Fe and is also regulated by Fe (Atkinson and Barton, 1998), may serve as a model for various diseases where interaction of the gene with Fe as a modifying factor may have a relatively small effect on familial risk, but a large effect on population risk. Similar effects can be expected for other genes involved in Fe metabolism, such as the hemochromatosis gene which has been identified as a modifier

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Fig. 1. Pedigree of the index patient (arrow) with SLC11A1 genotype 3/5 who was diagnosed with relapsing-remitting MS. Allele 5 was inherited from her unaffected mother. The same genotype was detected in her father's sister who was diagnosed with secondary progressive MS. MS=multiple sclerosis.

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locus for various conditions. These may include chronic liver disease (George et al., 1999), porphyria (Roberts et al., 1997), inherited anemias (Yaouanq et al., 1997), cystic fibrosis (Rohlfs et al, 1998), diabetes (Kwan et al., 1998), cardiovascular disease (Tuomainen et al., 1999; Roest et al., 1999), and Alzheimer's disease (Moalem et al., 2000). Even in conditions where Fe may not have a primary action, its potential secondary role cannot be negated. Hemochromatosis is an important paradigm for medical genetics because it offers an opportunity to explore the complexity of gene-gene and gene-environment interactions, and the same may apply to SLCllAl-associated disease.

2.2. Differential Expression of SLC11A1 Alleles The association detected between MS and the SLC11A1 gene does not exclude the possibility that the multiple pleiotropic effects associated with macrophage activation (Blackwell, 1996), and not the regulation of Fe transport per se, account for disease susceptibility. The effects on macrophage function include enhanced chemokine KC, tumour necrosis factor a, interleukin 1(3, inducible nitric oxide synthase, and MHC class II expression; all have potential importance in the induction/maintenance of autoimmune diseases and are crucially to the resistance to intramacrophage pathogens. Searle and Blackwell (1998) have used reporter gene constructs to demonstrate that the Z-DNA forming dinucleotide repeat in the promoter region of the SLC11A1 gene has endogenous enhancer activity. Different alleles showed a similar degree of enhancement of reporter gene expression in the presence of interferon-7 (IFN7), while addition of bacterial lipopolysaccharide (LPS) caused significant reduction in expression driven by allele 2 and enhanced expression driven by allele 3. These results were in accordance with the hypothesis that chronic hyperactivation of macrophages associated with allele 3 is functionally linked to autoimmune disease susceptibility, while the poor level of SLC11A1 allele 2 expression contributes to infectious disease susceptibility. The likelihood that allele 3 would conversely protect against infectious disease, and allele 2 against autoimmune disease, was supported by data from Kotze et al. (2001) who compared the allele frequencies of the promoter variant between different age groups. SLC11A1 alleles considered to be detrimental in relation to autoimmune disease susceptibility

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appear to be maintained in the population as a consequence of improved survival to reproductive age following infectious disease challenge. Ebers (1998) has highlighted the possibility that, within the context of susceptibility alleles being advantageous in one context and deleterious in another, no single allele sufficient for the development of MS may be found. In an attempt to demonstrate a possible direct relation of SLC11A1 alleles with cellular Fe status, functional studies were performed to test for differential expression of allelic variants of the promoter polymorphism upon Fe loading. We hypothesized that the expression of alleles 3 and 5 shown to be associated with autoimmune disease susceptibility would respond to treatment with ferric ammonium citrate (FAC). Figure 2 demonstrates that this, indeed, is the case. Similar to the results of Searle and Blackwell (1998), the addition of IFN7 and LPS as exogenous stimuli, separately or in combination, caused

n No stimuli • IFN^ m IFN-y+LPS • LPS

m

Allele 2

Allele 3 SLC11A1 promoter variants

FAC

Allele 5

Fig. 2. SLC11A1 promoter variants transfected into U937 cells. The effect of ferric ammonium citrate (FAC) on levels of luciferase reporter gene activity driven by three polymorphic alleles for SLC11A1 detected in the study cohort, in relation to interferon-7 (IFN7) and bacterial lipopolysaccharide (LPS) enhanced expression, is illustrated. The mean normalized values obtained over three independent experiments, including triplicate transfections for each experiment, are graphed for SLC11A1 constructs. The following comparisons were statistically significant: allele 2 = no stimuli versus IFN7, p < 0.05; no stimuli versus IFN7 + LPS, p < 0.05; allele 3 = no stimuli versus FAC, p < 0.05; IFN7 versus FAC, p < 0.015; IFN-v + LPS versus FAC, p < 0.03; allele 5 = no stimuli versus IFN7, p < 0.05; no stimuli versus IFN7 + LPS, p < 0.05; allele 2 versus allele 3 = IFN7 + LPS, p < 0.03; FAC, p < 0.006; LPS, p < 0.04. Similar results were obtained for these variants transfected into THP-1 cells.

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an enhancement of luciferase reporter gene expression for all three constructs. The differences in baseline activity between alleles 2 and 3 were statistically significant with the addition of LPS (p < 0.04), LPS superimposed on the background of IFN7 (p < 0.03), and FAC (p < 0.006). Notably, the most significant effect between different alleles is observed with FAC. While this results in enhanced promoter activity for alleles 3 and 5, which is shown to be over-represented in MS patients compared with population-matched controls (Table 1), reduced expression in relation to LPS and 1FN7 is obtained with respect to allele 2. With the addition of FAC to the U937 compared with no stimuli, only alleles 3 Q?. *•• .

