Role of Proteases in the Pathophysiology of Neurodegenerative Diseases

Role of Proteases in the Pathophysiology of Neurodegenerative Diseases

Role of Proteases in the Pathophysiology of Neurodegenerative Diseases Edited by

Abel Lajtha Nathan S. Kline Institute for Psychiatric Research Orangeburg, New York

and

Naren L. Banik Medical University of South Carolina Charleston, South Carolina

Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow

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CONTRIBUTORS Carmela R. Abraham Boston University School of Medicine Boston, Massachusetts 02118 M. Azuma Research Laboratories Senju Pharmaceutical Co. Ltd. Kobe, 6.51-2241 Japan Naren L. Banik Department of Neurology Medical University of South Carolina Charleston, S. C. 29425 Raymond T. Bartus Alkermes, Inc. Cambridge, MA 02139 Martin J. Berg Center for Neurochemistry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 Dieter Brömme Mount Sinai School of Medicine Department of Genetics New York, NY 10029 Sic L. Chan Laboratory of Neurosciences National Institute on Aging Baltimore, MD 21224 Jinyang Cong Muscle Biology Group University of Arizona Tucson, Arizona 85721

M.L. Cuzner Department of Neurochemistry Institute of Neurology University College London WClN 3BG, U.K. Dylan R. Edwards School of Biological Sciences University of East Anglia Norwich, Norfolk NR4 7TJ, England Dwaine F. Emerich Alkermes, Inc. Cambridge, MA 02139 Lawrence F. Eng Pathology Research Service Veterans Administration Hospital Palo Alto, CA 94304 Maria E. Figueiredo-Pereira Department of Biological Sciences Hunter College of the City University of New York New York, NY 10021 Peter A. Forsyth Oncology & Clinical Neurosciences University of Calgary and Department of Medicine Tom Baker Cancer Centre Calgary, Alberta T2N IN4, Canada C. Fukiage Research Laboratories Senju Pharmaceutical Co. Ltd. Kobe 651-2241 Japan Darrel E. Goll Muscle Biology Group University of Arizona Tucson, Arizona 85721 v

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Contributors

Gregor Guncar Jozef Stefan Institute Biochemistry and Molecular Biology Ljubljana SI-1000, Slovenia Edward L. Hogan Department of Neurology Medical University of South Carolina Charleston, SC 29425 Vivian Y.H. Hook Departments of Medicine and Neurosciences University of California, San Diego La Jolla, California 92093-0822 Janko Kos Jozef Stefan Institute Biochemistry and Molecular Biology and Krka, d.d. R&D Division Dept. of Biochemical Research and Drug Design Ljubljana SI-1000, Slovenia Marc A. LaFleur School of Biological Sciences University of East Anglia Norwich, Norfolk NR4 7TJ, England K.J. Lampi Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201 Hahn-Jun Lee Laboratory for Proteolytic Neuroscience RIKEN Brain Science Institute Saitama, 351 -0198, Japan Hongqi Li Muscle Biology Group University of Arizona Tucson, Arizona 85721 H. Ma Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201

Neville Marks Center for Neurochemistry Department of Psychiatry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 Mark P. Mattson Laboratory of Neurosciences National Institute on Aging Baltimore, MD 21224 Denise C. Matzelle Department of Neurology Medical University of South Carolina Charleston, SC 29425 Michael A. Moskowitz Stroke and Neurovascular Regulation Laboratory Neurology and Neurosurgery Service Massachusetts General Hospital Harvard Medical School Charlestown, MA 02129 Suzana Petanceska Department of Psychiatry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 Swapan K. Ray Department of Neurology Medical University of South Carolina Charleston, SC 29425 Patricia Rockwell Department of Biological Sciences Hunter College of the City University of New York New York, NY 10021 Takaomi C Saido Laboratory for Proteolytic Neuroscience RIKEN Brain Science Institute Saitama, 351 -0198, Japan

Contributors

Jörg B. Schulz Neurodegeneration Laboratory Department of Neurology University of Tübingen D-72076 Tübingen, Germany T.R. Shearer Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201 Donald C. Shields Department of Neurology Medical University of South Carolina Charleston, SC 29425 M. Shih Department of Oral Molecular Biology School of Dentistry Oregon Health Sciences University Portland, OR 97201

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Valery F. Thompson Muscle Biology Group University of Arizona Tucson, Arizona 85721 Boris Turk Jozef Stefan Institute Biochemistry and Molecular Biology Ljubljana SI-1000, Slovenia Vito Turk Jozef Stefan Institute Biochemistry and Molecular Biology Ljubljana SI-1000, Slovenia Kevin K.W. Wang Department of Neuroscience Therapeutics Parke-Davis Pharmaceutical Research A Division of Warner-Lambert Company Ann Arbor, MI 481 05

Franchot Slot Boston University School of Medicine Boston, Massachusetts 02118

Gloria G. Wilford Department of Neurology Medical University of South Carolina Charleston, SC 29425

Marion Smith Department of Neurology Stanford University School of Medicine and Veterans Administration Medical Center Palo Alto, CA 94304

V.W. Yong Oncology & Clinical Neurosciences University of Calgary and Department of Medicine Tom Baker Cancer Centre Calgary, Alberta T2N IN4, Canada

Koichi Suzuki Tokyo Metropolitan Institute of Gerontology Tokyo, 173-0015 Japan

PREFACE Researchers seeking problems that offer more hope of success often avoid subjects that seem to be difficult to approach experimentally, or subjects for which experimental results are difficult to interpret. The breakdown part of protein turnover in vivo, particularly in nervous tissue, was such a subject in the past – it was difficult to measure and difficult to explore the mechanisms involved. For factors that influence protein metabolism, it was thought that protein content, function, and distribution are controlled only by the synthetic mechanisms that can supply the needed specificity and response to stimuli. The role of breakdown was thought to be only a general metabolic digestion, elimination of excess polypeptides. We now know that the role of breakdown is much more complex: it has multiple functions, it is coupled to turnover, and it can affect protein composition, function, and synthesis. In addition to eliminating abnormal proteins, breakdown has many modulatory functions: it serves to activate and inactivate enzymes, modulate membrane function, alter receptor channel properties, affect transcription and cell cycle, form active peptides, and much more. The hydrolysis of peptide bonds often involves multiple steps, many enzymes, and cycles (such as ubiquination), and often requires the activity of enzyme complexes. Their activation, modification, and inactivation can thus play an important role in biological functions, with numerous families of proteases participating. The specific role of each remains to be elucidated. It seems that at least some of the proteolytic processes in the brain differ from those in other organs. Enzymes involved in neuropeptide metabolism, responsible for the formation and subsequently the inactivation of physiologically active peptides, for example, have a function specific to the brain. Other findings, such as the stability of brain proteins in severe malnutrition when most body proteins are greatly diminished, also indicate controls of protein metabolism in the brain that differ from such controls in other organs. The present book focuses on the role of proteases in pathological changes in the brain, an aspect of proteolysis that has recently gained increased importance and interest. Proteolytic enzymes have been implicated in the degeneration and destruction of tissue in a number of degenerative CNS diseases and trauma, and direct evidence of their involvement has been established. Among the first lysosomal proteinases (cathepsins) and uncharacterized neutral proteinases to be studied were in the tissues of patients with multiple sclerosis, and in animals with experimental demyelinating diseases. These studies suggested that cathepsins and carboxypeptidases and several neutral proteinases play a role in neurodegenerative diseases. Recent evidence suggests that neutral proteinases, including calpain (a calcium-activated neutral proteinase), calcium-independent metalloproteinase, multicatalytic proteinase complex or proteosome, and matrix metalloproteinase, are involved in autoimmune and other demyelinating diseases, such as multiple sclerosis, optic neuritis, amyotrophic lateral sclerosis, stroke, muscular dystrophy, and Alzheimer’s disease, also in ischemia, oxidative stress, and CNS trauma. The

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participation of both lysosomal and extra-lysosomal enzymes in tissue degeneration is noted in a growing number of diseases. Now that many of the enzymes are well characterized (and we can expect the characterization of many more), factors influencing their activities, such as inhibitors of specific enzymes, gain importance because they have great therapeutic potential in a number of diseases, for which some are already in clinical use. Currently, information on protease activity changes in the various neurodegenerative conditions is scattered. We hope that the excellent contributions of the authors of this volume will be helpful for investigators interested in the mechanisms of proteolysis in neurodegenerative diseases and in developing therapy for their damage. It is no longer necessary to think that research in this subject is difficult to interpret. The future for studies in this subject is bright. We would like to thank our authors for agreeing to contribute to this book, and for their excellent discussions of this interesting and important field. In their thoughtful summary of what has been achieved to date, they point out not only the significance of the findings so far, but also the yet unresolved problems, thereby indicating future tasks and approaches. The recent rapid expansion of our knowledge in this area should give us confidence and hope for further important advances in our understanding of the mechanisms involved, and in the use of our knowledge for improvements in therapy. Our thanks are also due to Ms. Denise Matzelle and Ms. Susan Foldi for their patience and efforts in putting this book into its final form. We are also grateful to Peter Sís, a fine artist who is always very imaginative and creative, for kindly agreeing to contribute for the cover of the book his drawing symbolizing our quest for knowledge. Naren L. Banik Abel Lajtha

CONTENTS 1.

2.

THE ROLE OF PROTEOLYTIC ENZYMES IN AUTOIMMUNE DEMYELINATING DISEASES: AN UPDATE Marion Smith.. .......................................................................

1

PROTEASES IN DEMYELINATION M.L. Cuzner...........................................................................

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3.

CALCIUM ACTIVATED NEUTRAL PROTEINASE IN DEMYELINATING DISEASES Donald C. Shields and Naren L. Banik.. ......................................... 25

4.

PAPAIN-LIKE CYSTEINE PROTEASES AND THEIR IMPLICATIONS IN NEURODEGENERATIVE DISEASES Dieter Brömme and Suzana Petanceska.. ........................................ 47

5.

THE ROLE OF THE CALPAIN SYSTEM IN NEUROMUSCULAR DISEASE Darrel E. Goll, Valery F. Thompson, Hongqi Li, and Jinyang Cong.. ................................................................................. 63

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THE ROLE OF CALPAIN PROTEOLYSIS IN CEREBRAL ISCHEMIA Dwaine F. Emerich and Raymond T. Bartus.. .................................. 75

7.

CALPAIN ISOFORMS IN THE EYE T.R. Shearer, H. Ma, M. Shih, K.J. Lampi, C. Fukiage, and M. Azuma.. ......................................................................

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METALLOENDOPEPTIDASE EC 3.4.24.15 IN NEURODEGENERATION Carmela R. Abraham and Franchot Slot. ........................................ 101

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CYSTEINE PROTEASES, SYNAPTIC DEGENERATION AND NEURODEGENERATIVE DISORDERS Mark P. Mattson and Sic L. Chan.. ..............................................

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THE UBIQUITIN/PROTEASOME PATHWAY IN NEUROLOGICAL DISORDERS Maria E. Figueiredo-Pereira and Patricia Rockwell.. .........................

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10.

11.

AMYLOID (TACE, BACE) AND PRESENILIN PROTEASES ASSOCIATED WITH ALZHEIMER'S DISEASE Neville Marks and Martin J. Berg.. ............................................... 155

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12.

13.

Contents

CASPASES IN NEURODEGENERATION Jörg B. Schulz and Michael A. Moskowitz.. ....................................

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THERAPEUTIC APPROACHES WITH PROTEASE INHIBITORS IN NEURODEGENERATIVE AND NEUROLOGICAL DISEASES Kevin K. W. Wang.. ..................................................................

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PATHOPHYSIOLOGY OF CENTRAL NERVOUS SYSTEM TRAUMA: PROTEOLYTIC MECHANISMS AND RELATED THERAPEUTIC APPROACHES Swapan K. Ray, Denise C. Matzelle, Gloria G. Wilford, Lawrence F. Eng, Edward L. Hogan, and Naren L. Banik.. ......................................... 199

15.

LYSOSOMAL CYSTEINE PROTEASES AND THEIR PROTEIN INHIBITORS Vito Turk, Janko Kos, Gregor Guncar, and Boris Turk.. ...................... 227

16.

PROTEASES AND THEIR INHIBITORS IN GLIOMAS Peter A. Forsyth, Dylan R. Edwards, Marc A. LuFleur, and V. W. Yong......................................................................

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17.

PROTEOLYSIS OF MUTANT GENE PRODUCTS ARE KEY MECHANISMS IN NEURODEGENERATIVE DISEASES Vivian Y.H. Hook ................................................................... 269

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MAMMALIAN PROTEINASE GENES Hahn-Jun Lee, Koichi Suzuki, and Takaomi C. Saido..........................

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AUTHOR INDEX .................................................................

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SUBJECT INDEX .................................................................

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Role of Proteases in the Pathophysiology of Neurodegenerative Diseases

THE ROLE OF PROTEOLYTIC ENZYMES IN AUTOIMMUNE DEMYELINATING DISEASES: AN UPDATE

Marion Smith Department of Neurology Stanford University School of Medicine and Veterans Administration Medical Center Palo Alto, CA 94304

Twenty-three years ago a review by this author (MES) summarized the evidence for the participation of proteolytic enzymes in myelin destruction in experimental allergic (autoimmune) encephalomyelitis (EAE)1. Because EAE was, as now, considered to be an animal model for multiple sclerosis (MS), the review described investigations up to that time, pointing to the involvement of proteolytic enzymes in EAE lesions and by analogy, in MS plaques. Since then, due to intensive investigations of proteolytic enzymes in these demyelinating conditions, it has become even more apparent that tissue destruction in EAE and MS, as well as in other degenerative diseases is dependent on proteolytic enzymes. These enzymes are contained in inflammatory cells such as macrophages, neutrophils, and lymphocytes that utilize proteases for their invasive mechanisms, as well as for tissue destruction. At the turn of a new century it is instructive to compare our former ideas with those of the present state of knowledge attained by the contributions of many investigators. One problem in comparing our present understanding of enzymic mechanisms with those of the past is that the nomenclature of the proteases has changed, with further purification and investigation of their properties. Thus, the calcium-activated neutral protease, originally described by Guroff 2 is now recognized as “calpain”, which exists in several forms. New proteinase families have been discovered, including the caspases, and the matrix-metalloproteinases, which encompass some of the proteases formerly known as collagenase, elastase, and gelatinase. Although the lesion of the EAE animal was described in detail by both light and electronmicroscopy in the 1960s, the use of immuno-methods and markers has allowed a much better delineation of the kinds of cells present, the timing of their invasion, their state of activation, as well as some insight into their function. In addition to the lymphocytes and small monocytes formerly mentioned, several classes of lymphocytes and activated macrophages have been identified, and microglia have been recognized as resident macrophages in the central nervous system. Microglia and macrophages are notable for their content of proteases, and their capability as secretors of proteases, including plasminogen activator 3,4, cysteine proteases 5 , calpain 6, and metalloproteinase-9 (gelatinase B)7. The latter has been shown to be augmented when microglia are activated in vitro8, and undoubtedly other proteases are similarly increased in activated phagocytes. In addition, a number of these enzymes appear to occur in greater than normal amounts in activated lymphocytes and astrocytes. Myelin proteins were formerly thought to be relatively few in number, but since 1978, many more myelin constituents have been identified. Some are enzymes, especially those involved in lipid metabolism, and may give rise to signaling molecules9. Furthermore, new structural proteins have been detected, including the myelin oligodenRole of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.

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drocyte glycoprotein (MOG) 10, and another basic protein, the myelin-associated oligodendrocytic basic protein (MOBP)11. Myelin basic protein and the myelin-associated glycoprotein, have both been shown to be susceptible to degradation by a number of proteases, and depleted in MS 12, while even proteolipid protein which is insoluble in water, is slowly degraded by calpain13. Other constituents of myelin may be vulnerable to enzymatic destruction, and some of the newly found active myelin constituents such as the signalling molecules, when injured, may cause metabolic collapse of the oligodendrocyte-myelin axis. In former years the ease of destruction of myelin basic protein in vitro by the acid protease cathepsin D was most emphasized (reviewed by Berlet)14. This lysosomal enzyme is probably not secreted, but may be a major effector of intracellular myelin degradation after phagocytosis of myelin in conjunction with other cathepsins such as B and L. Before myelin can be ingested, however, it must be disrupted into smaller fragments to facilitate its ingestion. This may be accomplished by several mechanisms, such as complement15, 16 and/or extracellular neutral proteases secreted by activated phagocytic cells. Lampert 17 first described areas of “vesicular degeneration” in demyelinating lesions of animals with EAE, and similar disruptive lesions have been noted in areas of the CNS in MS18, viruses19, or by various neurotoxic substances. Phagocytic cells may secrete these enzymes in the vicinity of the myelin sheath to disrupt the lamellae and to peel away the layers in MS and EAE. Traumatic damage, as in spinal cord injury, may result in an influx of calcium 20, which can activate calpain, thus causing vesicular myelin degradation. Therefore, both neutral and acidic proteolytic enzymes may be involved in myelin destruction, the former for disruption of myelin lamellae enabling phagocytic cells to ingest the droplets, then the acidic lysosomal cathepsins internally complete the protein digestion, while the myelin lipids are esterified or hydrolyzed. Evidence exists for the participation of a number of proteolytic enzymes in myelin destruction by autoimmune reactions, viral infection, and trauma. Most frequently mentioned are metalloproteinases 21, plasminogen activator 22, calpain 23, and the lysosomal cathepsins including cathepsin D, B, and L. Another proteolytic enzyme family, the caspases, may also be involved in cellular destruction of lymphocytes, phagocytic cells and oligodendroglia as activators of the apoptotic mechanisms shown to accompany EAE and MS 24. In this chapter and others, these enzymes and their roles in tissue destruction will be documented in detail. The 1978 review concluded “Further studies on proteinases and their role in disease will be of importance in devising a rationale for treatment. Many proteinase inhibitors have been identified, and it is possible that such inhibitors may be useful to intervene in the course of degenerative CNS diseases of myelin.” As of today, although protease inhibitors are standard treatment for other diseases, it is not clear whether these substances may be beneficial for MS. A number of these inhibitors appear to suppress EAE, including pepstatin, for cathepsin D25, inhibitors of plasminogen act ivator 26, neutral proteases such as leupeptin27 and others28. More recent work has suggested that metalloproteinase inhibitors may be useful as therapy for MS 29,30. We are further along than in 1978 in working out a treatment for MS with proteolytic inhibitors, but progress has been slow. As the newer aspects of these enzymes are described in this volume, the authors will undoubtedly point out possible new inhibitors as candidates for further investigation.

REFERENCES 1. M.E. Smith, The role of proteolytic enzymes in demyelination in experimental encephalomyelitis., Neurochem. Res. 2:233 (1977). 2. G. Guroff, A neutral, calcium-activated proteinase from the soluble fraction of rat brain, J. Biol. Chem. 239:149 (1964). 3. W. Cammer, B.R. Bloom, W.T. Norton, and S. Gordon, Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: A possible mechanism of inflammatory demyelination, Proc. Natl. Acad. Sci. U.S.A. 75:1554 (1978). 4. K. Nakajima, N. Tsuzaki, M. Shimojo, M. Hamanoue, and L. Kosaka, Microglia isolated from rat brain secrete a urokinase-type plasminogen activator, Brain Res. 577:285(1992).

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5. R.B. Banati, G. Rothe, G. Valet, and G.W. Kreutzberg, Detection of lysosomal cysteine proteinases in microglia: Flow cytometric measurement and histochemical localization of Cathepsin B. and L, Glia 7:183 (1993). 6. D.C. Shields and N.L. Banik, Pathophysiological role of calpain in experimental demyelination, J. Neurosci. Res. 55:553 (1999). 7. T. Yamada, Y. Yoshiyama, H. Sato, M. Seiki, A.Shinagawa, and M. Takahashi, White matter microglia produce membrane-type matrix metalloprotease, an activator of gelatinase A, in human brain tissues, Acta Neuropathol. 90:421 (1995). 8. P.E. Gottschall, X. Yu, and B. Bing, Increased production of gelatinase B (metalloproteinase-9) and interleukin-6 by activated rat microglia in culture, J. Neurosci. Res. 42:335 (1995). 9. J.N. Larocca, A. Cervone, and R.W. Ledeen, Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium, Brain Res. 436:357 (1984). 10. C. Linington, M. Webb, and P.L. Woodhams, A novel myelin-associated glycoprotein defined by a mouse monoclonal antibody, J. Neuroimmunol. 6:387 (1984). 11. Y. Yamamoto, R. Mizuno, T. Nishimura, Y. Ogawa, H. Yoshikawa, H. Fujimura, E. Adashi, T. Kishimoto, T. Yanagahara, and S. Sakoda, Cloning and expression of the myelin associated oligodendrocytic basic protein. A novel basic protein constituting the central nervous system myelin, J. Biol. Chem. 269:31725 (1994). 12. Y. Itoyama, N.H. Sternberger, H.DeF. Webster, R.H. Quarles, S.R. Cohen, and E.P. Richardson, Immunocytochemical observations on the distribution of myelin-associated glycoprotein and myelin basic protein in multiple sclerosis lesions, Ann. Neurol. 7:167 (1980). 13. N.L. Banik, D. Lobo-Matzelle, G. Gantt-Wlford, and E.L. Hogan, Calpain, A catabolic mediator in spinal cord trauma, in: Neurodegenerative Diseases, G. Fiscum, ed., Plenum Press, New York (1996). 14. H.H. Berlet, Degradation of myelin proteins by proteinases, in: Myelin, Biology and Chemistry, R. Martenson, ed., CRC Press, Ann Arbor (1992). 15. W. Cammer, C.F. Brosnan, C. Basile, B.R. Bloom, and W.T. Norton, Complement potentiates the degradation of myelin proteins by plasmin: Implications for a mechanism of inflammatory demyelination, Brain Res. 364:91 (1986). 16. P. Vanguri, C.L. Koski, B. Silverman, and M.L. Shin, Complement activation by isolated myelin. Activation of the classical pathway in the absence of myelin-specific antibodies, Proc. Natl. Acad. Sci. U.S.A. 79:3290 (1982). 17. P. Lampert, Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyelitis. Acta Neuropathol. 9:99 (1967). 18. H. Lassmann, H. Budka, and G. Schnaberth, Inflammatory demyelinating polyradiculitis in a patient with multiple sclerosis, Arch. Neurol. 38: 99 (1981). 19. M.C. Dal Canto, and H.L. Lipton, Primary demyelination in Theiler’s virus infection. An ultrastructural study, Lab. Invest. 33:626 (1975). 20. J.D. Ballentine, Spinal cord trauma. In search of the granular axoplasm and vesicular myelin. J. Neuropathol. Expt. Neurol. 47:77 (1988). 21. B.C. Keiseier, T. Seifert, G. Giovannoni, and H.-P. Hartung, Matrix metalloproteinases in inflammatory demyelination. Targets for treatment, Neurology 53:20 (1999). 22. M.L. Cuzner, and G. Opdenakker, Plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system, J. Neuroimmunol. 94:1 (1999). 23. D.C. Shields and N.L. Banik, Upregulation of calpain activity and expression in experimental allergic encephalomyelitis: a putative role for calpain in demyelination, Brain Res. 794:68 (1998). 24. R. Furlan, G. Martino, F. Galbiati, P.L. Poliani, S. Smiroldo, A. Bergami, G. Desina, G. Comi, R. Flavell, M.S. Su, and L. Adorini, Calpase-1 regulates the inflam- matory process leading to autoimmune demyelination, J. Immunol. 163:2403 (1999). 25. D.H. Boehme, H. Usezawa, G. Hashim, and N. Marks, Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (Pepstatin), Neurochem. Res. 3: 185 (1978). 26. C.F. Brosnan, W. Cammer, W.T. Norton, and B.R. Bloom, Proteinase inhibitors supress the development of experimental allergic encephalomyelitis, Nature 285:235 (1980). 27. Y. Nagai, Suppression of demyelination in acute EAE: New strategies for the therapy of EAE and MS. In: Proc. Asian Multiple Sclerosis Workshop, Y. Kurawa, and L.T. Kurland, eds., Kyushu University Press, Fukuoka, Japan (1982). 28. M.E. Smith, and L.A. Amaducci, Observations on the effects of protease inhibitors on the suppression of experimental allergic encephalomyelitis, Neurochem. Res. 7:541 (1982). 29. K. Gijbels, R.E. Galardy, and L. Steinman, Reversal of experimental autoimmune encephalomyelitis with a hydroxamate inhibitor of matrix metalloprotase, J. Clin. Invest. 94:2177 (1994).

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30. W. Liedtke, B. Cannella, R.J. Mazzaccaro, J.M. Clements, K.M. Miller, K.W. Wucherpfennig, A.J.H. Gearing, and C.S. Raine, Effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors, Ann. Neurol. 44:35 (1998).

PROTEASES IN DEMYELINATION

M.L. Cuzner Department of Neurochemistry Institute of Neurology University College London WC1N 3BG, U.K.

INTRODUCTION In both peripheral (PNS) and central (CNS) nervous systems a final common pathway of myelin breakdown is followed regardless of the mechanisms initiating demyelination. Myelin proteins and lipids are vulnerable to proteolytic and lipolytic action, following separation or splitting of the tightly compacted membrane lamellae. The hydrolytic agents are enzymes present in serum, secreted by inflammatory cells or lysosomal in origin acting on phagocytosed myelin. The spectra of enzymes include the endopeptidases, serine, carboxyl and thiol proteinases, the metalloproteinases, and a number of exopeptidases. This chapter will attempt to enumerate the enzymes most closely involved with myelin metabolism and breakdown and their sources, to highlight the relative vulnerability of the different myelin proteins and delineate the processes of demyelination.

PROTEASES IN METABOLIC PROCESSING OF MYELIN In all tissues proteases play an important role in protein turnover, in unmasking active sites of enzymes from proenzymes, in the regulation of intracellular protein concentration and in post translational modifications of newly synthesized proteins1. Additionally in the nervous system they have an important role in controlling the release of active neuropeptides and their inactivation at effector sites. Proteolysis is irreversible, unlike other post translational events and can be highly specific. Most proteinases are synthesized as precursors, with little or no proteolytic activity and can regulate their own activation. Thus large amounts of precursor can be present constitutively for activation on demand, Where there are proteinases, there are physiological inhibitors. The initiation of proteolysis is due to endopeptidase activity, located both in lysosomes (cathepsins acting

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at acid pH) and in the cytosol (mostly neutral proteinases). The final stage is the production of dipeptides which can then be cleaved to amino acids. However limited proteolysis is more frequent as, unlike prokaryotes that regulate cellular proteins via gene expression, eukaryotes do so more often by post translational modification. As cells in the adult CNS are generally post mitotic this is of central importance during the different stages of brain development. Myelin is an abundant, relatively metabolically stable membrane of the PNS and CNS2. The lipid composition of both myelins is similar and although they have only one protein in common (Table 1) their molecular architecture varies minimally. The selectivity of myelin loss in immunopathological conditions must be ascribed to fundamental differences in antigenic specificity of the proteins. There are also differences in the structural integrity of the two sheaths. The structure of the CNS depends on the presence of myelin basic protein (MBP), linking the cytoplasmic faces at the major dense line whereas that of the PNS appears to require the Po glycoprotein which spans the bilayer of the compacted lamellae3. Table 1. Protein composition of myelin (%)

Long-term metabolic studies designed to assess the turnover and catabolic rate of myelin proteins have highlighted three features of myelin metabolism4. Molecules entering myelin appear both to have half-lives comparable to those in other membranes and to exhibit long term metabolic stability. Myelin proteins leave the membrane at different rates, indicating that myelin is not degraded as a unit and lipids and proteins radiolabeled during myelination appear more stable than the same components labelled in the adult. Rapid turnover may represent exchange or proteolysis occurring shortly after deposition of a newly synthesized protein before lamellar compaction or localization in an outer cytoplasmic loop or at a nodal region. Slow turnover is probably representative of

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compacted myelin, when exposure to enzymes and metabolic pools occurs only at a limited number of sequestered sites. Turnover rates for individual myelin proteins vary, for example higher -molecular weight proteins localized near cytoplasmic loops or in the periaxonal space are accessible to intrinsic proteases. In contrast MBP of CNS myelin sequestered on the cytoplasmic face of the membrane is protected from proteolysis. Some proteins, such as proteolipid protein (PLP), are very stable in the membrane moving only slowly to areas where they would be exposed to degradation. Matrix metalloproteinases (MMPs), key effectors of extracellular matrix remodelling and regulators of neurite extension, have recently been reported to implement oligodendrocyte process extension during the initial phase of myelinations5. Specifically oligodendrocytes are found to utilize MMP-9 to encourage process extension in vitro along an astrocyte-derived extracellular matrix. In support of this data is the observation that a temporal increase in MMP-9 expression in murine white matter parallels signposts of myelination in vivo.

PROTEASES IN DEMYELINATION IN VITRO The relative metabolic stability of myelin might be expected to impart a significant degree of protection from proteolytic attack. However, there are a number of pathological conditions, which are characterized by demyelination, perpetrated generally by the proteolytic and lipolytic enzymes of phagocytic cells. While useful information can be gained from in vitro assays of proteolytic activity the potential enzymatic capacity may not bear a direct relationship to activity in vivo. Regulatory factors in vivo include cellular or subcellular compartmentation of proteases, their stereochemical and stoichiometric links with substrate, inhibitory and feedback signals and the ionic environment, including pH and specific metal activation 1 . Most tissue homogenates have proteolytic activity with pH peaks in the acid and neutral ranges. Brain is no exception and the inflammatory cells that constitute the hallmark of inflammatory demyelination in the CNS and PNS are also rich in proteases6. The best studied and most widely distributed acid (carboxy1) proteinase in the CNS is cathepsin D, a lysosomal enzyme with a pH optimum of 3.57; the stability however of brain lysosomes under normal circumstances limits autolytic action of lysosomal cathepsins in the CNS. A soluble, neutral Ca++-activated proteinase can be extracted from brain and is considered to be identical to that prepared from extraneural tissue, notably from cardiac and skeletal muscle8. A lysosomal thiol proteinase, cathepsin B9, and the intracellular exopeptidase carboxypeptidase A, have also been reported to be present in normal brain tissue10. MMPs and plasminogen activators (PAs) are constitutively expressed in CNS brain homogenates11. These enzymes all play a role in homeostasis in the nervous system, but the exceptional stability of brain lysosomes and the overall tightly compacted nature of the myelin sheath would not normally expose this membrane structure to high levels of proteolytic activity. Nonetheless myelin-associated neutral proteases have been reported12-14, and once myelin lamellae are disrupted due to entry of serum components or cellular infiltration following blood brain barrier (BBB) leakage, the local environment alters significantly and myelin proteins become vulnerable to attack. There is convincing evidence that electrostatic interactions between basic membrane proteins and acidic phospholipids, can result in changes of conformation resulting in increased stability with respect to proteolytic enzymes15. The constituent phospholipids of isolated myelin are hydrolyzed by crude snake venom and purified phospholipase A2,

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but the co-operative action of phospholipases and proteolytic enzymes, e.g. trypsin, results in a more extensive loss of the basic protein and proteolipid protein and conversion of myelin phosphoglycerides to the corresponding lysocompounds 16 . When injected under the perineurium of the sciatic nerve, both trypsin and phospholipase produce lesions in the myelin sheath, with lamellar splitting and expansion of the myelin structure as observed in electron micrographs. The product of lecithin hydrolysis, lysolecithin, has been demonstrated to produce extensive demyelination both in vivo and in vitro 17 . Not all myelin proteins are equally vulnerable to proteolysis and these will be individually addressed, initially in in vitro systems.

Myelin basic proteins The open conformation of CNS MBP, which is readily soluble as a purified molecule, predisposes it to the action of proteases. It is rapidly digested into a large number of peptide fragments by trypsin, pepsin, chymotrypsin and pronase, and is susceptible to the action of hydrolytic enzymes found in the brain and in inflammatory cells18. Depending on the incubation conditions up to a dozen peptide fragments can be produced from intact MBP19. Detailed peptide patterns are listed in an accompanying table (Table 2). The major acid protease cathepsin D, hydrolyses purified MBP in a limited manner initially into two components of approximately MW 4.5 and 13.5Kd, followed in time by the appearance of a new antigenic determinant when the larger peptide is further degraded20,21. Although the rate of digestion varies according to the cellular source of the enzyme, the peptide spectrum produced is common to all. Neutral proteolytic activity towards MBP is associated with myelin itself, as well as being present in brain cytoplasmic fractions, and both Ca++-dependent and Ca++-independent activity have been reported13. MBP that dissociates from myelin incubated at neutral pH is hydrolyzed, in a limited fashion, with the appearance of two/three fragments14 . The major neutral proteases of leukocytes are cathepsin G, elastase, collagenase and u-PA22,23 and although the rate of hydrolysis varies according to the cell type, whether macrophages in different states of activation or granulocytes the pattern of proteolytic fragments of MBP produced is similar reflecting a limited sequential hydrolysis24. Metalloprotease activity, which is generally associated with modelling of the extracellular matrix, has been documented in myelin and in both macrophages and microglia25,26. Guinea pig MBP is digested by MMP-9 into 6 major bands on electrophoresis with cleavage sites within encephalitogenic epitopes. Therefore, production of this enzyme in the CNS may constitute an important, if non-specific, pathogenic mechanism for both the disruption of the BBB and of the myelin sheath11. As all the enzymes are secreted as precursors and activation is initiated by plasmin, itself generated through proteolytic activation by PAs, regulation of the PA-MMP cascade of activity is stringent. There are at least 6 isoforms of MBP, whose relative susceptibilities to proteolysis have not been explored, although this could represent another level of control in the developing CNS. The P1 basic protein of PNS myelin is identical to the 18Kd MBP from CNS myelin while the tryptic peptide map of the 14Kd P2 protein is unique18. The P2 protein does however have similarities to the CNS MBP, in that it is highly basic, easily extracted with acid and digestible by the same proteolytic enzymes.

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Proteolipid proteins The hydrophobicity of the transmembrane PLP renders the molecule more resistant to enzymatic cleavage than MBP, but this resistance is not absolute and there are in vitro conditions, although somewhat unphysiological, under which the protein is digested. Crude PLP and the delipidated apoprotein are digested 10% and 40% respectively by trypsin, producing a peptide map with 17-18 ninhydrin-positive spots27. Elastase was found to be the only other protease capable of digesting crude PLP, but oxidized PLP and apoprotein were also susceptible to digestion by chymotrypsin and thermolysin. These observations highlight the way in which the microenvironment or the form in which the myelin proteins are presented to the enzymes will influence proteolysis. The major protein of PNS myelin is the Po protein, a glycoprotein with a 6% by weight carbohydrate content18. Like PLP it is very hydrophobic but trypsin digestion produces a 19Kd glycoprotein component from the 28-30Kd protein, removing the hydrophilic portion that represents approximately 30% of the total protein. The carbohydrate entity can also be released in soluble form by proteolytic digestion.

Wolfgram protein (CNP) The highly conserved Wolfgram protein constitutes two closely spaced molecules of approximately 46 and 48Kd and represents the activity of the myelin specific enzyme 2',3' -cyclic nucleotide 3' phosphodiesterase (CNP)28. CNP has a higher turnover than other myelin proteins, possible due to its asymmetric distribution in myelin and association with tongue processes and paranodal loops but is not as sensitive to proteolytic digestion as MBP. Following spinal cord compression, a progressive decrease in Wolfgram protein over 72 hours was matched by a comparable loss in CNP activity29. However, leukocyte neutral proteases have also been reported to cause degradation of the protein which is cleaved by treatment with elastase at carboxyl residues 149 and 385, the 26Kd elastase fragment retaining its CNP activity30.

Myelin glycoproteins Glycoproteins are quantitatively minor components of the myelin sheath but two well-characterised ones are reputed to play an important role in the molecular architecture and properties of the membrane. Myelin-associated glycoprotein (MAG) with a molecular weight of 100Kd constitutes 1% of myelin protein. A neutral proteinase associated with highly purified CNS myelin selectively degrades MBP and converts MAG to a smaller derivative, dMAG, with a molecular weight less than 100,000Kd12. Incubation of buffered human myelin at 25°C resulted in a conversion of half of the glycoprotein in 30 minutes, whereas degradation of half of the MBP required 18 hours. There was no significant loss of the PLP, the Wolfram doublet or other myelin proteins for up to 18 hr under these conditions. The endogenous proteolytic activity is not affected by protease inhibitors, which indicates a close association with the myelin membranes thus preventing soluble inhibitors from reaching the active site. All of the detectable degradation products of MBP were present in the supernatant, but no intact MAG was detected in the supernatant, and about half of the dMAG remained associated with the particulate fraction after a 30-min incubation.

14,25

MBP dissociated from rat, human and guinea pig myelin is hydrolysed with the production of two major polypeptides 1-73 and 74-170, the formation of which is inhibited by phenanthroline and DTT but not by inhibitors of serine or cysteine proteases

purified myelin

1.1.4Metalloendoprotease

1.2.1 Neutral proteases (proteinase 3, u-PA) (elastase, cathespsin G)10 (collagenase)

24,3 5,84

12

Incubation of rat or human myelin at pH 7.6 results in 50% degradation of MBP and 50% conversion of MAG in 30 minutes to dMAG with a molecular wt 10Kd less, which is stable and remains associated with myelin membrane

integral, myelin-associated

1.1.3 Neutral proteinases

neutrophils (PMN) PMN soluble extract hydrolyzes greater than 50% MBP and Wolfgram protein in isolated myelin. Plasmin degrades up to 70% of MBP in myelin with the appearance macrophage, microglia of 2 major proteolytic fragments. In the absence of plasminogen the decrease in MBP is less (39%). Purified MBP is hydrolyzed by cell homogenates of PMNs and activated macrophages to produce 3 major and 1 minor proteolytic fragment.

19

Sequential hydrolysis of crude MBP occurs with the production of 12 proteolytic fragments in molecular weight range 17.5 to 6Kd. Inhibitor pattern points to activity of cysteine, metallo - and/or serine proteases

acid extract of bovine brain

1.1.2 Neutral proteinases

1.2. Leukocyte - associated

13

Incubation of rat spinal cord myelin at pH 7.6 with or without Ca++ results in 60% or 30% degradation respectively of MBP over 24 hours, whereas incubation with Triton - X 100 leads to preferential loss of PLP (60%) and DM-20.

cytoplasmic myelin-associated

1.1.1. Neutral proteinases Ca++-independent and Ca++-activated

Ref.

Hydrolytic products

Enzyme source

1.1 CNS - associated

1. Neutral proteinases

Table 2. Proteolysis of myelin proteins

plasma

1.3.2.Thrombin

Lysosomes

Lysosomes

2.1 CNS - Associated

2.2 Leukocyte associated

2. Acid proteinase (Cathepsin D)

serum but not plasma

leukocytes microglia

1.3.1. Neutral proteases

1.3 Serum/plasma associated

1.2.2. Metalloproteinases (MMP-2, MMP-9)

The cathepsin activity in PMN and macrophages resembles that of brain, with the appearance of 3 major peptides 1-43,43-88 and 89-169. The ranking of specific activity is macrophage >> PMN > brain

MBP undergoes sequential but limited proteolysis initially with the appearance of two components, 1-42 and 43-169, and subsequently with peptides 1-36 and 43-88, -89, -92 and 89-, 92- 169, resulting in appearance of a new antigenic determinant 2.2.1. Leukocyte associated

Arg-X bonds at a single site of MBP, 95-96 are cleaved under mild conditions increasing to 8 sites under more stringent conditions.

Incubation of MBP in serum results in loss of encephalitogenicity and appearance of fragments similar to those produced by trypsin

Pure MBP is hydrolyzed by MMP-9 with two cleavage sites at residues 92-93 and 116- 117, both of which are within the encephalitogenic epitopes of SJL/J mice and guinea pig respectively

24

20,21

55

54,56

26,37

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The susceptibility of MAG and MBP to cleavage by the endogenous proteinase suggests that in addition to inflammatory processes, auto-degradation may have a role in demyelinating disease. The two proteins were found to break down more rapidly on incubation of multiple sclerosis (MS) myelin in comparison to myelin from control brain12. If periaxonally localized MAG is involved in interactions between myelinated axons or in maintenance of the cytoplasmic collar around myelin sheaths or in the promotion of neurite outgrowth, proteolytic alteration of MAG could disrupt these processes. MAG preparations from the PNS and CNS showed the same peptide maps after digestion with three different proteases18. Myelin oligodendrocyte glycoprotein (MOG) constituting only 0.1% of myelin protein and a member of the immunoglobulin superfamily31 was originally identified by a mouse monoclonal antibody against rat cerebellar glycoproteins, which on intravenous injection greatly augmented CNS-specific demyelination32. MOG localization on the outer surface of the myelin sheath provides an ideal target for hydrolytic attack, but there are no documented studies on its susceptibility to proteolysis or its fate in demyelinating pathologies.

PROTEASES IN DEMYELINATION IN VIVO The biochemical changes in MS and experimental allergic encephalomyelitis (EAE) have been documented extensively6,24. In the demyelinated plaque myelin proteins and lipids are almost completely replaced by the glial fibrillary acidic protein of astrocytic fibrils. In lesions with ongoing demyelination myelin is apparent within macrophages in the hypercellular zone between normal-appearing white matter and the plaque centre and histochemical, immunocytochemical and biochemical analyses show clearly the preferential loss of myelin basic protein and CNP-ase activity in the presence of both acid and neutral proteolytic activity. In the established chronic lesion lysosomal enzyme activity persists and CNP-ase is dramatically decreased. Even in normal-appearing white matter there is evidence of biochemical change, with increases in lysosomal hydrolase activity and a diffuse widespread decrease in myelin proteins, accompanied by gliosis. In the active MS plaque, in which proliferating astrocytes and macrophages are present, an increase is found in a wide spectrum of lysosomal hydrolases, cathepsin D, and acid phosphatase. ß-glucuronidase, arylsulfatase, plasmalogenase, phospholipase A2, and carboxypeptide A and B and in secreted neutral proteases, including PAs and MMPs11 . The finding of increased MMP-9 activity in the CSF in MS33,34 and of the vulnerability of myelin proteins such as MBP to digestion by proteases, notably plasmin35and MMP-936,37 has stimulated work on the cellular pathology of these enzymes in MS38-40. In the demyelinating MS lesion expression of t-PA and u-PA is prominent in foamy macrophages, as is that of components of the MMP cascade (MMP-2 and 9)39. Lymphocytes and macrophages appear strongly positive in the perivascular cuff in active plaques, the staining extending to macrophages and reactive astrocytes throughout the hypercellular zone radiating from the plaque center. u-PA forms a unique combination of enzyme and chemotactic factor upon interaction with its surface receptor u-PAR inducing focal pericellular proteolysis and promoting cell adhesion and migration41. The generation of plasmin, directed to the cell-matrix interface by the action of the C terminus of u-PA42 is a rate-limiting step in the activation of the MMP cascade. MMPs degrade basement membrane and the extracellular matrix and promote extravasation of leucocytes 43, but they

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13

also have the capacity within the CNS to cleave myelin basic protein (MBP) into fragments retaining encephalitogenicity 36,37,44 . Nine MMPs have been identified by cDNA cloning and sequencing, including forms of collagenase, gelatinase and stromelysin. Two separate but similar cDNAs encode a 72Kd gelatinase A and a 95Kd - gelatinase B and production of the latter by macrophages or neutrophils is regulated by cytokines 45. Serine proteases and MMPs are secreted as inactive precursors together with plasminogen activator inhibitors (PA1) and tissue inhibitors of metalloproteases (TIMPs), ensuring that local activation is stringently controlled 45,46 . Another family of proteinases which now attracts intense research interest are the caspases, cysteine proteases which execute apoptosis47. They are among the most specific of proteases, with an absolute requirement for cleavage after aspartic acid and substrates to date are proteins associated with the nucleus; there is no documented effect on myelin proteins. Based on higher than normal protease activity in areas of gliosis, astrocytes would appear to be an important source of proteolytic enzymes, while enhanced cathepsin A activities in MS brain are apparently associated with macrophages. In a microanalytical study 48 Hirsch found that enzymatic changes were not observed in grossly normal white matter despite the presence of patchy cellular infiltrates and areas of gliosis. The conclusion drawn was that a primary role should not be attributed to lysosomal or cytosolic CNS proteinases in plaque formation and that elevated activity was likely to be the result of inflammatory cell invasion of the tissue. More recent studies appear to contradict this conclusion as immunocytochemical studies suggest that the very early MS lesion consists of focal areas of activated microglia with internalised MBP but without obvious myelin loss around cells, preceding an inflammatory lymphocytic reaction49,50. The functional role of the PA - MMP cascade in cell migration may influence the development of these microglia foci and their progression to hypercellular demyelinating plaques. The increase in proteolytic enzyme activity in the CNS in EAE also appears to be primarily due to cellular infiltration as it is greatest in the hyperacute localized lesions observed in primates with EAE. The increments are greater in respect of cathepsin A than acid proteinase, reflecting the difference in levels of these enzymes between lymph nodes and brain stem, and are not seen until the onset of symptoms which corresponds to the time of cellular infiltration. Both acid and neutral proteinase levels are increased by approximately 130-180 percent, compared to unaffected areas, while cathepsin A is several-fold greater 51. In experimental models of CNS inflammation, in particular EAE, there is in general a correlation between mRNA levels and protein expression of MMPs which is localized to the sites of inflammation 11 . The spectrum of MMPs varies according to the cellular nature of the lesion, reflecting the proportions of neutrophils, lymphocytes and macrophages in the perivasculature and possibly the makeup of the extracellular matrix, although the majority of MMPs have a broad substrate specificity. Of the inhibitors TIMP-1 mirrors most accurately the expression of MMPs at sites of inflammation, the most prominent of which are matrilysin, MMP-9 and metalloelastase.

MECHANISMS OF DEMYELINATION The myelin sheath can be disrupted by soluble mediators from the circulation or

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secreted by inflammatory cells, through activation of enzymes associated with the myelin itself or by phagocytic processes. In autoimmune models of demyelination, such as EAE, mononuclear cell processes can be seen to penetrate between the myelin lamellae near the outer loop and phagocytosed myelin is observed within coated pits and vesicles 52. Vesiculation and swelling of myelin sheaths are also very early features of demyelination in EAE. This begins with a splitting at the intracellular apposition, followed by a curling of the lamellar fragments to form loops or vesicles 53. These are then phagocytosed by what appears to be a receptor-mediated mechanism. However demyelination is not extensive in EAE unless mediated by anti-MOG antibody or through chronic inflammation.

Systemic mediators of myelin damage In general the process of demyelination is preceded by permeabilization of the BBB and blood nerve-barrier (BNB). Serum and plasma contain proenzymes which upon conversion have the capacity to cause disruption of the myelin sheath despite the presence in blood of proteinase inhibitors which inactivate neutral proteinases 54,55. Incubation of isolated myelin with human or rabbit serum resulted in 50% loss of MBP, while no breakdown of other major myelin proteins was observed. A smaller loss of MBP was also observed upon incubation of brain slices with serum 56. Activation of complement may be responsible for myelin breakdown by serum as isolated rat and human myelin consume complement in the absence of specific antibodies and sera heated to inactivate complement only induce myelin swellings 57. Not only is complement activated by isolated rat CNS myelin but the activation proceeds via the terminal component C9 to the formation of the membrane attack complexes of complement58 and myelin is lysed following pore formation in the lamellae52. There is also evidence that tissue culture demyelination can result from nonimmunoglobulin activation of the alternate complement pathway. The demyelinating activity was heat labile at 50°C, which leaves the classic complement pathway intact but inactivates properdin factor B, a crucial component of the alternate pathway59.

Cell-mediated myelinolysis in CNS demyelination The predominant cellular route of myelin breakdown is via macrophages, although polymorphonuclear leukocytes participate in acute haemorrhagic lesions, and Schwann cells are involved in the PNS. Astrocytes probably also contribute, particularly in later stages as they are observed with ingested myelin in MS plaques 60. Activated macrophages possess a wide range of receptors which can effect myelin endocytosis 61. In vitro myelin is efficiently taken up by macrophages in the absence of specific antibody, and this uptake can be inhibited by zymosan, (a bacterial cell wall product), oxidized lipoprotein and by antibodies which block complement receptors, intimating the involvement of the mannose/fructose and scavenger receptors as well as the complement receptor, CR3. In serum-free media opsonization of myelin with a mixture of antibodies directed against individual myelin proteins results in a greater degree of phagocytosis but this can be blocked more efficiently by zymosan than by competing immune complexes. This suggests that Fc-receptor mediated mechanisms may not be the major route for demyelination in vivo. In view of the search for the antigenic specificity of the intrathecally produced immunoglobulins in MS, this is an important consideration. MS lesions are characterised by inflammatory macrophages 62 but as it is difficult to identify the earliest lesion, the mechanisms of demyelination are more difficult to decipher

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15

in the human disease. There is however evidence that macrophages containing material staining with antibodies against MBP epitopes and neoepitopes are detectable in areas of white matter with no apparent myelin loss and where there is no lymphocytic infiltration suggesting a primary role for macrophages in the demyelinating process in MS63. There are few reports of direct T cell-mediated demyelination, the emphasis being placed on recruitment of effector cells by lymphocytes sensitized to brain antigens. Intraocular injections of supernatants from non-brain specific activated T cells in the rabbit only cause retinal fibre demyelination in animals sensitized to spinal cord or in the presence of serum from sensitized animals64. However, damage to myelinated cultures produced by lymphocytes sensitized to peripheral nerve has been observed in vitro65, and in isolated rat optic nerve, MBP specific T lymphocytes alone were capable of blocking action potentials, providing both T cells and optic nerves were HLA compatible 66. When a particle is physically larger than the macrophage or microglia, internalization of myelin cannot occur and "frustrated" phagocytosis or reverse endocytosis results in discharge of lysosomal enzymes. In a system modelling extracellular phagocytosis in the CNS, lysosomal enzyme release is accompanied by production and secretion of lactic acid and a subsequent drop in pH 67. At local pockets of low pH interstitial fluid may penetrate the lamellae, leading to reversible disruption of the myelin ultrastructure. In the model system turbidity changes associated with acidification were consistent with an increase in the size of the multilamellar myelin particles (ie. swelling). Hence proteolytic attack on MBP could be secondary to myelin degeneration and the primary pathological process in inflammatory demyelination a spontaneous disruption of lamellae in response to localized macrophage hyperlactemia. Histochemical analysis of MS brain tissue has shown that lactic dehydrogenase activity is increased in plaques 68.

Cellular sources of proteases Transcriptional and translational control, activation of latent enzymes and specific inhibitors influence to a great extent the cellular distribution of proteinases. An important issue in the biological context is constitutive production versus induction. Some enzymes are constantly produced, although at low levels only. For instance MMP-2 is produced by most cell types in a constitutive way in contrast to MMP-9 which is induced by specific agonists11. Also relevant to the PA-MMP cascade action in vivo, is the ubiquitous presence of plasminogen, regulating plasmin-mediated conversion through the activators u-PA and t-PA. Astrocytes in culture produce u-PA constitutively while t-PA, initially high in the control, is downregulated by proinflammatory cytokines69 . Furthermore, proteinases can be targeted to specific membrane sites by protein sequences in the transmembrane domains of an enzyme as for example with membrane type-MMPs70 which activate MMPs downstream. Secreted proteases such as u-PA may be confined to the edge of migrating cells by specific receptors. Finally, proteinase activity may be localised to extracellular matrix, as in the case of the adamalysin-disintegrin metalloproteases71. Neutrophils are a rich source of proteolytic activity. Human neutrophils are rich in neutral proteinases, the two major components of which are elastase and cathepsin G72. Degranulation of preformed neutrophil MMP-8 and MMP-9 by CXC-chemokines is a fast phenomenon. MBP is highly susceptible to digestion at pH7.6 by neutrophils while there is negligible digestion by acid proteinases of these cells24.

16

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Macrophages alter their phenotype upon encountering inflammatory or immunologic stimuli becoming phagocytic and enriched in lysosomal hydrolases and secreting the neutral proteinases, elastase, collagenase and u-PA, which can be plasma membrane bound or in a secreted form72,73. The steps leading to activation may be initiated by the interaction of specific receptors with cytokines or components of complement or immunoglobulins. These can be early or late effects, the former including triggering of the respiratory burst and arachidonate metabolism. The late responses which require the generation of cytoplasmic messengers will include protease synthesis. Stimulated production by Fc receptor occupation is only seen when initial secretion rates are low. Complement coated particles bound, but not ingested, stimulated only elastase secretion transiently, which may reflect a release from intracellular stores, rather than stimulating accelerated enzyme synthesis. The acid proteinase, cathepsin D, also plays an important part in the digestion of proteins taken up by macrophages, in the acid environment of the lysosomal system7 . However, while the acid proteinase in guinea pig activated peritoneal macrophages is increased three-fold over that in resting cells, and is much higher than neutral proteinase activity with traditional substrates, the difference in the rate of digestion of purified MBP at neutral and acid pH is not significant24 . The rate of digestion of MBP by brain homogenates is greater at acid pH, possibly due to lysosomal hydrolase activity in astrocytes, and the high molecular weight peptide pattern is similar to that generated by phagocytic cell acid hydrolysis. Conditioned media from microglia has been found to contain an elastase-like protease which hydrolyzes MBP74 and a urokinase-type PA which markedly increases MBP degrading activity in the presence of plasminogen. Secretion of the PA from microglia was enhanced by interleukin-1 and basic fibroblast growth factor. Microglia secrete a wide range of MMPs, including MMPs-2 and 9 upon activation by chemokines and cytokines. Thus, a battery of proteinases at both acid and neutral pH with myelinolytic activity is present in phagocytic cells, while little digestion of myelin proteins is effected by human lymphocytes or products of activated rabbit lymphocytes at either pH75. As a result of its immunogenicity in EAE the processing of MBP by cells and proteolytic enzymes has been the most extensively studied. Nonetheless MBP is the myelin protein with by far the greatest sensitivity to proteolytic action and although all other myelin proteins are susceptible in vitro to hydrolysis they are considerably more resistant to attack in vivo.

PNS demyelination Macrophages also form an important part of the cellular response to peripheral nerve injury76,77. In the PNS during nerve fibre degeneration both Schwann cells and macrophages participate in the process of myelin degeneration. Initially the myelin sheath is interrupted by Schwann cell processes which then fragment into ovoids and ellipsoids. Phagocytic macrophages appear later, and are essential for myelin clearance, as shown elegantly in a study of transected nerve segments in intraperitoneal millipore chambers which regulate the entry of macrophages78. In vivo macrophages enter PNS nerve fibres during Wallerian degeneration and participate in myelin removal in a similar fashion to that in the CNS but at a much more rapid rate. The differences between the CNS and PNS may be reflected in the origin and timing of the signal that attracts the macrophage. They are recruited in significant numbers in the first 3-5 days, following nerve crush, restricted to the

Proteases in Demyelination

17

region containing degenerating axons, and then phagocytose myelin, to become foamy macrophages76 . When pieces of peripheral nerve are placed in chambers within the peritoneal cavity, or when teased fibres are placed in culture, the Schwann cells extrude their myelin through the basement membrane surrounding the fibre and thus allow macrophages to phagocytose myelin78 . A consensus points to macrophages playing the major role in phagocytosing PNS myelin in vivo. Extracellular myelin degradation must also be considered as macrophages recruited outside the nervous system are know to secret potent myelinolytic neutral protease activities. The slow rate of Wallerian degeneration in the CNS has been well documented, but there is little evidence to show why this might be the case. Results suggest that limited recruitment of macrophages after injury might be important because it is these cells that play a major role in myelin removal in the PNS79. However in a mutant mouse, in which Wallerian degeneration and monocyte recruitment are extremely slow the evidence suggests that the gene product of the autosomal dominant mutation affects the nerve per se. Nonetheless the paucity of regeneration may be ascribed to a decline in the synthesis of nerve growth factor which can be demonstrated in culture by the inclusion of macrophages. It is not clear why there should be such limited recruitment in the optic nerve but differences between the BNB and BBB.

PROTEINASE INHIBITORS OF INFLAMMATORY DEMYELINATION From the evidence that increased neutral and acid proteolytic activities are associated with demyelinating lesions in the CNS and PNS, probably originating from the macrophages in the cellular infiltrate, proteinase inhibitors have been tested both in the experimental model, EAE, and in MS. Proteolytic enzymes may be necessary at several steps of the sensitization process in the development of EAE including the activation of the immunologic process, the invasion of cells into the brain parenchyma, as well as the final dissolution of myelin. The first reports in the literature indicating some degree of success with proteolytic inhibitors in suppressing EAE showed that pepstatin, an inhibitor of acid proteinase will suppress the clinical signs as well as the incidence of lesions of EAE in the Lewis rats80 . In another report amino caproic acid, an inhibitor of plasminogen activator, given in large amounts suppressed both the paralysis and lesions of EAE and in Lewis rats 81 . A large series of experiments to identify proteolytic inhibitors and their effects on EAE have been carried out in two laboratories, those of Marion Smith82 and Celia The inhibitors tested were pepstatin, aprotinin, B r o s n a n 83. trans-4-(aminomethyl)-cyclohexanecarboxylic acid (AMCA), e-amino-n-caproic acid (EACA), nitrophenyl p-guanidinobenzoate (NPGB), leupeptin and antipain. Pepstatin is a peptide which has been found to inhibit pepsin and cathepsin D at very low concentrations. Aprotinin (trasylol), inhibits elastase, trypsin, chymotrypsin, and plasmin, but not plasminogen activator. Leupeptin inhibits various neutral proteases including trypsin, plasmin, papain, and cathepsin B; antipain, inhibits trypsin, papain, and cathepsins A and B. AMCA, EACA, and NPGB inhibit a variety of neutral proteases including plasminogen activators. Some inhibitors of proteases, particularly those active at neutral pH were effective in inhibiting the clinical symptoms of EAE and in some instances in decreasing the incidence and severity of CNS lesions. Three of the most effective AMCA, EACA, and NPGB act on the plasminogen activators system, a pointer to its

18

M. L. Cuzner

importance in the demyelinating process. Several enzyme systems are probably involved at various steps in the sensitization and demyelinative processes in EAE as partial protection is also provided by pepstatin. These results may be taken as evidence of a role for proteolytic enzymes in the primary attack on the myelin sheath, notably hydrolysis of basic protein. Despite observed increases in proteolytic enzyme activity in the CSF and inflammatory lesions of MS patients with active disease84 , no matching increase in the circulating inhibitors of neutral proteinases, a2-macroglobulin and a1 -antitrypsin has been observed 85 . In view of the putative role of plasminogen activator in promoting demyelination, an open trial of EACA was carried out in the U.S. and in Italy but inconclusive results discouraged continuance of this approach to therapy of MS86 . Renewed interest in the role of MMPs in promoting the traffic of leukocytes into the CNS has raised the possibility that inhibitors of these enzymes could modify the inflammatory process in the CNS. The metalloproteinase cascade is highly regulated by cytokines, which may also induce TIMPs 11 . For example IL- 1 can promote local synthesis of proteinases, while IL-6 and TGF-ß both induce TIMP production. Recent efforts to alleviate clinical signs of EAE have focused on synthetic inhibitors of MMPs. One hydroxamate inhibitor, GM6001, was found to suppress the development of EAE or reverse established clinical symptoms respectively, when administered prophylactically or therapeutically 87. A broad spectrum MMP inhibitor, BB- 1101 was effective in reducing the severity of EAE in the Lewis rat88 , and completely blocked the onset of EAE and reversed severe acute disease in the SJL/J mouse89 . Chronic relapsing EAE was also significantly modulated, with clinical improvement accompanied by a reduction in demyelination and glial scarring. The hydroxmate MMP inhibitor Ro31-9790 reduces the severity of EAE induced both by primary sensitization and following transfer of MBP-primed splenocytes in the Lewis rat, in both cases with a good correlation between clinical severity and histopathology 90 . In the actively induced model the beneficial effect was greatest in animals with moderate clinical signs, declining in those with more severe symptoms. MMP inhibitors might be expected to work at two levels - by blocking the extravasation to the CNS and effector properties of lymphocytes and macrophages in the initial stage of inflammation and by limiting the myelin loss in the established lesion. More specifically, the inhibitors appear to prevent degradation of extracellular matrix and basement membrane but seem to have no influence on the priming of MBP-specific T cells, as the course of clinical disease becomes essentially the same in inhibitor-treated as in vehicle-treated animals after cessation of treatment 87 . The clinical benefits of interferon (IFN)ß- 1b in the treatment of MS patients may be due, at least in part, to its ability to reduce the MMP-9 activity of T lymphocytes, resulting in their decreased migration91 . Results demonstrate that IFNß- 1 b treatment in vitro significantly decreases the migration of activated T cells through a fibronectin matrix. Migration of T cells was affected by IFNß-1b concentrations ranging from 10 to 1,000 IU/ml, concentrations which can be reached in serum following the systemic administration of 8 MIU of IFNß-1b in MS patients. All tested MMP inhibitors to date are broad spectrum and do not target individual metalloproteases hence the need to define selective and specific inhibitors of key enzymes.

Proteases in Demyelination

19

CONCLUSION Proteases in demyelination are effector molecules for leukocyte extravasation and in the myelinolytic process. As detailed in earlier sections myelin proteins, in particular basic protein are vulnerable to digestion by proteases acting at both neutral and acid pH, generally by limited sequential hydrolysis. In the case of MBP peptides in the molecular weight range 5- 10K are generated, and when isolated myelin is the substrate these peptides are released into the incubation medium. In contrast, the high molecular weight product of MAG proteolysis remains associated with the myelin membrane. In the inflammatory lesion macrophages are the most common source of these proteases, although neutrophils characterize the very acute demyelinating lesion, precipitated by administration of anti-MOG antibody. The neutral proteases elastase, cathepsin G and MMPs released from neutrophil granules are prime candidates for degrading myelin, on the assumption that BBB leakage has resulted in myelin lamellar splitting. Acid proteolytic activity would not be expected to play a large part initially, but as myelin is phagocytosed local changes in pH following fusion with lysosomes would enhance acid proteolytic activity. Macrophages have little proteolytic activity unless stimulated and the major neutral proteases with the capacity to hydrolyze myelin proteins are in a secreted form and would be predicted to act on myelin proteins extracellularly. In the situation when activated macrophages display increased expression of Fc, complement and scavenger receptors leading to myelin uptake in both a specific and non-specific manner lysosomal catheptic digestion of myelin proteins may supervene. Solubilized MBP would then be released into the cell cytoplasm. A limited amount of lysosomal hydrolase activity would be predicted to occur extracellularly in the presence of local acidic microenvironment. These reactions reflect the end point of the inflammatory demyelinating process and the mechanisms controlling the extent of demyelination in the CNS may be at the level of macrophage activation, whether specific or non-specific. Although myelin proteolysis is considered to be downstream from the priming of inflammation in immune-mediated diseases of the CNS and PNS, the metalloproteinases that influence the modelling of the extracellular matrix at the BBB and control entry of leukocytes into the CNS in the primary stage of immunological events are also capable of hydrolyzing myelin basic protein which could lead to release into the circulation of immunogenic peptides. Although some cytokines, notably tumour necrosis factor, are reported to act directly on myelin92, the major regulation of demyelination will be through the cytokines and other stimuli of macrophage activation, in this case most notably phagocytosis of myelin itself. Hence approaches to inhibiting of myelin breakdown per se are directed at specific inhibition of the proteases effecting demyelination.

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58. B.A. Silverman, D.F. Carney, C.A. Johnston, P. Vanguri, and M.L. Shin, Isolation of membrane attack complex of complement from myelin membranes treated with serum complement, J. Neurochem. 42: 1024-1030 (1984). 59. D.H. Silberberg, M.C. Manning, and A.D. Schreiber, Tissue culture demyelination by normal human serum, Ann. Neurol. 15:575-580 (1984). 60. J.W. Prineas, The neuropathology of multiple sclerosis, in: Handbook of Clinical Neurology, J.C. Koetsier, ed., Elsevier Science Publishing, pp.213-257 (1985). 61. K. Mosley and M.L. Cuzner, Receptor usage in myelin phagocytosis by microglia and macrophages, Neurochem. Res. 54:185(1994). 62. M.L. Cuzner, G.M. Hayes, J. Newcombe, and M.N. Woodroofe, The nature of inflammatory components during demyelination in multiple sclerosis, J. Neuroimmunol. 20:203-209 (1988). 63. H. Li, J. Newcombe, and M.L. Cuzner, Characterisation and distribution of phagocytic macrophages in MS plaques, Neuropathol. Appl. Neurobiol. 19:214-223 (1993). 64. C.F. Brosnan, G.L. Stoner, B.R. Bloom, and H.M. Wisniewski, Studies on demyelination by activated lymphocytes in the rabbit eye. II. Antibody-dependent cell mediated demyelination, J. Immunol. 118:2103-2110 (1977). 65. B.G.W. Arnason, G.F. Winkler, and N.M. Hadler, Cell-mediated demyelination of peripheral nerve in tissue culture, Lab. Invest. 21 : 1 -10 (1969). 66. Y. Yarom, Y. Naparstek, V. Lev-Ram, J. Holoshitz, A. Ben-Nun, and I.R. Cohen, Immunospecific inhibition of nerve conduction by T lymphocytes reactive to basic protein of myelin, Nature 303:246-247 (1983). 67. P.R. Young and A.P. Zygas, Secretion of lactic acid by peritoneal macrophages during extracellular phagocytosis - The possible role of local hyperacidity in inflammatory demyelination, J. Neuroimmunol. 15:295-308 (1986). 68. H.E. Hirsch, P. Duquette, and M.E. Parks, The quantitative histochemistry of multiple sclerosis plaques: acid proteinase and other acid hydrolases, J. Neurochem. 26:505-512 (1976). 69. A. Faber-Elman, R. Miskin, and M. Schwartz, Components of the plasminogen activator system in astrocytes are modulated by tumor necrosis factor-a and interleukin-lb through similar signal transduction pathways, J. Neurochem. 65:1524-1535 (1995). 70. H. Sato, T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki, A matrix metalloproteinase expressed on the surface of invasive tumour cells, Nature 370:61-65 (1996). 71. Z. Werb, ECM and cell surface proteolysis: regulating cellular ecology, Cell 91:439-442 (1997). 72. C.A. Owen and E.J. Campbell, The cell biology of leukocyte-mediated proteolysis, J. Leukocyte Biol. 65: 137-150 (1999). 73. C.G. Ragsdale and W.P. Arend, Neutral protease secretion by human monocytes - Effect of surface-bound immune complexes, J. Exp. Med. 149:954-968 (1979). 74. K. Nakajima, N. Tsuzaki, M. Shimojo, M. Hamanoue, and S. Kohsaka, Microglia isolated from rat brain secrete a urokinase-type plasminogen-activator, Brain Res. 577:285-292 (1 992). 75. H.M. Wisniewski, H. Lassmann, C.F. Brosnan, P.D. Mehta, A.A. Lidsky and R.E. Madrid, Multiple sclerosis: Immunological and experimental aspects, in: Recent Advances in Clinical Neurology, W.B. Matthews, G.M. Glack, eds., Churchill, London, pp. 95-125(1982). 76. V.H. Perry, M.C. Brown, and S. Gordon, The macrophage response to central and peripheral nerve injury, J. Exp. Med. 165:1218-1223 (1987). 77. G. Stoll, B.D. Trapp, and J.W. Griffin, Macrophage function during Wallerian degeneration of rat optic nerve: clearance of degenerating myelin, J. Neurosci. 9:2327-2335 (1989). 78. W. Beuche and R.L. Friede, The role of non-resident cells in Wallerian degeneration, J. Neurocytol. 13:767-796 (1984). 79. V.H. Perry and S. Gordon, Macrophages and the nervous system, Int. Rev. of Cytology 125:203-244 (1 99 1). 80. D.H. Boehme, H. Umezawa, G. Hashim, and N. Marks, Treatment of experimental allergic encephalomyelitis with an inhibitor of cathepsin D (pepstatin), Neurochem. Res. 3: 185-194 (1978). 81. W.A. Sibley, S. Kiernat, and S.F. Laguna, Modification of experimental allergic encephalomyelitis with EACA, Neurology 28:102-105 (1978). 82. M.E. Smith, Proteinase inhibitors and the suppression of EAE, in: The Suppression of Experimental Allergic Encephalomyelitis and Multiple Sclerosis, A.N. Davison, M.L. Cuzner, eds., Academic Press, New York, pp.211-226 (1980). 83. C.F. Brosnan, W. Cammer, W.T. Norton, and B.R. Bloom, Proteinase inhibitors suppress the development of EAE, Nature 285:235-238 (1980). 84. M.L. Cuzner, A.N. Davison, and P. Rudge, Proteolytic enzyme activity of blood leucocytes and CSF in multiple sclerosis, Ann. Neurol, 4:337-344 (1978). 85. P. Price and M.L. Cuzner, Proteinase inhibitors in cerebrospinal fluid in MS, J. Neurol. Sci. 42:251-259 (1979). 86. L. Amaducci, C. Arfaioli, R. Capparelli, D. Inzitari, D. Sita, P. Antuono, P. Zaccara, A. Doni, G. Lippi, and G. Leoncini, The clinical use of epsilon-amino caproic acid in MS, in: The Suppression

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CALCIUM ACTIVATED NEUTRAL PROTEINASE IN DEMYELINATING DISEASES

Donald C. Shields and Naren L. Banik Department of Neurology Medical University of South Carolina Charleston, S.C. 29425

INTRODUCTION The myelin sheath functions as an insulator to aid impulse conduction along axons. Myelin is synthesized by oligodendrocytes in the central nervous system (CNS) and by Schwann cells in the peripheral nervous system (PNS). Physical trauma or neurodegenerative disease processes affecting the white matter result in degradation of the myelin sheath with resulting impairment in axonal impulse conduction. In addition to sensory and motor function losses, biochemical changes including release of proteinases and lipases are commonly observed in the white matter or the myelin sheath following an insult. Although the mechanism(s) by which myelinolysis occurs has not been completely elucidated, the release of proteinases is believed to be at least one factor in this process. Various studies have provided evidence of proteolytic enzyme involvement in myelinolysis associated with demyelinating disorders. Myelin protein degradation has been implicated in chemical-induced demyelination, myelin degeneration in experimental allergic encephalomyelitis (EAE), and separation of myelin lamellae with splitting of the intraperiod line in Wallerian degeneration1-4 The incubation of myelin with trypsin in vitro resulted in ultrastructural alterations with a loss of myelin basic protein (MBP) which suggested involvement of proteinase in myelin breakdown5. This finding is firmly established by demonstration 6,7of increased acid proteinase activity in EAE and MS, concomitant with loss of MBP . Although acid proteinases were previously thought to be involved in the process, current evidence indicates neutral proteinases including calcium activated neutral proteinase (calpain), metalloprotease, multicatalytic proteinase complex (MPC), matrix metalloproteinases, and uncharacterized neutral proteinases also participate in the demyelinating mechanism8-12 . Some of these proteinases are found in myelin, suggesting myelin may be autodigestive in demyelinating diseases13 . Many of these proteinases degrade MBP while the peripheral nervous system (PNS) basic protein P2 is specifically digested by MPC and cathepsin D14,15 . Thus, in demyelinating

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diseases, degradation of myelin proteins is critical in progression of the disease process.

PROTEINS AND PROTEOLYTIC ENZYMES OF MYELIN Since this chapter deals essentially with the role of proteolytic enzymes in myelin breakdown, it is important to briefly review the structural proteins of CNS and PNS myelin which together with lipids maintain the integrity of the myelin sheath. Several important CNS proteinases partially responsible for destabilizing the myelin sheath in diseases will also be reviewed. Myelin is largely composed of lipids (70%) and proteins (30%). The two major proteins of myelin are myelin basic protein (MBP) and proteolipid protein (PLP) which constitute 30% and 50% of all myelin proteins, respectively. PLP (24kD molecular weight) is tightly bound to lipids while MBP (18kD molecular weight) is also complexed with lipids, but with less affinity than PLP16. Minor myelin proteins with important roles include MAG (myelin-associated glycoprotein) MOG (myelin oligodendrocyte-specific glycoprotein), MOBP (myelin oligodendrocyte-specific basic protein), and DM-20 of the PLP family of proteins. Proteinases, lipases, kinases, and peptidases are also present 13,17-22 . MBP is highly susceptible to proteolysis and is digested by cathepsin B and D, calpain, metalloproteinase, trypsin, and pepsin while PLP is resistant to trypsin. The latter however, is partially digested by elastase23, trypsin and calpain, if detergent is present23-26 . The intact MBP, PLP, and MOG proteins or their proteolyzed fragments when injected into susceptible animals result in an autoimmune demyelinating disease, experimental allergic encephalomyelitis (EAE) a model for human multiple sclerosis (MS). PNS myelin also contains MBP along with P0 protein, which is similar to CNS PLP. MAG and other minor enzyme proteins, proteinases, and lipases are present as well. One of the myelin basic proteins, P2 protein, is resistant to endogenous CNS and PNS proteinases. However, P2 can be degraded by cathepsin D and MPC, in the presence of a detergent. The P2 protein, intact or fragmented, has been found to cause an autoimmune demyelinating disease of PNS, experimental allergic neuritis (EAN), which has been used as a model for Guillain Barre Syndrome, the PNS demyelinating disease of humans. Proteinase Hypothesis Acid proteinase activity was demonstrated in CNS samples in the early 1930s and 1940s27,28. Subsequently lysosomal proteinases cathepsins A, B, and D were purified from brain samples with increased activities of these proteases observed in diseases29-32. Cathepsin D was found to degrade phenylalanine-phenylalanine linkages in MBP molecules. Acid proteinase activity was also found to be localized in neurons. In contrast to acid proteinases, the demonstration of non-lysosomal neutral proteinase activity in brain was difficult since they are more unstable than lysosomal proteinases. Nonetheless, in the 1950s and 1960s Ansell and Richter33 and Marks and Lajtha34 were able to determine neutral proteinase activity in brain samples35. Since then several neutral proteinases including MPC, calpain, and metalloproteinase activity were found in the brain and later purified 14,20-22,36-43 Aminopeptidases and arylamidases are also found in myelin 29,44-47 . In contrast to CNS, the characterization ofproteinases in PNS is less extensive. The histochemical demonstration of proteinases in PNS was shown in the early 1960s24 followed by findings of calpain activity in sciatic nerve and Schwann cells48,49. Later, an

Calcium Activated Neutral Proteinase

unidentified neutral proteinase capable of degrading the P0 protein of PNS was also demonstrated50. The characterization of these various proteinases suggested myelin may be metabolically active and even autodigestive in demyelinating diseases.

MECHANISMS OF MYELIN BREAKDOWN IN VITRO AND IN VIVO Since the process of myelinolysis in demyelinating diseases was poorly defined, in vitro models were used to investigate the degradative process. Several studies evaluated the effects of snake venom, proteinases, lipases, and lysolecithin in whole brain and/or purified myelin51-55. These studies indicated that lipases or proteinases alone are not adequate for destruction of the myelin sheath.

Effects of Proteolytic Enzymes on Myelin Since the detergent-like action of lysolecithin treatment did not change the myelin ultrastructure, it was hypothesized that myelin proteins play a significant role in maintaining the structure and stability of myelin. Subsequent experiments evaluating the effects of trypsin on purified5 4 - 5 7myelin demonstrated a loss of phospholipids and MBP but no change in PLP . PLP in vitro is digestible with trypsin only in the presence of detergents such as Triton X-100 since it is embedded and protected in the membrane by lipids23,58. In order for PLP to be degraded, lipases were needed to expose it to proteinases. Thus, incubation of myelin with trypsin and phospholipase together resulted in the digestion of MBP and PLP with 58. ultrastructural dissolution of myelin into vesicles This suggested that the dual actions of proteinases and lipases were essential components in the mechanism of myelin breakdown in demyelinating disease. Studies with nerve extracts of Wallerian degeneration on purified myelin at 37°C also revealed that proteinases are important for myelin alterations59. There was greater loss of MBP and some loss of lipids when myelin was incubated with Wallerian Degeneration nerve extracts compared to controls. Acid proteinases have been implicated in these studies. From these and other studies it was suggested that MBP digestion is the initial step in myelin breakdown. Since activated inflammatory cells, including macrophages and lymphocytes have been shown to secrete proteinases into a culture medium, CNS and PNS myelin proteins were incubated in this medium with subsequent digestion10,60. Although these neutral proteases were not characterized at that time, subsequent studies from other laboratories identified calpain and metalloproteinase secreted by activated lymphocytes and macrophages capable of degrading purified myelin and MBP61,62.