B

Fig. 3. Cerebral gray matter stained with anti-microtubule-associated protein (MAP) antibody. (A) Case control negative for anti-MAP. (B) Positivity for MAP antibody in the pyramidal neurons of an uremic subject. (Magnification 500X)

in the pyramidal neurons of an uremic subject (Fig. 3B). Intra- and extra-neuronal amyloid is a characteristic feature of several neurodegenerative diseases. Likewise, anti-Ap4 antibody recognized senile plaques in some of the uremic patients who underwent dialysis, but scored negative in the case control. Figures 4A and 4B show two distinct types of A|34-positive senile plaques detected in an uremic patient.

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Fig. 4. Two examples of positivity for amyloid-f34 antibody in the gray matter of an uremic subject: presence of diffuse/immature plaque-like structures. (Magnification 500X)

4. REMARKS Renal failure commonly affects the nervous system, and the effects of kidney failure on the nervous system are more pronounced when failure is acute. The neurologic manifestations of renal failure are variable, nonspecific, and most likely result from multiple metabolic derangements. Dialysis itself is associated with different and distinct disorders of the central nervous system, and patients on maintenance hemodialysis are at risk for several neurological implications. Nervous system dysfunction remains a major cause of disability in patients with ESRD. In this connection, cerebral atrophy was observed in the majority of subjects on long-term hemodialysis treatment but, notably no significant correlation was found between the degree of atrophy and relevant hematoseric parameters (Savazzi et aI., 1999). Uremic encephalopathy is associated with problems in cognition and memory, and may progress to delirium, convulsions, and eventually coma (Burns and Bates, 1998). Although clinical, epidemiologic, toxicologic, and electroencephalographic features of dialysis encephalopathy have been throughly described, the neuropathologic and clinicopathologic aspects of the disease have remained rather obscure. In spite of an abnormal accumulation of aluminum, some uremic subjects treated by dialysis are not affected by dialysis encephalopathy

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(Andriani et al., 1995). The relevance of this observation, in terms of the connection between aluminum and neurological manifestations, remains unclear. In our study, the most typical aspects found in uremic brains were an enhanced expression of MT-I-II and a reduced expression of synaptobrevin and phosphorylated neurofilaments. Reactive astrocytosis is a well-recognized general phenomenon where astrocytes can change in phenotype. Glial changes are invariably accompanied by structural synaptic remodeling which result in an increased number of neurotransmitter afferents (Theodosis and Poulain, 1999). One of the functions attributed to the activation of astrocytes is the restoration of damaged tissue. However, the role played by astrocytes in inflammatory reactions in several neurological diseases associated with uremia has not received much attention, and hence our interest in establishing the status of astrocytes' activation in the brains of uremic patients. MTs are low molecular weight zinc-binding protein thiols whose function remains unclear. In primates and humans, MTs are mainly found in the astrocytes (Hidalgo et al., 2001; Mocchegiani et al., 2001). In the mammalian central nervous system, MTs are found in pia-arachnoid, ependymal cells and astrocytes, and both furnish essential elements such as Cu(II) and Zn(II) and protect neurons against toxic ions (see Chapter 11). It has been hypothesized that MTs can act as antioxidants intercepting free radicals by forming complexes with redox metals. MTs can be secreted by glial cells in response to the presence of xenobiotics and dismetabolic metal ions (Nordio et al., 1998). MTs may, thus, serve to protect cells from the toxic effects of heavy metals and oxidative stress. MTs are highly expressed in uremic subjects undergoing dialysis, indicating activation of astrocytes as a result of alterated metal ions' homeostasis and oxidative stress. Synaptic vesicles contain neurotransmitter molecules that are predominantly proteic in character. One set of these proteins, synaptophysin and synaptobrevin, appear to be specific to the formation of a fusion complex of small vesicles (Savazzi et al., 1995). Synaptophysin and synaptobrevin are integral proteins that cross the membrane of vesicles. Both can bind Ca 2+ and may be phosphorylated at a tyrosine residue. Alterations in the expression of these proteins are an important marker for the functionality of neurons and axonal current. While synaptobrevin does not appears

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to be altered in our uremic subjects (data not reported), synaptophysin levels appear to be modestly reduced with respect to the controls. Uremic encephalopathy may initially worsen with dialysis as a result of impaired synaptic functions and metabolic alterations, such as relevant ionic changes (Burns and Bates, 1998), probably related to ionized Ca 2+ cellular content. The positive expression of A(34 in four uremic brains, although not uniform, suggests some interesting considerations. Treatment of experimental animals with aluminum salts produces a striking and long-lasting accumulation of amyloid-(3 protein precursor (Af3PP) and an activation of microglial cells (Shigematsu and McGeer, 1992). In this connection, it is known that uremic subjects under dialysis are at risk for aluminum accumulation in the brain (see Chapter 4). It is worth noting that in Alzheimer's disease (AD), perivascular microglial cells are involved in the production of A(34 fibrils and accumulation of amyloid in the capillary wall. In addition, microglial cells are loosely arranged in primitive plaques in AD (Wisniewski and Wegiel, 1995). In our study, we observed that microglial cells are all activated to some extent (Fig. 1). Classical and primitive plaques are complex deposits that include amyloid, microglial cells, dystrophic neurons, and activated astrocytes. According to some authors, microglial cells represent the site of amyloid formation (Wegiel and Wisniewski, 1992). It is worth noting that, in our uremic subjects, microglial cells are not only activated (Fig. 2), but also tested positive for A(34 (Fig. 4). These findings confirm previous observations reported by other authors (Brun and Dictor, 1981). In addition, a study of patients with chronic renal failure revealed nuclear immunostaining with an antibody to the N-terminal region of the A(3PP in 14 of 15 cases studied (Candy et al., 1992), with five of these patients having amorphous senile plaques containing A(34. These plaques were of the same so-called "immature" type observed in AD. On the other hand, neurofibrillary tangles, a characteristic feature of AD, are never, or very rarely, observed in dialysis patients. In this regard, Edwardson et al. (1991) concluded that Al(III) may induce some AD-like neuropathological changes in uremic subjects through mechanisms involving transferrin-mediated uptake of Al(III) by neurons or decreased turnover of the A(3PP.