Myelinolysis in Experimental Animal Models The role of proteolytic enzymes in myelinolysis associated with experimental allergic encephalomyelitis (EAE), Wallerian degeneration, diphtheritic (toxin) degeneration, and CNS injury were later studied. Of these animal models, demyelination has been studied most widely in EAE. Myelin breakdown also has been examined in several viral models, including canine distemper, scrapie, measles, and in cuprizone intoxication, i.e. non-infective and non-autoimmune perturbation. Both biochemical and histochemical studies revealed increased acid and neutral proteinase activity during the first week of degeneration. Results form these studies suggested loss of myelin basic proteins was responsible for changes in myelin structure. Subsequent in vitro studies showed losses of P0, P1, and P2 myelin proteins

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following incubation of PNS myelin with trypsin63-66. These studies suggested that the removal of proteins by various proteinases is responsible for structural changes in nerve degeneration. Increased proteinase activity was also associated with diphtheritic toxin-mediated demyelination67-69. In this model, increases in acid proteinase activity are found much earlier than that of neutral proteinase activity suggesting initial involvement of lysosomal proteinases in diphtheritic neuropathy50,67,68. Although the neutral proteinase found in this model was uncharacterized, recent studies demonstrated increased calpain activity in diphtheritic neuropathy70. The increased calpain activity has been correlated with degradation of calpain substrates (neurofilament proteins [NFP]), and elevated intracellular free calcium concentrations. CNS Wallerian degeneration was studied in optic nerve where macrophages, astrocytes, oligodendrocytes and microglia were implicated in myelin breakdown during nerve degeneration71-75. Long term intervals of optic nerve degeneration following enucleation of the eye resulted in almost complete loss of myelin proteins, while short term degeneration showed no apparent myelin lipid or protein loss76. Optic nerve degeneration caused by retinal destruction also demonstrated losses of myelin proteins MAG and MBP with structural alteration of myelin77,78, implicating involvement of proteinases. Recent in vitro and in vivo studies using experimental allergic optic neuritis (EAON) models revealed de adation of axon and myelin proteins concomitant with elevated calpain activity79 In viral animal models of demyelination, there was increased lysosomal hydrolases (e.g., ß-glucuronidase and cathepsin A) suggesting neuronal degeneration and inflammatory infiltration80 82. Increased glucuronidase and cathepsins A and D are also found in toxic cuprizone demyelination, often associated with proliferative glial cells (astrocytes and microglia). In these models increased proteinase activities concomitant with a loss of myelin proteins have been implicated in the mechanism of myelinolysis in demyelinating process. Alterations in the axon/myelin structural unit, at the morphological and biochemical levels are also common in brain and spinal cord injury83-87. In spinal cord injury lesions there is splitting of myelin lamellae with concomitant axon/myelin protein loss87,88. Various enzyme activities included calpain and cathepsins B and D are increased in and around the lesion site89-91. Use of calpain inhibitors in vivo in these injury models has been shown to prevent axon and myelin protein degradation92-95. THE CALPAIN FAMILY In autoimmune demyelinating diseases such as multiple sclerosis and EAE, the corresponding animal model, degradation of myelin proteins in CNS lesions suggested a role for calpain since all major myelin proteins are substrates of this enzyme. Calpain is a cytosolic cysteine endopeptidase (EC 3.4.22.17) that retains characteristics of the thiol proteinase, papain and calcium binding protein, calmodulin. The proteinase has been localized in every mammalian cell type studied, and calpain homologues have been identified in lower order organisms including nematodes, insects, yeast, and fungi96. The calpain family consists of at least six homologous members divided into two classes according to tissue distribution, i.e., ubiquitous or tissue specific. Recently discovered tissue specific calpains, such as p94 and nCL-2 (localized in muscle), exist as monomers or oligomers of the 80 kD catalytic subunit. Ubiquitous calpain is distributed in every cell, most often as a heterodimeric complex composed of 80 kD catalytic and 30 kD

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regulatory subunits. Both ubiquitous calpain isoforms, millicalpain (mcalpain) and microcalpain (µcalpain), share similar biochemical and catalytic properties with the exception of calcium concentrations required for activation. Approximately 1 -20µM and 250-750µM calcium levels are required for half-maximal activity of µcalpain and mcalpain, respectively, because calcium binding domains of the two isoforms differ in affinity for calcium97,98. Prior to activation, proenzyme calpain is normally associated with the endogenous inhibitor calpastatin. This association also requires calcium levels similar to the calcium activation requirement for each isoform. After calpain activation, calpastatin is also degraded by the active enzyme98,99. Calpain has been described as a biomodulator because it degrades substrates in a limited fashion, resulting in alteration rather than destruction of the ‘substrate. Calpain degrades a wide array of substrates including cytoskeletal and myofibrillar proteins, histones, enzymes, myelin proteins (myelin basic protein, MBP) and Since calpain activation often occurs along the cell receptor protein13,100-104. membrane, many membrane or membrane-associated proteins (actin-binding proteins such as fodrin, talin, filamin, a-actinin, and microtubule-associated proteins; growth factor receptors such as EGF receptors; adhesion molecules such as integrin, cadherin, N-CAM; and ion transporters such as Ca2+ -ATPase) are calpain substrates105.

CALPAIN ACTIVITY AND EXPRESSION IN ALLERGIC ENCEPHALOMYELITIS

EXPERIMENTAL

EAE is an autoimmune inflammatory disease induced in animals - most commonly rabbits, guinea pigs, monkeys, mice and Lewis rats - by injection of an emulsified suspension of whole CNS tissue, white matter, myelin, PLP or MBP, together with Freund’s complete adjuvant (FCA)106-110. The animals progressively lose weight followed by development of paralysis at 10 to 12 days after challenge. Perivascular cuffing with infiltrating lymphocytes, monocytes and plasma cells, can be observed by light microscopy 106,109,111. Electron microscopic examination has shown lamellar separation and splitting of the major dense and intraperiod lines of the myelin lamellae followed by vesicular degeneration of myelin which is ultimately phagocytozed112,113. Blood-brain barrier permeability is increased in EAE, possibly by free-radicals produced by inflammatory cells since permeability is reduced by antioxidant enzymes in EAE114. The hypothesis that MBP degradation is the initial step in myelin breakdown in demyelinating diseases59 is supported by findings of substantially increased acid and neutral proteinase activities in EAE and MS tissue6,115-123. Neutral proteinase activity is increased in EAE lymph nodes and serum124,125. Several early studies demonstrated significant increased acid proteinase activity in the lesion in rabbits and monkeys with acute EAE119. This greatly increased proteinase activity was later confirmed by Smith and colleagues66. These investigators demonstrated increased activity was due to at least two proteolytic enzymes, an uncharacterized neutral proteinase and an acid proteinase, cathepsin A. The activity of cathepsin A was found to be less than that of the neutral proteinase. Later, the increased neutral proteinase activity was shown to be elevated by 250% in the EAE lesion compared to controls. Cathepsin A activity was also found to be several fold greater in the lesion than controls80,118. Subsequently, increased activities of cathepsins B and D and neutral proteinase were observed in the lesion of monkeys with EAE66. In all these studies, MBP was preferentially degraded in the lesion.

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In contrast to monkeys with EAE, both cathepsins A and D and neutral proteinase activities are increased in rats with acute EAE. Cathepsin A activity was found to be greater than other proteinases118,127 while Boeheme et al128 reported no change in cathepsin A activity in EAE. Hirsch and colleagues reported increased cathepsin A and D in the lesion of rats with acute EAE, suggesting lymphocytic infiltration127. MBP was selectively degraded by EAE lymph node homogenate66 and later an uncharacterized neutral proteinase was partially purified from lymph nodes66 . Subsequent studies revealed an increased neutral proteinase activity in the serum of rats with acute EAE125. These studies indicated that elevated proteinase activity plays an important role in demyelination and they may derive not only from infiltrating cells, but also from endogenous cells. To examine the specific role of calpain in EAE, experiments in our laboratory were designed to evaluate and localize calpain activity and expression in the spinal cords of Lewis rats with an acute form of the disease129-132. These studies were also carried out in white matter tissue sections from human patients with various neurodegenerative diseases. Calpain activity was measured indirectly by evaluating the degradation of known calpain substrates such as fodrin, 68 kD axonal neurofilament protein (NFP) and the myelin protein MAG131. Western blot analysis revealed a 43% loss (p=0.041) of 68 kD NFP in spinal cords from animals with EAE compared to controls (Fig. 1A). This loss of NFP in EAE animals confirms earlier findings of NFP degradation in patients with MS, which suggests axonal degeneration may be present in autoimmune demyelinating diseases133,134. Although other proteases are involved in myelin degradation, recent studies suggest activated calpain plays a major role in this process since NFP and myelin protein degr adation is significantly decreased when calpain is inhibited following CNS injury 86,93,124,135142 . Similarly, spinal cords from rats with EAE demonstrated a 40% loss (p=0.014)

Figure 1. Loss of 68 kD neurofilament protein (A) and 96 kD myelin associated glycoprotein (B) in spinal cords of animals with EAE compared to adjuvant controls. Western blots (top) of samples from both groups were quantified via densitometry and analyzed by one way ANOVA (+S.E.M.). The dMAG band in (B) is marked with an (ortex>epithelium, show highest concentrations in the insoluble fraction, are both activated during lens maturation and cataract formation, and both disappear with lens aging. Due to these similarities, even partially purified Lp82 preparations contain 20% Lp85. However, independent, transient expression of each in COS-7 cells shows that Lp82 and Lp85 are proteolytically active independent of each other19. The three most urgent questions regarding Lp82/Lp85 mixtures are their calcium requirements, substrate specificity, and biologic functions.

Calcium Requirement The concentration of calcium needed for activation of Lp82 is important because the calcium concentration in normal lens is approximately 0.1- 0.2 µM. Yet calpains are somehow activated during normal rodent lens maturation. The same calpain cleavage sites found in cataract are also found in normal maturing rodent lenses. Our preliminary data suggested that 25 µM caused 50% activation of Lp82 (Fig. 9), and this is approximately 5 to 10 times lower than the published value for m-calpain16.

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In contrast to the autolysis involved in the activation of µ-calpain, our immunoblotting results indicate that Lp82 can be active without the appreciable autolysis20. This makes sense because the IS1 region, missing in Lp82, was shown to be the cleavage site for p94 autolysis4. However, 25 µM is still approximately 50 times higher than the physiologic levels of calcium in normal lens. We speculate that in vivo activators lower calcium activation requirements of both Lp82 and m-calpain in normal lens. Lp82 was also found to be less sensitive to calpastatin than m-calpain21, and this would also help promote Lp82 activity under lower calcium conditions found in maturing lenses.

Substrate Specificity Discovery of the in vivo substrates for Lp82 is important because such information might suggest physiological functions. Ail calpain isoforms discovered so far in the eye contain the papain-like active site found in m-calpain. Thus, we expected cleavage sites produced by Lp82 to be similar to those produced by m-calpain. Initial experiments showed this to be only partially true. In vitro proteolysis of EB1 and EA3-crystallin by Lp82 or mcalpain appeared to produce the same truncated polypeptide depending on the substrate (Fig. 10A). However, against DA-crystallin, Lp82 produced a unique cleavage site five amino acids in from the C-terminus, which was not produced by m-calpain (Fig. 10B). Production of this unique cleavage site may serve a specific function and may also be useful in identifying Lp82 activity within specific regions of the lens.

Figure 9. Calcium activation curve for partially purified Lp82 from 12 day old rat lens. fluorescence units released from hydrolyzed substrate.

FU = relative

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A. EB1 crystallin

B. DA crystallin AcMDVTIQHPWFKRALGPFYPSRLFDQFFGEGLFEYDLLPFLSSTISPYYRQSLFRT VLDSGISEVRSDRDKFVIFLDVKHFSPEDLTVKVLEDFVEIHGKHNRQDDHGYIS REFHRRYRLPSNVDQSALSCSLSADGMLTFSGPKVQSGLDA

Figure 10. (A) SDS-PAGE showing in vitro proteolysis of recombinant βBl by Lp82 or m-calpain. E64 = control inhibited by cysteine protease inhibitor. Molecular weight markers on left in kDa. (B) Cleavage sites on isolated bovine DA crystallin produced by L82 and m-calpain as revealed by mass spectrography. (From Nakamura et al., submitted)14.

Functions of Lens-Specific Calpains Because Lp82 is found in high concentrations in young lenses and then it disappears with lens maturation, we believe that the major physiological function of Lp82 is for lens development. Other roles are also possible, but they are speculative. For example, redundancy of calpain activities may be another function of Lp82. This was suggested in a recent experiment using a transgenic mouse harboring a mutant gene for inactive m-calpain. The strategy was that an over abundance of inactive m-calpain would suppress wild type mcalpain activity by binding to calcium and substrates. As yet we do not know if this dominant negative was effective in knocking out wild-type m-calpain because m-calpain activity was decreased (Fig. 11B, lane marked "3") and BSO cataracts formed equally well in normal and transgenic mice (Fig. 11A). Rather than an autolytic decrease in m-calpain, this decrease could have been due to breakdown of m-calpain by the large amounts of Lp82. Note that Lp82 was also decreased (Fig. 11B). Thus, lenses have apparent redundancy in the case of calpain isoforms. Even if m-calpain activity were to be totally knocked out, cataracts may still form due to Lp82-induced proteolysis. Another consequence of over-activation of Lp82 may be in light scattering. Calpaininduced in vitro light scattering was abolished when mature rat lenses were used as a source of soluble proteins22. We previously reasoned that this was due to the fact that maturation of lenses is accompanied by normal proteolysis of E-crystallins and slow insolubilization. Essentially no more precipitation-susceptible crystallins remained in the older lenses. However, this age-related loss in ability to undergo in vitro precipitation (Fig. 12) is also well correlated with age-related loss in Lp82 (Fig. 12 insert). Partially purified Lp82 is able to cause in vitro light scattering23. Thus, loss in ability to undergo in vitro precipitation may be partially related to the specific cleavages caused by Lp82.

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Figure 11. (A) Lenses in transgenic mice (inactive m-calpain) and in normal mice (FBV/NJ). Both groups received BSO injections. Dark areas indicate cataract. (B) Casein zymograms of the soluble proteins from transgenic and normal mice receiving BSO injections, showing activities (white areas) for Lp82, mcalpain, µ-calpain, and their activated forms.

Figure 12. Attenuation of light scattering with maturation of mouse lens. The casein zymogram (above) shows normal maturational loss of Lp82 (upper band)

CONCLUSIONS Retina and lens from rodents contain at least five newly discovered isoforms of calpain (Lp82, Lp85, Rt88, Rt88’ and Rt90), in addition to the ubiquitous calpains. Unexpectedly, these isoforms were related to muscle-preferred p94. Although some of their biochemical characteristics are known, the current challenge is to discover the functions of calpain isoforms under normal and pathological conditions. Further, because of stop codons, orthologues of the isoforms discussed above do not exist in man. Another challenge is to relate the rodent data to the human situation. For example, do undiscovered

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calpain isoforms performing the same functions as Lp82, Lp85, Rt88 and Rt90 exist in human eye?

Acknowledgements Partially supported by NIH grants EY03600, EY05786 to TRS and EY12239 to KJL. REFERENCES 1. M. Herasse, Y. Ono, F. Fougerousse, E. Kimura, D. Stockholm, et al., Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events, Mol. Cell. Biol. 19(6):4047 (1999). 2. H. Ma, M. Shih, I. Hata, C. Fukiage, M. Azuma, and T.R. Shearer, Protein for Lp82 calpain is expressed and enzymatically active in young rat lens, Exp. Eye Res. 67(2):221 (1998). 3. M. Azuma, C. Fukiage, M. Higashine, T. Nakajima, Y. Kawamoto, et al., Identification of a retinaspecific calpain (Rt88) from rat, Curr. Eye Res. (submitted). 4. K. Kinbara, S. Ishiura, S. Tomioka, H. Sorimachi, S.Y. Jeong, et al., Purification of native p94, a muscle-specific calpain, and characterization of its autolysis. Biochem. J. 335:589 (1998). 5. T.R. Shearer, H. Ma, M. Shih, C. Fukiage, and M. Azuma, Calpains in lens of the eye, In: CALPAIN: Pharmacology and Toxicology of Calcium-Dependent Protease, K.K.W. Wang and P.-W. Yuen, EDS., Philadelphia, Taylor & Francis (1999). 6. T.R. Shearer and L.L. David, Role of calcium in selenium cataract, Curr. Eye Res. 2(11): 777 (1982). 7. L.L. David, M. Azuma, and T.R. Shearer, Cataract and the acceleration of calpain-induced ß-crystallin insolubilization occurring during normal maturation of rat lens, Invest. Ophthalmol. Vis. Science 35(3):785 (1994). 8. M.J. Kelley, L.L. David, N. Iwasaki, J. Wright, and T.R. Shearer, alpha-Crystallin chaperone activity is reduced by calpain II in vitro and in selenite cataract, J. Biol. Chem. 268(25): 18844 (1993). 9. T.R. Shearer, M. Shih, T. Mizuno, and L.L. David, Crystallins from rat lens are especially susceptible to calpain-induced light scattering compared to other species, Curr. Eye Res. 15(8):860 (1996). 10. Y. Nakamura, C. Fukiage, M. Azuma, and T.R. Shearer, Oxidation enhances calpain-induced turbidity in young rat lenses, Curr. Eye Res. (19): 33 (1999). 11. L.L. David, M.D. Varnum, K.J. Lampi, and T.R. Shearer, Calpain II in human lens, Invest. Ophthalmol. Vis. Sci. 30(2):269 (1989). 12. M.S. Ajaz, Z. Ma, D.L. Smith, and J.B. Smith, Size of human lens beta-crystallin aggregates are distinguished by N-terminal truncation of betaB1, J. Biol. Chem. 272:11250 (1997). 13. J. Dillon, UV-B as a pro-aging and pro-cataract factor, Doc. Ophthalmol. 88(3-4):339 (1994). 14. K.J. Lampi, J. Oxford, T.R. Shearer, L.L. David, H.P. Bachinger, and D.M. Kapfer, Human bB1 crystallin structure and altered structure by truncation and deamidiation, (Submitted). 15. Y. Nakamura, M. Azuma, and T.R. Shearer, Calpain-induced light scattering in young rat lenses is enhanced by UV-B, Exp. Eye Res. (2000) In press. 16. L.L. David, and T.R. Shearer, Purification of calpain II from rat lens and determination of endogenous substrates, Exp. Eye Res. 42(3):227 (1986). 17. H. Ma, C. Fukiage, M. Azuma, and T.R. Shearer, Cloning and expression of mRNA for calpain Lp82 from rat lens: splice variant of p94, Invest. Ophthalmol. Vis. Sci. 39(2):454 (1998). 18. H. Ma, M. Shih, C. Fukiage, Y. Nakamura, M. Azuma, and T.R. Shearer, Lp82 is the dominant form of calpain in young mouse lens, Exp. Eye Res. 68:447 (1999). 19. H. Ma, M. Shih, I. Hata, C. Fukiage, M. Azuma, and T. Shearer, Lp85 is an enzymatically active rodent-specific isozyme of Lp82, Curr. Eye Res. 20(3):183 (2000). 20. I. Shih, H. Ma, and T.R. Shearer, unpublished. 21. Y. Nakamura, C. Fukiage, H. Ma, M. Shih, M. Azuma, and T. Shearer, Decreased sensitivity of lensspecific calpain Lp82 to calpastatin inhibitor, Exp. Eye Res. 69:155 (1999). 22. C. Fukiage, M. Azuma, Y. Nakamura, Y. Tamada, and T.R. Shearer, Calpain-induced light scattering by crystallins from three rodent species, Exp. Eye Res. 65(6):757 (1997). 23. Y. Nakamura, C. Fukiage, M. Shih, H. Ma, L.L. David, et al., Contribution of Lp82-induced proteolysis to experimental cataractogenesis in mice, Invest. Ophthalmol. Vis. Sci. 41: 1460 (2000).

METALLOENDOPEPTIDASE EC 3.4.24.15 IN NEURODEGENERATION

Carmela R. Abraham and Franchot Slot Boston University School of Medicine Boston, Massachusetts 02 118

INTRODUCTION The metalloendopeptidases represent a fascinating class of enzymes involved in neurodegenerative diseases. Many metalloendopeptidases are integrally involved in brain processes. This family boasts enkephalinase (24.11), neurolysin (24.16), and others. One of the most important members of this family is 24.15, also known as Thimet oligopeptidase or ThopI. 24.15 has been strongly implicated in Alzheimer’s disease and a multitude of analgesic pathways and is a regulator of major reproductive hormones. In addition, it has been recently suggested to serve important roles in antigen presentation and immunity. Here, 24.15 will be the focus, but in discussing 24.15 there will be reference to 24.11 and 24.16, each of which shows some overlap in function and activity. While direct links have been made between 24.15 activity and Alzheimer’s disease, numerous suggestive links have been considered as well. These may be contributors to Alzheimer’s disease pathology and may play roles in other neurodegenerative phenomena as well. Both the direct and indirect links will be explored here. One of the prevailing aspects of this enzyme that will become apparent is the large number of very different activities. Coupled to this is the fact that these activities don’t necessarily quickly form an integrated picture of the physiological “role” of the enzyme. Finding that role has been and continues to be elusive. Even the direct links to disease leave many questions open. Its importance in many pathways is clear and there are many hints that suggest the beginnings of a coherent function for the enzyme. Nevertheless, as will be seen, much more work is necessary to come to a clear understanding of the full and precise influence of this enzyme. The common practice of naming an enzyme by its cleavage assay, (i.e. Pz-peptidase is the enzyme cleaving the Pz peptide) has led to multiple names for the metalloendopeptidases. In some cases more than half a dozen names refer to the same molecule. The Enzyme Commission (EC) classification system was developed, in part, to unite these many names under a common description. We will use this system where possible for the sake of clarity and refer, therefore, to the metalloendopeptidase as 24.15, rather than the ThopI or the full EC 3.4.24.15.

Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.

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HISTORY OF 24.15 When collagen was first sequenced, the repetitive nature of the amino acids led to the design of a synthetic peptide with a similar sequence. Synthesized in 1963, the peptide was called the Pz-peptide for the N-terminal, yellow, exopeptidase-blocking group, “Pz” (phenylazobenzyloxycarbonyl)1. The degradation and subsequent extraction of the Pzpeptide fragments were used as a marker for collagen breakdown. The assumption here was that the enzyme responsible for the Pz-peptide degradation would be the same enzyme responsible for degrading collagen itself. As a result of this assumption Pz-peptidase was considered nearly synonymous with collagenase. Research into collagen mechanics, maintenance, and modeling used this assay extensively2. Indeed, it seemed to be a good marker system since it was shown to correlate well with tissue breakdown in a variety of systems. In 1972, Aswanikumar and Radhakrishnan purified the Pz-peptidase from rat granuloma tissue (known to be high in Pz-peptidase activity)3. It was given the number EC 3.4.99.31. The same authors later showed Pz-peptidase to be a 56 KDa protein inhibited by EDTA, with an optimum pH of 7.04. Further characterization, however, showed that Pz-peptidase was a poor candidate as a collagenase. The enzyme was not able to cleave native mammalian or avian collagen5, nor was it even effective in breaking down denatured collagen (gelatin), or gelatin constituents4,6,7. In fact, it was determined that the purified Pz-peptidase had a very narrow substrate specificity, almost exclusively restricted to small peptides less than 18 amino acids in length8. In addition, further work confirmed that “Pz-peptidase” was probably not a single enzyme. This has resulted in some confusion in the literature. The ability to cleave the Pz-peptide is not a specific effect of a single enzyme. Later purifications of the Pz-peptide cleaving activity resulted in preparations with different molecular weights, inhibitor profiles, and cofactor responses4,9,10. Data used here under the aegis of Pzpeptidase, therefore, will be specified as such. Obviously we have excluded data from consideration that derive from irrelevant “Pz-peptidases” (judged by physico-chemical attributes). Camargo and colleagues in 1972, isolated Endo-oligopeptidase A (originally called neutral endopeptidase) from rabbit brain11. Endo-oligopeptidase A (EOPA) was deemed to be a cysteine protease and in keeping with that was given the number EC 3.4.22.19 (3.4.22.19 refers to the 19th enzyme purified in the cysteine endopeptidase family). Subsequent to the purification of EOPA, Orlowski and colleagues identified “Soluble Metalloendopeptidase” in 198312. Accordingly their enzyme was labeled EC 3.4.24.15 (metalloendopeptidase family). The International Union of Biochemistry (IUB) recommended name for 24.15 became Thimet oligopeptidase, for its thiol and metal dependence as well as its restriction to small peptides less than 18 amino acids8,13. At the outset Pz-peptidase, Endo-oligopeptidase A, and Soluble Metalloendopeptidase appeared to be very different enzymes. In 1989, however, Tisljaf et al. determined by “substrate swapping” that Pz-peptidase and EOPA were identical.7 Shortly thereafter it was also shown that 24.15 and Pz-peptidase were identical14 . By extension, it was naturally argued that 24.15 and EOPA were the same as well. This proved to be problematic though. The enzyme had been identified alternatively as a cysteine, metallo-, or simply unclassified peptidase based on inhibitor profile. As a result of the inhibitor differences in addition to substrate specificity differences, it was convincingly argued that these were in fact different molecules15. Significant differences in assay design and animal models confounded results and comparisons. Publications in 1989 and 1991, though, argued that EOPA and 24.15 were indeed the same. They attributed the apparent differences in inhibitor profile and substrate specificity, to a cysteine near the metallopeptidase active site, and to species differences, respectively7,13,14. These enzymes were all believed to be the same until EOPA was very recently cloned. Sequence analysis 76 of EOPA has indicated finally that the two enzymes are separate . With the resolution of the nomenclature, there was continuing evidence for 24.15 as an important neuropeptidase. Other evidence also came to light linking it with Alzheimer’s disease (AD). The first of these links indicated that 24.15 was responsible for the cleavage of a synthetic peptide mimicking the E-secretase cleavage16,17,18 of the amyloid precursor protein (APP). APP gives rise to the Amyloid beta (AE) peptide, which accumulates and is thought to be causative for AD pathology. Subsequently, we

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determined that 24.15 activated a serine protease, which in turn, degraded AE19. Our work has also indicated that D -antichymotrypsin (ACT) inhibits this serine protease. ACT has long been identified as an important factor in AD20 . The role of 24.15 in AD may be a critical one and will be discussed later. In addition to these important and direct roles in AD for the metalloendopeptidase 24.15, the last decade has shown multiple indirect associations with other disease pathology. In at least five different pain and analgesia pathways 24.15 has been noted as a key regulator. The array of neuropeptides processed by this one enzyme is intriguing. Many of these transmitters have proven to be directly involved with a host of neuropathologies, Since 24.15 is critical for the regulation of these transmitters and peptides, one may speculate that these pathways are interrelated through the enzyme. It invites a search for a unifying and coherent role of the enzyme in the organism. Neuropeptides may be neurotransmitters or hormones or both. As a primary regulator of both, 24.15 hints at integrating these and other physiologic systems. Early and continuing work has identified the enzyme in the clearance of Gonadotropin releasing hormone (GnRH)21,22. This master reproductive hormone in turn controls the levels of a number of other hormones in the body. The potential interplay between these and the wide variety of other activities of 24.15 is fascinating and will also receive more attention later. The most recently discovered activity of 24.15 is in its coordination with the function of the proteasome23,24. Peptides processed from this complex seem to associate tightly with the 24.15 peptidase, (even without being cleaved), and serve an as yet unclear role in delivering the “cargo” to the MHC antigen presenting molecules at the cell surface. Other work has strongly implicated 24.15 in related immune functions and as a potential player in the inflammatory response. The history, therefore, of this single metallo-endopeptidase is very colorful. The many seemingly disparate effects and potential roles are both extraordinary and perplexing. Each of them, however, may be readily associated with aspects of neurodegeneration. It is the goal of this chapter to continue our growth toward a comprehensive and coherent understanding of the overarching role of this important peptidase.

BIOCHEMICAL CHARACTERIZATION Molecular Weight Molecular weights reported for Thimet oligopeptidase (24.15) vary from species to species and tissue to tissue. In fact, even within a single cell there seems to bemore than one form. The enzyme is found in the nucleus, cytosol, on the cell surface, and even secreted. Selective trafficking may be the result of differential splicing, proteolytic processing, or alternative starts of transcription. Such processes would be expected to result in size changes. In addition, a particular tissue or species may selectively use the numerous potential glycosylation and phosphorylation sites. Lastly, there are some reports of the enzyme forming multimers under certain conditions25,26. This may explain some of the larger sizes reported, especially since some of these analyses were done in SDS-free systems9,10,27 The molecular weight determined from amino acid composition of a rat clone is approximately 73 kDa2 and the human is 78.5 kDa28. Most preparations fall within a 70-85 kDa range. This 32 includes rabbit skeletal muscle (74 kDa),29 rat testis (70 kDa)31, rat epidermis (80 kDa) , bovine brain (75 kDa)33, chicken liver (80 kDa)31, human brain (85 kDa)17, monkey brain (80 kDa)34,35, and others. The high molecular weight form was seen in bovine dental follicle and adrenal gland (220 kDa)9,27 and rabbit serum10. Human erythrocytes showed a molecular weight of 75 kDa36. In addition, numerous lower molecular weight forms have been seen. Most of these center around 50 kDa. Indeed, the first size determination of Pz-peptidase by Aswanikumar and Radhakrishnan from monkey tissue sizes were human brain (55 kDa),34,37 monkey brain (55 kidne was 56 kDa4. Other kDa)35 human testis (55)38, rat brain (43)39, etc. There have also been many lower molecular weight bands noted in the 20-30 kDa range, however, it is unclear whether these are degradation products or isolation artifacts. Multiple forms of the enzyme have been seen in the same tissues and are often seen in the same preparation. We consistently find two molecular weight forms in AD brain that migrate at 85 kDa and 43-55 kDa17,34,35.

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Only a few of the isozymes noted have been tested for activity. When the primary form found has been in the 55 kDa range, it has been active37.

Isoelectric Point Most reports have found isoelectric points ranging between 4.8 and 5.2. One report using a preparation from rabbit uterus notes a 4.4 pI40, and another in human pituitary is as high as 5.538. In rat liver mitochondria, two isoelectric forms were separated, one at 4.9 and another at 5.241

pH optimum Reports of pH optima have ranged between 6.0 and 8.5 nearly exclusively. Two papers showed dual pH optima with 6.5 and 8 reported by one group9 and 7.1 and 7.9 by another10. Both of these were looking at Pz-peptidase activity, however, and it is likely that these activities reflect enzymes other than 24.15. (As mentioned previously, several different enzymes cleaving the Pz-peptide have been found – only one of them being 24.15). The few studies discussing pH inactivation show significant loss of 24.15 activity both below and above these optima. A 50% loss of activity was seen below 6 and above 8.5 in one study32. We showed a pH optimum closer to 6 with inactivation below pH 5 and above 7.617. A further study determined that irreversible inactivation was achieved at a pH less than 3.542. These results are not consistent with data indicating a role for 24.15 in the low pH endosomal vesicles43. It is possible that immunohistochemical cross-reactivity is responsible for this. It may be also that this is one route to degradation of 24.15, and its presence in endosomes may not reflect activity.

Thiol Activity It is a consistent finding that 24.15 requires a low level of thiol content for full activation. This is found to be sufficient at concentrations less than 0.5mM dithiothreitol. On the other hand concentrations above 5mM reversibly block activity25. The reason for this inactivation is not entirely clear. One group showed that the low thiol content was necessary to convert the enzyme from a multimeric to a monomeric form25 . This study has been neither confirmed nor refuted in the literature but seems that it might fit well with some other indications of complex formation discussed. Thiol activation is thought to be the result of reduction of cysteine residues that lead to intermolecular bridges and complex formation, which block substrate access to the active site25,44 The inhibitory effect of high thiol concentrations may, on the other hand, be a result of the proximity of a cysteine residue near the active site. This cysteine has also been implicated as the reason for inhibition by N-ethylmaleimide, iodoacetate, and iodoacetamide; known cysteine protease inhibitors25. This effect of thiol-sensitive inhibition is pronounced and explains the original classification as a cysteine protease (EC 3.4.22.19), as well as the confusion with Endo-oligopeptidase A.

Inhibitor Profile Table 1 indicates the inhibitor profile of Thimet oligopeptidase (24.15). By virtue of its inhibition by EDTA, EGTA, and 1,10-phenanthroline, and the ability to restore activity with a variety of metals, 24.15 has been classified as a metallo-protease. In keeping with this, the enzyme displays the characteristic HEXXH motif for coordinating a zinc ion and zinc has been seen in pure preparations to be a constituent of the protein41. As mentioned, the inhibition by certain cysteine protease inhibitors has been suggested to be due to a cysteine residue 5 amino acids from the active site (Cys-483 in rat)25. The failure of inhibition by E-64, a universal cysteine peptidase blocker, indicates also that the thiol inhibition is an effect of Cys-483 rather than 24.15 being a cysteine peptidase, In keeping with 24.15 as a metallopeptidase, a variety of metals have been shown to restore enzyme activity subsequent to EDTA treatment. Zn fully restores activity at the lowest concentrations (50uM). Ca, Sr, Mg, Ba, Mn, and Cd are also effective in varying degrees of reactivation. 2mM Mg seems to be important for full enzymatic activity17. Ca and Mn in concentrations approaching 20mM can actually induce an activity above and

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beyond what is normally seen in an unaltered preparation35,45. This depends in part on the preparation and species used but has been seen for the recombinant enzyme as well35. Importantly, both aluminum and iron salts can completely inhibit recombinant 24.15 at low millimolar and micromolar concentrations, respectively35 . Aluminum and iron have both been shown to accumulate in AD brain46,47. Specific inhibitors to 24.15 have also been developed. Orlowski and colleagues produced the first set of these. They were peptide analogues capable of coordinating the active site zinc with a carbonyl on the C-terminus. The best of this group displayed a Km of 16.6nM48. Later, Jiracek et al. reported the creation of phosphinic peptide moieties (which were also peptide analogues). These studies showed that alanine was preferred in the Pl’, an aromatic residue in the P1, arginine or lysine in the P2’, and methionine in the P3’ positions. The phosphinic peptides are the most specific and effective inhibitors known to date with a Km in the picomolar range (the best being 70 picomolar). One of these inhibitors displayed a 1,000 fold selectivity for 24.15 over the very closely related metallo-endopeptidase, neurolysin (24.16.)49

Substrate Specificity The synthetic substrates and inhibitors for 24.15 have provided considerable insight into the specificity of the enzyme. Nevertheless, there are no good rules for cleavage yet, and it is impossible to predict where the peptidase will cleave. Table 1 lists both the endogenous and synthetic substrates that are known for the enzyme along with the cleavage sites. It should be noted here, however, these cleavage sites are specific and well defined even though the rules guiding the cleavage are neither specific nor well defined. The cleavage sites noted are the only sites utilized, and only in a small number of peptides is there more than one. Processing of the peptides by 24.15 is clearly not a promiscuous activity. Most of these cleavages occur with Km values in the low micromolar range and a random peptide population is not cleaved23.

Table 1. Substrates for EC 3.4.24.15 Peptide

Peptide Structure

Reference

Synthetic Substrates Pz-peptide (for continuous rate) analysis (flourescent) “E-secretase peptide” “E-secretase peptide” (recomb.)

Pz-PL+GP-D-R Suc-G-PL+GP-MCA Bz-G+AAF-pAB DNP-PL+GPW-D-K Mcc-PL+GP-D-K(DNP) HSEVKM+DAEF ISEVK+M+D+AE+FRHDS

2 2 2 2 2 17 68

NATURAL SUBSTRATES Bradykinin Neurotensin GnRH Angiotensin I Angiotensin II Somatostatin Substance P Dynorphin A 1-8 Dynorphin B D- Neoendorphin E- Neoendorphin Orphanin FQ Cholecystokinin-8

RPPGF+SPFR pELYGNKPR+RPYIL pEWSY+GLRPG DRVY+IHP+FHL DRVY+IHPF AGCK+NFFW+KTFTSC RPKPQQF+F+GLM YGGFL+RRI YGGFL+RRQF+KWT YGGF+M+TSELSE+TPLVT YGGFL+RKYP FGGFTGA*RKSA*R*KLANQ DYMGW+MDF- NH2

12,26,96 12,26,85,96,33 26,12,85,100 97 85,97 85,97 12,85 26,84,99 96,33 84,99 26,84,99 98 101

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Of the few guidelines for substrate specificity, Orlowski reported that there was selectivity for hydrophobic residues in the P1’ and P3’ sites48. 24.15 seems to be able to cleave peptides with a proline in the P1 position as well50. 24.15 is strictly an endo-peptidase. The enzyme does not cleave N or C-terminal residues from a peptide unless the peptide is reduced to a minimal size. The peptide-binding pocket accepts approximately 5 amino acid residues48 and the enzyme is incapable of cleaving peptides smaller than 4 amino acids12. Conversely, no peptide chain longer than 18 amino acids has been shown to be reproducibly cleaved (APP serving as a possible exception). These data, then, ensure 24.15’s definition as an endo-oligopeptidase.

Active Site Mechanism Thimet oligopeptidase is a zinc-metalloendopeptidase. The coordination of zinc and the reaction mechanism of the active site are performed by the HEXXH motif. In 24.15, this is seen as HEFGH. In this mechanism, zinc is coordinated by Histidines-473 and 477 and by the distant Glutamate-502. Glu-474 then forms a hydrogen bond with water and coordinates the zinc with the water’s oxygen. This creates a nucleophilic center, which makes an attack on the carbonyl carbon of the peptide bond. The carbonyl carbon is released, and the enzyme is freed for another reaction51. Gene Structure, RNA, Protein Motifs Our lab determined that Thimet oligopeptidase was located on Chromosome 19q13.3 in 199652. This was very promising because of the proximity of the risk factor for AD, Apo E. Since then we went on to identify its localization on the p-arm at position 13.353 . Because Apo E is on the q-arm it is unlikely that there is any connection between these two genes from a chromosomal regulation standpoint. Recently, the Lawrence Livermore National Laboratory has read through this region of human chromosome 19 and the entire genomic sequence is now known. Previous to this sequencing, however, the cDNA sequence and intron/exon sites were identified in the pig. It was found that 24.15 bears a strong homology and very close relationship to 24.16. The two enzymes were over 80% identical and had the great majority of intron splice sites in common. Exons 5-1 6 of 24.16 matched exactly to exons 2- 13 of 24.15 despite varying lengths of introns54. The chromosomal localization of human 24.16 is currently unknown. Substrate specificities differ and the two enzymes cleave at distinct locations (even when they work on the same peptide). We routinely see a single mRNA transcript of 2.5Kbp for Thimet oligopeptidase (aside from the heterogeneous nuclear transcript) in human and monkey brain35. Kato et al. report the porcine 24.15 RNA to exist as a single transcript, in contrast to 24.16 with six transcripts which span 45 kbp in 13 exons54. The fact of a single transcript is interesting considering that 24.15 seems to be located in both the cytosol and nucleus as well as secreted to the extracellular milieu. One might expect different transcripts to cover this function. Pierotti et al. found three potential 5’ starts by primer extension. The size differences of these are small and may explain the single band seen by the relatively poor resolution of Northern blot55. Porcine 24.16 has been shown to make use of alternative splicing and multiple starts of transcription to direct its trafficking to the mitochondria, cytosol, or cell surface54. The different 5’ ends may be alternative start sites and therefore may be responsible for the alternative localization of the enzyme. 24.15 does contain a consensus nuclear localization sequence but its functionality and relationship to our observations and previous studies that have seen 24.15 in the cell nucleus is not known. 18,34,35,56 This may in part be explained by some confusion in the promoter sequence of 24.15. Original Genbank sequences for human 24.15 and other data showed neither a TATA box nor an initiation region for the enzyme (Accession # U29367)55. Porcine 24.15 is also reported to contain no TATA box54. The more recent sequencing by the Lawrence Livermore National Laboratory, however, indicates the presence of a TATA box (Accession # AC006538). There are multiple potential transcription factor consensus sequences but until the actual start of transcription is determined by primer extension analyses, or any discussion of the nuclear regulation of the enzyme is speculation. The protein sequence of 24.15 reveals a host of potential phosphorylation and glycosylation sites. It is currently unknown which, if any, of these are utilized. Other potential protein motifs have not been tested or published.

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CONSERVATION AND TISSUE DISTRIBUTION Assayed by the ability to cleave a test peptide, 24.15 has been found in varying concentrations in nearly every tissue tested. It has been seen in humans, monkeys, rabbits, rats, cows, pigs, plants, and a close homologue exists in yeast57. The greatest amount of study has been in human, rabbit, and rat species. In all organisms, 24.15 seems to be highly associated with rapidly dividing cells, or in tissues undergoing high turnover. Although its role as a mammalian collagenase has been disproven, (Pz-peptide is not a substrate)5, clearly the association of the enzyme to these high-turnover areas and its utility as a marker for tissue re-modeling is strong58,59. The only organ where this does not seem to fit is in the brain, where ironically it is high in all species tested. In fact, the brain contains such high levels of 24.15 that in most animals it is second only to the levels in the testis. These two tissues, the brain and testis have often been seen to display concentrations an order of magnitude higher than all others. The reason for this is unknown. Certainly, the enzyme is not playing a critical role in the brain for cell division, though tissue remodeling leading to memory formation or synapse plasticity is a possibility. Generally, however, the role in the brain is assumed to be as a neuropeptidase. Why 24.15 is found in such high concentrations in the testis is also less than clear. Here its role in cell division/tissue modeling is easier to justify. Equally plausible, though, is the strong connection the enzyme has with hormonal regulation. This will be discussed later. At any rate, the enzyme has been highly conserved over evolution and is likely to serve an important function beyond those already identified. It has been suggested that 24.15 and 24.16 diverged approximately 500 million years ago60. The enzymes have changed remarkably little, it would seem, even since the early eukaryotes. The yeast yscD gene shares nearly 35% open reading frame homology and very similar substrate and inhibitor profiles with 24.1557. Our recent studies have shown that 24.15 is very important to cells in culture. Growth rate and morphology are altered when 24.15 expression is disturbed23,35. Clearly, in culture, cleavage of neuropeptides is not of preeminent importance and so the question remains open as to what else 24.15 may be doing for the cell. Recent data in our laboratory and in Silva et. al.61 provides clues to an important role in cell division itself.

ALZHEIMER’S DISEASE Alzheimer’s disease is a neurodegenerative disorder that takes a tremendous toll on the aging population around the world. In nations with longer life expectancies, and therefore, where a larger percentage of the population is aged, it is seen to be a major health care crisis. Beyond the tragic effect it has on families and individuals, as well as the loss of productivity, costs of health care alone have been estimated to surmount 100 billion dollars in the United States alone. Alzheimer’s disease accounts for 60-70% of dementia cases in Europe as well62. First diagnosed by Alois Alzheimer in 190763, the pathology of the disease is characterized by amyloid plaques, neurofibrillary tangles, dystrophic neurites, microgliosis, and astrocytosis. The amyloid plaque is an extracellular protein deposit composed of the Amyloid E peptide (AE) and surrounded by dystrophic neurites, activated microglia and astrocytes. The neurofibrillary tangle is an intracellular deposition of hyperphosphorylated tau protein that occurs in selected neurons. A review of these deposits is available64 . The Amyloid precursor protein has long been seen as a major factor in AD. The single-pass transmembrane protein has been shown to give rise to the neurotoxic AE, a 3942 amino acid peptide that aggregates to form an SDS insoluble amyloid plaque65. Amyloid is defined as an insoluble protein precipitate of E-pleated sheet structure, which stains with Congo Red to give a classic apple-green birefringence under polarizing light. A large number of proteins have been shown to form amyloid, however, and these lead to the diseases termed amyloidoses. The mechanism for this conversion is not completely understood.

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In the case of the APP, two cleavages must be made in the 100-135 KDa protein to liberate the AE peptide. The enzymes responsible for these cuts have been termed secretases. The E- and J-secretases generate the N- and C-termini of the AE-peptide, respectively. The sequencing of AE in 1984 by Glenner66 paved the way for the cloning and sequencing of the APP molecule. With this, the E-secretase and J-secretase cleavage sites were made apparent. Armed with this sequence, our lab constructed a 10 amino acid synthetic test peptide which flanked the N-terminus of AE . This peptide, known as P1, recapitulated the E-secretase cleavage site. The P1 peptide was used to assay for Esecretase activity in AD brain. The P1 peptide cleavage activity occurring between the methionine and aspartic acid (leaving the aspartic acid as the common N-terminus of Aβ) was purified from AD brain and partially characterized in 1991 67 . In 1992, by means of an inhibitor study, McDermott et al. showed that the cleavage of a shorter test peptide indicated 24.15 to be the enzyme generating the N-terminus of Aβ16. Our purification of the enzyme confirmed these results. We showed that a homogeneous preparation of 24.15 was indeed active against the test peptide and also reported that the protease cleaved recombinant APP17 in vitro. It was shown, in addition, that 24.15 cleaved APP in multiple sites including the β-secretase site68, which may suggest 24.15 is a decision point between an Aβ and a non-Aβ generating event. The data that 24.15 cleaves APP, unfortunately, has not been reliably reproduced by other groups. Koike et al., nevertheless, recently reported that 24.15 was capable of cleaving APP, citing insensitive fluorescent assay schemes for the previous failure of reproducibility18. While this data is compelling, the reports that claim 24.15 is not functional in cleaving native APP make strong arguments69. These issues travel beyond the simple failure to detect 24.15-induced APP cleavage. The largest of these is the repeated observation that 24.15 is severely restricted by substrate size8,70 . Thompson et al. showed no increase in AE formation when 24.15 was transfected with APP into HEK-293 cells68. Specific 24.15 inhibitors did not block AE formation in the same cell line71. When normally cleaved peptides have been increased to beyond 15 or 18 amino acids in length, 24.15 loses all ability to process them. Indeed, no peptide larger than this has ever been shown to be cleaved by the peptidase other than APP. (Studies indicating the cleavage of collagen and gelatin by Pz-peptidase have been disproven). Because of this substrate size restriction it seems unlikely that the APP protein being greater than 100kDa would be cut. Still, as has been discussed previously, the cleavage specificity of 24.15 is far from understood. It is known that the enzyme is active in the presence of a proline-induced chain bend in the substrate50. Therefore, it may be possible that under certain conditions there is a looping out of the β-secretase site of APP either freely or induced by another protein that makes this area uniquely accessible to the action of 24.15. The cloning of a major β-secretase (β-site APP-cleaving enzyme) in 1999, makes 24.15’s role in this process more suspect. Nevertheless, this β-secretase seems to be responsible for only approximately 70% of Aβ-generating activity72,73,74. So it may still be that the remaining E-secretase fraction (or β-secretase activity in other cell types) is a result of 24.15. We sought to further explore the role that 24.15 may have in AD. Our lab generated unique and specific monoclonal antibodies to the enzyme. Strikingly, two antibodies used in immunohistochemistry were shown to strongly label neurofibrillary tangles (NFT), a major pathologic feature of AD. In addition to labeling neurofibrillary tangles, the antibodies were seen to stain neurites of senile plaques as well as neuropil threads34. Given the suggestions for a role of 24.15 in tissue remodeling (as Pz-peptidase), it is interesting that it is found associated with several disturbances of cell shape and microtubule structure seen in AD. Additionally, the metallopeptidase co-localizes with APP in the dystrophic neurites surrounding senile plaques as well34. Therefore the enzyme is importantly, and intimately, involved with both of the two cardinal features of AD. In order to identify the role of 24.15 in cell culture, we used a human neuroblastoma cell line, SKNMC, shown to have certain features in common with neurons. Three stable transfectants were created with this cell line. The transfectants were established with a plasmid alternatively expressing 24.15 cDNA (giving an overexpressing cell line), antisense cDNA to 24.15 (giving an underexpressing cell line), or simply the plasmid vector used for each of the other two (giving the mock cell line)19. Since 24.15 had been implicated as being a possible β-secretase, we expected to see that the overexpressing cell line would produce a higher level of Aβ. Similarly we

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believed the antisense transfectant would show little to no Aβ. The actual results were exactly contrary to our expectations. Conditioned medium from overexpressing cells in fact showed significantly lower levels of Aβ and that of the antisense cells showed higher levels19. It was unclear why this would be. Conditioned medium (CM) of the three cell lines was then incubated in vitro with iodinated synthetic Aβ peptide. The results were consistent. Aβ-degradation was increased after incubation with sense CM and decreased after incubation with the antisense CM. Beyond this, we found that the degradation of Aβ1-42 proceeded more slowly than did the degradation of Aβ 1-40. Furthermore, aggregated Aβ 1-42 showed almost no degradation whatsoever19. Aβ 1-42 has been previously shown to aggregate more quickly than 1-40, therefore it is more amyloidogenic64 . These results were intriguing. We attempted to show absolute specificity by adding a 24.15-specific inhibitor to the incubations. However, we only saw a slight reduction with this inhibitor (approximately 30%). As a result a panel of other protease inhibitors was tested. Again we were surprised, for pretreatment of the CM with serine protease This was done initially with 4-(2inhibitors entirely suppressed Aβ-degradation. aminoethyl)benzenesulfonyl flouride (AEBSF) and diisopropylfluorophosphate (DFP) 19. With a serine protease invoked in a mechanism of Aβ clearance we wondered about the possible effect of α1-antichymotrypsin (ACT). ACT is a serine protease inhibitor of the serpin family. Abraham et al. 1988, showed that ACT mRNA was highly elevated in AD brain75, serum, and cerebrospinal fluid77. ACT was shown to not only co-localize with both diffuse and neuritic amyloid plaques but also to be tightly associated with them. It is also known that ACT increases the rate of Aβ 1-42 fibril formation78,79. Consistently, APP/ACT doubly transgenic mice display a plaque load that is increased over APP singly transgenic animals further implicating ACT in Aβ clearance80. ACT is an acute phase reactant, as well, and rises significantly in an inflammatory response. Inflammation is currently an intensively studied area in Alzheimer’s disease research and seems to play a very significant role in the downstream pathology of AD. Until now, however, the role that ACT might be playing has often been hinted at but remained unclear. This is despite its involvement in Alzheimer’s being depicted as early as 1988. It is nevertheless interesting to speculate that this molecule seems to play a role in several aspects of AD. We therefore sought to determine whether it might also have a role in Aβ-clearance. Our results showed that ACT blocked up to 60% of Aβ-degradation in the assay system. In addition, incubation of ACT with the conditioned medium of overexpressing cells led to the formation of an SDS-resistant complex with the inhibitor. Recombinant 24.15 alone, on the other hand, was not capable of degrading Aβ. Yet if CM of overexpressing cells was treated with radiolabeled diisopropylfluorophosphate there were more resultant serine proteases labeled. In all, the conclusion is that 24.15 activates possibly several serine proteases that are functional in degrading Aβ19. The mechanism of activation of the serine proteases is not currently understood. It is possible that 24.15 cleaves a proenzyme to activate it. Of course for the same reasons that 24.15 is unlikely to be the β-secretase, namely the substrate size restriction of the enzyme, it does not seem reasonable that this is the explanation. A better possibility is that 24.15 cleaves a peptide inhibitor of the one or more serine proteases and therefore activates them. Perhaps also, in a converse manner, 24.15 cleaves a peptide precursor to generate an activator of the serine protease. Currently the mechanism of serine protease activation remains unclear. We are seeking now to isolate the serine protease(s), activated by 24.15, likely to be a final step in Aβ clearance. The possibility exists, of course, that if the serine protease can be induced (perhaps by the induction of 24.15), Aβ levels can be reduced. This would lead to a lower plaque load and reduce the severity and progression of the disease. Similarly, if ACT indeed plays as important a role as it seems to, the increased activation of the serine protease may draw ACT away from its role in inflammatory mediation and Aβ deposition by mass action. With less Aβ, there will be an additional decrease in the resultant microgliosis and astrocytosis. With fewer reactive astrocytes, ACT release is likely to be slowed thus ending the vicious cycle of Aβ deposition and pathology as described81. It has been shown that 24.15 declines significantly in AD82. If so, it would be reasonable to expect that the serine protease declines as well. This may or may not be a causative event but it would seem likely to at least make a contribution to the pathology.

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OTHER POTENTIAL ROLES AS A NEUROPEPTIDASE The fact that 24.15 cleaves Somatostatin, Substance P, Neurotensin, Neoendorphins, Dynorphin, Metorphinamide, GnRH, and other neuropeptides is striking. Even more striking are the roles of these peptides in many degenerative diseases including AD. Somatostatin, for example, has been shown to be significantly deficient in AD83. It is important for memory and experimental lowering triggers a severe impairment of memory71. Therefore, if connections can be drawn between these many disparate peptides acted upon by 24.15, they can lead to functional clues. These insights may indicate the specific overarching role that 24.15 is playing. We expect this function to be critical to both normal and pathologic physiology. We will take a brief look here at some of the more interesting of these connections.

Pain and Analgesia It has been noted already that 24.15 acts upon α - and β-Neoendorphin, Metorphinamide, and Dynorphin A. It has also been shown that by selectively inhibiting the metallopeptidase, rats display an increased pain threshold and tail-flick latency84a as would be expected. These effects can be blocked by administration of the opiate antagonist, naloxone. So it may be assumed, by this alone, that 24.15 plays an important role in analgesia. In the degradation of these peptides by 24.15, however, the enzyme in turn generates both Met-and Leu-enkephalins. These latter two peptides display analgesic properties of their own including antinociception84b. These different opioids are suspected of mediating different types of analgesic effects in the brain. Because 24.15 is capable of converting one type of opioid peptide into another, it may be in part responsible for tailoring the analgesic response to match the organism’s need. There is greater significance to 24.15 than its action on opioid peptides alone however, for the enzyme acts on at least four other distinct analgesia-mediating pathways. The Neurotensin/Neuromedin system and the Orphanin system both point to 24.15 as a major regulator. Similarly, Bradykinin and Substance P mediate pain sensation and processes and are effectively degraded by the same enzyme32,85. What might 24.15 be doing that it is so intimately linked to at least 5 different pain and analgesia systems? Clearly, it begs the question whether some or even all of these pathways are linked. It is possible that regulation of this single peptidase could be in part responsible for a coordinated analgesic response. It is also not unreasonable to suggest that these peptides may be intimately linked with learning and memory processes. Therefore it may be a foregone conclusion that 24.15 plays a unique role in mediating these learning and memory functions. Looking at its role in Aβ-clearance may be enough in itself to make this case. Soluble Aβ has been seen to be associated with synaptic loss86. Additionally, it takes only a small leap to argue that a misregulation or dysfunction of this enzyme could be a serious contributing factor to memory loss, learning difficulties, and neurodegeneration. Estrogen, which is under the influence of 24.15 as discussed below, has a clear role in learning as well87. Or, on the other side of the coin, that 24.15 could be a valuable target for the remedy of these ailing systems. Remarkably, little work has explored these possibilities.

Hormonal Implications Prolyl Endopeptidase and 24.15 have been long known to be the two principal posttranslational regulators of Gonadotropin Releasing Hormone (GnRH.). This critical peptide is responsible for determining the secretion of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH). LH and FSH, in turn, determine the pattern of secretion of all the major sex hormones of the body including estrogen, progesterone, testosterone and their derivatives. For this reason GnFW has been called the “Master Hormone”. The involvement of 24.15 here is compelling for a variety of other indications surrounding the enzyme in both a reproductive as well as neural sense. 24.15 is routinely seen at highest concentrations in the testis. Why? It is unclear, but it is known that both enkephalins and endorphins (generated and degraded by 24.15, respectively) are potent activators of smooth muscle tone in the male reproductive tract.

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Similarly, Substance P (also degraded by 24.15) has been shown to have a role in increasing contractility of vas deferens smooth muscle88 . In addition, 24.15 (studied then as Pz-peptidase), was shown to double in activity in uterine tissue during the course of gestation and then double again during labor. It has been long known that estradiol plays a very large role in neural remodeling and cognition. Studies of post-menopausal women who are given estradiol exogenously routinely fare better on tests for cognitive function and decline. Pertinent to our own research in AD, women who received estradiol post-menopausally demonstrated an increased age of disease onset and a slower rate of progression towards dementia89. The role of estradiol is still unclear but the effect is pronounced. One very interesting side note to the possible involvement of 24.15 in a hormonally regulatory role is found with female athletes. High performance female athletes frequently experience amenorrhea90. This is believed to result from a lack of GnRH pulsatility.91 GnRH pulsatility has been suggested to be mediated by an autofeedback loop whereby the GnRH breakdown products (after Prolyl Endopeptidase and 24.15 processing) may act to modulate GnRH release92. Endorphin and enkephalin release are pronounced in these types of activities. Therefore 24.15 is in a key position on each side of the issue. Does the athleticism and toll on the body result in a change in 24.15 levels such that greater enkephalins are produced and consequently GnRH is shut down? Or is it that the enkephalin/endorphin-release trigger a 24.15 response that reacts consequently on GnRH This is also unknown. But here we see as well that the enzyme is uniquely poised to be a key player in this physiology.

Inflammation role Inflammation has been repeatedly proven to be a critical player in a number of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis, Multiple Sclerosis, and many others. Strangely, this diverse metallopeptidase 24.15 has a number of potential connections to the role of inflammation as well. Substance P, Neurotensin, endorphins93 and Bradykinin32 (all degraded by 24.15 as discussed) are primary mediators of the inflammatory response. Substance P induces cytokine release and activates mast cells93a. Neurotensin has been seen to bind to peritioneal macrophages and modulate phagocytic activation93. Enkephalins and endorphins stimulate interferon release, chemotaxis, superoxide production, antibodydependent cytotoxicity and a variety of other functions93 . Bradykinin is markedly increased in human skin during severe inflammation. It induces vasodilation, prostacyclin synthesis, and leads to intracellular calcium influx32. Also at least one paper reported that PGE2 upregulates 24.1594. It is known that chronic users of non-steroidal antiinflammatory drugs (NSAIDS) are consistently seen to have a reduced incidence of AD, and a consequent diminished rate of progression to dementia. Therefore, the effect of PGE2 on 24.15 (which is in turn inhibited by NSAIDS) is another possible connection that travels hand in hand with the degradation of the other neuropeptides that are also strong inflammatory mediators. Most intriguing of all, however, is the recent 1999 data by Silva et al. In their studies of the potential role of 24.15 in antigen presentation, this group performed an experiment theoretically similar to our own with Alzheimer’s disease. In one series of experiments they used a liposome transfection method to transfer 24.15 directly into T-cells. T-cells which were transfected with the enzyme had a diminished doubling time (ie divided more rapidly), whereas introduction of 24.15 specific inhibitors had the opposite effect. They also showed that the 24.15-containing immune cells were more efficient at killing a mycobacterium-containing macrophage. Again, the 24.15 inhibitor blocked this effect61. This correlates very well to our own work where we see neuroblastoma cells transfected with 24.15 antisense RNA grow at a significantly reduced rate compared to overexpressing cells35. In another set of experiments published side by side with this one they showed that 24.15 bound tightly to a set of randomly generated peptides23 . However, the enzyme only actually cleaved a very small fraction of them. This led the group to hypothesize that 24.15 may be instrumental in trafficking these peptides. Instead of cleaving them, it acts to protect the majority of them from cleavage. The antigens are transported intact to the antigen presenting machinery of the cell. Rather than modifying

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the post-proteasomally processed peptides to make them suitable for presentation, 24.15 may somehow select the appropriate antigens and then protect them on route to the cell surface. Other work in our own laboratory with the protease Bleomycin Hydrolase (BH) has suggested that both BH and 24.15 may be considered as “chaperases”. BH and 24.15 both serve on the one hand as a protease or peptidase, and on the other hand with the ability to act as a chaperone to a protein or peptide, respectively95. The specifically coordinated role of 24.15 in inflammation and antigen presentation is being actively pursued. Obviously much work here remains and speculation still dominates the field.

FUTURE DIRECTIONS Much work needs to be done to clarify all of these issues and fully define the role 24.15 is playing. The connections it makes to a wide variety of disease processes are exciting. Currently, its greatest potential role is in halting or reversing the causes and effects of neurodegeneration. But beyond this, great discoveries are forthcoming in the fields of immunology and endocrinology. The possibilities for useful interventions and therapies through this enzyme are significant. The very nature of the ties it makes between physiological systems suggest that it may serve to draw together a more unified understanding of the interplay and integration of the mind and health and the mediation by the messengers that travel between.

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16. J.R. McDermott, J.A. Biggins, and A.M. Gibson, Human brain peptidase activity with the specificity to generate the N-terminus of the Alzheimer beta-amyloid protein from its precursor, Biochem. Biophys. Res. Comm. 185:746 (1992). 17. G. Papastoitsis, R. Siman, R. Scott, and C.R. Abraham, Identification of a metalloprotease from Alzheimer's disease brain able to degrade the beta-amyloid precursor protein and generate amyloidogenic fragments, Biochemistry 33: 192 (1994). 18. H. Koike, H. Seki, Z. Kouchi, M. Ito, T. Kinouchi, S. Tomioka, H. Sorimachi, T.C. Saido, K. Maruyama, K. Suzuki, and S. Ishiura, Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor protein at a beta-secretase cleavage site in COS cells, J. Biochem. 126:235 (1999). 19. R. Yamin, E.G. Malgeri, J.A. Sloane, W.T. McGraw, C.R. Abraham, Metalloendopeptidase EC 3.4.24.15 is necessary for Alzheimer's amyloid-beta peptide degradation., J. Biol. Chem. 274: 18777 (1999). 20. C.R. Abraham, D.J. Selkoe, and H. Potter, Immunochemical identification of the serine protease inhibitor alphal-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease, Cell 52:487 (1988). 21. A.I. Smith, T. Tetaz, J.L. Roberts, M. Glucksman, I.J. Clarke, R.A. Lew, The role of EC 3.4.24.15 in the post-secretory regulation of peptide signals, Biochimie 76:288 (1994). 22. B. Horsthemke and K. Bauer, Characterization of a nonchymotrypsin-like endopeptidase from anterior that hydrolyzes luteining hormone-releasing hormone at the tyrosyl-glycine and histidyl-tryptophan bonds, Biochemistry 19:2867 (1980). 23. F.C. V. Portaro, M.D. Gomes, A. Cabrera, B.L. Fernandes, C.L. Silva, E.S. Ferro, L. Juliano, and A.C.M. Camargo, Thimet oligopeptidase and the stability of MHC class I epitopes in macrophage cytosol, Biochem. Biophys. Res. Comm . 255:596 (1999). 24. I.A.York, A.L. Goldberg, X.Y. Mo, K.L. Rock, Proteolysis and class I major histocompatibility complex antigen presentation, Immunol. Rev. 172:49 (1999). 25. C.N. Shrimpton, M.J. Glucksman, R.A. Lew, J.W. Tullai, E.H. Margulies, J.L. Roberts, and A.I. Smith, Thiol activation of endopeptidase EC 3.4.24.15. A novel mechanism for the regulation of catalytic activity, J. Biol. Chem. 272:I7395 (1997). 26. M. Orlowski, S. Reznik, J. Ayala, and A.R. Pierotti, Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its toward synthetic and natural peptides, including enkephalin-containing peptides, Biochem. J. 261:951 (1989). 27. K. Mizuno, A. Miyata, K. Kangawa, H. Matsuo, A unique proenkephalin-converting enzyme purified from bovine adrenal chromaffin granules, Biochem. Biophys. Res. Comm. 108:1235 (1982). 28. A. Thompson, G. Huber, and P. Malherbe, Cloning and functional expression of a metalloendopeptidase from human brain the ability to cleave a beta-APP substrate peptide, Biochem. Biophys. Res. Comm. 213:66 (1995). 29. U. Tisljar and A.J. Barrett, Purification and characterization of Pz-peptidase from rabbit muscle, Arch. Bioch. Biophys. 274:138 (1989). 30. A.C.M. de Camargo, H.Caldo, and P.C. Emson, Degradation of neurotensin by rabbit brain endooligopeptidase A and endo-oligopeptidase B (proline-endopeptidase), Biochem. Biophys. Res. Comm. 116:1151 (1983). 31. A.J. Barrett, A new look at Pz-peptidase, Biol. Chem. Hoppe-Seyler 371: Suppl:311 (1990). 32. M. Kikuchi, K. Fukuyama, and W.L. Epstein, Purification and characterization of bradykininhydrolyzing enzyme from 2-day-rat epidermis, Biochim. Biophys. Acta 965: 176 (1988). 33. A.C.M. de Camargo, E.B. Oliveira, O. Toffoletto, K.M. Metters, J. Rossier, Brain endo-oligopeptidase A, a putative enkephalin converting enzyme, J. Neurochem. 48: 1258 (1987). 34. K.J. Conn, M. Pietropaolo, S.-T. Ju, and C.R. Abraham, Monoclonal antibodies against the human metalloprotease EC 3.4.24.15 label neurofibrillary tangles in Alzheimer's disease brain, J. Neurochem. 66:2011 (1996). 35. F. Slot, and C.R. Abraham, unpublished results 36. P.M. Dando, M.A. Brown, A.J. Barrett, Human thimet oligopeptidase, Bioch. J. 294(2):451 (1993). 37. S.R. Sahasrabude, A.M. Brown, J.D. Hulmes, J.S. Jacobsen, M.P. Vitek, A.J. Blume, J.S. Sonnenberg, Enzymatic generation of the amino terminus of the beta-amyloid peptide, J. Biol. Chem. 268: 16699 (1993). 38. M.S. Medeiros, N. Iazigi, A.C.M. Camargo, and E.B. Oliveira, Distribution and properties of endooligopeptidases A and B in the human neuroendocrine system, J. Endocrinol. 135:579 (1992). 39. E. S. Oliveira, P.E.P. Leite, M.G.Spillantini, A.C.M.Camargo, and S.P. Hunt, Localization of endooligopeptidase (EC 3.4.22.19) in the rat nervous tissue, J. Neurochem. 55:1114 (1990).

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Carmela R. Abrahamand Franchot Slot K. Sakyo, J. Kobayashi, A. Ito, and Y. Mori, Partial purification and characterization of gelatinase and metal dependent peptidase from rabbit uterus and their synergistic action on gelatin in vitro, J. Biochem 94:1913 (1983). U. Tisljar and A.J. Barrett, A distinct thimet peptidase from rat liver mitochondria, FEBS Lett. 264:84 (1990). B. A. Lessley and D.L. Garner, Purification and characterization of Pz-peptidase B, a neutral metalloendopeptidase from bovine spermatozoa, Biol. Reprod. 43:643 (1990). J.-M. Chen, A. Changco, M.A. Brown, and A.J. Barrett, Immunolocalization of thimet oligopeptidase in chicken embryonic fibroblasts, Exper. Cell Res. 216:80 (1995). A.J. Barrett, M.A. Brown, P.M. Dando, C.G. Knight, N. McKie, N.D. Rawlings, and A. Serizawa, Thimet oligopeptidase and oligopeptidase M or neurolysin, Methods Enzym. 248:529 (1995). A. J. Wolfson, C.N. Shrimpton, R.A. Lew, and A.I. Smith, Differential activation of endopeptidase EC 3.4.24.15 toward natural and synthetic substrates by metal ions, Biochem. Biophys. Res. Comm. 229:341 (1996). G. Bartzokis, D. Sultzer, J. Cummings, L.E. Holt, D.B. Hance, V.W. Henderson, J. Mintz, In vivo evaluation of brain iron in Alzheimer disease using magnetic resonance imaging, Arch. Gen. Psychiatry 57:47 (2000). G.A. Trapp, G.D. Miner, R.L. Zimmerman, A.R. Mastri, L.L. Heston, Aluminum levels in brain in Alzheimer’s disease, Biol. Psych. 13:709 (1978). M. Orlowski, C. Michaud, and C.J. Molineaux, Substrate-related potent inhibitors of brain metalloendopeptidase, Biochem. 27:597 (1988). J. Jiracek, A. Yiotakis, B. Vincent, A. Lecoq, A. Nicolaou, F. Checler, V. Dive, Development of highly potent and selective phosphinic peptide inhibitors of zinc endopeptidase 24-1 5 using combinatorial chemistry, J. Biol. Chem. 270:2 1701 (1995). A.C.M. Camargo, M.D. Gomes, A.P. Reichl, E.S. Ferro, S. Jacchieri, I.Y. Hirata, and L. Juliano, Structural features that make oligopeptides susceptible substrates for hydrolysis by recombinant thimet oligopeptidase, Biochem J. 324:517 (1997). P.M. Cummins, A. Pabon, E.H. Margulies, M.J. Glucksman, Zinc coordination and substrate catalysis within the neuropeptide enzyme endopeptidase EC 3.4.24.15. Identification of active site histidine and glutamate residues, J. Biol. Chem. 274: 16003 (1999). B. Meckelein, H.A.R. de Silva, A.D. Roses, P.N. Rao, M.J. Pettenati, P.-T. Xu, R. Hodge, M.J. Glucksman, and C.R. Abraham, Human endopeptidase (THOP1) is localized on chromosome 19 within the linkage region for the late-onset alzheimer disease AD2 locus, Genomics 3 1:246 (1996). F. Slot, A.S. Olsen, P.N. Rao, and C.R. Abraham, Localization of human endopeptidase EC 3.4.24.15 (THOP1) to chromosome 19p13.3, Alzheim. Rep. 1:327 (1998). A. Kato, N. Sugiura, Y. Saruta, T. Hosoiri, H. Yasue, and S. Hirose, Targeting of endopeptidase 24.16 to different subcellular compartments by alternative promoter usage, J. Biol. Chem. 272: 153 13 (1997). A. Pierotti, K.-W. Dong, M.J. Glucksman, M. Orlowski, and J.L. Roberts, Molecular cloning and primary structure of rat testes metalloendopeptidase 3.4.24.15, Biochem 29:10323-10329 (1990). D. P. Healy and M. Orlowski, Immunocytochemical localization of endopeptidase 24.15 in rat brain, Brain Res. 571:121 (1992). M. Buchler, U. Tisljar, and D.H. Wolf, Proteinase yscD (oligopeptidase yscD). Structure, function and relationship of the yeast enzyme with mammalian thimet oligopeptidase (metalloendopeptidase, EP 24.15), Eur. J. Biochem. 219:627 (1994). J.F. Woessner Jr., Collagen remodeling in chick skin embroygenesis, in: Chemistry and Molecular Biology of the Intercellular Matrix, Vol. 3, E.A. Balazs, ed., Academic Press, New York (1970). E. Keiditsch and L. Strauch, Peptidase and collegenase activities in invasion zones of tumors of the brease, in: Chemistry and Molecular Biology of the Intercellular Matrix, Vol. 3, E.A. Balazs, ed., Academic Press, New York (1970). A. Serizawa, P.M. Dando, and A.J. Barrett, Characterization of a mitochondrial metallopeptidase reveals neurolysin as a homologue of thimet oligopeptidase, J. Biol. Chem. 270:2092 (1995). C.L. Silva, F.C.V. Portaro, V.L.D Bonato, A.C.M. de Camargo, and E.S. Ferro, Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class I antigen presentation, Biochem. Biophys. Res. Comm. 255:591 (1999). L.J. Launer, L. Fratiglioni, K. Andersen, M.M. B. Breteler, R.J.M. Copeland, J.-F. Dartigues, A. Lobo, J. Martinez-Lage, H. Soininen, and A. Hofman, in: Alzheimer’s disease and Related Disorders, Iqbal et al. Eds., John Wiley and Sons, LTD. (1999). A. Alzheimer, Ueber eine eigenartige Erkrankung der himrinde. Algemeine Zeitschrift fuer Psychiatrie und Psychisch-Gerichtliche Medizin. 64:146 (1907).

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84b. B. Kest, M. Orlowski, R.J. Bodnar, Increases in opioid-mediated swim antinociception following endopeptidase 24.15 inhibition, Physiol. Behav. 50:843 (1991). 85. R. Mentlein, P. Dahms, Endopeptidases 24.16 and 24.15 are responsible for the degradation of somatostatin, neurotensin, and other neuropeptides by cultivated rat cortical astrocytes, J. Neurochem 62:27 (1994). 86. L.F. Lue, Y.M. Kuo, A.E. Roher, L. Brachova, Y. Shen, L. Sue, T. Beach, J. H. Kurth, R.E. Rydel, J. Rogers, Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease, Am. J. Pathol. 155:953 (1999). 87. R.B. Gibbs, Estrogen replacement enhances acquisition of a spatial memory task and reduces deficits associated with hippocampal muscarinic receptor inhibition, Horm. Behav. 36:222 (1 999). 88. B.V. R. Sastry, V.E. Jenson, L.K. Owens, and O.S. Tayeb, Enkephalin- and substance P-like immunoreactivities of mammalian spenn and accessory sex glands, Biochem. Pharm 31:3519 (1982). 89. A.J. Slooter, J. Bronzova, J.C. Witteman, C. Van Broeckhoven, A. Hofman, C.M. van Duijn, Estrogen use and early onset Alzheimer's disease: a population-based study, J. Neurol. Neurosurg. Psych. 67:779 (1999). 90. C. De Cree, Comment on health issues for women athletes: exercise-induced amenorrhea, J. Clin. Endocrinol. Metab. 84:4750 (1999). 91. G.A. Laughlin, S.S. Yen, Nutritional and endocrine-metabolic aberrations in amenorrheic athletes, J.Clin. Endocrinol. Metab. 81:4301 (1996). 92. C. Yamanaka, M.C. Lebrethon, E. Vandersmissen, A. Gerard, G. Purnelle, M. Lemaitre, S. Wilk, J.P. Bourguignon, Early prepubertal ontogeny of pulsatile gonadotropin-releasing hormone (GnRH) secretion: I. Inhibitory autofeedback control through prolyl endopeptidase degradation of GnRH, Endocrin. 140:4609 (1999). 93. M. Lesser, K. Fung, H.S. Choi, O.H. Yoo, C. Cardozo, Identification of two zinc metalloendopeptidases in alveolar macrophages of rats, guinea pigs, and human beings, J. Lab. Clin. Med. 120:597 (1992). 93a. J.C. Ansel, C.A. Armstrong, I. Song, K.L. Quinlan, J.E. Olerud, S.W. Caughman, N. W. Bunnett, Interactions of the skin and nervous system, J. Invest. Dermatol. Symp. Proc. 2(1):23 ( 1997). 94. T. Chikuma, Y. Ishii, T. Kato, H. Kodama, Y. Hakeda, M. Kumegawa, Effect of prostaglandin E2 on PZ-peptidase and several other peptidase activities in a clonal osteoblast-like cell line derived from newborn mouse calvaria, J. Biochem. Tokyo 97: 1533 (1985). 95. W.T. McGraw, R. Yamin, E.A. Berg, M. Gartner, S. Keve, E.M. Schaefer, R.E. Fine, and C.R. Abraham, Bleomycin Hydrolase Modulates the Maturation and Trafficking of the Amyloid Precursor Protein and the Secretion of Ab in a Dose-dependent Manner, (submitted). 96. M.A. Cicilini, M.J. Ribeiro, E.B. de Oliveira, R.A. Mortara, A.C. de Camargo, Endooligopeptidase A activity in rabbit heart: generation of enkephalin from enkephalin containing peptides, Peptides 9:945 (1988). 97. T. G. Chu, M. Orlowski, Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides, Endocrinology 116:1418 (1985). 98. J.L. Montiel, F. Cornille, B.P. Roques, F. Noble, Nociceptin/orphanin FQ metabolism: role of aminopeptidase and endopeptidase 24.15, J. Neurochem. 68:354 (1997). 99. G.R. Acker, C. Molineaux, M. Orlowski, Synaptosomal membrane-bound form of endopeptidase-24.15 generates Leu-enkephalin from dynorphin1-8, alpha- and beta-neoendorphin, and Met-enkephalin from Met-enkephalin-Arg6-Gly7-Leu8, J. Neurochem. 48:284 (1987). 100. C.J. Molineaux, A. Lasdun, C. Michaud, M. Orlowski, Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo, J. Neurochem. 51:624 (1988). 101. M.G. Oakes, T.P. Davis, The ontogeny of enzymes involved in post-translational processing and of neuropeptides, Br. Res. Dev. Br. Res. 80: 127 (1994).

CYSTEINE PROTEASES, SYNAPTIC DEGENERATION AND NEURODEGENERATIVE DISORDERS

Mark P. Mattson and Sic L. Chan Laboratory of Neurosciences National Institute on Aging Baltimore, MD 21224

INTRODUCTION Neurons communicate with each other at highly specialized structures called synapses and, accordingly, receptors for neurotransmitters, neurotrophic factors and some cytokines are concentrated in synaptic terminals. Signaling at synapses plays or controls all of our behaviors including processes such as learning and memory, and is also critical for the growth and survival of neurons. Recent findings suggest that synapses are sites where the neurodegenerative process begins in disorders ranging from Alzheimer’s, Parkinson’s and Huntington’s diseases, to stroke. Apoptosis (a form of programmed cell death) and excitotoxicity (resulting from overactivation of glutamate receptors) may occur in neurons in such disorders. Recent findings indicate that apoptotic and excitotoxic biochemical cascades are activated in synaptic terminals in experimental models of neurodegenerative disorders. Proteases of the caspase and calpain families are implicated in the neurodegenerative process, as their activation can be triggered by calcium influx and oxidative stress. Caspases cleave a variety of substrates including cytoskeletal proteins, kinases, cell surface receptors, members of the Bcl-2 family of apoptosis-related proteins, preseniliis, amyloid precursor protein, and DNA-cleaving enzymes. Calpains degrade cytoskeletal and associated proteins, kinases and phosphatases, membrane receptors and transporters, and steroid receptors. Many of these substrates are located in pre- and/or postsynaptic compartments of neurons wherein they play roles in modulating synaptic transmission and plasticity. Emerging data suggest that excessive cleavage of synaptic proteins by cysteine proteases mediates synaptic degeneration and neuronal cell death in several different neurodegenerative disorders. Accordingly therapeutic strategies are being developed that are aimed at preventing caspase and/or calpain activation or inhibiting the activated proteases. CHARACTERISTICS OF CASPASES Caspase-like proteases were first discovered in studies of genes that regulate programmed cell death in the nematode C. elegans1. The first mammalian caspase identified was interleukin-1b converting enzyme (ICE), a homolog of the C. elegans cell death protease CED-3, which cleaves the 31 kDa pro-form of interleukin-1b to produce a 18 kDa active form of interleukin- 1b2. ICE is now called caspase- 1, and is one member of a family of at least 14 cysteine proteases that share the general function of controlling the process of programmed cell death or apoptosis. As is the case with other cysteine proteases, the proteolytic activity of caspases is dependent on protein dimerization and a catalytic dyad Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.