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5. CONCLUSIONS Histological modifications that occur in the brains of dialysis patients who died for reasons apparently not related to dialytic treatment are unspecific (Burks et al., 1976). Our study shows morphological alterations sometimes coherent with clinical and neurological manifestations. It is notable that morphological changes observed in neurons and glial cells are rather different from those observed in AD. This is an important issue in that some authors hypothesized that the abnormal accumulation of Al(III), observed in the brains of uremic subjects in dialysis, could be a high risk factor for AD. In agreement with other authors (Reusche and Seydel, 1993), we did not observe any neurofibrillary tangles or "mature" senile plaques in the brain tissue of uremic patients, and detected a limited number of plaques of the "immature" and diffuse type in only a few cases. Thus, the Ap4 detected in some of our patients could indicate that amyloidosis is more likely a general feature than a specific property. As dialysis itself is associated with a variety of neurological syndromes, including disequilibrium syndrome, subdural hematoma, and Werniche's encephalopathy, the involvement of neurologists in a tight collaboration with nephrologists is highly advised in order to prevent the aggravation of neurological, clinical symptomatology. Patients under dialysis show histochemical alterations that suggest the need for careful attention in the clinical practice in a collaborative approach among different medical specialties.

ACKNOWLEDGMENT The authors thank Ms Cristina Renesto for her excellent technical assistance.

REFERENCES Alfrey AC, LeGendre GR, Kaehny WD. The dialysis encephalopathy syndrome. Possible aluminum intoxication. N Engl J Med 1976; 22:184-188. Andriani M, Nordio M, Zambenedetti P, et al. L'emodialisi induce malattia? In: Saporiti E, Marchini E, editors. L'alluminio: Dalla Storia Alia Ricerca, Milan: Wichting Publishing, 1995: 9-21. Beecker A, Drenckhahn A, Pahner I, et al. The synaptophysin-synaptobrevin complex: A hallmark of synaptic vesicle maturation. J Neurosci 1999; 19:1922-1931.

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Brun A, Dictor M. Senile plaques and tangles in dialysis dementia. Acta Pathol Microbiol Scand 1981; 89:193-198. Burks JS, Alfrey AC, Huddlestone J, Norenberg MD, Lewin E. A fatal encephalopathy in chronic hemodialysis patients. Lancet 1976; 1:764-768. Bums DJ, Bates D. Neurology and the kidney. J Neurol-Neurosurg Psychiatr 1998: 810-821. Candy JM, McArthur FK, Oakley AE et al. Aluminum accumulation in relation to senile plaques and neurofibrillary tangles formation in the brains of patients with renal failure. Neurol Sci 1992; 107:210-218. Edwardson JA, Ferrier IN, McArthur FK et al. Alzheimer's disease and the aluminum hypothesis. In: Nicolini M, Zatta P, Coram B, editors. Aluminum in Chemistry, Biology and Medicine. New York: Raven Press, 1991: 85-96. Fraser CL, Arieff Al. Nervous system and complications in uremia. Ann Intern Med 1988; 109:143-153. Hidalgo J, Aschner M, Zatta P, Vasak M. Roles of the metallothionein family of proteins in the central nervous system. Brain Res Bull 2001; 55:133-146. Kawahara T, Markert M, Waiters JP. Neutrophil radical production by dialysis membrane. Nephrol Dial Transpl 1988; 3:661-665. Loockwood AH. Neurologic complications of renal disease. Neurol Clin 1989; 7:617-627. Mocchegiani E, Giacconi R, Cipriano C et al. Zinc-bound metallothioneins as potential biological markers of aging. Brain Res Bull 2001; 55:147-154. Nordio M, Andriani M, Gerotto M. et al. Serum concentration of trace elements during different stages of chronic renal failure. ItalJ Min Electrol Metabol 1998; 12:81-86. Reusche E, Seydel U. Dialysis-associated encephalopathy: Light and electron microscopic morphology and topography with evidence of aluminum by laser microprobe mass analysis. Acta Neuropathol 1993; 86:249-258. Savazzi GM, Cusumano F, Bergamaschi E et al. Hypertension as an etiopathological factor in the development of cerebral atrophy in hemodialysed patients. Nephron 1999; 81:17-24. Savazzi GM, Cusumano F, Musini S. Cerebral imaging changes in patients with chronic renal failure treated conservatively or in hemodialysis. Nephron 2001; 89:31-36. Savazzi GM, Cusumano F, Vinci S, Allegri L. Progression of cerebral atrophy in patients on regular hemodialysis treatment: Long-term follow-up with cerebral computed tomography. Nephron 1995; 69:29-33. Shigematsu K, McGeer PL. Accumulation of amyloid precursor protein in damaged neuronal processes and microglia following intracerebral administration of aluminum salts. Brain Res 1992; 593:117-123. Smogorzewski MJ. Central nervous dysfunction in uremia. Am J Kidney Dis 2001; 38:S122-S128. Suzuki Y. Genes, cells and cytokines in resistance against development of toxoplasmic encephalitis. Immunobiol 1999; 201:255-271. Theodosis DT, Poulain DA. Contribution of astrocytes to activity-dependent structural plasticity in the adult brain. Adv Exp Med Biol 1999; 468:175-182.