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composed of a cysteine residue in close proximity to a histidine residue. Amino acids from both caspase subunits contribute to this active site, and subunits of different caspase family members can combine to form novel tetramers with divergent substrate specificities3,4,5 Caspases 1-7 and 11-14 contain the highly conserved pentapeptide sequence QACRG at the catalytic dyad. Substrate recognition depends on the sequence of four amino acids Nterminal to the critical aspartate residue at the PI position of a consensus cleavage site in all known caspase substrates6. The phosphorylation of amino acids adjacent to the caspase cleavage site in substrate proteins can modify the ability of the protease to cleave the substrate7. The caspase family can be subdivided into the ICE-like proteases (caspases 1, 4, 5, 11, 13 and 14) which prefer substrates with bulky hydrophobic amino acids at P4 and have the consensus cleavage sequence (YWL)EHD. Caspases 2, 3 and 9 belong to the CPP32like subfamily, which shows preference for aspartate at the P4 position and the DXXD consensus sequence. The third subfamily (caspases 6, 8, 9 and 10) prefers branched-chain aliphatic amino acids at P4 and prefers the consensus sequence (IVL)(QE)XD. There is considerable overlap in the substrates cleaved by members of the different subfamilies of caspases, which may ensure rapid and effective substrate processing in cells that express multiple caspases. The pivotal role of the PI and P4 residues to substrate specificity of caspases has permitted the design of specific synthetic tri- and tetrapeptide aldehyde caspase inhibitors. Experiments with such caspase inhibitors have proven invaluable in establishing the roles of caspases in various physiological and pathological processes. For example, zVAD-fmk and YVAD-fmk peptides are capable of inhibiting most caspases, DEVD-fmk specifically inhibits caspases 3 and 7, and VEID-fmk inhibits caspase 68 . Although caspases are present at high levels in the cytoplasm, they can also be localized in the nucleus, mitochondria and endoplasmic reticulum9,10, The inactive procaspase forms of caspases I and 2 contain nuclear localization signals11,12, suggesting that they can be transported to the nucleus. Subcellular compartmentalization of caspases could be an important determinant not only of their activation, but also substrate specificity (see below) and the ultimate cellular response. Caspases may also be associated with membranes, and a membrane-associated form of procaspase-3 was recently described13. Moreover, some caspases may act extracellularly as indicated by their association with cell surfaces14. Different caspases exhibit different patterns of cellular expression, and increasing data suggest that caspase expression is regulated spatially and temporally depending on cell type and developmental stage. Levels of mRNAs encoding caspases 2,3,10 and 14 mRNAs are quite high in many different embryonic tissues including the brain, and then decrease markedly during postnatal development15,16,17,18 . In comparison with many other types of cells, mature neurons express lower levels of caspases 2,6, 7 and 8 under basal conditions. As is the case in most cells, levels of caspase mRNA and protein are increased in neurons in response to various environmental insults. Thus, levels of mRNAs for caspases 1, 2 and 3 are increased in cortical and hippocampal neurons following cerebral ischemia19,20,21 and traumatic brain injury22. Levels of caspase-3 mRNA are increased in cultured cerebellar granule neurons incubated in medium containing a low concentration of potassium (a nondepolarizing condition that induces apoptosis in these cells)17; inhibitors of RNA and protein synthesis can prevent apoptosis in this paradigm23,24 suggesting a role for increased caspase production in the cell death process. As is the case with other cell types, most neurons express multiple caspases that may act at different stages of the apoptotic process. Moreover, because one type of caspase can cleave and thereby activate proforms of other caspases, the presence of multiple caspases in a single cell may provide an amplification mechanism to ensure rapid death and elimination of the cell. Caspases are synthesized as an inactive polypeptide with one large and one small subunit joined by a small spacer and a variable N-terminal prodomain which varies in size and sequence. The prodomain serves the function of preventing unwanted protease activation in healthy cells and controls protease activation by interacting with cofactors during apoptosis. When a cell receives an apoptotic stimulus, autoproteolytic processing removes both the spacer and prodomain resulting in a heterodimer comprised of small and large subunits. X-ray crystallographic analyses have revealed the structures of caspases 1 and 3 in complex with peptide-based inhibitors; in both cases the active protease is a tetrameric complex formed by two self-associating heterodimers interacting via the small subunits25,26. This tetrameric structure of the active enzyme complex suggests a requirement

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for multimerization of pro-caspase molecules during processing and activationz27. Additional studies have shown that the long prodomains in caspases 8, 9 and 10 are able to mediate dimerization of procaspase molecules and promote autoprocessing28. Accordingly, overexpression of these pro-caspases induces cell death that is dependent on their autocatalytic activity. Co-factors that modulate pro-caspase multimerization may therefore act as regulators of apoptosis29.

REGULATION OF CASPASE ACTIVITY There appear to be many different mechanisms whereby cells regulate caspase activity, positively or negatively. From the perspective of their positioning in apoptotic biochemical cascades, caspases can be classified as either initiator caspases (caspases 2, 8, 9, and 10) or effector caspases (caspases 3,4, 5, 6,7, 11, 12, and 13). One mechanism for positive modulation of caspase activity involves autocatalysis which was first suggested by the observation that initiator caspases have substrate specificities that are similar to caspase recognition sites present in their own sequence. Several pathways for activation of initiator caspases have been identified (Fig. 1). One pathway involves receptor-mediated activation of Fas-activated death domain (FADD)-associated caspase 8, which is activated by trimerization of death receptors such as Fas and tumor necrosis factor receptors (TNFR). Receptor engagement results in recruitment of death effector domain (DED)-containing procaspases (caspases 8 and 10) into a complex with the death receptor via the adapter protein FADD/MORT. This results in multimerization of pro-caspases followed by autoprocessing via transproteolysis. Activation of caspases 8 and 10 by this mechanism thus initiates an autocatalytic cascade because both of these caspases are capable of activating themselves and other caspases30. A second pathway of caspase activation involves postmitochondrial (cytochrome c-mediated) activation of caspase-9, a death-receptor independent process. Data suggest that cytochrome c, together with Apaf- I and cas ase-9, mediates the proteolytic activation of caspase-3 in an ATP-dependent manner31,32,33. The amount of caspase-8 generated at the receptor, which may vary between cell types, determines whether a mitochondria-dependent pathway is required for amplification of the caspase cascade34. Cytochrome c release is amplified by a caspase-8 dependent mechanism that involves cleavage and translocation of a protein called Bid to mitochondria35. Two prominent triggers of neuronal apoptosis that may play central roles in an array of neurodegenerative disorders are reactive oxygen species and calcium36,37. Exposure of cultured neurons to agents that induce calcium influx through plasma membrane channels (e.g., glutamate) or calcium release from endoplasmic reticulum (eg, thapsigargin) can induce apoptosis, and drugs that suppress calcium influx can prevent neuronal apoptosis38,39. Similarly, agents that induce oxidative stress (e.g., Fe2+ and amyloid b-peptide) can induce neuronal apoptosis, and antioxidants prevent such cell death40. Several endogenous caspase inhibitors have been identified including FADD-like ice (FLICE)-inhibitory proteins (FLIPS), inhibitor of apoptosis proteins (IAPs), CrmA and Bcl2. FLIPS are cytosolic caspase-like proteins that have two death domains at their aminoterminus and a caspase-like domain with significant homology to caspase-841. FLIPs inhibit caspases 8 and 10 by binding to the death domains of the caspase which interferes with the recruitment of caspases 8 and 10 to FADD. FLIPS are differentially expressed among cell types, and their expression can be regulated in ways suggesting an important role for changes in FLIP expression in differential susceptibility to apoptosis mediated by death receptors42,43,44. Another mechanism of caspase inhibition involves roduction of different splice variants of caspases that serve to inhibit caspase activity 45,46,3,47,18. For example, a form of caspase-9 which lacks the large subunit containing the catalytic domain functions as an anti-apoptotic protein by specifically blocking the Apaf-I-mediated activation of caspase 348. This form of caspase-9 may also 49form a heterodimeric complex with caspase-9 that would result in a nonfunctional enzyme . Short forms of caspases 2 and 6 can protect transfected cells from apoptosis by acting as dominant inhibitors of caspase activity45,4. Interestingly, analyses of mice lacking caspase-2 have shown that germ cells, but not motor or sympathetic neurons, are resistant to apoptosiss50, suggesting that activity of specific caspase splice variants modulates cell death and survival in the absence of caspase-251.

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Figure 1. Mechanisms of caspase activation and inactivation. Engagement of "death receptors" such as those for CD95 and tumor necrosis factor (TNF) receptor 1 stimulates the Fas-activated death domain (FADD), which then recruits caspases 8 and 10. The latter caspases, which contain death domains, are then proteolytically activated and released from the receptors and are then able to activate effector caspases such as caspases 3 and 7. Caspase 8 can also cleave Bid (l), which then translocates to mitochondria and induces cytochrome c release3. Cytochrome c combines with Apaf- I to activate caspase 95, which then cleaves downstream procaspases 3 and 7. Receptor-independent apoptotic signals, such as calcium and reactive oxygen species (ROS), can also induce cytochrome release (2) or can act in the nucleus to release yet unknown apoptogenic factors (7). Bc1-2 and Bc1-XL can block caspase activation at various steps. Calcium induces a conformational change in calpains, which subsequently translocate to the plasma membrane where both subunits undergo autocatalytic conversion at the N-termini to form the 76- and 18-kDa subunits. Activated calpain can interact with calpastatin in the presence of calcium and become inactivated. Caspases can cleave calpastatin which results in increased calpain proteolytic activity.

Members of the Bcl-2 and IAP families of apoptosis-regulating proteins can interact with caspases. Bcl-2 is an anti-apoptotic protein that can protect neurons against different apoptosis-inducing stimuli includin tro hic factor withdrawal, glutamate, oxidative insults and DNA-damaging agents52,53,54,39,40. Bcl-2 acts at a premitochondrial stage to prevent apoptosis and may do so by either preventing cytochrome c release from mitochondria or by binding to Apaf-1 (a protein that activates caspase-9) and thereby reventing multimerization of Apaf-I28. In addition, Bcl-2 blocks activation of nuclear55 and membrane-bound13 caspase-3. On the other hand, Bcl-2 is itself a substrate for caspase-3 and cleavage of Bcl-2 by caspase-3 may convert Bcl-2 to a pro-apoptotic protein56,57. IAPs are a family of caspase inhibitors that were identified based on homology to baculovirus. IAPs may suppress apoptosis, in part by inhibiting the activation of pro-caspase 958 and the activities of caspases 3 and 759. Many viruses produce proteins that inhibit caspases. For example, overexpression of p35 and CrmA in sympathetic neurons prevents cell death induced by trophic factor deprivation, staurosporine and Fas60,61,62. The mechanism whereby p35 suppresses apoptosis involves inhibition of caspases 1, 2, 3, 4, 6 and 1063,64, whereas CrmA inhibits caspases 1, 3, 7 and 865,66, CrmA and p35 act as competitive substrate

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inhibitors of caspases such that following cleavage their fragments remain bound to the active site of the caspase67,66. As is the case with many other proteins that regulate physiological processes such as apoptosis, caspase activity is subject to modulation by phosphorylation. For example, caspase-9 is inactivated when phosphorylated by Akt, a kinase that mediates anti-apoptotic actions of some growth factors68. Evidence suggests that several other kinases also modulate the activity of one or more caspases; the list of such kinases includes mitogenactivated protein kinase kinase69,70,71, protein kinase C72 and Akt71. Nitric oxide and other reactive oxygen species can also affect caspase activity73,74. NO inhibits apoptosis in some cell types75,76,77,78 by a mechanism involving reversible inhibition of caspases by direct Snitrosylation of the catalytic cysteine residue that is essential for enzyme activity75,79 . On the other hand, oxidative stress and nitric oxide can induce apoptosis in other cell types including neurons, and caspases mediate such oxidative stress-induced apoptosis 80,81,40,82,83. Table 1. Examples of caspase and calpain substrates that may mediate effects of the enzymes in synaptic plasticity and cell death.

Caspase substrates E-Catenin DII Spectrin (D -Fodrin) Actin Amyloid precursor protein Bcl-2/Bl-xL DNA-dependent protein kinase catalytic subunit (DNA-PKcs) DNA-repair enzyme PAPR Focal adhesion kinase (pp125FAK) Gelsolin Glutamate receptor (AMPA subunits) Inositol trisphosphate receptors (IP3R1 and IP3R2) Lamins A, B 1, C NFκB p50 and p65 subunits Presenilins 1 and 2 Protein kinase B Protein kinases G Protein phosphatase A2 (PP2a)

Calpain substrates Actin Amyloid precursor protein Bax Ca2+ATPase Focal adhesion kinase Glucocorticoid receptor IκBD Ionotropic glutamate receptors L-type Ca2+ channel MAP2 P53 Phospholipase C (PLC) Protein kinase A Protein kinase C Ryanodine receptors Spectrin (DII and EII) Tau

There are many different substrates cleaved by caspases, and their specific roles in apoptosis are being revealed (Table 1). Because nuclear DNA condensation and fragmentation are not observed in cells of caspase-3 knockout mice, and caspase-3 inhibitors also prevent DNA fragmentation, substrates cleaved by this enzyme are required for the alterations in nuclear DNA84. The nuclear envelope protein lamin and the actin-severing protein gelsolin play a role in mediating these nuclear fragmentation events. An enzyme called caspase-activated DNAase is activated by caspase-3 and effects internucleosomal cleavage of DNA leading to DNA laddering, a hallmark of apoptosis85. The activity of the DNAase is also dependent on caspase 3-mediated processing of an inhibitory protein associated with the DNAase86, which is a homologue of human DNA defragmentation factors87. Other nuclear proteins that are substrates for caspases include poly ADP-ribose polymerase (PAW), DNA-dependent protein kinase, and Ku protein88,89,90. Caspases can also proteolyze cell cycle-related proteins including cyclin D, several protein components of the RNA splicing complex, and the retinoblastoma tumor suppressor protein91-92. Some caspase substrates are activated upon cleavage. For example, several stress response proteins are activated by caspases and may play a role in triggering apoptosis93. Overexpression of the caspase-3-generated catalytically active fragments of PKC or PAK alone contribute to certain features of the apoptotic phenotype. It has been suggested that cleaved signaling proteins act to turn on death-promoting and/or turn off survival pathways

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that may differ from one cell type to another. Caspase-3-mediated cleavage of MAPK/ERK kinase kinase I (MEKKI) leads to the constitutive activation of Jun no-terminal kinase (JNK)/SAPK, which promotes the death pathway induced by a variety of cellular stresses including ultraviolet and J-irradiation, heat-shock, and camptothecin94. In addition, antiapoptotic signaling cascades may be turned off by caspases. For example, caspases can cleave and inactivate) the p50 and p65 subunits of NF-kB95, an anti-apoptotic transcription factor36,96, and caspases also cleave AKUPKB, a kinase that normally inactivates the proapoptotic Bad protein17. PAK2 has been shown to activate the JNK pathway and may be involved in JNK activation during apoptosis signaling. Phosphorylation of c-jun increases the transcriptional activity of the AP-I complex and is essential for apoptosis in cerebellar granule neurons and differentiated PC- 12 cells following withdrawal of surviving factors97. Interestingly, an aldehydic product of membrane lipid peroxidation called 4-hydroxynonenal, which is implicated in various experimental models of neurodegenerative disorders37, activates AP-1 by a mechanism involving caspase activation98. Cleavage of the regulatory subunit of protein phosphatase 2A (PP2A) increases its activity and leads to altered phosphorylation states of several substrates including MAP kinase, whose activity is dependent on phosphorylation. These studies suggest that caspases induce cell death not only by protein degradation but also by proteolytic activation of specific downstream effector molecules. The data further suggest roles for caspases in modulating these signaling kinases and transcription factors under physiological (nonapoptotic) conditions. Other cell survival proteins such as anti-apoptotic members of the Bcl-2 family (Bcl-2, and Bcl-xL) are cleaved by caspase-3 at functional domains that reverse their roles so as to promote cell death rather than survival56,57. This may result in the opening of the mitochondrial permeability transition (PT) pore because Bcl-2 has been reported to associate with the PT pore constituents and regulate the function of this entity in both isolated mitochondria and intact cells99,100,10.

CASPASES IN NEURONAL APOPTOSIS AND NEURODEGENERATIVE DISORDERS Three major lines of evidence support roles for caspases in neuronal apoptosis that occurs during normal development of the nervous system and in neurodegenerative disorders. The first line of evidence is that caspase activation is increased in neurons prior to their demise during development of the nervous system in mammals, and in humans with neurodegenerative disorders. The second line of evidence is that caspase activation occurs prior to, and in association with, neuronal death in cell culture and in vivo models of neurodegenerative disorders. The third line of evidence is that caspase inhibitors or genetic “knockout” of caspases prevent neuronal death. Caspase activity is typically assessed by determining whether caspase substrates are cleaved, and by employing “reporter” caspase substrates in either in vitro or in situ assays101,102. Analyses of neuronal populations in which programmed cell death occurs during development have revealed evidence for caspase activation. For example, caspase-3 is activated in CNS neurons that undergo apoptosis in developing mice103. Examination of tissue sections from brains of Alzheimer’s patients immunostained with an antibody that specifically recognizes activated caspase-3 reveals evidence for caspase activation in vulnerable neuronal populations in the hippocampus and cerebral cortex. Masliah et al.104 stained brain sections from Alzheimer’s disease and age-matched control patients with antibodies against activated caspase-3 and reported that, compared to agematched controls, Alzheimer’s patients exhibited greatly increased numbers of neurons with caspase-3 immunoreactivity. Chan and coworkers102 showed that overall levels of activated caspase- 1 activity are increased in hippocampal tissue from Alzheimer patients and that many neurons exhibit immunoreactivity with an antibody against activated caspase-3. The latter study also provided evidence for caspase-mediated degradation of the AMPA-type glutamate receptor subunits in Alzheimer’s brain tissue and in cultured neurons exposed to amyloid bpeptide. Immunostaining of brain sections from Alzheimer’s disease and Down Syndrome patients using an antibody against activated caspases revealed labeling of approximately 0.02 —0.1 % of neurons, while no neurons were labeled in tissue from control patients105. The involvement of individual caspases in programmed death of neurons has been studied in mice using caspase inhibitors and gene-targeting methods to specifically disrupt a caspase gene. Overexpression of the caspase-1 inhibitor crmA prevents programmed cell

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death of dorsal root ganglion neurons in the developing chick60. Examination of caspase knockout mice has revealed interesting tissue-specific roles for individual caspases106,107,108,50. For example, mice lacking caspase-3 have severe defects in nervous system development, but no apparent abnormalities in apoptosis of lymphocytes. Caspase-3 knockout mice die within five weeks of birth and display profound abnormalities in the cerebral cortex and forebrain owing to failed apoptosis in the proliferative Caspase-9 knockout mice exhibit a phenotype similar to that of neuroepithelium106,109. caspase 3-deficient mice, but die at a younger age. Cells lacking caspase-9 are highly resistant to apoptosis induced by radiation, but are susceptible to death induced by Fas ligation107. Mice lacking caspase-8 suffer early lethality and are resistant to Fas- and TNFinduced cell death, indicating that despite the association of these receptors with caspase 10, they depend on caspase 8 for cell death induction in vivo110. Caspase-1 knockout mice have no major defects in apoptosis and are developmentally normal, indicating that this caspase does not play a critical role in cell death during development111. Nevertheless, mice lacking caspase- 1 do have a defect in interleukin-lb (IL- 1b) processing in response to lipopolysaccharide and are resistant to endotoxic shock112. Moreover, the production of inflammatory cytokines (TNF, IL-Ib and IL-6) is impaired consistent with a role for caspase1 in inflammation113. Thus, caspase activation does not necessarily result in apoptosis, as cytokine processing is observed during a normal immune response without apoptosis of the secreting cells114. Studies of experimental models of neurodegenerative disorders have provided further evidence for a causal role for caspase activation in the neurodegenerative process (Fig. 2). Exposure of cultured hippocampal neurons to amyloid β-peptide induces caspase-3 activation, and treatment of the neurons with caspase inhibitors protects them against apoptosis induced by amyloid E-peptide115,116. When expressed in knockin mice, mutations in presenilin- 1 that cause early-onset inherited Alzheimer’s disease result in increased neuronal vulnerability to apoptosis which is associated with increased caspase activation117.

Figure 2. Roles of caspases and calpains in neuronal degeneration in Alzheimer's disease. See text for discussion. Ab, amyloid b-peptide; ApoE, apolipoprotein E; APP, amyloid precursor protein; ER, endoplasmic reticulum; PAW, poly-ADP-ribose polymerase; ROS, reactive oxygen species; RyR, ryanodine receptor; VDCC, voltage-dependent calcium channel.

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Transgenic mice expressing exon 1 of the huntingin gene with an expanded polyglutamine repeat, a model of Huntington's disease, exhibit increased caspase- 1 activation in association with progressive neurodegeneration. Expression of a dominant negative caspase-1 mutant delays development of neurodegenerative changes and motor dysfunction118. Degeneration of dopaminergic neurons in the substantia nigra of rats induced by 6-hydroxydopamine, a model of Parkinson's disease, is greatly reduced in animals pretreated with the caspase inhibitor zVAD-fmk119.

CHARACTERISTICS AND REGULATION OF CALPAINS Calpains are calcium-activated neutral (cysteinyl/thiol) proteases that are widely expressed in tissues throughout the nervous system. The two major isoforms of calpain that are widely expressed in mammals and have been most intensively studied are µ-calpain and m-calpain120,121. µ-calpain (also called calpain-I or CANP- I) binds Ca2+ with relatively high affinity (micromolar), whereas m-calpain (Calpain-II or CANP-11) binds Ca2+ with relatively low affinity (millimolar). Information on the structure and biochemical properties have been thoroughly reviewed previously122.121 and will therefore not be covered in the present article. Calpains I and II have distinct subcellular distributions suggesting different physiological roles for these two enzymes123. Within the central nervous system calpain I is localized mainly in neurons wherein its levels are higher in dendrites and the cell body than in axons. In contrast, calpain II is present at higher levels in axons and glial cell124,125 Whereas calpain I exhibits a relatively high level of basal activity, calpain II is generally responsive to stimuli, such as glutamate, that elevate intracellular Ca2+ levels. Calpains are expressed as proenzyme heterodimers consisting of an 80-kDa catalytic subunit, unique to each isozyme and encoded by a separate ene, and a 30-kDa regulatory subunit shared by both isozymes126,121. As with other Ca 2+-binding proteins, each subunit of the calpain heterodimer contains an EF hand Ca2+-binding domain127. The 30-kDa subunit also contains a hydrophobic glycine-rich domain that allows the enzyme to associate with cell membranes, whereas the catalytic site containing the critical cysteine and histidine residues is located on the 80-kDa subunit. Calpains are activated in response to increased levels of intracellular Ca2+ and inhibited by binding to the protein calpastatin. Calpain-I is activated in the cytosol or when bound to the cell membrane, whereas calpain-II activation occurs primarily at membrane sites. When calpains bind to Ca2+ they undergo a conformational change and translocate to phospholipid membranes where limited autolysis of the N-terminus of both subunits occurs. Attachment to plasma membrane sites serves to increase Ca2+ sensitivity, facilitating autocatalytic conversion of calpain at physiological concentrations of Ca2+ (0.1 – 1 µM). During this process, the 80-kDa subunit is processed to a stable 76-kDa form through a 78kDa intermediate, while the 30-kDa subunit is cleaved to an 18-kDa subunit128. An important consequence of autocatalytic proteolysis is that the cleaved protease requires a lower Ca2+ concentration for its activation, and is able to degrade membrane proteins directly or is freed to the cytoplasm where it may further proteolyze cytosolic substrates or become inactivated by combining with calpastatin. Calpastatin is a 110 kDa protein that is the only known endogenous inhibitor of calpains129. Calpastatin is widely expressed122 and is generally found at a higher concentration than calpains130,131. It contains four 140 amino acid repeat calpain inhibitory domains132. Calpastatin may be regulated by environmental signals because it can be phosphorylated by several different kinases including cyclic AMPdependent kinase133. Phosphorylation of calpastatin has been shown to alter its interactions with µ and m forms of calpain134, suggesting that phosphorylation of calpastatin may be a mechanism for modulating calpain activity.

CALPAINS IN NEURODEGENERATIVE DISORDERS The involvement of calpains in neurodegenerative conditions has been most intensively studied in experimental models of excitotoxic and ischemic brain injury135,136. It is well-established the disruption of neuronal Ca2+ homeostasis occurs as the result of acute ischemic insults, epileptic seizures and traumatic brain injury. Data suggest that calpains are activated in vulnerable neuronal populations in each of these conditions137,135,138.

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Administration of calpain inhibitors to animals in stroke models reduces neuronal damage and improves functional outcome139,135. Several chronic neurodegenerative disorders are also characterized by altered levels of calpain and/or calpastatin in vulnerable neuronal populations, including Alzheimer's In the case of disease140, amyotrophic lateral sclerosis141 and Parkinson's disease142, Alzheimer's disease, considerable data indicate that perturbed cellular calcium homeostasis contributes to the neurodegenerative process. In addition to evidence for activation of calpains in neurons in Alzheimer's patients, experimental studies have shown that: amyloid ß-peptide increases intracellular calcium levels in cultured hippocampal and cortical neurons and thereby promotes apoptosis and excitotoxicity143,144; mutations in the amyloid precursor protein that are responsible for some cases of Alzheimer's disease result in increased production of neurotoxic amyloid ß-peptide and decreased production of the neuroprotective (and calcium-stabilizing) secreted form of amyloid precursor protein (Fig. 2)145; mutations in presenilin- 1 that are responsible for many cases of early-onset inherited Alzheimer's disease perturb endoplasmic reticulum calcium homeostasis and thereby increase vulnerability of neurons to apoptosis and excitotoxicity39,146,117. It was recently shown that µ-calpain interacts with presenilin-2147, further suggesting a contribution of calpains to the pathogenic mechanism of presenilin mutations. While the majority of data suggest that calpains may contribute to the neurodegenerative process in Alzheimer's disease, it was shown that calpain inhibitors may alter proteolytic processing of the amyloid precursor protein in a manner that increases amyloid ß-peptide production148, suggesting that the contributions of calpains to the neurodegenerative process in Alzheimer's disease may be quite complex. Calpains are believed to play important roles in both apoptosis and necrosis in neurons149. Treatment with calpain inhibitors can protect neurons against apoptosis and necrosis in several experimental models150. In cultures of ciliary neurons deprived of trophic factors, inhibitors of calpain are at least as effective as caspase inhibitors in preventing DNA fragmentation and neuronal death151. However, because calpains normally subserve a variety of important physiological functions (see below), it is unclear how these proteases are recruited to the tightly regulated process of apoptosis. Calpains may work in concert with, or independently of, the process of apoptosis.

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Receptors for neurotransmitters, neurotrophic factors and cell adhesion proteins are concentrated in synaptic terminals. Accordingly, synapses are sites at which signal transduction pathways are highly activated. Many of these same signal transduction pathways that mediate adaptive changes in neuronal structure and function are also involved in degeneration of synapses and neuronal cell death during development and in pathological settings152. The two synaptic signaling pathways that have been most extensively studied from the perspectives of neuronal plasticity and death are those activated by the excitatory neurotransmitter glutamate and by neurotrophic factors152. Glutamate is the major excitatory neurotransmitter in the central nervous system, and activation of glutamate receptors is required for both short- and long-term changes in the structure and function of neuronal circuits153. For example, glutamate regulates neurite outgrowth and synaptogenesis in the developing hippocampus153, and long-term potentiation of synaptic transmission (a correlated of learning and memory) in the adult hippocampus154. Activation of glutamate receptors and reduced activation of neurotrophic factor receptors can induce caspase activation in neurons36,37. Recent findings suggest that caspases and calpains, in addition to their roles in neuronal death, play important roles in synaptic plasticity. Before considering the roles of caspases and calpains in synaptic plasticity and degeneration, it is of obvious importance to establish that these enzymes are present in synaptic compartments. Immunohistochemical studies of hippocampal neurons in cell culture and in vivo have shown that both calpains and some caspases (caspases 3 and 8) are present in dendrites and axons155,36,37. Experiments performed in cerebrocortical synaptosomes have shown that caspase-3 can be activated in synaptic terminals, and can mediate apoptotic changes in those terminals including mitochondrial alterations36,37. Excitotoxic and ischemic insults can induce calpain activation in dendrites following

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excitotoxic in cell culture and in vivo156,157, and calpains can also be activated in synaptosomes wherein they proteolyze spectrin155. In addition to their being activated in response to apoptotic and/or excitotoxic insults, caspases and calpains can also be activated by physiological activity in neurons. For example, caspases can be activated in response to membrane depolarization in cultured hippocampal neurons (M. P. Mattson, unpublished data) and calpains are activated in response to stimulation of hippocampal slices at frequencies that induce long-term potentiation200. Intraventricular administration of caspase inhibitors to adult rats impairs their performance in a water maze spatial learning task (Fig, 3), suggesting that caspases may normally play a role in this form of synaptic plasticity.

Figure 3. Caspases are activated in neurons in response to electrical activity, and may serve important functions in learning and memory processes. A. Rat hippocampal neurons in culture were exposed to saline (control), 30 mM KC1, or 100 mM KC1 for the indicated time periods and levels of caspase-3 activity in neuronal cell bodies were then quantified. Values are the mean and SEM. B. Saline (control) or the caspase inhibitor zVAD-fmk (10 pg) was administered to adult male rats via injection into the lateral ventricles, and goal latencies were determined in the Morris water maze one hour later as described previously. Values are the mean and SEM (n=4).

What are the protein substrates of caspases and calpains that might mediate effects of these enzymes on synaptic plasticity? A large number of caspase substrates have been identified and can be placed into several categories including structural proteins, proteins involved in signal transduction pathways, and nuclear proteins. Many cytoskeletal proteins are cleaved by caspases including those that form dynamic complexes involving actin filaments, membranes, and cell adhesion proteins. Interactions of actin and spectrin with membranes play major roles in regulating growth cone behaviors in developing neurons and synaptic structures such as dendritic spines in mature neuronal circuits. The cleavage of such cytoskeletal substrates likely contributes to the alterations in cell morphology that occur in cells undergoing apoptosis (cell rounding and membrane blebbing). Calpains plays a role in calcium-mediated changes in the actin-spectrin system199 and caspases, which are also responsive to elevations of intracellular calcium levels158,159, may also effect cytoskeletal changes when calcium levels are increased. One function of caspase and/or calpain-mediated cleavage of actin and spectrin may be to regulate neuronal calcium homeostasis. For example, it has been shown that actin-depolymerizing agents such as cytochalasin D reduce calcium influx through voltale-dependent calcium channels and N-methyl-D-aspartate (NMDA) receptor channels160,161,162. Studies of gelsolin knockout mice have shown that this calcium-activated actin-severing protein plays an important role in modulating the activity of voltage-dependent calcium channels and NMDA receptors162. Geloslin itself is a caspase substrate, and it will therefore be of considerable interest to elucidate the role of its cleavage in synaptic plasticity. Calpain activity may regulate the shape of dendritic spines in hippocampal neurons163, and data further suggest that NMDA and AMPA (a-amino-3hydroxy-5-methyl-4-isoxazole propionate) receptor subunits are substrates for calpains164,165,166, and that AMPA receptor subunits are substrates for caspases102. Cleavage

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of AMPA receptor subunits by caspases reduces AMPA responses, and may be a mechanism to prevent excitotoxic necrosis102. Proteins involved in signal transduction pathways are substrates for caspases and/or calpains. For example, several different membrane-associated and soluble kinases are cleaved by calpain including focal adhesion kinase and protein kinase C (PKC)167,168,169. Similarly, caspases can cleave focal adhesion proteins, certain PKC isozymes, and mitogenactivated protein kinases93,170,171,172,173. Phosphatases such as calcineurin are also substrates for cysteine proteases174. Physiological roles for cleavage of such substrates remain to be established, but seem very likely based on the wide array of physiological processes (ion channel activity, transcription, neurotransmitter release, etc.) that are regulated by phosphorylation and dephosphorylation. Indeed, long-term potentiation of synaptic transmission involves several caspase substrates including calcineurin, calcium/calmodulindependent protein kinase and protein kinase C. Both calpain inhibitors175 and caspase inhibitors (Fig. 3) have been reported to impair LTP and/or spatial learning. Cleavage of kinases and phosphatases by caspases and calpains may also influence neuronal cell death by altering neuroprotective signaling pathways. Synapses are sites where focal adhesion complexes are concentrated in neurons. Adhesion complexes consist of integral membrane proteins, such as neural cell adhesion molecules, cadherins and integrins that interact with similar cell adhesion molecules on other cells or the extracellular matrix; integrins also interact with cytoskeletal proteins on the cytoplasmic side of the membrane176. Integrins appear to play roles in modulating synaptic plasticity in the hippoccampus177,178 and integrin-mediated signaling may promote cell survival through anti-apoptotic signaling upon activation by ECM proteins179. Recent studies suggest that integrin engagement by laminin activates a neuroprotective signaling pathway involving PI3 kinase and Akt kinase in hippocampal neurons180. Several proteins in the integrin signaling pathway are substrates for caspases including Akt181. On the other hand, activation of the PI3 kinase – Akt athway can suppress caspase activation in several different paradigms of apoptosis182,183. The collective data therefore suggest intriguing reciprocal interactions between caspases and integrin signaling pathways exist that are presumably involved in controlling the various physiological processes regulated by integrin signaling. Studies of the molecular and cellular underpinnings of Alzheimer's disease have revealed additional caspase substrates that may play roles in regulating synaptic plasticity and degeneration. The ß-amyloid precursor protein (APP) is an axonally transported integral membrane protein that is present in synaptic terminals145. APP contains a 42-amino acid peptide called amyloid b-peptide, which is the major component of the insoluble amyloid "plaques" that accumulate in the brains of Alzheimer's patients. Amyloid b-peptide is liberated from APP by enzymatic cleavages at each end of the peptide. An alternative processing pathway, effected by an enzyme activity called (a-secretase, cuts in the middle of the amyloid ß-peptide and releases a large extracellular domain called sAPPa from synaptic terminals184,145. Studies of synaptic plasticity in hippocampal slices have shown that sAPPa can shift the frequency dependence for induction of long-term depression, and can enhance sAPPa can also protect neurons against apoptosis and long-term potentiation185. excitotoxicit in various cell culture and in vivo models186,187. APP is a substrate for caspases188,189 and calpains190,192, and it will be of considerable interest to determine the consequences of such cleavage on APP processing and the normal functions of APP. On the other hand, amyloid b-peptide can induce caspase activation in dendrites and synaptic terminals116, whereas sAPPa can stabilize intracellular calcium levels and suppress activation of apoptotic pathways that involve caspases186,201. Thus, changes in APP processing can indirectly affect caspase activity levels. Many cases of early-onset inherited forms of Alzheimer's disease are caused by mutations in the presenilin-1 gene191. Presenilin-1, an integral membrane protein with 8 transmembrane domains localized primarily in the endoplasmic reticulum, plays important roles in development and appears to interact with the Notch signaling pathway. Neurons expressing presenilin-I mutations exhibit increased vulnerability to apoptosis and excitotoxicity146,117 which appears to result from an adverse effect of the mutations on endoplasmic reticulum calcium regulation192. Presenilin-I is a substrate for caspase-3193,194, and its cleavage could conceivably contribute to neuronal apoptosis in Alzheimer's disease. Because calcium regulation by the endoplasmic reticulum is increasingly recognized as playing a role in synaptic plasticity195, caspase-mediated cleavage of presenilin-I might also serve a physiological role in synapses. In addition to APP and presenilin-1, several other

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proteins linked to neurode enerative disorders are caspase substrates. For example, the Huntingtin gene product196, the dentatorubral pallidoluysian atrophy (DRPLA) protein197, and the androgen receptor198 are each caspase substrates. The latter proteins contain elongated polyglutamine repeats caused by CAG expansion, which upon cleavage by caspase-3 generate fragments that aggregate intracellularly198.

CONCLUSIONS Caspases and calpains act on many different protein substrates in neurons, and cleavage of these substrates results in a variety of physiological and pathophysiological changes in the structure and function of neuronal circuits. In addition to playing central roles in the process of neuronal apoptosis, caspases appear to regulate synaptic plasticity and may be involved in synaptic degeneration and remodeling. The calcium sensitivity of calpains suggests that they are important effectors of changes in neurons brought about by calcium influx, an important physiological and pathological signal in neurons. Many different neurodegenerative disorders involve excessive activation of caspases and calpains including Alzheimer’s, Parkinson’s, Huntington’s diseases and stroke. Experimental findings suggest that caspase and/or calpain inhibitors can attenuate neuronal degeneration in models of these neurodegenerative disorders.

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Yamada, Dentatorubral pallidoluysian atrophy (DRPLA) protein is cleaved by caspase-3 during apoptosis, J. Biol. Chem. 272:29238 (1997). 198. C.L. Wellington, L.M. Ellerby, A.S. Hackam, R.L. Margolis, M.A. Tfifiro, R. Singaraja, K. McCutcheon, G.S. Salvesen, S.S. Propp, M. Bromm, K.J. Rowland, TZhang, D. Rasper, S. Roy, N. Thomberry, L. Pinsky, A. Kakizuka, C.A. Ross, D.W. Nicholson, D.E. Bredesen and M.R. Hayden, Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract, J. Biol. Chem. 273:9158 (1998). 199. G. Lynch and M. Baudry, Brain spectrin, calpain and long-term chances in synaptic efficacy, Brain Res. Bull. 18:809 (1987). 200. T. Suzuki, K. Okumura-Noji, A. Ogura, R. Tanaka, K. Nakamura and Y. Kudo, Calpain may produce a Ca(2+)-independent form of kinase C in long-term potentiation, Biochem. Biophys. Res. Commun. 189:1515 (1992). 201. K. Furukawa, B.L. Sopher, R.E. Rydel, J.G. Begley, D.G. Pham, G.M. Martin, M. Fox and M.P. Mattson, Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain, J. Neurochem. 67:1882 (1996).

THE UBIQUITIN/PROTEASOME PATHWAY IN NEUROLOGICAL DISORDERS

Maria E. Figueiredo-Pereira and Patricia Rockwell Department of Biological Sciences Hunter College of the City University of New York New York, NY 10021

INTRODUCTION Proteolysis is an important cellular event involving tightly regulated removal of unwanted proteins and retention of those that are essential. The ubiquitin/proteasome pathway plays a major role in the quality control process by eliminating mutated or abnormally folded proteins by degradation to prevent their accumulation as aggregates that often form intracellular inclusions. In many neurological disorders, aggregates of ubiquitin protein conjugates are detected in neuronal inclusions but their role in neurodegeneration remains to be defined. However, it has become increasingly evident that functional changes in the ubiquitin/proteasome pathway are critical to the neurodegenerative process. The aims of this chapter are to provide an overview of the: (1) components of the ubiquitin/proteasome pathway, (2) relationship between the ubiquitin/proteasome pathway and mechanisms such as oxidative stress, inflammation, and apoptosis, which are thought to participate in neurodegeneration, (3) recent information suggesting that the ubiquitin/proteasome pathway plays a role in hereditary forms of neurodegenerative disorders, and (4) current knowledge on the biogenesis of ubiquitin protein inclusions (aggresomes).

THE UBIQUITIN/PROTEASOME PATHWAY The ubiquitin/proteasome pathway is a proteolytic mechanism with broad specificity, cleaving peptide bonds after basic, acidic and hydrophobic amino acids. To function efficiently, this pathway requires proteins to be tagged by ubiquitin to target them for degradation. Therefore, proteolysis by the ubiquitin/proteasome pathway involves two major steps: ubiquitination followed by degradation. A de-ubiquitination step also plays an important role in this pathway.

Ubiquitination/de-ubiquitination Ubiquitin (Ub) is a small protein of 76 amino acids which can form polyubiquitin chains by the successive attachment of monomers. These are linked by an isopeptide bond most frequently formed between the side chain of Lys48 in one ubiquitin molecule and the carboxyl group of the C-terminal Gly76 in another ubiquitin molecule. Polyubiquitin chains thus formed are attached to lysine residues on a protein substrate resulting in at least a 1 0-fold increase in its degradation rate1. Polyubiquitin chains with linkages involving lysine residues on Ub other than Lys48 were found to play distinct roles2. In humans, there Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.

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are three Ub genes. Two of these contain heat-shock promoters, namely polyubiquitin B and C that code for four and nine copies of ubiquitin, respectively. The ubiquitin A gene codes for Ub fused to ribosomal proteins, and its function is not well understood 3

.

Figure 1. Protein ubiquitination. First, a high energy thioester bond is formed between ubiquitin (Ub) and a ubiquitin-activating enzyme (El). This reaction requires ATP hydrolysis. Secondly, the activated ubiquitin is transferred to a ubiquitin conjugating enzyme (E2). Thirdly, the activated ubiquitin is ligated, via an isopeptide bond, to the protein substrate by a ubiquitin ligase (E3). Lastly, the ubiquitin chain is elongated, by an ubiquitin-chain elongating factor (E4) which drives polyubiquitin chain (poly Ub) assembly.

Ubiquitination of proteins (Figure 1) is a complex process involving the following sequence of events: (1) formation of a high energy thioester bond between Ub and a ubiquitin-activating enzyme (El) in a reaction that requires ATP hydrolysis; (2) formation of a thioester bond between the activated ubiquitin and ubiquitin-conjugating enzymes (E2); (3) covalent attachment of the carboxyl terminal of ubiquitin, usually to the H-amino group of a lysine residue on protein substrates via an isopeptide bond mediated by ubiquitin ligases (E3); and (4) assembly of polyubiquitin chains carried out by a novel family of ubiquitination factors (E4) which promote the production of longer Ub-chains4. In some cases, ubiquitin can be transferred directly to the protein substrate by ubiquitinconjugating enzymes (E2). There are many different E2 and E3 enzymes, indicating that this pathway may operate through selective proteolysis5. There are at least 30 E2s identified in humans. They share a common 150-amino acid catalytic core, whereas each subfamily possesses affinity for a different class of E3 enzymes. E3s recognize specific protein substrates for ubiquitination, and at least four classes have been described. (D enzymes bind protein substrates with basic or hydrophobic N-terminal amino acids5. The HECT-E3s (h omologous to E6AP carboxyl-terminus) form ubiquitin-thioester intermediates and ubiquitinate substrates directly. So far, 20 members of this class (HECT-E3s) have been identified6. Other E3 classes, such as Skpl-Cullin-F box complexes (SCF) and anaphase pomoting complexes (APC) do not form a ubiquitinthioester intermediate6. Several RING-finger-containing proteins were found to be E2dependent ubiquitin ligases (E3)7,8. Ubiquitin is removed from ubiquitinated proteins by de-ubiquitinating enzymes which also disassemble polyubiquitin chains. More than 90 genes coding for de-ubiquitinating enzymes have been identified, making them the largest family of enzymes involved in the ubiquitin pathway9. There are two major classes of de-ubiquitinating enzymes: (1) Ubiquitin carboxyl-terminal hydrolases (UCHs) that remove small amides, esters, peptides and small proteins at the carboxyl terminus of ubiquitin, and (2) ubiquitin-specific processing proteases (UBPs) which disassemble the polyubiquitin chains and may edit the ubiquitination state of proteins10.

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Proteasome degradation Covalent binding of ubiquitin to proteins marks them for degradation by the 26S proteasome (Figure 2), an enzymatic complex with a native molecular weight of approximately 2,000 kDa11. The 26S proteasome includes two major particles: a 20S particle, known as the 20S proteasome, which is the catalytic core, and a 19S particle, known as PA700, which is the regulatory component. Association between the two particles in the cell is a dynamic process and requires ATP-hydrolysis. The 20S proteasome can associate with other regulatory members, such as PA28, but this combination is not known to degrade ubiquitinated proteins11.

Figure 2. The 26S proteasome. Its two major particles, the 20S particle (20s proteasome) which is the catalytic core, and the 19S particle (PA700) which is the regulatory component, require ATP hydrolysis to assemble into the 26S proteasome. The PA700 lid confers ubiquitin/ATP-dependency to proteasome proteolysis. The PA700 base has ATPase and chaperone-like activity. The midlongitudinal view of the 20S proteasome was drawn from Iryp.pdb12.

The 20S particle is composed of 28 subunits arranged in four heptameric-stacked rings forming a cylindrical structure with a hollow center in which proteolysis takes place12. The 20S proteasome hydrolyses most peptide bonds present in a protein13, and the rate of this hydrolysis is influenced by proteasome subunit composition14. Assembly of this particle from precursor subunits is a complex process and was shown to require the assistance of a short-lived chaperone15. The 19S particle (PA700) contains at least 17 subunits, including ATPases, a deubiquitinating enzyme and polyubiquitin-binding subunits. It confers ubiquitid/ATPdependency to proteolysis by the 26S proteasome11. PA700 can also stimulate proteasomal degradation of non-ubiquitinated proteins such as ornithine decarboxylase, which requires only ATP hydrolysis for its proteasomal breakdown16. The subunits in PA700 are distributed into a lid and base arrangement, with the lid required for ubiquitin/ATPdependent proteolysis17. The base, containing the ATPases, exhibits chaperone-like activity18. The 26S proteasome is found in the cytosol next to intermediate filaments of the cytoskeleton19. It also resides in the nucleus and in association with the cytosolic side of ER membranes20,21. Localization studies with fluorescently labeled subunits of the 20S and 19S particles demonstrated that proteasomal proteolysis occurs mainly at the nuclear envelope/rough ER site22. An important function of such proteolysis is to eliminate abnormal secretory proteins residing in an EWpre-Golgi compartment11. Functionally inefficient, misfolded or unassembled ER proteins leave this intracellular compartment by retrograde transport through the Sec61 translocation channel, whereupon, they are ubiquitinated by ubiquitin-conjugating enzymes associated with the cytosolic side of the ER membrane, and then degraded by the cytosolic 26S proteasome23. Although this ER degradation pathway appears to be non-essential for viability, its importance is underscored by its evolutionary preservation "despite strong negative selection" since disruption of this mechanism seems to be associated with many diseased states23. The 20S proteasome was detected in all areas of the rat CNS, but higher levels were found in pyramidal cortical neurons of layer 5 in the brain and the motor neurons of the ventral horn of the spinal cord24.

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Substrate recognition Three key characteristics target proteins for ubiquitination/degradation: (1) misfolding due to mutation or damaging events; (2) constitutively active ubiquitination signals; and (3) post-translational modifications such as phosphorylation/dephosphorylation events or co-factor binding25. The unfolding of normal substrates precedes their degradation. This step is required to allow entry into the proteolytic chamber of the 20S proteasome through its narrow openings26. Unfolding activities may be provided by ATPase subunits in the PA700 base or by extraproteasomal chaperones. Degradation of ubiquitinated proteins is enhanced when more than one ubiquitin is attached to the target protein. The minimal signal for efficient degradation is a tetraubiquitin chain26. Removal of two ubiquitins from a tetraubiquitinated substrate by deubiquitinating enzymes, such as UCH37, can decrease substrate/26S proteasome affmity by approximately 100-fold, allowing the substrate to escape degradation. Longer chains do not increase substrate/26S proteasome affinity, but optimize their interaction time26. The interaction of the polyubiquitin chain with the 26S proteasome involves hydrophobic patches on the surface of the tetraubiquitin chain, generated by Leu8, I1e44, and Val70 in each ubiquitin moiety, and two hydrophobic sequences with the motif LeuAlaLeuAlaLeu in the PA700 subunit S5a27. Additional ubiquitin-binding subunits on the 26S proteasome must exist since S5a is not an essential protein in yeast27. The rate at which protein substrates of this pathway are degraded depends on the interplay between their deubiquitination and their unfolding26.

Ubiquitin-like proteins Two types of ubiquitin-like (Ubl) proteins have been identified: type 1 and type-2 Ubls28. Type 1 Ubls, such as SUMO1 (small Ub-related modifier) and NEDD8 (neural precursor cell-expressed developmentally down-regulated gene), are small and are covalently attached to proteins in a manner similar to ubiquitination, although they require their own enzymatic components29. Some SUMO1-modified proteins seem to assist nuclear translocation of other proteins28. NEDD8-protein interaction is important in cell cycle regulation28. Type-2 Ubls, such as RAD23, Parkin and ElonginB, are not ligated to other proteins. Instead, they occur as fusion proteins with a ubiquitin-like domain located at their N-terminus, in the central portion, or at the C-terminus. The physiological significance of these fusion proteins remains uncertain, although they may function in DNA repair (RAD23) or as ubiquitin ligases (ElonginB).28

THE UBIQUITIN/ROTEASOME PATHWAY AND NEURODEGENERATIONINDUCING MECHANISMS Although selective sets of neurons are affected in different neurodegenerative disorders most of them share an intriguing morphological feature, namely, the accumulation of ubiquitinated proteins30. These diseases are, therefore, associated with an inability of the neuron to degrade ubiquitinated proteins, and may be classified as ubiquitinopathies31. In general, high levels of ubiquitinated proteins do not accumulate in healthy cells as they are rapidly degraded by the ubiquitin/proteasome pathway. The inability to eliminate these modified proteins may result from a functional failure of the ubiquitin/proteasome pathway or from structural changes in the protein substrates which render them inaccessible to the degradation component. The ubiquitin/proteasome pathway may, therefore, play a role in mechanisms such as oxidative stress, inflammation and apoptosis, all of which are implicated as mediators of abnormal protein deposition and cell death in neurodegeneration.

Oxidative Stress The involvement of oxidative stress in neurodegeneration has gained support from increasing evidence of its role in neuronal death in disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Studies with autopsied brains of AD patients showed a co-localization of high levels of oxidative stress products with neurofibrillary tangles and senile plaques32. Signs of oxidative stress, such as lipid peroxidation and a decline in

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reduced glutathione, were also detected in the substantia nigra in brains of PD patients33 The production of free radicals and lipid peroxidation by oxidative stress promotes partial unfolding of cellular proteins, resulting in exposure of previously buried hydrophobic domains to proteolytic enzymes34,35, and to ubiquitin-conjugating enzymes.36 Therefore, one important cellular anti-oxidant mechanism is an increase in intracellular proteolysis by the ubiquitin/proteasome pathway. Oxidative stress can affect components of the ubiquitin/proteasome pathway as well as its substrates, leading to an increase in the intracellular levels of ubiquitinated proteins. Covalent attachment of the lipid peroxidation product 4-hydroxy-2-noneal (HNE) to the 20S proteasome decreases its activity37,38. The chaperone HSP90, however, can prevent the inactivation of the proteasome under such conditions39 . In addition, HNE-modification of proteins results in their accumulation as ubiquitin-conjugates, confirming that the metabolism of HNE-altered proteins involves the ubiquitin/proteasome pathway40. Cadmium- or zinc-induced oxidative stress results in protein thiolation and inhibits the activity of the ubiquitin/proteasome pathway41,42. The increased levels of protein mixed disulfides produced in cadmium- and zinc-treated neuronal cells was found to be accompanied by an accumulation of ubiquitinated proteins, suggesting that thiol-modified proteins are broken down by the ubiquitin/proteasome pathway. Oxidative stress induced by hydrogen peroxide also affects the ubiquitin/proteasome pathway, either by directly decreasing the activity of the 20S or 26S proteasome43-45 or by increasing the expression and activity of at least two members of the ubiquitination machinery, namely E1 and E2 enzymes46. In addition, H2O2-induced oxidative stress increases intracellular levels of protein-bound carbonyls. Such modified proteins are removed by the proteasome47. The H2O2-effects are dependent on changes in the redox status of the cell, manifested by a decrease in reduced glutathione (GSH) and an increase in oxidized glutathione (GSSG). Reestablishment of the GSSG:GSH ratio allows recovery from the oxidative stress insult. The stability and, therefore, the activity of two transcription factors namely iron regulatory protein2 (IRP2)48 and hypoxia-inducible factor1 alpha (HIF1 D  were found to be dependent on the oxidation level of iron- or oxygen-degradation-dependent domains on each protein, respectively. In iron- or oxygen-replete cells, IRP2 and HIF1 D are rapidly and selectively turned over by the ubiquitin/proteasome pathway. However, they are transcriptionally active only under conditions of iron depletion, in the case of IRP2, or oxygen deprivation, in the case of HIF1 D Degradation of these transcription factors by the ubiquitin/proteasome pathway appears to involve recognition of specific oxidatively modified amino acids on these proteins by ubiquitin protein ligases. Just as phosphorylation of selective amino acids dictates turnover of proteins like ,N%D so may oxidation of particular amino acids target proteins, such as IRP2 and HIF1D for degradation. Furthermore, stabilization of HIF1D is not only elicited by hypoxia but also by transition metals, iron chelators, and several anti-oxidants50. Together, these results indicate that the ubiquitin/proteasome pathway plays a key role in the intracellular antioxidant defense mechanism, because it removes oxidatively damaged proteins and modulates the activity of oxidation-dependent transcription factors.

Inflammation Many neurodegenerative disorders, including AD, are associated with chronic inflammation, as shown by the presence of more than 40 immunoprotective proteins in AD brains at autopsy 51 . These immunoprotective proteins cannot be detected in normal brains. In addition, epidemiological studies involving 1686 participants in the Baltimore Longitudinal Study of Aging demonstrated that the use of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, decreases the relative risk for AD, and that this decrease is proportional to the duration of the treatment.52 The protective effect of NSAIDs may correlate with their inhibition of the enzymatic activity of a pro-inflammatory and inducible cyclooxygenase, known as COX-2. Up-regulation of this enzyme causes tissue damage through prostaglandin and reactive oxygen species production53. While COX-2 protein levels are almost undetectable in normal brains, its expression increases after focal ischemia in infarcted human brains52 . This enzyme was shown to be an immediate early gene transiently induced in hippocampal neurons after injection of the excitotoxin kainic acid into rat brains54. In patients with Fukuyama-type congenital muscular dystrophy, a

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neurodegenerative disorder transmitted through autosomal recessive inheritance, upregulation of COX-2 precedes appearance of neurofibrillary tangles (NFT)-containing neurons and neurodegeneration55. No E-amyloid deposits or senile plaques are detected in this disorder, but COX-2 immunoreactivity was found to co-localize with NFT-containing neurons. Similarly, both NFT-containing and damaged neurons in Down's syndrome and AD were found to exhibit high expression of COX-256 These findings suggest that the spatial and temporal association of COX-2 with neuropathological changes, such as NFTformation, correlates with neurodegeneration in these diseases. Recent studies with neuronal cells also provided evidence that a relationship may exist between COX-2 induction and the accumulation of ubiquitinated proteins. Neuronal cell death resulting from inhibition of the ubiquitin/proteasome pathway was preceded by an accumulation of ubiquitinated proteins in conjunction with increased expression levels of the stress-inducible protein HSP70, COX-2 and its pro-inflammatory product, prostaglandin PGE257. In addition, these studies showed that COX-2 turnover was mediated by the ubiquitin/proteasome pathway. Other investigations demonstrated that prostaglandins act as neurotoxins by increasing the levels of ubiquitin-conjugates and Eamyloid production in differentiated neuroblastoma PC 12 cells58 . Thus, the metabolic products induced by pro-inflammatory responses in neuronal cells may create a mechanism of self-destruction via an autotoxic loop59. This event could intensify the fundamental pathology of some neurodegenerative disorders such as AD.

Apoptosis The causes of neuronal cell death in many neurodegenerative disorders remain unclear. Hereditary forms of neurodegeneration can be attributed to specific gene mutations, but the underlying mechanisms responsible for loss of selective neuronal populations in these diseases have yet to be identified, although apoptosis appears to play a role60 . In particular, members of a family of proteases known as caspases, specifically caspase 1 and 3, were implicated as mediators of neuronal apoptosis61. In primary neuronal cultures, proteasome inhibitors were found to induce apoptosis, manifested by activation of caspase 3 proteases, disruption of mitochondrial membrane potential, and release of mitochondrial cytochrome C into the cytosol62. Stimuli-induced Bcl-2 turnover by the ubiquitin/proteasome pathway was shown to be preceded by its dephosphorylation63,64. It is now clear that the ubiquitin/proteasome pathway plays an important role in apoptosis, upstream of the caspase cascade, because it regulates the levels of the anti-apoptotic protein Bcl-2 (B-cell lymphoma-related protein).65 This view is supported by the finding that proteasome inhibitors prevent cerebellar granule neuronal death caused by a reduction in extracellular potassium, if they are administered before the onset of this process66.

POSSIBLE ROLE OF THE UBIQUITIN/PROTEASOME PATHWAY IN HEREDITARY FORMS OF NEURODEGENERATIVE DISORDERS The neurofibrillary tangles (NFT) in AD were the first neuropathological intracellular lesions found to immunostain with antibodies against ubiquitin conjugates67. Since then, ubiquitin conjugates were identified in innumerable neuronal inclusions68. The discovery that many neurodegenerative disorders are associated with mutations in genes other than those linked to the ubiquitin/proteasome pathway, suggested that the causal relationship between ubiquitin conjugate deposition and neurodegeneration is indirect. However, recent findings that mutations in ubiquitin and other components of the ubiquitin/proteasome pathway are associated with certain neurodegenerative diseases, indicate that the ubiquitin aggregates may hold a clue to the pathological process in neurodegeneration.

Alzheimer's disease Most cases of AD result from sporadic changes in neuronal cell metabolism whereas a small percentage (up to 10%) is genetic and occurs as autosomal dominant mutations. Familial AD is associated with mutations in the amyloid precursor protein (APP) and

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presenilins but the exact functions of these proteins are not known. In sporadic cases, individuals carrying two copies of the allele 4 of apolipoprotein E (ApoE4) have an increased risk of contracting the disease.69 The accumulation of dysfunctional frameshift proteins in neurons is fairly common and may occur as a result of incorrect editing of RNA transcripts. Frameshift mutants of ubiquitin B (UbB+l) and APP (APP+l) were found to co-localize with NFT and senile plaques in the cerebral cortex of patients with sporadic AD. The genes encoding the two proteins contain one or more GAGAG motifs that are prone to GA deletions during transcription. A single dinucleotide deletion (GA) in the first GAGAG motif of UbB mRNA, produces UbB+1, which lacks the C-terminal glycine, an amino acid essential for ubiquitination31. UbB+1 molecules may impair degradation of ubiquitinated proteins by competing with wild type ubiquitin for the interaction with the 26S proteasome. Presenilins (PS) are transmembrane proteins. They may regulate APP maturation, as certain mutations in PSs seem to increase production of one of the products of APP, Aβ, a peptide present in senile plaques of AD brains and postulated to be neurotoxic70. One of the presenilins, PS1, was found to interact directly with subunits of the 20S proteasome71. The demonstration that proteasome inhibitors promote PS1 accumulation as highmolecular weight ubiquitin conjugates provided evidence that the ubiquitin/proteasome pathway degrades this presenilin.71,72 The ubiquitin/proteasome pathway, therefore, seems to play an important role in PS1 turnover. APP and ubiquitin were found to co-localize in AD brain extracts subjected to electrophoresis on non-denaturing gels, suggesting that their interaction is non-covalent73. In addition, in vitro studies demonstrate that Aβ,1-40, but not its reverse peptide Aβ40-1, can enter the catalytic chamber of the 20S proteasome and inhibit the degradation of ubiquitinated proteins.74,75 The in vivo importance of these interactions remains to be established. Neurofibrillary tangles (NFT) in AD brains contain not only ubiquitin conjugates, but also neurofilaments and tau, a cytoskeleton protein required for stabilization of microtubules in the polymerized state. In NFTs, tau is hyperphosphorylated and forms paired helical filaments, losing its microtubule-stabilizing properties. Mutations in the tau gene, some of which lead to an increase in intracellular levels of normal tau, were found to cause frontotemporal dementia and parkinsonism linked to chromosome 1776,77. Transgenic mice overexpressing the four-repeat human tau in neurons, mimicking tau mutations in intron 10, developed axonal degeneration, astrogliosis and accumulation of ubiquitinated proteins in a transgene-dose-dependent fashion. These effects appeared without formation of intraneuronal neurofibrillary tangles78. Transgenic mice overexpressing the smallest tau isoform developed inclusions mostly in the spinal cord, but these inclusions lacked detectable ubiquitin/conjugates79. These findings indicate that higher than normal levels of a protein become cytotoxic when they accumulate in aggregates, if their rate of synthesis far exceeds their rate of degradation.

Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis is the dominant motor neuron disease. Familial ALS is associated with mutations in the human Cu/Zn superoxide dismutase gene (SOD1).80 Transgenic mice expressing certain SOD mutations develop a motor neuron disease (MND) that is phenotipically similar to ALS. Lewy body-like inclusions containing crosslinked neurofilaments and ubiquitinated proteins are detected in motor neuron of FALS patients and MND mice.80 Both protein modifications are thought to result from oxidative stress induced by the SOD-mutations.80 Exacerbation of this insult in affected motor neurons may impair degradation of the modified proteins by the ubiquitin/proteasome pathway. Spinal cord injury in humans and spinal cord compression injury in rats are followed by accumulation of ubiquitinated proteins and of neuronal PGP9.5, an ubiquitin C-terminal hydrolase. These findings implicate a role for the ubiquitin/proteasome pathway in the recovery process.81

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Angelman's syndrome Sporadic and familial cases of Angelman's syndrome (AS) are associated with severe motor dysfunction and mental retardation82 . AS is one of the first hereditary disorders in humans shown to result directly from a loss of the maternal copy of a gene that codes for a component of the ubiquitin/proteasome pathway and which is located on chromosome 1583-85. Thus, positional cloning revealed that loss-of-function mutations in ube3, which encodes the E3 ubiquitin ligase E6AP, are the cause of the disease84,85. E6AP belongs to the HECT class of E3s. The crystal structure of its catalytic HECT domain revealed that most AS mutations map to the catalytic cleft and affect ubiquitin-thioester bond formation.6 While the protein substrates critical for AS have yet to be identified it is known that E6AP ubiquitinates p53, several Src family protein kinases, the human homologue of Rad23 and MCM7, a protein that plays a role in chromosomal replication.6 In a mouse model of AS, animals inheriting E6AP mutations in the maternal chromosome, have a severely impaired long-term potentiation (LTP), although their baseline synaptic transmission and neuroanatomy remain normal86. These findings suggest a role for E6AP-mediated ubiquitination in LTP. Interestingly, PGP9.5, a neuronal ubiquitin C-terminal hydrolase, was found to be an immediate early gene product involved in long-term facilitation (LTF) in the marine snail, Aplysia87. LTF in Aplysia involves prolonged activation of the CAMP-dependent protein kinase, which in turn requires the ubiquitin/proteasome pathway for degradation of its regulatory subunit.88 Persistent kinase activity is needed to induce the CREB-mediated transcriptional cascade for synthesis of selective proteins that participate in new synapse growth.87 The Aplysia transcription factor ApC/EBP, active early in LTF, was also found to be degraded by the ubiquitin/proteasome pathway only when dephosphorylated.89 In neuroblastoma cells, proteasome inhibitors promote neuritogenesis and NGF-treatment causes an increase in the levels of ubiquitinated proteins and of Ub-E1 and Ub-E2 thioesters.90,91 These data suggest that stabilization, rather than degradation of substrates of the ubiquitin/proteasome pathway must play a role in neurite outgrowth. Other studies showed that differentiation induced by retinoic acid in human neuronal progenital cells resulted in changes in proteasome activity and composition92.

CAG/polyglutamine expansion diseases At least eight neurodegenerative diseases are caused by polyglutamine (polyQ) repeats in specific proteins: DRPLA (dentatorubral pallidolusian atrophy), HD (Huntington's disease), SBMA (spinal and bulbar muscular atrophy), and the spinocerebellar ataxias SCA1, SCA2, SCA3, SCA6, and SCA7.93 The expanded polyQs of the mutant proteins participate in the formation of toxic intranuclear inclusions within the neuron. This may lead to cell death and neurodegeneration.94 In HD, mutant huntingtin, with 36 to 120 glutamine(Q)-repeats at its N-terminus, forms intranuclear inclusions containing ubiquitin-conjugates in affected brain regions, such as the striatum and the cerebral cortex.95 Wild type huntingtin, with only 6 to 34 Qrepeats at its N-terminus, is localized in the cytosol. The toxic nature of the intranuclear inclusions is a controversial issue. The first HD transgenic mouse model was established by inserting 141-157 CAG/glutamine repeats into exon 1 of the human huntingtin gene.96 The onset of the disease in these mice is at approximately 8 weeks of age, and prior to this stage, no ubiquitin staining could be detected in the neuropil aggregates. An inclusion analysis demonstrated that intranuclear lesions were formed in these mice by selfaggregation of the mutant protein into amyloid-like fibrils prior to the onset of symptoms, possibly triggering manifestations of the disease.93 Only upon onset of the disease could ubiquitin-aggregates be detected, suggesting that ubiquitination represents a final attempt to remove the aggregates by proteolysis,96,97 Abundant nuclear inclusions in cellular models of HD do not correlate with cell death.98-100 These investigations, however, test only the short-term effects of overexpressed mutant huntingtin, and may not mimic the diseased state. 100 Other studies associate toxicity with mutant huntingtin only when it is expressed with a nuclear targeting signal and not with a nuclear export signal, suggesting that only nuclear mutant huntingtin plays a role in the pathogenesis of the disease.95,101,102 Properties of mutant huntingtin independent of its potential for aggregation may be more directly linked to the disease. The function of the normal huntingtin protein is not

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well understood, but it seems to interact with cytoskeleton elements and to be required for neurogenesis.103 Huntingtin interacts with the ubiquitin-conjugating enzyme E225K, also known as Hip-2 (huntingtin-interacting protein), and its interaction occurs independently of the number of polyQ repeats in huntingtin.103,104 Studies on mouse brain development indicate that the mRNA expression of huntingtin and E225K is spatiotemporally related during neuronal maturation, suggesting that the interaction between the two proteins is required for normal development and the onset of HD.105 Huntingtin turnover is carried-out by the ubiquitin/proteasome pathway.103,104 Mutant huntingtin, however, is ubiquitinated but not degraded, suggesting that polyQ repeats may block protein binding or access to the proteolytic core of the 26S proteasome for degradation. 103 Like huntingtin, the mutant proteins ataxin1 and ataxin3 associated with spinocerebellar ataxias SCA1 and SCA3 (also known as Machado-Joseph disease), exhibit polyQ repeats and accumulate in intranuclear inclusions containing ubiquitin-conjugates. In both diseases, the nuclear environment is essential for aggregate toxicity. These aggregates also stain positive for components of the 20S and 19S particles of the 26S proteasome, a finding that strongly suggests a role for the ubiquitin/proteasome pathway in aggregate biogenesis.106-108 Further studies support this view, since proteasome secific inhibitors in cellular models of SCA3 were shown to increase aggregate formation.109 Both wild type ataxin1 with two glutamines (Q) and mutant ataxin1 with 92 Qs were found to be polyubiquitinated, but proteasomal degradation of mutant ataxin1 was significantly impaired.110 In addition, transgenic mice expressing mutant ataxin1 and lacking the ubiquitin ligase E6AP, do not develop nuclear inclusions but exhibit accelerated SCA1 pathology.110 Although nuclear inclusions may not be essential to trigger neurodegeneration in these diseases, a loss of selective proteasomal degradation by a lack of E6AP-activity may be pivotal to the process. 111 This may be the case in SCA6, an ataxia due to a small polyQ expansion on a calcium channel which does not form detectable ubiquitin intranuclear inclusions. 109

Parkinson's disease The etiology of Parkinson's disease remains unknown. In the substantia nigra of Parkinson's diseased brains, ubiquitin-conjugates accumulate in cytosolic inclusions known as Lewy bodies. These inclusions also contain a protein of unknown function, Dsynuclein, an ubiquitin C-terminal hydrolase, UCH-L1 also known as PGP9.5, and proteasome subunits. These findings clearly implicate the ubiquitin/proteasome pathway in the etiology of this disease.112 Mutations in D-synuclein were found to cause familial PD in four different families.113 In vitro studies demonstrated that mutant D-synuclein (A53T) is cleaved by the ubiquitin/proteasome pathway at a slower rate than wild type, an event that provides a basis for its aggregation in intracellular inclusions.112 Deletions in the exon regions of the parkin gene were found to be associated with an autosomal recessive juvenile parkinsonism.114 Parkin, the protein product, is abundant in the brain and its N-terminal sequence is moderately similar to ubiquitin. The function of this newly identified ubiquitin-like protein remains unknown. A missence mutation (Ile93Met) in the uch-l1 gene, which codes for a deubiquitinating enzyme, was identified in a German family with PD.115 This ubiquitin Cterminal hydrolase is very abundant in the brain and the mutation described was shown to decrease its catalytic activity.115 Genetic analysis in other Caucasian families failed to detect a similar mutation.116-118 An in-frame deletion including exons 7 and 8 of the uch-l1 gene was described as the cause of gracile axonal dystrophy (gad ) in mice. This mutation results in a truncated protein lacking 42 amino acids including a possible active site histidine119. This genetic model is characterized by a retrograde accumulation of amyloid E-protein and ubiquitinconjugates in sensory and motor neurons, as seen in certain inherited human neurodegenerative diseases119,120. The gad mouse is the first mammalian model of a hereditary neurodegenerative disorder that results from a mutation in a component of the ubiquitin/proteasome pathway. Future studies may reveal defects in other proteins that function in the ubiquitin/proteasome pathway as further evidence is acquired to substantiate the role of this pathway in pathogenesis of neurodegeneration.

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Prion diseases Prion diseases occur as either genetic disorders or as sporadic forms some of which are acquired by infection. Both types share a central pathogenic event in which a conformational change is elicited in the wild type prion protein (PrPc), a 209- amino acid glycoprotein res linked to the plasma membrane121. This results in the conversion of PrPc to an isoform (PrP ) that is protease-resistant and, in some cases, transmissible by unknown mechanisms. Twenty three pathogenic mutations are thought to cause spontaneous conformational changes in PrPc and are manifested as three phenotypes: Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), and Gerstmann-Straussler-Scheiker (GSS) syndrome121. One of the PrPc mutations associated with GSS involves replacement of tyrosine (TAT) at codon 145 with a stop codon (TAG), designated Y145stop, to yield a truncated PrP protein (PrP145). Neuroblastoma cells transfected with the gene encoding PrP145 showed expression of an unstable mutant protein that was rapidly degraded by the ubiquitin/proteasome pathway121. These studies are the first to show degradation of a prion protein by the ubiquitin/proteasome pathway. Proteasome inhibition led to accumulation of PrP145 in aggregates that could be extracted as detergent insoluble and soluble fractions, both forms displaying resistance to proteinase K treatment121. Zanusso et al121 suggest that decreases in proteasomal activity with advanced age are responsible for accumulation of the mutant protein. Hence, this age-related decrease in proteolysis may increase the levels of the highly amyloidogenic PrP fragments and cause the formation of amyloid deposits in cerebral parenchyma and vessels detected in the variant of the GSS disease expressing PrP145.

Wilson disease Copper is a trace element and maintenance of its homeostasis is essential for the nervous system to function properly122. Its importance is underscored by the discovery of Wilson disease, a hereditary human disorder caused by a deficiency in copper metabolism that leads to neurodegeneration and hepatic cirrhosis122. The Wilson protein is a coppertransporting ATPase which resides in the trans Golgi network and its absence or dysfunction causes neurodegeneration by disrupting copper homeostasis. Mutations in this protein result in its misfolding and retention in the ER, followed by a retrograde transport out of the ER and its degradation by the ubiquitin/proteasome pathway23.

BIOGENESIS OF UBIQUITIN PROTEIN INCLUSIONS (AGGRESOMES) The hallmark of many neurodegenerative diseases is the presence of intraneuronal inclusions consisting of ubiquitin protein conjugates. The mechanisms leading to formation of such abnormal aggregates remain unclear and their role in the progression of the disease has yet to be elucidated123. It is possible that inclusions arise from a cellular attempt to compartmentalize accumulated proteins, and prevent their interference with normal cell function. Their presence may also confer cytotoxic effects that can contribute to cellular damage associated with neurodegeneration. Aggregate size may be a pivotal determinant in their toxicity92. As the ubiquitin protein aggregates expand they may confer fatal effects by chokin the cell as the cytosolic or nuclear space is ultimately filled by the abnormal aggregates 124 . The fact that many components of the ubiquitin/proteasome pathway, such ubiquitin C-terminal hydrolases and 26S proteasome subunits, are found together with ubiquitin conjugates within inclusions strongly supports a role for this pathway in inclusion biogenesis. A broad range of diseases, including cystic fibrosis and neurodegenerative disorders such as prion diseases, Wilson disease, and AD, are associated with defective proteins lacking the capability for correct ER processing125. Gene mutations in a transmembrane conductor regulator, a prion glycoprotein (PrP) associated with the plasma membrane, and a copper-transporting ATPase, are responsible for such disorders as cystic fibrosis, GSS syndrome and Wilson disease, respectively. The improper transport of these mutant proteins results in their accumulation in the ER, followed by their targeting for cytosolic

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degradation by the ubiquitin/proteasome pathway. It is postulated that in AD there is an association between deficiencies in APP processing in the ER and presenilin mutations and tau hypophosphorylation, the latter being a component of NFTs. All these deficiencies may contribute to aggregate formation125. Centrosomes, which are microtubule-organizing centers, were found to be deposition sites for ubiquitinated proteins that escape degradation by the ubiquitin/proteasome pathway, and were named accordingly as "aggresomes"126. Ubiquitin protein aggregates shown to be deposited in the aggresome resulted from either overexpression of mutant cystic fibrosis transmembrane conductor regulator (CFTR) or presenilin1(PS1) or from impaired protein degradation induced by treating cells with proteasome-specific inhibitors126-128. Centrosomes were shown to be associated with high levels of 26S proteasomes and also with de-ubiquitination activity129. While some studies demonstrated that the retrograde transport of ubiquitin protein aggregates to the centrosome is dependent on the integrity of microtubules126,126 , others found that this process does not require intact microtubules129. The mechanism by which ubiquitin protein aggregates are deposited in the aggresome may mimic the formation of intraneuronal inclusions found in many neurodegenerative diseases. It is unclear why neurodegeneration associated with hereditary forms of neurodegenerative disorders only becomes symptomatic in the adult or at an advanced age despite the congenital presence of specific mutant proteins. Zanusso et al121 postulate that the ubiquitin/proteasome pathway degrades mutant proteins shortly after they are produced, thus preventing their aggregation. Malfunction of this pathway caused by harmful conditions, such as oxidative stress or inflammation, may mediate a decrease in the degradation rate of abnormal proteins, bringing about their accumulation as protein aggregates which may form inclusions. An aging-induced decrease in proteasome function may also contribute to stabilization and aggregation of mutant proteins that are normally turned over by the ubiquitin/proteasome pathway. A micro-array analysis of the expression of 6347 genes in mouse skeletal muscle revealed age-dependent decreases in the expression of genes encoding stress factors and proteins involved in the ubiquitin/proteasome pathway, including 26S proteasome subunits and ubiquitin thioesterases130. Most of the identified changes could be reversed by caloric restriction diets130. These findings strongly support the influence of the aging process on the regulation of transcriptional activation of genes involved in the turnover of damaged and misfolded proteins, such as those encoding components of the ubiquitin/proteasome pathway.

CONCLUSIONS Recent advancements in gene cloning techniques and gene expression analyses provide compelling evidence linking the ubiquitin/proteasome pathway with the turnover of many proteins required to maintain neuronal homeostasis. These findings address new and exciting questions concerning the impact of a deregulation in proteolysis on cellular function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in neurodegeneration. Genetic data revealed that components of the ubiquitin/proteasome system are far more complex in number and function than previously thought. Findings from studies on hereditary forms of neurodegeneration, such as Angelman's disease in humans and the gad phenotype in mice, provide direct evidence that the manifestation of these disorders results from genetic defects in enzymes that are essential components of the ubiquitin/proteasome pathway. Consequently, these findings support the notion that malfunctions in this system may be critical events that trigger the initiation of the neuodegenerative process. Under harmful conditions, such as those induced by oxidative stress, inflammation, and genetic mutations, the cell may rely on the ubiquitin/proteasome pathway to remove abnormal proteins produced under such conditions, thus promoting neuronal homeostasis. An age-dependent decline in the activities of this pathway may be critical to the neurodegeneration process. This explanation provides an alternate interpretation to the view that ubiquitin conjugate deposition is merely an indirect consequence induced by other factors involved in the disease. The need for more research to identify and define components associated with the ubiquitin/proteasome pathway underscores the potential for new targets of therapeutic

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intervention in neuronal diseases as well as diagnostic markers for individuals at risk for these disorders

ACKNOWLEDGMENTS We thank Ms. Romia Bull for editorial comments and Ms. Tine Herreman for preparing the 20S proteasome structure shown in Figure 2. National Institutes of Health Grants NS34018 (to M.E.F.-P.) and RR03037 (Research Centers in Minority Institutions) supported this work.

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AMYLOID (TACE, BACE) AND PRESENILIN PROTEASES ASSOCIATED WITH ALZHEIMER'S DISEASE

Neville Marks1,2 and Martin J. Berg1 Center for Neurochemistry Department of Psychiatry New York University Nathan S. Kline Institute for Psychiatric Research Orangeburg, NY 10962 1

2

INTRODUCTION This overview emphasizes recent findings on proteolysis of amyloid precursor protein (APP) and presenilins (PS1/PS2). Interest in these components stems from their association with AD/FAD resulting in the overproduction of fibril forming amyloid peptides (Aβ). These accumulate in AD neuritic plaques and are thought to be etiological factors in neurodegeneration. Aβ is formed on shedding of an APP ectodomain followed by cleavage of cell-associated fragments by 'secretases', a term denoting enzymes for secretion of soluble metabolites. Until recently secretases were not available in purified form and data were descriptive and inferential. Recent isolation of Asp-proteases and metalloendoproteases provide new insights in mechanisms involved in APP turnover and a basis for synthesis of inhibitors or probes with therapeutic potential. Mutated presenilins differentially increase the secretion of C-terminally extended forms of Aβ (Aβx) indicating a shift in processing to account for altered composition of neuritic plaques in familial AD (FAD-PS) and in transgenic brains co-expressing FADPS/APPs. Cultured cells expressing FAD genes provide in vitro assays to monitor this genetic autosomal dominant gain-of-function. Converging on these themes are questions on the functional significance of PS itself since this component was discovered and then named only in the context of presenile pathologies. Clues for function arise from morphological similarities between phenotypes for Notch or PS knockouts, and from complementation assays using mutant flies or worms deficient in PS-homologs. These provide evidence for PS playing roles in Notch-signaling pathways including eye/wing maturation in Dps mutant Drosophila, or egg laying in sel-1/hop-1 deficient nematodes1-10 , and form a basis to examine effects of PS in turnover of other proteins containing a single TM domain. An example considered here is Notch-receptor (Notch-r), a Type-1 protein having its C-terminus in the cytosol, that is processed following binding to ligands. Other examples include protein components of the unfolding protein response (UPR) signaling

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pathway Type-1 IreI, and ATF6, a Type-2 protein having N-terminus in the cytosol. Because of space limitations we emphasize here recent developments since earlier findings are extensively documented elsewhere11-13. PROCESSING ENZYMES AND COMMENTS ON STRUCTURE OF APP/PS Structure and post-translational modifications are factors relevant to processing putative membrane components by tissue hydrolases (Table 1). APPs occur as glycosylated isoforms with Mr ~100-140 kDa formed by alternative splicing with some having a serine protease inhibitory sequence (KPI) and other domains, although several of these are absent in the major neuronal form, APP695 (Fig. 1). The variable sugar content and other post-translational modifications contribute to APP heterogeneity, and probably markedly, to their turnover as illustrated for catabolism of soluble rAPP versus axolemmal-bound precursor by purified brain cathepsin B14. Therefore it is necessary to evaluate action of putative secretases on membrane-bound precursor in addition to the use of shorter peptide surrogates. Earlier studies on isolated cells or crude membranes show sequential breakdown of APP with removal of soluble ectodomains followed by cleavage of cell-associated products (Fig. 2). Importantly, cleavage at K16L17 by D-secretase is nonamyloidogenic since this effectively destroys the fibrillar sequence. Cleavage of the residual fragment C-89 in this case results in formation of P3 fragment rather than AE, and the labile C-7 product that is undetected in tissues. The K16L17 and M-1D1 D- and E- sites are accessible to soluble hydrolases/proteases since they are downstream from the putative

Table 1. Secretases. Dresenilinases. and type-1/2 substrates discussed in this review

a, IC3, batimisat, marimastat, GI-120471, SE-205, TAPI, KD-1X-73-4, chelators42-44 b, Used for affinity purification19; c, ALLN, MG132 (calpain I), ALLM (calpain II); d, lactacystin, Abbr.: TACE, Tumor Necrosis Factor-α-Converting Enzyme; ADAM, A Disintegrin and Metalloendopeptidase; KUZ, Kuzbanian Protease; BACE, β-Amyloid Converting Enzyme; UPR, Unfolding Protein Response; A23187, Ca2+-ionophore; α-1PDX, α1-antitrypsin; L685,458, (1 S-benzyl-4R-(1-( 1Scarbamoyl-2-phenylethylcarbamoyl)- 1S-3-methyl-butylcarbamoyl)-2R-hydroxy-5-phenylpentyl) carbamic acid tert butyl ester, a transition state aspartyl-like inhibitor.