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Trznadel K, Lucinak M, Pawlicki L. Superoxide anion generation and lipid peroxidation processes during hemodialysis with reused cuprophan dialyzer. Free Rad Biol Med 1990; 8:429^132. Vukicevic S, Kracun I, Vukelic Z. 24R,25-dihydroxyvitamin D3 prevents aluminuminduced alteration of brain gangliosides in uremic rats by keeping the metal within perivascular astrocytes of the blood-brain barrier. Neurochem Intern 1992; 20:391-399. Wegiel J, Wisniewski HM. Tubuloreticular structures in microglial cells, pericytes and endothelial cells in Alzheimer's disease. Acta Neuropathol 1992; 83:653-658. Winkelman MD, Ricanati ES. Dialysis encephalopathy: Neuropathological aspects. Human Pathol 1986; 17:823-833. Wisniewski H, Wegiel J. Non-neuronal cells involved in (3-amyloid deposition in Alzheimer's disease. In: Zatta P, Nicolini M, editors. Non-neuronal Cells in Alzheimer's Disease. Singapore: World Scientific, 1995: 194-203.

CHAPTER 22

Clinical Neurotoxicity of Metals and Neurodegenerative Disorders Marcello Lotti

ABSTRACT While clinical neurotoxicity of metals rarely results in neurodegeneration, the pathophysiology of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis is characterized by the accumulation of certain metal ions in the central nervous system, which eventually leads to free radical-mediated oxidative stress and neuronal death. However, it is not clear if metal accumulation is a primary or secondary event, whether it is pivotal in driving the progression of the disease, and how it may interact with genetic factors. Example only one suggests analogies between clinical neurotoxicity of metals and neurodegenerative diseases. Clinical, histopathological, and toxicological characteristics of manganese poisoning overlap with those of two metal accumulation diseases, Wilson's disease and Hallervorden-Spatz disease. The understanding of mechanistic analogies and differences among toxic and nontoxic diseases may help to clarify the role of metal accumulation in neurodegeneration. Keywords: Neurotoxicity; neurodegeneration; Alzheimer's disease; Parkinson's disease; amyotrophic lateral sclerosis.

1. INTRODUCTION The asymmetry between the way we understand etiology and pathophysiology of neurodegenerative disorders is striking. It is also one of the most disturbing. While heredity has been established in a few, the etiology of 495

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the vast majority of cases is still unknown. Conversely, various pathways of neurodegeneration have been explored and the key role of metal ions has been highlighted by several studies, as has been described in the various chapters in this book. In recent years, the search for the etiologies of neurodegenerative disorders has resulted in a focus on genetic factors known to play important roles in the pathogenesis of Alzheimer dementia (Lendon et al., 1997), Parkinson's disease (PD) (Polymeropoulus et al., 1997), and amyotrophic lateral sclerosis (ALS) (Rosen et al., 1993). However, only about 1% to 10% of such cases are hereditary often as autosomal dominant traits (Martin, 1999). A possible role of environmental toxins in causing or precipitating neurodegenerative disorders has been actively explored for a long time now, and the report that l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) causes Parkinsonism in humans has led the hunt, unsuccessful, so far for candidate environmental chemicals (Ben-Shlomo, 1996). In the case of metals, this possibility has revolved largely around the putative role of some ions and, in particular, that of aluminum in Alzheimer's disease (AD) (Alfrey et al., 1976; Neri and Hewitt, 1991; Exley, 2001). However, the pathological role of metals, if any, remains unclear and there is no compelling evidence that they serve as primary toxins which can initiate these diseases. The outbreak of a neurodegenerative disorder characterized by any combination of dementia, Parkinsonian features, and motor neuron disease, which occurred between 1945 and 1980 in the indigenous population of Guam and other islands in the Western Pacific (Spencer et al., 2000), is an example of the qualified failure to clarify genetic-environmental relationships in neurodegeneration. Investigators hoped that this syndrome would represent the Rosetta Stone of neurodegenerative disorders, and early suggestions indicated that the disease may have been related to the consumption of seeds of cycas circinalis (Kurland, 1963). Aluminum was also considered an etiological factor because the metal had accumulated in neurofibrillary tangles found in brains of affected subjects, but no association was found between the incidence of Guam disease and the geographical distribution of aluminum in the island's waters (Zhang et al., 1996). The disease has rapidly declined since, though some cases of dementia with rigidity continue to be seen. The epidemiological evidence

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supports a probable environmental cause, in particular that of Cycas consumption (Spencer, 2000), but it remains unsolved what role toxicity had in this cluster of atypical neurodegenerative disorders, considering that the possibility of a contributing genetic factor has not been excluded (Zhang et al., 1996; Bird, 2001). This book, as well as a large body of clinical and experimental literature, indicates that several metals are involved in the neuropathology of disorders such as AD, ALS, and PD (Campbell et al., 2001). Increased concentrations of metal ions have been found in the nervous tissues of these patients and the underlying cause of neuronal cell degeneration is thought to be a measurable increase in free radical-mediated oxidative stress at some stage during disease progression (Bains and Shaw, 1997). Chapter 1 summarizes the central role of metals in oxidative damage and response in neurodegenerative diseases. Nevertheless, fundamental questions remain unanswered, such as whether these mechanisms represent a primary initiating event or a secondary phenomena, if they are involved in the progression of the disease, and if and how metals may interact with genetic factors. Moreover, transition metals are normally tightly regulated to prevent free radical formation, and several additional factors have been identified which could either account or exacerbate the toxic potential of brain-associated metals in neurodegenerative disorders (Bains and Shaw, 1997; Patrick et al., 1999; Hironishi et al., 1999; Oshiro et al., 2000; Montoliu et al., 2000; Horning et al., 2000; White et al., 2001; Rajan et al., 2001; LaFerla, 2002; see Chapter 17). The vastness of the subject matter is daunting, the literature is monumental, and each week's journals publish many new items of interest. Consequently, the following commentary has to be selective. Its purpose is to discuss some of the above questions in light of the clinical characteristics of metal poisoning as they compare with those of neurodegenerative disorders.

2. SYSTEMIC DISEASES AND NEUROTOXICITY OF METALS As a result of changes in metal homeostasis, neurotoxicity of metals may be involved in some neurological complications of systemic diseases.

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Neurotoxicity of copper is thought to occur in Wilson's disease WD" which is a rare autosomal recessive disorder of copper metabolism, as indicated by a decrease in the rate of copper incorporation into ceruloplasmin, an altered hepatic copper uptake, a prolonged copper turnover, and its accumulation in several organs including the brain (Sternlieb, 1978). The deposition of copper in the basal ganglia, mainly putamen and pallidum, may result in the clinical manifestations of tremor, diskinesias, and rigidity, whereas wider deposition in the brain may account for mental deterioration and varied neurological signs (Menkes, 1989). The WD gene, ATP7B, is a membrane-bound P-type copper transporting ATPase containing six copper-binding sites (Bull et al., 1993; Tanzi et al., 1993). Mutations of ATP7B result in reduced biliary excretion of copper and low incorporation of copper into ceruloplasmin. It is postulated that copper toxicity in the brain may result from accumulation of copper in neurons whose ability to efflux copper is restricted by the loss of ATP7B activity (see Chapter 7). Another event in WD may be abnormal iron metabolism, as suggested by the linkages between iron and copper metabolism (Siegel et al., 1999). However, hemochromatosis, a disease caused by altered iron homeostasis, is not associated with basal ganglia pathology. Moreover, exogenous copper and iron, which are involved in the events connected with neurodegenerative diseases, do not affect the nervous systems of poisoned patients (Ellenhorn, 1997). Liver failure is thought to cause accumulation of manganese in the basal ganglia, thus explaining the extrapyramidal symptoms frequently encountered in cirrhotic patients, such as tremor and rigidity. Blood manganese concentrations are increased in these patients and a positive correlation was found between blood manganese concentrations and the magnitude of pallidal signals on magnetic resonance images (MRI) (Spahr et al., 1996). These patients display several clinical similarities with those poisoned by manganese (see below).

3. DIFFERENCES BETWEEN METAL POISONING AND NEURODEGENERATION Clinical features of neurotoxicities show some differences with those of neurodegenerative disorders. A dose-related selectivity is a cardinal tenet

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of neurotoxicity caused by chemicals triggering off structural damage to the nervous system (Schaumburg, 2000a). For instance, lower doses of lead and methylmercury may cause peripheral neuropathy, while higher doses are associated with acute encephalopathy and cerebellar ataxia, respectively (Ellenhorn, 1997). Even at higher doses, neurotoxicities of metals show a remarkable selectivity. Thus, high doses of methylmercury may cause selective degeneration of the calcarine cortex and cerebellum (Bakir et al., 1973), and high doses of manganese cause degeneration of certain basal ganglia where the metal accumulates (Yamada et al., 1986). On the contrary, most neurodegenerative disorders display much more complex manifestations due to a wide range of neuronal deaths and losses of connectivity. For instance, pure pyramidal syndromes are rare in neurology, since a few neurodegenerative diseases (such primary lateral sclerosis and hereditary spastic paraparesis) are confined to the upper motor neuron cell bodies or their descending cortico-spinal tracts. Examples of a more widespread involvement of the central nervous system include PD, which is often associated with dementia (Lang and Lozano, 1998a), and AD which may be associated in some cases with cortical blindness (Bird, 2001). While neurodegenerative diseases are progressive, most neurotoxic disorders improve or disappear after cessation of exposure, unless a significant neuronal loss or irreparable damage of the brain or spinal cord occurred. In such a case, the disease is usually not progressive and possible worsening of clinical signs over time may be due to the concurrent physiological loss of neurons during aging (Spencer et al., 1987). However, this general concept has recently been challenged by the findings of gliosis in the brains of MPTP-poisoned patients whose exposure halted several years before death (Langston et al., 1999). It is concluded that a self-limited toxic insult to the nigrostriatal system of these patients had triggered off a self-perpetuating process of neurodegeneration. Since manganese neurotoxicity may also be progressive, as shown in a controlled study of five patients (Huang et al., 1993), it is far from clear why some neurotoxicities are progressive while others are not.