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Fig. 1: Domain structure of APP770. KPI ( ) and OX-2 ( ) domains deleted in neuronal APP695. Large open arrowheads show sites of major secretase cleavage in Aβ (bold type), with minor cleavage at Y10E11 shown with smaller arrowhead. Familial mutations are indicated in Italics and sites of substitution by arrowheads. The TM domain is shown within Sites of glycosylation shown by .

Fig. 2: Pattern for fragmentation of APP by secretases yielding soluble or cell-associated fragments. Numbers at bottom refer to AE sequence shown in Fig. 1.

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TM domain (Fig. 1). In contrast, formation of the Aβ moiety requires cleavage within the TM-domain implicating action by a membrane-bound TM-endoprotease referred to as J _secretase. Presenilins (PS) exist as two genes coding for proteins with ~70% homology (hPS1, 467 residues maps to chr 14q.24.3; hPS2, 448 residues maps to chr lq.42.1). Most mutations are found for PS1 (~50) with fewer (2) for PS2. Together these mutated genes comprise most of the reported cases for early-onset FAD or 10% of all cases of AD-like neurodegeneration. Presenilins are rapidly processed with residual full-length protein associated with the ER and NTF (28-34 kDa) and complementary CTF (18-20 kDa) fragments with Golgi membranes45. The deduced sequence contains 10 putative hydrophobic regions (HR) with 6-8 as TM4,46-48: the model of Nakai proposes 7 TM and one intramembranal HR48 and that of Li and Greenwald has 8 TM with the C-terminus as cytosolic4 as illustrated in Fig. 3A (solid black and dashed gray lines respectively). Hydropathy plots resemble the nematode homolog sel-12, a feature that may be consistent with comparable functional properties (Fig 3B). The NTF and CTF are formed via primary cleavage within the cytosolic loop along with minor metabolites as shown in Fig. ) although the "pressenilinase' enzyme(s) responsible have not been , 3A ( ' identified. It is unknown if one or more proteases account for variability of Mr and Ctermini of fragments. Importantly, FAD-mutations do not significantly alter turnover rates except for the PSl' exon9 lacking the 290-319 domain spanning the cleavage sites (Fig. 3A). The fact this mutant retains potency indicates breakdown is not obligatory for gain-offunction. Location of aspartyl residues postulated to participate in PS-mediated proteolysis are indicated in Fig. 3A (gray boxed).



DSECRETASES (Kl6L17 CLEAVAGE) Tissues contain metalloendopeptidases of the disintegrin family (ADAM) with putative α-secretase properties (Table 1). ADAM family members are widely distributed in tissues, and play diverse roles in a number of tissue functions by shedding ectodomains from a variety of components including TGFα, EGF, proTNFα, Fas-L, TNFR, L-selectin, ACE, Delta, Notch, erbβ4/HER4, and interleukin-6, thus acquiring the term 'sheddases' 1517,39,49-56 . Two groups independently identified TACE (ADAM-17) as a 501 polypeptide for conversion of 26 kDa ProTNFa to form active 17 kDa cytokine, later shown also to shed the APP ectodomain by cleavage at K16L15,16,39,50. Black et al.15 purified this enzyme from detergent-extracts of stimulated human monocyte cell line THP-1 cells and assayed fractions with Ac-SPLAQAVRSSR-amide, and Moss, et al.16 using an affinity derivative termed GW9471 purified enzyme from porcine spleen cleaving N-flagged ProTNFα and also the same peptide surrogate. Buxbaum et al.50 provided evidence for cleavage by TACE at the relevant APP site for the surrogate Ac-VHHQKLVFFA-amide, and release of sAPPα or Aβ by CHO cells expressing APP751. Cleavage was blocked by the inhibitor designated IC3, along with reduced secretion of sAPPβ for fibroblasts from TACE knockout mice. The most compelling evidence for ADAM-17 acting as a putative αsecretase is reduction of sAPPα secretion on transfection of K293 cells with a dominant negative (DN) mutant lacking the Zn-binding motif17. The deduced sequence of ADAMs show existence of the Zn-binding motif HEXXH (see Fig 4), providing a potential target for design of anti-inflammatory agents notably hydroxamates (Table 1), and the creation of the DN mutant. Comparison of the hydroxamate inhibitor batimastat shows 100-700 fold higher potency towards TACE and collagenase compared to ACE secretase and a-secretase. The analog marimastat is ~4 fold more potent towards APP than ACE but still retains considerable potency towards

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Fig. 4: Domain structure of h TNFa Convertin Enzyme (TACE) zymogen (see GenBank Accession Nos. U69611, U86755)15,I6. Cleavage by Furin at R 211VKRR214 leads to release of active enzyme through removal of proenzyme domain containing motif PKVC 184 GYL forming a 'cysteine switch' coordinately binding to and inhibiting the metalloendopeptidase catalytic center H405ELGH. For other details see Fig. 1 and text.

collagenase and thus is not specific for α-secretase42,44. Studies on COS and neuroblastoma cells show hydroxamates affect stimulated cells more potently than basal APP turnover although in the case of PKC the effects are unrelated to APP phosphorylation suggesting it acts on other processing events43,57. CHO cells expressing APPSW treated with phorbol esters and the hydroxamate TAPI show α- competes with βsecretase-like activity in the TGN providing another example of reciprocal relationships between these processing enzymes (see comments on BACE localization below)58. ADAM-10 cleaves Aβ11-28 or APP at the K16L17 site with reduced hydrolysis for the peptide substrate bearing the non-AD Dutch mutation although mutations associated directly with FAD were not examined (Fig. 1)17. TACE and ADAM-10 act at K16L17 compared to MCD9 (γ-meltrin) which cleaves at H14Q15 that may be the preferred site in hippocampal neurons59,60. MCD9 has α-secretase-like properties on transfection in phorbol ester-treated COS and K293 cells61. While there is scope to develop inhibitors62, since α-secretase destroys the putative amyloidogenic domain, activation rather than inhibition is more desirable provided this does not alter other secretases (see above for effects of TAPI)58. Testosterone-treated N2A cells or rat primary neurons increases the secretion of sAPPα and secretion of Aβ63. There is evidence for participation of other factors for conversion of APP or ProTNFα in -/stimulated TACE-/- fibroblasts, or after their fusion with PKC CHO cells64,65. ProTACE itself requires activation by a furin-like convertase and the removal of a Cys residue coordinately blocking the active Zn2+ motif ('cysteine switch', see Fig. 4)66. Overexpression in K293 cells of the prohormone convertase PC7, a furin enzyme, increases sAPPα thereby lowering Aβ via a pathway inhibited by α-1-PDX67.

Yeast Asp-proteases (Yapsins) Similarities between APP turnover in insect or yeast cells suggest one or more aspartyl proteases act as putative α-secretases68-70. Interestingly, these were discovered using an approach similar to that for identifying mammalian furins by use of yeast Kex mRNA probes, a method also found useful for recent purification of β-secretase71. Asp proteases Yap3 and McK7 (Yapsins) increase in yeast strains defective in vacuolar transport providing a basis for their characterization. Yapsins restore APP processing to sec 17 or 18 deficient mutants providing evidence for their α-secretase-like properties. Interestingly, some assays utilize an internally quenched fluorescent substrate AcRE(Edans)VHHQKLVPFK-(dabcyl) based on the α-secretase site. The relevance of yeast Yapsins or if these are expressed in mammalian tissues remains to be clarified.

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E-SECRETASE (M1D1 CLEAVAGE) Tissues contain acidic Asp-like proteases with putative β-secretase properties (BACE1/2, Asp 1-4, memapsins 1/2) although there is no agreed terminology. These resemble, enzymes predicted earlier as present in cells, membranes, extracts, or inferences based on analyses of body fluids (Table 2). Such studies provided evidence for processing in acidic compartments, roles for endocytosis, and with higher activity in neurons compared to astrocytes. Effects of serine protease inhibitors are questionable because of high concentrations used72.

Table 2. A. Examples of β-secretase type cleavage in diverse cell lines.

,

,

I

*Transfection with APPwt unless as noted. a , CM; conditioned medium b, NSE; neuronal-specific enolase c , GFAP; glial fibrillary acidic protein

Aspartyl proteinases Vasser et al.18 constructed a directional cDNA expression library using a CMV promoter to transfect HEK 293 cells overexpressing APP to identify a 501 polypeptide sharing homology to pepsin-like aspartyl proteases (Fig. 5). BACE has a 21 mer signal peptide (SP), a 24 mer pro-domain (Pro Pep), a lumenal 414 mer catalytic domain, a single 17 TM plus a 24 mer cytoplasmic tail. A furin-like convertase cleavage may be responsible for maturation of the BACE proenzyme83.

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Fig. 5. Domain structure of β-APP Converting Enzyme (BACE-l/Asp-2) (see GenBank Accession No. NM 012104 for sequence). Glycosylation sites ( ), Cys residues( )form intralumenal disulfides capable of for altering the conformation of the (Asp)-catalytic centers.

Lack of inhibition by pepstatin indicates presence of a unique lumenal catalytic center D*S/TGS/T 84-86 with six cysteines forming three potential intramolecular disulfides. This enzyme acts also at Glu11, consistent with earlier predictions on whole cells or membranes78,87, while cleavage at Val-3 and Ile-6 reported for intact cells probably represents processing by other tissue proteases/peptidases12. hBACE- 1 mRNA is present in all adult peripheral tissues and especially the pancreas. In brain, detection in hippocampus and cortical membranes is of interest to pathology since these regions are vulnerable to neuronal loss. Also high levels of BACE-1 in Golgi, TGN, and secretory vesicles occur at sites linked to APP processing: this was confirmed by co-localization of a hemagglutinin-tagged enzyme with sAPPβ and C99 in cells overexpressing APP. BACE-1 overexpression results in cleavage at Met-1 and Tyr10: antisense probes in APPSW expressing cells reduce formation of products associated with β-secretase. Specificity studies show BACE-1 cleaves the 30 mer T-21-K8(dnp)G9 at the MD site. BACE-1-IgG fusion protein catalyzed APPSW better than wt or was blocked using the M-1/V mutant. There will be interest in knockouts to evaluate roles of this unique Asp protease in APP turnover and on morphology. Sinha et al.19 designed transition-state analogs to directly purify from human brain a similar 501 polypeptide with pH maxima 5.5. The inhibitor used for affinity chromatography was prepared using an APP sequence with the Swedish mutation and Leu at P1: KTEEISEVNLstatineVAEF, ID50 of 30 nM (Table 1). Enzyme converted C-125-MBP fusion protein at the M-1D1 site. Sequence analysis and lack of effects by inhibitors indicated a unique aspartyl protease lacking typical serine or cysteinyl catalytic centers. Acetylation of the statine hydroxyl or replacement of (S)- with (R)-statine enantiomer blocked action of the inhibitors. The enzyme is largely neuronal, and not readily detected in peripheral tissues. Co-transfection with APPwt or APPsw increased formation of Aβ and SAPPβ. Yan et al.21 scanned the genome of C. elegans to isolate candidate protease genes to subsequently isolate mammalian homologs. Four new human sequences termed Asp- 1 to – 4 were identified with -3 and -4 comparable to napsins 1 and 288. Asp-l/2 have C-terminal extensions containing a single TM domain. Asp-2 (BACE-1) maps to chr 11q23.2 while Asp-1 (BACE-2) maps to chr 21 .q22.2-.3 linked to Down's syndrome89-91: co-localization of BACE-2 with the trisomic region of chr2 1 may have implications in Down's pathology. There is a 52% homology between BACE-1 and -2, with both containing a single TM and requisite Aspartyl motifs; they are divergent only at the C and N-termini. BACE-1 is the major β-secretase in HEK 293 cells since BACE-2 does not compensate for loss of BACE1 in these cells which can be blocked by antisense probes. This is consistent with low expression of BACE-2 mRNA in most human tissues, and especially fetal and adult brain, along with a distribution that does not match that predicted for β-Secretase92. In IMR-32 neuroblastoma cells, antisense probes reduce Asp-2 mRNAs and formation of C9921. Murine Asp-2 cDNA is 98% homologous to hBACE- 1. Enzyme acts on SEVKMDAEFR

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and more avidly on peptide bearing the Swedish KM/NL mutation. Pepstatin, leupeptin, E64, or EDTA do not inhibit this aspartyl-like protease. BACE is a potential target for drug design since inhibition prevents formation of intermediates containing Aβ. It will be of interest if BACE knockouts can be created and the morphological or behavioral consequences on overexpression of APPs. Hussain et al.22 obtained a cDNA clone from a proprietary EST database termed βsecretase Asp-2, but did not divulge methods of cloning although the enzyme appears to be homologous to BACE- 1. Transient expression of Asp-2 in different cell lines increases Aβx secretion confirming its role as a β-secretase. This is supported by Asp site mutations at the catalytic center (Asp-Ser/Thr-Gly-Ser/Thr)84 abrogating secretase properties. Tissues also contain alternative membrane bound Asp proteinases (memapsins 1/2) with memapsin 2 having putative β-secretase activiy towards wt or Swedish APP in cotransfected HeLa cells93. The PS1 mutation (V1717I, see Fig. 3) enhances formation also of N-truncated AEpyroGlu3-42 and 4-42 in situ. Unless these 'alternative' Esecretases are definitively characterized, such fragments may arise by N-terminal trimming by aminopeptidases or suggest PS mutations influence sites cleaved by BACE94. Scope exists to examine localization of BACE in endosomal or other pathways as factors in APP turnover95,96. Comments on cathepsins or other candidates Transfection of cathepsin D, a prototypic aspartyl protease, does not promote Aβ z secretion in cells 2. Since Aβ secretion by cathepsin D knockouts continues, this lysosomal aspartic protease is not essential for its formation97. Nevertheless, cathepsin D polymorphisms may constitute a risk factor for AD but play an alternative role in the clearance or turnover of other neurodegenerative proteins98-100. Cathepsin D is reported present in AD neuritic plaques, but its function is unknown especially since these deposits lack APP holoprotein or its CTFs101. Cathepsin S or the serine protease cathepsin G active at physiological pH also degrade APP but do not generate directly Aβ11,102-104. Similarly, the neuronal metalloendopeptidases such as phosphoramidon-sensitive 24.1 1 or insensitive 24.15 do not have secretase properties14,105, although this property is reported for platelet or leukocyte derived enzymes106. Overexpression of thimet oligopeptidase in COS cells is reported to enhance secretion of sAPPβ107. Roles are implicated for a GPI-anchor or caveolin-3 to facilitate β-secretase activity108,109. One study shows a 68 kDa serine proteinase processes lymphocyte but not brain APP, and attributes the differences to states of glycosylation110. γ-SECRETASES (VV40IA42A43TVIV) In cell models, processing of APP-CTFs (C-89/-99) within the TM results in release of P3 along with labile C-7 fragments (Fig. 1, Table 1)111-115 representing an example of regulated intramembrane proteolysis (RIP), applicable to several animal and bacterial proteins116. A common feature is removal of the bulk of the extracytosolic domain prior to action within the TM. Depending on the Typel/2 protein used as substrate, cleavage occurs in the ER-lumen, in a post-ER compartment, or at the cell surface. The putative γsecretase does not resemble the 'site-2 ‘ enzyme converting Sterol Regulatory Element Binding Protein (SREBP) acting within the TM since CHO cells deficient in this enzyme still secrete Aβ117,118 Similarly, putative γ-secretases do not resemble catabolism of APP by other lysosomal hydrolases119. Purification of γ-secretases is awaited to establish their localization, properties, specificity, and number120. There currently is little coherence on

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effects of site-specific agents as a method for identification (Table 1): this is attributable to overlap in specificity of such agents and discordant results especially on comparing effects on stimulated versus basal secretion for Aβ27,121-123. Rank order potencies for putative calpain and proteosome inhibitors on HeLa-pNAN8 cells expressing an APP C-103–YCFA construct suggest a single γ-secretase accounts for the secretion of Aβ/Aβx123. This also appears to be the case for a 2.0 x 106 kDa complex extracted from HeLa cells using CHAPS or CHAPS0 containing iR N- and C-termini of PS1: these degrade a Met-C-100-flag (DYKDDPPK) fusion protein to release Aβ1-40 and 142 by a mechanism sensitive to pepstatin, the transition-state inhibitor L685,458 containing a hydroxyethylene dipeptide isostere, but with a conformation opposite to HIV protease inhibitors, or other similar anologs23,124-127 . However, a recent comparison using E64 and peptidyl aldehydes proposes cysteine proteinases account for Aβ40 while calpains account for Aβx122. A lower Aβ/Aβx ratio for cells exposed to ALLN or MG132 (calpain I) or ALLM (calpain II) points to a Ca2+-activated protease for conversion Aβ/TM 26-57 domain see Fig. 1) flanked by N-hemagglutinin and C-c-myc epitopes when used as a substrate26. Effects of leupeptin, pepstatin, phosphoramidon, Z-LL-CHO, Z-VL-CHO, ZLL-leucinal, and lactacystin in vitro yield conflicting data (Table 1). Studies using APPs or PS1 constructs in transfected cells are difficult to interpret because of presence of multiple proteases/peptidases yielding major and minor products112.120. This is illustrated for APPSW with I637 F/P substitutions favoring cleavage at G38V, G37G, and V40I. In contrast, insertion of a repeat G625AII sequence favors cleavage at G33L or G38G. Deletion of this tetrapeptide altogether from the native holoprotein favors formation of C-terminally extended products112. In another study, transfection of a Leu-Glu-C-99 fusion protein in COS-7 cells shows T43A-V46F or T43G-V46F yields Aβx, while I45E increases the Aβx/Aβ ratio by 34 fold compared to V44F that yields AE38111. Processing of a C100 carrying a trans-Golgi sorting signal to yield Aβ40/42 was increased whereas this fragment bearing an ER sorting signal was decreased in FAD-PS 1 transfected N2A cells by a pathway inhibited by brefeldin but not monensin128.

PRESENILINS Mutated presenilins co-segregate with the majority of early onset presenile dementias and lead to a differential increase in Aβ42/43 in neuritic plaques. Replication of this feature in transgenics or isolated cells co-expressing FAD-PS/APPs points to a shift in γ-secretase processing to favor C-terminally extended forms11,129,130 . However, whether an increase of 0.5-1.0 fold in secretion of Aβx versus Aβ in isolated cells is sufficient for AD pathology in situ is debatable, although this is the basis of assays to monitor gain-of function. Transfection leading to overexpression of PS/APPs in cells is likely to distort the endogenous pools of metabolites13, thus complicating the interpretation. Overproduction of Aβx ma be pathogenic since this fragment readily aggregates in solution, and in vitro is cytotoxic12,311.

Presenilinase Presenilins are labile and occur as NTF/CTF fragments in Golgi membranes or as residual holoprotein in ER45. The enzyme(s) responsible for forming major metabolites are unknown although tissues contain candidates that may account for minor products. Surprisingly, labile mutants retain toxicity comparable to those of stable mutants or constructs. The Mr and immunoreactive profiles indicate cleavage in the cytosolic loop at Met292 of PS1 or Met298 of PS2 to account for the formation of NTF (~30 kDa) and CTF

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(~20 kDa) in a stoichiometric ratio of 1:1 (Fig 3)30,132,133 . Sites of cleavage are confirmed by metabolic stability following Met292 substitutions of a FAD-PS1 . Interestingly, this construct retains Aβ toxicity on transfection in APPSW expressing HEK 293 cells132. In contrast, the comparable stable PS2-M298D construct lacks potency in neuroblastoma cells, but rescues egg laying in sel-12 defective worms133. This disparity has not been explained but may reflect different athogenicities for the two PS genes or lack of concordance between assay procedures3,134. Stable hPS1 D257 or D385, hPS2 D263 or D366, and zebrafish PS1 D374 mutants lack potency leading to the hypothesis that the Asp groups facilitate catalysis31,135,136. This hypothesis is supported by studies showing that co-transfection of PS1 and PS2 Aspdeficient mutants reduced Aβ secretion in CHO cells expressing hAPP, or lowered Notch cleavage and translocation to the nucleus135,137,138. These data suggest there is no PSindependent pathway for production of Aβ/Aβx. PSI or 2 can substitute for each other for restoration of secretion of sAPPβ on transfection in PS1-/- fibroblasts139. Mutagenesis of putative catalytic Asp residues, plausibly, may be beneficial by reducing processing of APP intermediates. Binding of APP to PS in ER, followed by PS and C83/C99 in Golgi/TGN points to processing within these compartments for vectorial transport and production of Aβ/Aβx140. N2a cells doubly transfected with PS1' exon 9 and APPSW results in co-localization of Aβ42 with rab8, a marker of TGN vesicles141; also, PSI N-terminal binds rab GDI (GTP dissociation inhibitor), a component that decreases 2-fold in PSdeficient neurons142, Processing of APP lacking the C-terminal consensus sequence for internalization by PS1-transfected CHO cells indicates endocytosis is not obligatory (Fig 2)143.

Properties of PS fragments and complexes The ~10 fold increase in half life from 1.5 to 12-24 h for NTF/CTFs compared to holoprotein suggests stabilization of fragments on binding to cell accessory proteins144. Binding proteins themselves may be rate limiting since overexpression of PS in cells or in transgenics, leads to accumulation of holoprotein145. Also lack of toxicity on transfection of FAD-NTF ± the complementary CTF provides clues on events that occur prior to binding146. Current data on PS suggest N- and C- fragments share the consensus sequence required for toxicity. Structure-activity relationships show C-terminal third of PS2 (FADN141I) is critical since its removal, or modifications by addition of five His residues, or replacement of hydrophobic residues, reduce toxicity while the N-terminal 25-75 residues are dispensable147,148. Chimeras consisting of PS1/2 fragments also are active, in line with contribution from the N and C-termini146. The C-terminus of PS2 may contain a signal for entry into the processing pathway149. Complexes with Mr of ~150-250 kDa vary with cell-type and methods of extraction using detergents. Such complexes contain ir-NTF/CTF and one or more of the following components: catenins, Ca2+ - binding cadherins, calsenilins or other calcium-binding proteins, bcl-2/bclx, cavelolin-3, or syntaxin 1A among others109,150-163 . Recently, a 2.0 x 106 kDa complex from HeLa cells containing iR N- and C-terminal fragments of PS1 was shown to degrade an APP C-100 fusion protein to yield Aβ40 and 42 in a process inhibited by aspartyl protease inhibitors124. In HEK 293 or other cells, PS1 binds to C-APPs by a process facilitated by a 708-mer polypeptide Nicastrin164: it is speculated that nicastrin promotes pseudocatalytic properties of PS165. The conspicuous absence of mixed PS 1/PS2 heterodimers suggests catabolism of PS holoprotein occurs only on binding to access0 proteins thereby providing a 'scaffold' to retain the pathogenic signature of mPS146. Mutations may alter PS configuration in a subtle manner to account for the genetic gain-offunction although this remains conjectural. Other binding components include 42-mer

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armadillo repeat proteins (ARD) p0071 and neuronal B6P-plakophilin, capable of binding to the 372-399 region of PS1-CTF, but their role(s) in pathogenesis is unclear154,166. PS processing by caspases and proteosomes. Caspases are conserved Asp-specific cysteine proteases linked to apoptotic cell death. In peripheral cells expressing FasL/TNFα receptors, interaction with antigens recruits proximal caspases and adaptor proteins containing ‘death-effector domains’ for activation of distal effector forms167. Alternative pathways in non-mitotic neurons result in release of cytochrome c from mitochondria, and conversion of proximal caspase-9 for sequential processing of distal procaspases. These degrade PSI at D345 (PS1) or D329 (PS2) to yield alternative NTFs/CTFs compared to other putative presenilinases32,33. Stability of mutant PSID326/PS2-D329 or effects of N- and C-blocked tetrapeptide inhibitors -DEVD-, -YVAD-,or . the -D345SYD- recognition sequence of PS1 provides evidence for cleavage32. Caspase activation resulting from induction of apoptosis probably precedes formation of Aβ and its cytotoxic actions. In H4 neuroglioma, treatment with etoposide or staurosporine to activate caspases results in degradation of PS2 to form a ~20 kDa CTF33. Caspases differ in rates of hydrolysis of an ENDD329 PS1 sequence without effect by five PS missense mutations168. Independent1y, caspases degrade the CTF by a mechanism that is decreased via phosphorylation of Ser169,170. Caspase-12 present in ER, on activation is thought to contribute to turnover of amyloid peptides: mice knockouts resist apoptosis resulting from ER-stress [see section ‘Notch-signaling and the Unfolded Protein Response (UPR) ’ below] 171 . Proteosomes, a family of cytosolic ~700 kDa proteases, with mixed chymotryptic-, tryptic-, and ostglutamyl-like protease activities also degrade PS to yield alternative metabolites36,172. Cleavage occurs within the Met288-Glu299 domain using a purified 20 S proteosome and a synthetic substrate, but is inhibited by lactacystin or other similar agents36,173. However, roles for proteosomes are considered unlikely since they require ubiquinated substrates, harsh conditions for activation, and in situ a variety of accessory factors172.

NOTCH-SIGNALING AND THE UNFOLDED PROTEIN RESPONSE (UPR) Presenilins recently were found to influence turnover of Notch-r and components of the ‘Unfolded Protein Response’ thus reinforcing their roles as putative Asp-proteases (see Fig. 6). While they are spatially separated in cells, the binding of PS to Notch at the ER membrane provides a potential pathway for targeting to the plasma membrane, the site for Notch processing. Mutation of PS aspartyl residues, while not blocking trafficking, prevents Notch processing174,175. Notch-r is formed by proteolytic processing of a large multidomain precursor several fold larger than APP (Fig 6). Like APP it contains a single TM, and, in addition, 29-36 epidermal growth factor (EGF) repeats, 3 copies of a Lin-12/Notch/Glp motif in the ectodomain, and six CDC10/Ankyrin repeats among other motifs (Fig. 6). The shedding of the ectodomain by furin and/or ADAMs ( KUZ/TACE ) yields funtional receptors38,39,176 for binding ligands, resulting in further proteolysis at or within the TM55,177 to release a downstream activator (NICD) translocated to the nucleus where it acts on C/S/L genes (CBF-1/Suppressor of Hairless/LAG-1)178. Ligands identified in Drosophila include Delta and Serrate, in C. elegans are LAG-2 and APX-1 , and in rodents and humans are Delta1 and Jagged1/2179. NICD in neuronal nuclei influences rates of differentiation; this pathway is downregulated in DN mutants. Developing cortical neurons also express inhibitors of Notch-r of the Numb family (Numb , Numb-like, Deltex) influencing neuronal differentiation180. These resemble adaptor/scaffold proteins and include a phosphotyrosine

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binding (PTB) domain and proline rich (PRR) C-terminal containing Src and EH binding motifs181 . Interestingly, Notch-3 mutations are associated with CADASIL ( cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) and those of Jagged linked to Alagille syndrome, a mild mental retardation associated with multiple developmental disorders178,182. Recent studies implicate wt/mPS in the turnover of TM-containing UPR components Ire1α and ATF640,183,184, and reinforces roles for PS acting as or in concert with a transmembrane protease. UPR results from ER-stress triggering synthesis of hsp proteins GRP (glucose regulated protein) 78/94, and a Growth Arrest of DNA damage product GAD D153, also known as CHOP (c/ERP homologous protein)185,186. ER-stress results in phosphorylation of components within the UPR signaling pathways, in some cases resulting in apoptosis. GRP78 increases on exposing isolated hippocampal neurons to Glu excitotoxicity, Fe2+, or Aβ, and in NG108-15 exposed to ethanol as examples187,188. Antisense GRP78 promotes cell death of NGF-deprived PC-12 cells. This is reduced by dantrolene, an agent blocking ER-Ca2+ release, or by Z-VAD-fmk, a pan-caspase inhibitor187. Mammalian cells contain UPR ER-resident proteins Ire1α and β (Ern I and 2), and PERK, an interferon-induced PKR-like ER kinase186. Ire1 D is a type- 1 protein containing a sensor domain responsive to calcium ionophores, or thapsigargin that releases stores of ER Ca2+, or tunicamycin, an inhibitor of glycosylation. These induce Ire1 D phosphorylation and dimerization with cleavage at the TM domain to release a fragment having dual kinase and nuclease activity (K/N in Fig. 6). This fragment on translocation to the nucleus results in synthesis of GRP78/94, calreticulin, and protein disulfide isomerase to counteract ER protein misfolding (Fig.6)40,189,190. ER-stress also results in phosphorylation but not cleavage of PERK via a parallel pathway with phosphorylation of eIF2 α kinases ; this reduces protein synthesis and alleviates ER protein overload186. Roles for UPR in pathology are suggested by decrease in GRP78 mRNA on mPSl transfection for SK-N-SH neuroblastoma, and their enhanced response to tunicamycin by processes reversible on overexpression of this chaperone183. In PS1-/- HeLa cells, defective IreI processing points to significant roles for PS in conversion40. Significantly, a higher level of GRP78 in CNS neurons spared in AD suggests this chaperone is neuroprotective191. In line with this property, GRP78 levels are lower in temporal cortex of sporadic AD and FAD-PS1 brains183. The binding of ER-Ire1 to mPSl may prevent synthesis of GRP78 and protein refolding. GRP78 (T37G), a tightly binding ATPase mutant192 on transfection in HEK 293 overexpressing APP, reduces secretion of sAPP and Aβ40/42 points to novel therapeutic applications. Pathways in yeast for UPR show a requirement for DNA-binding protein hac1p acting as a co-transcriptional activator for protein folding. The use of yeast ER-Stress Response Element (ERSE) CCAATN9CCAGG recently lead to purification of ATF6, a Type-2 90kDa mammalian homolog that responds to ER-stress via proteolytic cleavage within the TM. This releases the biologically active N-terminal 50 kDa fragment containing a leucine-zipper domain (bZIP) binding to the ERSE and acting as a co-transcriptional activator (Fig. 6)184. Among many unresolved questions are how J-secretases act within TM, and the role of adaptor or chaperone proteins. Generally, scissile bonds are protected by hydrogen bonding within an α-helical conformation. However the removal of extracytosolic domains by D-type secretases (sheddases) followed by interaction with PS may facilitate unfolding of D-helices, and formation of random coils that may render these more susceptible to proteolysis, resulting in enhanced generation of fibril-forming or aggregated Thus, processing of UPR components may be relevant to AE peptides116. folding/misfolding of APP or intermediates implicated in AD/FAD pathology.

Fig. 6.: Postulated roles for PS modulating a TM-endopeptidase (J -secretase). See text for abbreviations and other details.

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CONCLUDING COMMENTS Interest in secretases arises from their roles in turnover of APP or intermediates to form senile plaque Aβx. Rapid advances within the past year have lead to successful purification of a new class of novel aspartyl proteases including BACE-1 (Asp-2 ) having Esecretase specificity at pH 4.5-5.5. Metalloendopeptidases of the disintegrin family ADAM-17 (TACE ), and ADAM-10 (KUZ ) act as putative D-secretases, but cleavage within the fibril-forming domain of APP yields non-amyloidogenic products. There has been less progress in characterizing γ-secretase(s) essential for final processing of C-terminal APP to form Aβ/Aβx found in AD deposits. Interest in PS stems from an autosomal dominant gain-of-function conferred by single point or other mutations: these promote generation of Aβx by shifting sites targeted by γsecretase. Presenilins are labile although enzymes forming major metabolites remain to be characterized. Proteolysis is not mandatory since FAD-PS or constructs lacking key protease sites retain toxicity. Gain of function for labile PS mutants may arise by binding of fragments to accessory proteins to form ‘stabilized’ complexes that retain a pathological signature. Recent studies implicate PS for conversion of other TM proteins including Notch-r, and UPR (unfolding protein response) components Ire1 D and ATF6. Potential roles for PS itself as J-secretase with unique aspartyl protease properties, or as an adjunct protein necessary for its activity remain to be explored, and this may require novel concepts that cannot be explained by classical enzymology.

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CASPASES IN NEURODEGENERATION

Jörg B. Schulz1, Michael A. Moskowitz2 Neurodegeneration Laboratory Department of Neurology University of Tübingen D-72076 Tübingen, Germany 2Stroke and Neurovascular Regulation Laboratory Neurology and Neurosurgery Service Massachusetts General Hospital Harvard Medical School Charlestown, MA 02129 1

INTRODUCTION Caspases are the mammalian cell-death-effector proteins. They may have an important role in acute and chronic neurodegenerative diseases, exemplified by stroke, head trauma, Huntington's, Parkinson's and Alzheimer's disease. They execute cell death but may also be linked to the initiation of chronic neurodegenerative diseases. Peptide or protein inhibitors of caspases protect neurons in vitro or in animal models of neurological disorders. Although preclinical results are promising, clinical studies have not been performed because of the lack of synthetic caspase inhibitors that cross the blood brain barrier. Such agents are a major focus in current programs of drug development and will hopefully become available soon.

APOPTOSIS Apoptosis is an important form of cell death characterized by a series of distinct morphological and biochemical alterations suggesting the presence of a common execution machinery in different cells. Condensation and fragmentation of nuclear chromatin, compaction of cytoplasmic organelles, a decrease in cell volume and alterations to the plasma membrane are classically observed resulting in the recognition and phagocytosis of apoptotic cells. The nuclear alterations are often associated with internucleosomal cleavage of DNA, recognized as DNA laddering on conventional agarose gel electrophoresis. Internucleosomal cleavage of DNA is a relatively late event in the apoptotic process, which in some models of neuronal cell death may be dissociated from early critical steps1-3. In fact, apoptosis is not restricted to nucleated cells4. Nevertheless, detecting DNA fragmentation is simple and often used as a criterion to determine whether or not a cell is dying by apoptosis. Unfortunately, it is often overinterpreted and not without shortcomings.

Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.

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CASPASES Caspases are the major executioners of apoptosis but some caspases are also involved in cytokine processing and inflammation. A family of at least 14 related cysteine proteases are known, named caspase-1 to caspase-14, depending upon their sequence of discovery. The family includes two murine homologues (caspase-11 and – 12) that have no known human counterparts yet. Caspases are synthesized and stored as inactive proenzymes. They contain an N-terminal prodomain together with one large (p17 to p20) and one small (p10 to p12) subunit. The activation of caspases requires cleavage (usually by other caspases) to liberate one large and one small subunit, which associate into a heterotetramer, containing two small and two large subunits5. Due to their differential substrate specificities they may be divided into three major groups , which also provides insight into their biological roles in inflammation and apoptosis6,7. The three groups can be largely distinguished by their P4 preferences, a crucial determinant in caspase specificity. Group-I enzymes (caspases-1, -4, -5 and -13) prefer hydrophobic residues at P4 and are involved in the maturation of multiple proinflammatory cytokines. Group-II enzymes (caspases-2, -3, and-7 and) have a strict requirement for Asp at P4 and will cleave DxxD apoptotic substrates. The cleaved substrates will disable cellular repair, halt cell cycle progression, inactivate inhibitors of DNA fragmentation, dismantle structural elements and mark dying cells for engulfment. GroupIII enzymes (caspases-6, -8, -9, -10) prefer branched-chain aliphatic amino acids in the position P4 and will activate group-II caspases and other group-III caspases.

EVIDENCE FOR APOPTOSIS AND CASPASE ACTIVATION IN HUMAN DISEASES The development of therapeutic targets for acute and chronic neurodegenerative diseases depends in part upon identifying specific mechanisms of cell death in humans and animal models. In sporadic and inherited neur egenerative disorders like Huntington's disease (HD) and Alzheimer's disease (AD) 8,9 , the presence of chromatin condensation and DNA fragmentation suggests that cells are dying by an apoptotic-like mechanism. The results are more controversial for Parkinson's disease (PD): two studies reported that 5-8% of neurons in the substantia nigra pars compacta (SNpc) of PD patients show DNA-end labeling, a third study reported 6% of the melanin-containing neurons with chromatin changes upon electron microscopy 10-12 On the other hand, others have failed to detect apoptotic changes in the SNpc13-15, possibly because apoptotic DNA fragments have a relatively short half-life. While the significance of morphologic features suggestive of apoptosis remains controversial in human postmortum tissue, the detection of molecular apoptotic markers in human brain tissue and in animal models supports the pathological evidence. In PD16, HD17 and AD18 , activation of caspases as well as appearance of substrate cleavage products support the hypothesis that apoptosis and processed caspases are important mediators of neuronal cell death in neurodegenerative diseases. In brain tissue taken during surgical decompression for acute intracranial hypertension following trauma, cleavage of caspase- 1, upregulation and cleavage of caspase-3 were found along with DNA fragmentation with both apoptotic and necrotic morphologies19. Evidence for apoptotic cell death in human stroke is scant at the present time. In two autopsy cases, a significant number of TUNEL-positive granule cells were found in the cerebellum after global ischemia20 ; the importance of such changes to postmortem interval was not clarified. Unlike rodents, human cerebral neurons reportedly exhibit little or no caspase-3 immunoreactivity under normal conditions21. However, during ischemic degeneration, caspase-3 protein expression increases.

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Figure 1: cascade of apoptotic events in acute and chronic neurodegenerative diseases. The panel depicts our current understanding of intracellular events leading to the activation of effector caspases, e.g. caspase3. Different apoptosis-triggering pathways employ distinct signal transduction pathways that culminate in the release of cytochrome c from mitochondria. Alternatively, caspase-8 which contains two death effector domain-like molecules (DED), reacts with FADD (Fas-associating protein with death domain), and is recruited for activation at either the CD95 death-inducing signaling complex (DISC) or the tumor necrosis factor-α receptor-1 (TNF-R). However this mechanism has not clearly been show to occur in mature, differentiated neurons (Induction phase ). Two general mechanisms for release of cytochrome c (or other caspase-activating proteins) have been proposed: one involves osmotic disequilibrium leading to an expansion of the matrix space, organellar swelling, and subsequent rupture of the outer membrane; the other envisions opening of channels in the outer membrane, thus releasing cytochrome c from the intermembrane space of mitochondria into the cytosol. Members of the Bcl-2 family may perform double duty, controlling cytochrome c release from mitochondria and also possibly binding Apaf-1. Cytochrome c activates caspases by binding to Apaf-1 causing it to associate with initiator procaspases (e.g., procaspase9). Apaf-1 shares sequence similarity with the prodomain of Ced-3 and other initiator caspases with long prodomains including caspase-1, -2, -8, -9 and -10. This domain may serve as a caspase recruitment domain (CARD complex) by binding to caspases that have similar CARDS at their NH2 termini. Upon reception of a death stimulus, the complex might dissociate, freeing Apaf-1 and thereby triggering the activation of initiator caspases (Propagation phase ). Active initiator caspases may activate effector caspases (e.g. caspase-3), initiating the proteolytic cascade that culminates in apoptosis (Execution phase ). Potential sides for peptide (zIETD-fmk, caspase-8 inhibitor; zLEHD-fmk, caspase-9 inhibitor; DEVD-fmk, caspase-3 inhibitor) and protein inhibitors (IAP, p35) of caspases to interfere with this pathway are noted.