4. METAL POISONINGS Neurotoxicity of metals of clinical relevance is nowadays a rare condition and most information derives from old case reports and epidemiological

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studies in work environments. Several metals have been associated with various forms of neurotoxicity, which either mimick or represent real neurodegenerative disorders. Examples include aluminum (Mahurkar et al., 1973), lithium (Smith and Kocen, 1988), bismuth (Jungreis and Schaumburg, 1993), mercury (Tsubaki and Takahashi, 1986; Adams et al., 1983), manganese (Chu et al., 2000), and lead (Boothby et al., 1974; see Chapter 18). Metals that have also been etiologically associated with neurodegenerative disorders in patients without clinical evidence of poisoning include selenium (Kilness and Hochberg, 1977), manganese (Mitchell et al., 1991), and lead (Boothby et al, 1974). Conclusive evidence of neurotoxicity associated with neurodegeneration is only available for a few metals and two opposite examples will be discussed. Correlations between exposure to manganese, a defined clinical syndrome, and basal ganglia pathology are well-established. Human manganism is characterized by extrapyramidal symptoms and signs, and was the consequence of occupational exposures in miners and industrial workers (WHO, 1981; Wang et al., 1989). The disease is now rare because of improved hygienic conditions in work places. The syndrome reveals some clinical features different from those observed in patients affected by PD and include psychotic symptoms (referred to as locura manganica), dystonia, rigidity associated with normal diadochokinesis, and failure of levodopa therapy (Beuter et al., 1994; Calne et al., 1994; Lu et al., 1994). Neuropathology is also different because lesions are selective to the globus pallidus, where the metal accumulates, whereas normal histology is observed in the substantia nigra (Yamada et al., 1986). Interestingly, the clinical and neuropathological features of manganese toxicity display striking similarities with Hallervorden-Spatz disease. This disease is a progressive autosomal recessive disorder characterized by extrapyramidal signs associated with changes of the medial segment of globus pallidus and less severe neuronal loss in the rest of the basal ganglia. Increased amount of iron and other metals, such as copper and zinc, are found in the affected tissues (Rapin, 1989). Extrapyramidal signs, histopathology, and selective metal accumulation in both WD and manganism are, therefore, overlapping with those of Hallervorden-Spatz disease. Given these similarities in the clinical features and regional distribution of metals, it was suggested that such selective kinetics of metal access to the basal ganglia may have a central

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role in the pathogenesis of metal accumulation diseases (Chu et al., 2000). This example suggests that information derived from commonality between diseases that is not induced by chemicals and that elicited by toxicants may help to understand both. Unlike manganism, the clinical and histopathological features of aluminum neurotoxicity indicate that the hallmark of the disease is not neurodegeneration. Dialysis encephalopathy was seen in patients after years of parenteral aluminum exposure from an aluminum-contaminated dialysate (Alfrey et al., 1976; Flendrig et al., 1976). No signs of neurological disease have been observed in individuals with normal renal function who were occupationally exposed to aluminum (Spencer et al., 2000). Dementia and other neurological disturbances, including seizures, characterize aluminum intoxication and evolve gradually over the course of the disease. However, it is not clear what the neurophysiological bases of dementia are and to what extent the encephalopathy is progressive. Histological examination of brains of patients who had died of dialysis encephalopathy revealed mild spongiform pathology in the cerebral cortex associated with nonspecific neurological changes (Burks et al., 1976) and without marked loss of neurons (Winkelman and Ricanati, 1986; see Chapter 4). Although aluminum has been associated with a variety of neurodegenerative diseases, the analysis of a wide range of neurological disorders has found elevated aluminum in some cases, but this was not a consistent finding within a given disorder (Traub et al., 1981; Zatta, 1993; Exley, 2001).

5. DISCUSSION Accumulating evidence suggests that copper and/or iron have an important role in the pathophysiology of neurodegenerative disorders (Campbell et al., 2001; see Chapters 12 and 14), including AD (Huang et al., 1999; see Chapters 6 and 8), PD (Riederer et al, 1989), ALS (Andrus et al., 1998; see Chapter 9), and prion disease (Wadsworth et al., 1999; see Chapter 10). Evidence includes increased redox-reactive copper and iron levels (Sayre et al., 2000), changes in the expression of metalbinding proteins (Aschner, 1996; Adlard et al., 1998; see Chapters 5 and 11), promotion of amyloid-3 aggregation (Atwood et al., 1998), and free radical generation by amyloid (Bondy et al., 1998), indicating that

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excessive production of damaging free radicals is associated with neurodegeneration. Nevertheless, exceptions have been observed and an exploration of the reasons for such discrepancies and the significance of metal accumulation in neurodegenerative disorders may be better understood. For instance, manganese has been shown to mediate opposite effects in the nigrostriatal system since it causes both neurodegeneration and an antioxidative effect, as opposed to the exclusively pro-oxidant properties of iron in brain tissues (Sziraki et al., 1998). Consequently, manganism may be produced with mechanisms other than those of oxidative stress (Graham, 1984; Brouillet et al., 1993; Desole et al., 1994; Lloyd, 1995; Chang and Liu, 1999; see Chapter 16). Lithium may also display opposite effects (Smith and Kocen, 1988; see Chapter 19). Whether metal accumulation and oxidative stress represent the primary event in neurodegeneration is unclear. Increased iron and reduced complex I activity were not found in the brains of patients with incidental Lewys body disease, suggesting that these may be later or secondary changes (Jenner and Olanow, 1996), a hypothesis which is also supported by evidence from animal experiments (Double et al., 2000). On the contrary, MRI studies indicated increased manganese in the basal ganglia of intoxicated patients, but the abnormal MRI signals decrease or disappear after exposure (Ejima et al., 1992), suggesting that neurodegeneration may be a sequela of the initial phase of manganism. Based on the mechanism of oxidative stress in triggering off neurodegeneration, the hypothesis was made that inadequate intake of antioxidant might have predisposed patients to PD. This was shown in a study where patients had an intake of vitamin E lower than the controls' (de Rijk et al., 1997). However, these studies were generally inconclusive (Lang andLozano, 1998a). Whether deposition of transition metals in the brain (see Chapter 1) increases the oxidative load on the cells, thereby driving neurodegeneration and its progression, is also unclear. In the attempt to slow the progression of neurodegeneration, clinical trials with antioxidant therapy were initiated. They have shown some benefits from treatment (Parkinson Study Group, 1996; Schneider et al., 1997), though, on the whole, these results are not convincing (Lang and Lozano, 1998b). Nevertheless, it should be stressed that the clinical course of neurodegenerative disorders

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may be quite bizarre because it is characterized by both slow progression and fast acceleration, thereby making the assessment of therapeutic effects quite difficult. Several links between genetic factors and metal toxicity in the development of neurodegeneration and other neurological diseases (see Chapter 15) have been suggested. Mutations of the copper/zinc superoxide dismutase gene in familial ALS are one example (Rosen et al., 1993; see Chapter 9). Superoxide dismutase mutations are thought to cause a gain-of-function that is selectively lethal to motor neurons. Thus, excessive levels of hydroxyl radical can be formed through the reaction of hydrogen peroxide with a transition metal, such as iron (Martin, 1999). An example of a loss-of-function disorder is Friedreich's ataxia, a hereditary neurodegenerative disease. It has been linked to a defect in the mitochondrial protein frataxin (Campuzano et al., 1997), eventually leading to abnormal accumulation of iron in the mitochondria (Babcock et al., 1997). Elucidation and characterization of the interactions between aberrant proteins and metals may help in understanding how affected cells die and whether chelation and other therapies may inhibit or prevent these diseases. In this respect, the discovery that deposition of amyloid-@ is accelerated by metals, notably copper and zinc (Atwood et al., 1998; Huang et al., 1999; Cuajungco et al., 2000) offers some promising perspectives on chelation therapy (see Chapter 4). However, chelation may remove unwanted metals and it seems somewhat paradoxical that a recent candidate for chelating therapy is iodochlorohydroxyquin (Cherny et al., 2001), an old and well-known antibiotic that was responsible for a myelopathy epidemic (Schaumburg, 2000b). In conclusion, the clinical toxicology of metals offers some, though limited, clues to understand neurodegeneration. Nevertheless, it is increasingly appreciated that the mechanisms of many forms of toxicity and the pathophysiology of common diseases may involve a few stereotypical reactions, such as the mechanism of apoptosis (Lotti and Nicotera, 2002). In this respect, the mechanisms of metal neurotoxicity and neurodegeneration could be explored within a common frame. One example of this approach is to study the analogies and differences in protein-protein interactions, as well as the complexes that proteins form at various stages of metal neurotoxicity and neurodegenerative diseases. The study of such interactions, called the sociology of proteins (Abbott, 2002), is now possible (Gavin et al., 2002;

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Ho et al., 2002) and may help in understanding the role of metal ions in the initiation and/or progression of various forms of neurodegeneration.

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Lotti M

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Index 6-hydroxydopamine

5, 7

a-synuclein 357 •y-aminobutyric acid

82

A(3 aggregation 4 aceruloplasminemia 220 adenylate cyclase 149 alkyl peroxides 7 alpha synuclein 312, 313 aluminum 118 aluminosilicates 118 Alzheimer's disease 1, 100 Amnion's horn 18, 23 amygdala 23, 53, 370 amyloid (3 protein precursor 4 amyloid-beta 3, 101 amyotrophic lateral sclerosis 10 anorexia 158 anterograde transport 73 apomorphine 336 arteriovenous malformation 379 astrocytes 6,77 axonal transport 75 basal ganglia 22 Bcl-2 226,473 bilirubin 388 blood-brain barrier 88, 149, 208, 298, 308, 336, 367, 382 Bowman's glands 69 brain homeostasis 186 brainstem 20 bulimia 158 Camelford 120 canalicular membrane 220 catalase 2 catecholamines 77 centrum semiovale 21 cerebellum 17,75 cerebral malaria 382

ceruloplasmin 6, 186 Chamorro 46 chelation therapy 104 citrate 118 clioquinol 314 copper foundries 442 copper-zinc superoxide dismutase 2 corpus callosum 19 corpus mamillare 20 Creutzfeldt-Jacob syndrome 121 Creutzfeldt-Jakob disease 9 cuprizone 282 CuZnSOD 8, 10 CuZnSOD gene 10 CuZnSOD knockout mice 9 cytokines 101 desferrioxamine 100,315 diabetes 405 dialysis encephalopathy 120, 121 dopamine 7 electrocorticograms 380 electrocorticography 369 epiphysis 24,53 experimental autoimmune encephalitis 309 extrapyrimidal syndromes 7 familial AD 184 Fenton reaction 5, 352 Fenton 7 Feralex-G 100, 108 ferritin 72, 345, 354 flavonoids 336 fluidity 437 Freidreich's ataxia 335 gamma-(p-y) secretase 101 glial fibrillary acidic protein 77 globus pallidus 17 glutathione 7 509

510

Index

glycation 273 granulomas 81 Guam island 17, 45, 46, 49 Guamian dementia 326

Morin stain 122 Mossbauer 355 multiple sclerosis 309 muscimol 374 myelinogenesis 408

head injury 195 hemochromatosis 402,405 hemodialysis 120 hippocampus 16, 19, 22, 23, 52, 53,74 histocompatibility complex 402 Huntington's disease 9, 325, 345 hypothalamus 20, 24, 74

nerve growth factor 292 neurinoma 132 neurofibrillary tangles 3 neuromelanin 329, 354, 355, 356 New Guinea 46 nitecapone 336 nitric oxide 77 nucleus basalis of Meynert 32 nystagmic jerks 455

inductively coupled plasma mass spectroscopy 247 inflammation 4 inherited anemias 405 instrumental neutron activation analysis 36,247 interleukin-l(3 101 iron regulatory protein 3

olfactory pathways 68 olfactory bulb 22 olfactory mucosa 70 olfactory neuron 69 otoacoustic emissions 449 oxidative stress 2, 3, 4, 5, 6, 7, 8

kainic acid 310 Kii Peninsula 46 Kuru 318 lactotransferrin 6, 326 laser microprobe mass analysis (LAMMA) 82, 129, 132 Lewy bodies 334, 345, 355, 356 lipid peroxidation 6 lipofuscinosis 122 locus coeruleus 22 Lou Gehrig's disease 263 manganese superoxide dismutase 2 manganoproteins 417 Mclntyre powder 120 melanotransferrin 6 melatonin 152,371 mental lethargy 158 metalloproteinases 248 microglia 5 microparticle-induced X-ray emission MnSOD 8

Parkinson's disease 1 peroxidase 3 peroxynitrite 2 pesticides 430 PIXE 246 pons 18,24 porphyria 405 positron emission tomography 416 presenilin 5 presenilin-1 and -2 101 prion diseases 1 pro-oxidant 7 pulvinar 18 putamen 17, 18, 20, 22, 23, 45, 53 quinone

7

Romberg test

3

451

selegiline 336 senescent red blood senile plaques 3 silicon 118

401

511

Index somatostatin 151 spin trap 268 spinal cord 75 sprouting 147 substantia nigra 7, 76 synaptic plasticity 249 temporal lobe 17 thalamus 18,19,21,74

transferrin 76, 118, 186, 408 transgenic mice 10 transgenic mouse 5, 6, 8 veratridine

375

xenobiotics

88

ZnTl-4

144

PAOLO ZATTA is the Director of the "Metalloproteins" Unit of the CNR-lnstitute for Biomedical Technologies at the University of Padua, Italy. Graduated in Chemistry, he received his doctoral degree in Biological Sciences at the University of Padua. He was a post doctoral fellow at the University of California at San Francisco, and was a visiting scientist at the University of Leeds in UK, University of Hamburg in Germany, University of California at San Francisco and University of Georgia in USA, University of Tel Aviv in Israel and University of Concepcion in Chile. Prof. Zatta's scientific interests concern the Physiopathological Role of Metal Ions in Neurodegenerative Disorders. He has published about 200 full papers and several reviews and books as an editor. He is also the organizer and co-organizer of several international conferences, including Aluminum in Chemistry, Biology and Medicine, Padua, Italy, 1992-1994; Second International Conference on Alzheimer's Disease and Related Disorders, Padua, 1992; First and Second International Conference On Metal Ions and the B r a i n : From N e u r o c h e m i s t r y t o Neurodegeneration, Padua, Italy, 2000; Fez, Morocco, 2002. More information on Prof. Zatta's scientific activities can be found at http://www.bio.unipd.it/-zatta/aluminum.html

Numerous studies have established a clear connection between neuronal oxidative stress and several neurodegenerative diseases, with consequential damages to lipids, proteins, nucleic acids, etc. In addition, several modifications indicative of oxidative stress have been described in association with neurons, neurofibrillary tangles and senile plaques in Alzheimer's disease, including advanced glycation end products and free carbonyl oxidation. Oxidative damage and antioxidant responses are now well characterized, but sources of damaging free radicals are yet to be fully understood. Evidences of alteration in metal ions metabolism have been reported in various diseases like Alzheimer's, Wilson, Menkes, Prion, Pick, Huntington disease, epilepsy and other pathological events. Thus, metal ions play a pivotal role in neurodegenerative phenomena. Chelation therapy is still in the early days of its development, but research in this area could lead to new products that could revolutionize treatment. Two international conferences on "Metals and the Brain: From Neurochemistry to Neurodegeneration" (Padova, Italy, 2000 and Fez, Morocco, 2002) were recently held to discuss the role of metal ions in neurophysiopathology. A third will be held in 2005 in Johannesburg, South Africa. This book follows the same train of thought as those conferences, in order to highlight the unquestionable importance of metal ions in the research on the neurophysiopathology of neurodegenerative diseases. The excellent reputation of the scientists who have contributed to this project ensures the quality of the chapters presented here, and hopefully this will help spur new research initiatives in the field, which is still in its infancy.

Metal Ions and Neurodegenerative Disorders World Scientific

ISBN 981-238-398-0

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