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T-cell mediated inflammation may play a key role in the pathogenic mechanism sustaining multiple sclerosis. At nearly all stages of multiple sclerosis, apoptoti cells bearing myelin markers, presumably oligodendrocytes, are present in brain 22,23. Multiple sclerosis plaques show a pronounced expression of Fas/Apo-1/CD95 and Fas ligand death signaling molecules on glia cells, including oligodendrocytes, suggesting that the Fas signaling pathway may be pathogenetically relevant to multiple sclerosis23,24.

STUDIES IN ANIMAL MODELS Because post mortem brains often contain artifacts due to autopsy delay, and typically show end stage disease rather than an evolving disease process, the best clues to mechanisms underlying neurodegenerative diseases come from animal studies.

Stroke Morphological and biochemical characterization of central neurons following global or focal ischemia suggests that apoptosis contributes to ischemic death of neuron25,26. Two lines of evidence indicate that caspase-3 activation plays a key role following transient forebrain ischemia. Firstly, immunohistochemical and biochemical studies show that caspase-3 activation occurs in susceptible cortical and hippocampal neurons following temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery or four vessel occlusion for 12 min and global ischemia, respectively27,28. Secondly, intracerebral administration of selective caspase peptide inhibitors reduce cellular and behavioral deficits following transient focal (30 min to 2 h filament insertion into the carotid artery) or global ischemia (bilateral carotid artery occlusion for 5 min)29-31. Moreover, neuroprotection can still be achieved when intracerebral administration of a caspase-3-specific (DEVD-cmk) or a pan-specific (zVAD-fmk)-caspase inhibitor was delayed by 6-9 hr after mild transient (30 min) focal ischemia29,32 or after chemically-induced hypoxia33. In both models the N-methy-Daspartic acid receptor antagonist dizolcipine (MK-801) is only efficacious when administered less than 1 hour after the initial insult. The prolonged therapeutic window makes caspase inhibitors particularly attractive for the treatment of stroke. The observation that distinct mechanisms of cell protection reduce neuronal injury in ischemia suggests the possibility that, when combined, these treatments may act in synergy. Pretreatment with subthreshold doses of MK-801, and delayed treatment with subthreshold doses of zVAD-fmk, provide synergistic protection compared with either treatment alone. Moreover, both treatment xtend the therapeutic window for caspase inhibition for an additional 2 to 3 hours33,34. The data suggest the potential value of combining treatment strategies to reduce potential side effects and to extend the treatment window in cerebral ischemia. Inhibitors of apoptosis proteins (IAPs) are a family of proteins which confer resistance to neuronal apoptosis35 by caspase inhibition36,37. Adenovirally-mediated overexpression of neuronal apoptosis inhibitory protein (NAIP) and of X-chromosomal IAP (XIAP) attenuates ischemic damage in the hippocampus er global ischemia induced by four vessel occlusion for 12 min and behavioral deficits 27,38 . Spinal cord ischemia activates caspases-8 and -3 which colocalize in neurons with cells showing DNA fragmentation. The Fas receptor expressed in neurons coexpressing caspase-8, may provide one upstream mechanism for caspase activation39.

Trauma The inhibition of caspases may offer therapeutic potential in the treatment of traumatic brain or spinal cord injury. Caspases-1 and -3 are cleaved and activated after fluid percussion-, impact- or cold injury-induced brain trauma40-42 and impact spinal trauma43 in neurons and oligodendrocytes. Intracerebroventricular injection of zVADfmk, a panspecific caspase inhibitor, zDEVD-fmk, a caspase-3 specific inhibitor, or YVAD- fmk, a caspase-1 specific inhibitor, markedly reduces posttraumatic apoptotic

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cell death and significantly enhances neurological recovery40-42. Intraocular application of caspase inhibitors reduces delayed cell death of retinal ganglion cells caused by transection of the optical nerve44.

Multiple Sclerosis Inhibition of oligodendrocyte apoptosis in autoimmune demyelinating diseases may block or attenuate the neurological manifestations of the disorder. Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis. Oligodendrocytes from transgenic mice that express the baculovirus anti-apoptotic protein p35 (inhibits multiple caspases), were resistant to cell death induced by TNF-D  agonistic anti-Fas antibody and INF-J Further, cre/p35 transgenic mice were resistant to EAE induction by immunization with the myelin oligodendrocyte glycoprotein. The numbers of infiltrating T cells and macrophages/microglia in the EAE lesions were significantly reduced, as wer the numbers of apoptotic oligodendrocytes expressing the activated form of caspase-345.

Huntington’s diseases This disease is characterized by the presence of mutated Huntingtin protein containing extended repeats of the amino acid glutamine; this mutated protein appears to be neurotoxic, but proteolytic cleavage may be needed to generate a neurotoxic fragment from the full-length, mututated Huntingtin protein. Caspase-3 cleaves Huntingtin in vitro and in apoptotic cells46,47, although an in vivo role for caspase-3 in generating Huntingtin fragments has not yet been established. Caspases are synthesized as pro-enzymes that are activated by proteolytic cleavage. According to conventional theory, procaspases are not active; recently, however, several groups have shown that procaspases may also have catalytic activity, albeit at a level much lower than that of active caspases48. Mutant, full-length Huntingtin with extended polyglutamine tracts may provide a suitable substrate for basal procaspase activity in the absence of apoptosis generating neurotoxic Huntingtin fragments. Ona and colleagues have shown recently, that a dominant-negative mutant of interleukin1-1 E-converting enzyme (caspase-1), delays the onset and progression of pathology in a transgenic model of Huntington’s disease expressing a mutant human Huntingtin exon 1 encoding an expanded polyglutamine repeat49. Further, intracerebroventricular administration of zVAD-fmk delays mortality in this model.

Parkinson’s disease 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical and neuropathologic changes reminiscent of those occuring+ in idiopathic Parkinson’s disease (PD). The toxicity of its active metabolite MPP involves the activation of caspases in vitro 50 and in vivo51. In mice chronic administration of MPTP induces apoptotic cell death in dopaminergic substantia nigra neurons. Transgenic mice expressing a dominant-negative m tant of interleukin-1 E converting enzyme are relatively resistant to MPTP toxicity52. Further, the overexpression of the antiapoptic protein, Bcl-2, prevents activation of caspases and provides protection against MPTP toxicity51.

CASPASE INHIBITION AND INFLAMMATION Until recently, the brain was considered immunologically privileged and unable to develop inflammation unless the blood-brain barrier was disrupted. We now know that the brain is capable of sustaining its own endogenous inflammatory reaction, and the evidence in Alzheimer’s disease is particularly strong53, but information occurs in other neurodegenerative diseases as well. It has been hypothesized that this reaction contributes heavily to progressive neuronal death.

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Non-specific caspase inhibitors block group-II and III caspases involved in apoptosis, and caspase- 1, the enzyme that cleaves pro-interleukin- 1E to mature interleukin-1E Since interleukin-1 receptor antagonists prevent damage after focal ischemia induced by permanent middle cerebral artery occlusion54, the first study using pan-caspase inhibitors was in ended to show that blocking ICE activity prevents ischemic damage in the same model55. In fact, transgenic mice expressing a dominant negative caspase-1 are protected in animal models of stroke induced by 3 h of cerebral artery occlusion followed by 24 h of reperfusion56, impact-induced head trauma 41, Parkinson’s disease52, amyotrophic lateral sclerosis57 and Huntington’s disease58. It remains to be elucidated whether both, inhibition of inflammation and inhibition of caspase-mediated neuronal apoptosis contribute to the protective effects of caspase inhibitors. Of note, murine caspase-11, with homology to human caspase-4, promotes both caspase-1 and caspase-3 processing, thereby enhancing both apoptosis and cytokine maturation59.

CASPASE INHIBITORS UNDER DEVELOPMENT The development of non-peptide selective caspase inhibitors which cross the blood brain barrier has become a major goal of drug discovery. One example, L-826791 is under development by Merck for the treatment of cerebrovascular ischemia60. L826791 has an IC50 value of only 8.0 nM and in vivo limits cerebral cortical damage following acute occlusion of the middle cerebral artery in rats. The caspase inhibitor IDN-6556 is under investigation by IDUN Pharmaceuticals for the treatment of alcoholic hepatitis, inflammation, neurodegenerative diseases and ischemia. Its IC50 values for maximal efficacy against recombinant activity and in cell culture are 0.5 nM and 1.8 µM, respectively 61. Cytovia is investigating caspase inhibitors for the potential treatment of degenerative diseases, hepatitis, sepsis and cerebral ischemia. The lead compound, CV1013 shows good efficacy in animal models and has low toxicity and favorable PK profile62. Other companies including Vertex Pharmaceuticals, Texas Biotechnology Corp., Novartis and Aventis are developing new and specific caspase inhibitors but no further published information is currently available.

LIMITATIONS AND CAUTIONS Caspases and related proteins are emerging as important therapeutic targets in a variety of acute and chronic CNS diseases. Preclinical evidence in stroke supports the need to investigate anti-apoptotic treatment strategies, particularly because caspase inhibitors reduce tissue injury when administered many hours after mild ischemia. In the clinical setting, anti-apoptotic strategies might become useful to treat brief episodes of brain ischemia or be given in advance of risky surgical procedures (e.g. cardiopulmonary by-pass) or combined with thrombolytics or agents in which synergy has been documented (e.g. glutamate receptor antagonists). Furthermore, caspase inhibitors may provide promising opportunities for other neurological conditions in which cell death is prominent. Although the results of treatment in animals with caspase inhibitors are promising, clinical studies have not yet been performed because of the lack of synthetic caspase inhibitors that cross the blood brain barrier. Such agents are a major focus in current programs of drug development and will hopefully become available soon. In addition, therapies that lead to the increased expression of IAPs are a potential avenue for treatment of chronic neurodegenerative disorders.

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21.M. Krajewska, H.-G. Wang S. Krajewski, J.M. Zapata, A. Shabaik, R Gascoyne, and J.C. Reed, Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease, Cancer Res. 57:1605-1613 (1997). 22. P. Dowling, G. Shang, S. Raval, J. Menonna, S. Cook, and W. Husar, Involvement of the CD95 (APO1/Fas) receptor/ligand system in multiple sclerosis brain,J. Exp. Med 184:1513-1518 (1996). 23. K. Ozawa, G. Suchanek, H. Breitschopf, W. Bruck, H. Budka, K. Jellinger, and H. Lessmann, Patterns of oligodendroglia pathology in multiple sclerosis, Brain 117:1311-1322 (1994). 24.S.D. D'Souza, B. Bonetti V. Balasingam, N.R Cashman, P.A. Barker, A.B. Troutt, C.S. Raine, and J.P. Antel. Multiple sclerosis: Fas signaling in oligodendrocyte cell death, J. Exp. Med. 184:2361-2370 (1996). 25. J.P. MacManus, A.M. Buchan I.E. Hill, I. Rasquinha, and E. Preston, Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain, Neurosci. Lett. 164:89-92 (1993). 26.J.P. MacManus, LE. Hill, Z.G. Huang I. Rasquinha, D. Xue, and A.M. Buchan, DNA damage consistent with apoptosis in transient focal ischaemic neocortex, NeuroReport 5:493-496 (1994). 27. D. Xu, Y. Bureau, D.C. McIntyre, D.W. Nicholson, P. Liston, Y. Zhu, W.G. Fong, S.J. Crocker, R.G. Korneluk, and G.S. Robertson, Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus, J. Neurosci. 19:5026-5033 (1999). 28. S. Namura, J. Zhu, IC. Fink, M. Endres, A. Srinivasan, K.J. Tomaselli, J. Yuan, and MA. Moskowitz, Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia, J. Neurosci. 18:3659-3668 (1998). 29. M. Endres, S. Namura, M. Shimizu-Sasamata, C. Waeber, L. Zhang, T. Gómez-Isla, B.T. Hyman, and M.A. Moskowitz, Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family, J. Cereb. BIoodFIow Metab. 18:238-247 (1998). 30.H. Hara, RM. Friedlander, V. Gagliardini, C. Ayata, K. Fink, Z. Huang M. Shimizu-Sasamata, J. Yuan, and MA. Moskowitz, Inhibition of interleukin 1β converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage, Proc. Natl. Acad Sci. USA 942007-2012 (1 997). 31. T. Himi, Y. Ishizaki, and S. Murota, A caspase inhibitor blocks ischaemia-induced delayed neuronal death in the gerbil, Eur. J. Neurosci. 10:777-781(1998). 32.K. Fink, J. Zhu, S. Namura M. Shimizu-Sasamata, M. Endres, J. Ma, T. Dalkara, J. Yuan, and M.A. Moskowitz, Prolonged therapuetic window for ischemic brain damage caused by delayed caspase activation, J. Cereb. Blood Flow Metab. 18: 1071-1076 (1998). 33. J.B. Schulz M. Weller, RT. Matthews, M.T. Heneka, P. Groscurth, J.C. Martinou, J. Lommatzsch, R von Coelln, U. Wüllner, P.-A. Löschmann, M.F. Beal, J. Dichgans, and T. Klockgether, Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia, Cell Death Diff. 5:847-857 (1998). 34. J. Ma, M. Endres, and MA Moskowitz, Synergistic effects of caspase inhibitors and MK-801 in brain injury after transient focal cerebral ischemia in mice, Br. J. Pharmacol. 124:756-762 (1998). 35.M. Simons, S. Beinroth, M. Gleichmann, P. Liston, R.G. Komeluk, A.E. MacKenzie, M. Bähr, T. Klockgether, G.S. Robertson, M. Weller, and J.B. Schulz Adenovirus-mediated gene transfer of IAPs delays apoptosis of cerebellar granule neurons, J. Neurochem. 72:292-301 (1999). 36.Q.L. Deveraux, and J.C. Reed, IAP family proteins-suppressors of apoptosis, Genes Dev. 13:239-252 (1999). 37.G.S. Robertson, S.J. Crocker, D.W. Nicholson, and J.B. Schulz, Neuroprotection by the inhibition of apoptosis, Brain Pathol. 10:283-292 (2000). 38.D.G. Xu, S.J. Crocker, J.-P. Doucet, M. St-Jean, K. Tamai, A.M. Hakim, J.-E. Ikeda, P. Liston, C.S. Thompson, RG. Komeluk, A. MacKenzie, and G.S. Robertson, Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus, Nature Med 3:997-1004 (1997). 39.K. Matsushita, Y. Wu, L. Lang-Lazdunski, L. Hirt, C. Waeber, B.T. Hyman, J. Yuan, and M.A. Modtowitz, Caspase-8 and caspase-3 activation and neuronal cell death after spinal cord ischemia, J. Neurosci. in press (2000). 40. A.G. Yakovlev, S.M. Knoblach, L. Fan, G.B. Fox, R Goodnight, and A.I. Faden, Activation of CPP32like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury, J. Neurosci. 17:7415-7424 (1997). 41.K.B. Fink, L.J. Andrews, W.E. Butler, V.O. Ona, M. Li, M. Bogdanov, M. Endres, S.Q. Khan, S. Namura, P.E. Stieg, M.F. Beal, MA. Moskowitz, J. Yuan, and RM. Friedlander, Reduction of post-traumatic brain injury and free radical production by inhibition of the caspase-1 cascade, Neuroscience 94: 12 13 -12 18 (1 999).

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42. Y. Morita-Fujimura, M. Fujimura, M. Kawase, K. Murakami, G.W. Kim, and P.H. Chan, Inhibition of interleukin-1beta converting enzyme family proteases (caspases) reduces cold injury-induced brain trauma and DNA fragmentation in mice, J Cereb Blood Flow Metab 19:634-642 (1999). 43. J.E. Springer, R.D. Azbill, and P.E. Knapp, Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury, Nat Med 5:943-946 (1999). 44. P. Kermer, N. Klöker, M. Labes, and M. Bähr,, Inhibition of CPP32-like proteases rescues axotomized retinal ganglion cells from secondary cell death in vivo, J. Neurosci. 15:4656-4662 (1998). 45. S. Hisahara, T. Araki, F. Sugiyama, K. Yagami, M. Suzuki, K. Abe, K. Yamamura, J. Miyazaki, T. Momoi, T. Saruta, C.C. Bernard, H. Okano, and M. Miura, Targeted expression of baculovirus p35 caspase inhibitor in oligodendrocytes protects mice against autoimmune-mediated demyelination, EMBOJ. 19:341-348 (2000). 46. Y .P. Goldberg, D. W. Nicholson, D.M. Rasper, MA Kalchman, H.B. Koide, R.K. Graham, M. Bromm, P. Kazemi-Esfarjani, N.A. Thornberry, J.P. Vaillancourt, and M.R. Hayden, Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract, Nature Genet. 13:442-449 (1996). 47.C.L. Wellington, L.M. Ellerby, AS. Hackam, R.L. Margolis, R.L. Trifiro, R. Singaraja, K. McCutcheon, G.S. Salvesen, S.S. Propp, M. Bromm, K.J. Rowland, T. Zhang, D. Rasper, S. Roy, N. Thornberry, L. Pinsky, A. Kakizuka, C.A. Ross, D.W. Nicholson, D.E. Bredesen, and M.R Hayden, Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract, J. Biol. Chem. 273:9158-9167 (1998). 48. H.R. Stennicke, Q.L. Deveraux, E. W. Humke, J.C. Reed, V.M. Dixit, and G.S. Salvesen, Caspase-9 can be activated without proteolytic processing, J. Biol. Chem. 274:8359-8362 (1999). 49. V.O. Ona, M. Li, J.P. Vonsattel, L.J. Andrews, S.Q. Khan, W.M. Chung A.S. Frey, A.S. Menon, X.J. Li, P.E. Stieg, J. Yuan, J.B. Penney, A.B. Young, J.H. Cha, and R.M. Friedlander, Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease, Nature 399:263267 (1999). 50. R.C. Dodel, Y. Du, K.R. Bales, Z.D. Ling, P.M. Carvey, and S.M. Paul, Peptide inhibitors of caspase3-like proteases attenuate 1-methyl-4- phenylpyridinum-induced toxicity of cultured fetal rat mesencephalic dopamine neurons, Neuroscience 86:70 1-707 (1998). 51. L. Yang, R.T. Matthews, J.B. Schulz, T. Klockgether, A.W. Liao, J.C. Martinou, J.B. Penney Jr., B.T. Hyman, and M.F. Beal, MPTP neurotoxicity is attenuated in mice overexpressing Bc1-2, J. Neurosci. 18:8145-8152 (1998). 52.P. Klevenyi, O. Andreassen, RJ. Ferrante, J.R. Schleicher, Jr., R.M. Friedlander, and M.F. Beal, Transgenic mice expressing a dominant negative mutant interleukin-lbeta converting enzyme show resistance to MPTP neurotoxicity, NeuroReport 10:635-638 (1999). 53.P.L. McGeer, and J. Rogers, Anti-inflammatory agents as a therapeutic approach to Alzheimer’s disease, Neurology 42:447-449 (1992). 54. S.A. Loddick, and N.J. Rothwell, Neuroprotective effects of human recombinant interleukin-1 receptor antagonist in focal cerebral ischemia in the rat, J. Cereb. Blood Flow Metab. 16:932-940 (19%). 55. S.A. Loddick, A. MacKenzie, and N.J. Rothwell, An ICE inhibitor, z-VAD-DCB attenuates ischaemic brain damage in the rat, NeuroReport 7:1465-1468 (19%). 56. H. Hara, K. Fink, M. Endres, RM. Friedlander, V. Gagliardini, J. Yuan, and M.A. Moskowitz, Attenuation of transient focal cerebral ischemia injury in transgenic mice expressing a mutant ICE inhibitory protein, J. Cereb. Blood Flow Metab. 17:370-375 (1997). 57. R.M. Friedlander, RH. Brown, V. Gagliardini, J. Wang, and J. Yuan, Inhibition of ICE slows ALS in mice [letter], Nature 388:31 (1997). 58.V.O. Ona, M. Li, J.P. Vonsattel, L.J. Andrews, S.Q. Khan, W.M. Chung, AS. Frey, A.S. Menon, XJ. Li, P.E. Stieg, J. Yuan, J.B. Penney, A.B. Young, J.H. Cha, and R.M. Friedlander, Inhibition of caspase-1 slows disease progression in a mouse model of Huntington's disease, Nature 399:263267 (1999). 59. S.-J. Kang, S. Wang H. Hara E.P. Peterson, S. Namura, S. Amin-Hanjani, Z. Huang, A. Srinivasan, K.J. Tomaselli, N.A. Thornberry, M.A. Moskowitz, and J. Yuan, Dual role of caspase-11 in mediating actrivation of caspase-1 and caspase-3 under pathological conditions, J. Cell Biol. 149:613-622 (2000). 60.Programmed cell death regulation: basic mechanisms and therapeutic opportunities (Part II)., IDDB Meeting report (2000). 61. Caspases in apoptotic death, Exp Opin Invest Drugs 8:37-50 (1999). 62. BioPartnering - Cytovia Inc, Ann Biopartnering Europe Meeting (1999). 18-19 October

THERAPEUTIC APPROACHES WITH PROTEASE INHIBITORS NEURODEGENERATIVE AND NEUROLOGICAL DISEASES

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Kevin K.W. Wang Department of Neuroscience Therapeutics Parke-Davis Pharmaceutical Research A Division of Warner-Lambert Company Ann Arbor, MI 48 105

INTRODCUTION TO THE CLASSES OF PROTEASES IMPLICATED IN NEURODEGENERATIVE AND NEUROLOGICAL DISEASES There are five major classes of mammalian proteases identified to date: serine proteases (EC 3.4.21), cysteine proteases (EC 3.4.22), aspartate proteases (EC 3.4.23) metalloproteases (EC 3.4.24) and theronine proteases (EC 3.4.25)1. Interestingly, members of all five protease classes have been implicated at contributing factors in various neurological or neurodegenerative disorders (Table 1). Due to the differences in how these proteases are activated and how they function, different strategies of inhibition are required. Cysteine proteases have cysteine; histidine and asparagine residues that form the catalytic triad involved in the hydrolysis of protein peptide bonds. Due to the requirement of the reduced cysteine sulfhydroyl group, a reducing intracellular environment is needed. Calpain is a heterodimeric cytosolic cysteine protease that is also regulated by free Ca2+ and it has been implicated in contributing to cell death in stroke, traumatic brain injury (TBI) as well as Alzheimer’s disease (AD)2,3 (Table 1). Another subfamily of cysteine proteases called caspases has been implicated in apoptotic neuronal death. Caspases-3 which is activated by caspase-8 via receptor-linked pathway (e.g. TNF-alpha receptor) or by caspase-9 via a mitochrondria-dependent pathway. The cytosolically located caspase-3 then goes on and attacks various cellular proteins and executes the programmed cell death (apoptosis)3-6 Thus, caspase-3, -8 and -9 contribute to the apoptotic cell death components in various neurological (stroke, TBI, spinal cord injury (SCI)) and neurodegenerative disorders (AD, amyotrophic lateral sclerosis (ALS)) (Table 1). Their related cousin Caspase-1 (interleukin converting enzyme or ICE), on the other hand, is more likely to be involved in the inflammatory responses upon neuronal injury. It does so by processing and activating the pro-inflammatory cytokine pro-interleukin beta to its mature form7. Cathepsin B is a lysosomal cysteine protease that is also implicated in ischemic strokes8,9 Its selective and cell-permeable inhibitor CA074Me10 is neuroprotective11,12 Both cathepsin B and cathepsin D immunostatining appear to intensified in AD brain13. Cathepsin D, unlike cathepsin B, is an aspartate protease which has been linked to cerbral ischemia14. It has also been implicated as a candidate beta-secretase of amyloid precursor protein (APP) 15-17. Also recently, a fury of activity in the literature have pointed to the identification of two potential asparate proteases as candidate beta-secretases (BACE or Asp-2 and BACE2 or Asp-118-21 (Table 1). They are unique in that they have

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transmembrane helix in the C-terminal which localized them to golgi and endosome membrane where APP protein is also located. Thrombosis is one of the major causes of ischemic stroke in human. Anticoagulants such as heparin have demonstrated beneficial effects in reducing brain edema and infarct volume in rats subjected to thrombotic middle cerebral artery occlusion (MCAO), a model for human thrombotic strokes22. Following the same rationale, thrombin (a serine protease) is also implicated in thrombotic stroke23- 25 . In a rat thrombotic MCAO model, where a platelet-rich thrombus was used for occlusion, a selective thrombin inhibitor argatroban, was found to decrease the size of the cerebral infarction and improved neurological deficits26. A recent study shows that post-ischemic subcutaneous injection of argatroban (5 mg/kg) significantly attenuated cell damages in the cerebral cortex, attenuated of brain edema, increased cortical cerebral blood flow after reperfusion and also attenuated during 14 days' observation27. But it is important to point out that antithrombotic agents are generally contraindicated in hemorrhagic stroke. Matrix metalloproteases (e.g. gelatinases, collagenases and stromelysins) are zincrequiring proteases that are secreted into intercellular space (MMP-1 through MMP-12)28. Their major targets are extracellular matrix (ECM) proteins, such as collagen. Some of them are inducible when the cells are stimulated. MMPs also have a reputation of capable of refolding back into active enzyme following removal of denaturing detergent such as SDS. A zymography technique is commonly used. MMP (such as MMP2 (gelatinase A) or MMP9 (gelatinase B) or MMP3 (stromeysin-1)) preferred substrates such as collagen, Because of their matrix-degradative capability, MMP-2, -3 -7 and -9 have been implicated in cerebral ischemia29-31 multiple sclerosis (MS) or in its animal model EAE (experimental autoimmune encephalomyelitis) (Table 1) 32-35. Proteasome is a very complex multi-subunit and large oligomeric proteases36. Its is composed of both small regulatory subunits and protease subunits (with distinct substrate specificity). It is thus sometimes called multi-catalytic protease (MCP). It also contains a regulatory component that confers its ATP-dependence as well as ubiquitin-dependence. Interestingly ATP is hydrolyzed during peptide hydrolysis. Also, protein substrate must first be conjugated at lysine-residues with multimers of a protein called ubiquitin before its is recognized by proteasome. All the proteasome catalytic subunits belong to a novel class of protease (theronine proteases). It utilizes two theronine residues at the N-terminal for the hydrolysis of peptide bonds. Proteasome has also been implicated in stroke)37 as well as MS38. Proteasome inhibitor (CVT-634) was recently found to be neuroprotective by suppressing the NF-kB activation pathway39 (Table 1). Table 1 Known proteases implicated in neurodegenerativeand neurological diseases and reference inhibitors Protease

Class

Disorder(s)

Ref. Inhibitors

Calpain

Cysteine

Stroke, TBI, AD

MDL28170, PD150606, SJA6017

Caspase-3, 8,9

Cysteine

Stroke, TBI, SCI, AD

Z-VAD, Z-D-DCB, Ac-DEVD-fink

ICE (Caspase-I)

Cysteine

Stroke

Ac-YVAD-CHO, VE-18858, VX-740

Cathepsin B

Cysteine

Stroke, AD

CA074-Me

Thrombin

Serine

Stroke, TBI

Argatroban

Cathepsin D

Aspartate

AD

Pepstatin A, CEL5-A, EA-1

MMP2 3,7 & 9

Metalllo -

MS

CP-4- 7 1,474 (broad-spectrum)

BACE

Aspartate

AD

KTEEISEVN(Statine)DAEF

Proteasome

Theronine

MS, stroke

lactacystin, MG132, PSI

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CURRENT INHIBITORS OF PROTEASES The use of inhibitors to study the role of a protease in a particular neurodegenerative and neurological disease is a very powerful technique. But the most common limitations are the lack of cell permeability and selectivity of the protease inhibitor. Sometimes, by applying higher concentration of a peptide inhibitor with low cell permeability, one can get sufficient amount of the inhibitor into the cell. Selectivity is a more important issue. For example, several calpain inhibiting peptide aldehydes (such as calpain inhibitor I and II) also cross-inhibit theronine protease proteasome. Yet, a calciumbinding site directed calpain inhibitor PD1 50606 (Calbiochem Co.) does not inhibit proteasome40 (Table 1). Lactacystine is naturally occurring beta-lactone that specifically and colvantly modifies Thr and is thus highly selective in inhibiting proteasome38. Other proteasome inhibitors including Cbz-Leu-Leu-Leu-CHO (MG132), and Cbz-IleGlu(OtBu)-Ala-Leu-H (PSI), are also more selective for proteasome than for calpain (Calbiochem Co.) (Table 1). Thus, the use of more than one inhibitor is strongly advised. Iodinated calpain inhibitors have been used successfully in labeling activated calpain in activated platelets41. Similarly, Biotin-conjugated cas ase inhibitor. Recently, two biotinlabeled caspase inhibitors (Ac-YVK(Biotin)D-amk42 and Cbz-VK(Biotin)D-fmk43 were utilized successfully to label caspase-3 in apoptotic cell lysate. For the caspases family, the apoptosis-effector caspases (caspase-3 and 7) are readily inhibited by Ac-DEVD-CHO or DEVD-fmk, while the apoptosis upstream caspases (caspase-8, -9 and -10) are readily inhibited by Z-VAD. Pan-specific caspases inhibitors (Z-D-DCB, Boc-Asp-fmk) also exist and are quite useful neuroprotectant in vitro and in vivo3,44-49. On the other hand, inflammation-linked ICE (Caspase-1) are selectively inhibited by Ac-YVAD-CHO and Ac-WVAD-CHO. Vertex pharmaceutical have also developed potential clinical ICE inhibitor candidates ( VE- 18858, VX-740)3,50,51. Among the cysteine protease cathepsins K, L and B, there are now documented inhibitors that show subclass selectivity52-54 (Table 1). Aspartic protease inhibitor pepstatin A has been used successfully in inhibiting cathepsin D activity in intact cells55. Bi et al.56 recently reported several potent cathepsin D inhibitors (CEL5-A, CEL5-G, EA-1). Lastly, argatroban is a well established selective thrombin inhibitor57.

PROTEASE TARGET VALIDATIONS Assuming that there are some data in the existing literature suggesting that a specific protease might be involved in a particular neurological disease, it is important to provide further “proof-of concept” type of compelling evidence that the protease target is in fact valid and relevant to the disease of interest. Table 2 outlines a number of evidence that have been used in the past. Co-localization of protease protein to the site of disorder (e.g. a specific CNS region or specific neuronal cell types) gives a positive correlation of the protease to the disorder. Similarly, increased mRNA an/or protein level of the protease of interest in the disease state compared to control is powerful evidence. Sometimes, the specific protease activity can be monitored either by direct assaying or by following the integrity of endogenous protein substrate(s) in animal disease model. Should this protease activity increased significantly in disease state, it is compelling evidence for the involvement of the protease. It also applies to cell culture models (if available) of a neurological disorder) if increase of a protease activity can be detected reproducibly. It is also common to approach target validation by suppression of the protease of interest or intervention of its processing / activation. This is most commonly achieved pharmacologically by applying selective inhibitors to influence the outcome in the animal disease or in neuronal cell culture (e.g. neuroprotection against ischemic injury in vivo or in situ). Alternatively, genetic manipulation could be employed to knockout the protease gene and then study the k/o mice to see if they show different susceptibility compared to control mice. Similarly, overexpressing a protease using CNS neuron-specific promoters such as PDGF-B, Thy-1, prion or elonase promoters in transgenic mice is also very powerful technique that might produce a phenotype which mimics or exaggerates the disease state of interest.

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Table 2 Target protease validation checklist Key studies or evidence Localization of protease to site of disorder/disease Increased mRNA and protease levels in animal disease model or in human patients Increased protease activity or protease substrate breakdown product accumulation in neuronal cell culture model of disease Increased protease activity or protease substrate breakdown product accumulation in animal disease model or in human patients Selective reference inhibitors improve outcome in animal disease model or cell culture model Protease knockout mice have improved outcome in mouse disease model Protease-overexpressing transgenic mice have worsened outcome in mouse disease model

PROTEASE INHIBITOR SCREENING AND DEVELOPMENT STRATEGY Generally, for a pharmaceutical institute to discover and develop a selective patent-able protease inhibitor as a small organic molecule drug to treat a neurodegenerative and or neurological disease, various steps are required.

1. Source of enzyme and in vitro protease assays After the “proof of concept” stage, one must have either (I) purified native proteases from human and/or other species or (II) clone and express recombinant proteases (Fig. 1). In the latter case, one can either express the full length protease or in some cases a truncated form that preserves the catalytic protease domain is sufficient. Of course, the recombinant protease must have activity and substrate specificity similar to the native enzyme counterpart. An in vitro robust protease assay must then be established to determine inhibitory potency of compounds, as measured by inhibitory concentration that causes 50% inhibition (IC50) or kinetically by inhibitory constant (Ki) (Fig. 1). This primary assay can then be optimized and sometimes miniaturized to run through high volume and high throughput screening of random compound library (# entries usually needed to be at least 200,000300,000 to be successful in identifying structural leads). This strategy is designed to identify novel patentable inhibitor(s) simply by chance. Once structural leads are identified, additional medicinal chemistry support is required to further structure-activity relationship (SAR) to improve potency. Alternatively, if the protease can be crystallized as a complex with a reference inhibitor in an early stage, then rational drug design by medicinal chemists in collaboration with computer aided design (CAD) specialists can be launched to discover new patentable inhibitors. It is extremely desirable to have certain selectivity assays using other related protease within the same class and in other protease classes. Sometimes, even non-protease enzymes as selectivity screens are also included (Fig. 1). The lack of selectivity of a protease inhibitor drug usually contribute to undesirable side effects in vivo.

(b) Cell-based assays An important screening step for intracellularly located protease is the establishment of a cell-based protease assay (Fig. 1). To truly test the permeability and efficacy of an inhibitor in cells, it is not good enough to assay cell lysate but rather, the inhibitor must be

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introduced to intact cell culture medium directly and examine if there are active in inhibiting its target protease in intact cells! A powerful means to monitor intracellular protease activity is in fact not to introduce an artificial substrate, but rather monitoring the integrity and/processing of the protease as well as its endogenous protein substrates. The general technique calls for standard cellular protein extraction, SDS-PAGE, Western blots and then the detection with a specific antibody (Fig. 1). Most proteases exist as zymogens that are proteolytically or autolytically activated (e.g. cathepsin B, L, D, calpains, caspases and matrix metalloproteases (MMPs))1. Similarly, distinct endogenous protein substrates have been identified for a number of intracellular proteases such as calpain (alphaspectrin), caspases (poly(ADP)ribose polymerase or PARP) and proteasome (e.g. ikappaBalpha)58. With calpain and caspase, the substrates are generally cleaved into fragments with smaller molecular weight, readily distinguished from the intact proteins. The proteasome generally degraded proteins into very small peptides. So the disappearance or intensity reduction of the intact protein band, rather than protein fragments, is expected. Also, inhibition of proteasome (e.g. lactacystin) would result in an accumulation of the high molecular weight ubiquitin-conjugates of its protein substrates59 since ubiquitinization is the biochemical step preceding proteasome-mediated degradation . For certain proteases, it is possible to introduce a small fluorogenic peptide that is permeable to cell membrane. Thus, once the peptide is introduced to the cells, the protease can be activated by the addition of stimulus and the protease activity is tracked either continuously or as end-point measurement using a fluorometer. The use of chromogenic substrates (e.g. peptide-p-nitroanalide) usually does not have a strong enough signal for detection and is therefore not recommended. The choice of cell type is obviously very important. The primary criterion is the presence of reasonably high level of the protease of interest. Other favorable considerations are the lack of other proteases that could hydrolyze the same substrate and low levels of endogenous inhibitor(s) of the protease of interest. (Such as calpastatin for calpain, serpin for chymase and other serine protease, Inhibitor of apoptosis proteins (IAPs) for caspase60, and tissue inhibitor of matrix metalloprotease (TIMPs) for matrix metalloproteases). The choice is peptide substrate is also very important. The primary goal is to select a substrate that would be (i) selectively hydrolyzed by the protease of interest and (ii) cell membrane permanent. An example is the study of the calcium-activated protease (calpain). A cell-permanent fluorogenic peptide substrate such as succinyl-Leu-Leu-Val-Tyr-7-amino-4-chloromethylcoumarin (SLLVY-AMC) can be introduced and then the protease is activated by a calcium channel opener maitotoxin or calcium ionophore (e.g. A23 187), the fluorescence derived from the release of AMC is monitored in a 12-well plate format40. A similar peptide (tbutoxycarbonyl-Leu-Met-AMC) which is conjugated with glutathione intracellularly (used as cell trap) can be used61. A secondary cell-based assay or a cell-based assay to track cytotoxicity is also important to give an indication of the selectivity of the compound. Also, it is sometimes possible to establish a tissue-based assay (e.g. hippocampal slices) to monitor protease activity or other functional endpoints that can be influenced by the inhibitors (e.g. cell viability or conductivity).

(c) In vivo studies A desirable pharmacokinetic (PK) profile often makes the difference in whether the inhibitor will eventually become a drug or not, thus it is important to profile PK at an early stage. Acceptable plasma level, rat of clearance, metabolic stability (based microsome studies), cell membrane permeability, oral bioavailabilty (e.g. for chronic disorders) as well as brain penetration are important features. It is sometimes possible for the medicinal chemists to modify the inhibitor structures to improve their PK profile while maintaining potency of the compounds. Drug formulation is also an important issue. Depending on the route or administration (i.v,, p.o.) the criteria involved are quite diverse. Some experimentation is usually required to find an optimal formulation for in vivo studies. Ideally, the same formulation to be used in animal studies can be eventually used in humans. Any excipients used in such formulations (co-solvents, stabilizers, etc.) and the quantities used must meet Food and Drug Administration (FDA) guidelines. The physical stability of the compound upon storage also needed to be determined and potentially optimized.

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For chronically administrated drug, it is desirable to test compound in a small-scale acute toxicity study to ensure that the compound is reasonably safe before investing too much efforts in other in vivo studies (Fig. 1). Usually, PK data can help determine or recommend a therapeutic dose range to be used in vivo. Here, we assume that an animal model for the disease of interest is available. If a biomarker that tracks with the protease activity can be identified (e.g. a specific protein substrate fragment), it is greatly desirable to perform an initial studies to examine if the inhibitor can suppress the target protease in vivo and at what dose (mechanistic endpoint). Once this is established, it is time to conduct inhibitor studies with the optimal dosing regime and to determine if it would alter the efficacy endpoint in a positive direction (e.g. neuroprotection). If a single dose of a drug proves to be efficacious, it is often important to repeat the efficacy studies and establish a dose-response (efficacy) relationship. The data obtained will be extremely helpful to guide pre-clinical toxicology studies as well as possible clinical trails for the neurological or neurodegenerative disease of interest.

Figure 1. Flow chart for protease inhibitor screening and discovery strategy

PERSPECTIVES In this chapter, we gave an overview on various classes of proteases that might contribute to one or more neurological or neurodegenerative disorders. We also discussed the practical issues regarding how to discover and characterize new and selective protease inhibitors as potential drugs. It was shown that the inhibitory compound not only has been a great inhibitor pharmacologically, it also must have good drug-like features regarding pharmacokinetical and safety profile. Lastly, the ultimate test is to see if the compound of interest is robustly efficacy in animal disease model.

REFERENCES 1. A.J Barrett, N.D. Rawlings, and J.F. Woessner, (eds) Handbook ofProteolytic Enzymes, Academic Press, London (1998).

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PATHOPHYSIOLOGY OF CENTRAL NERVOUS SYSTEM TRAUMA: PROTEOLYTIC MECHANISMS AND RELATED THERAPEUTIC APPROACHES

Swapan K. Ray, Denise C. Matzelle, Gloria G. Wilford, Lawrence F. Eng*, Edward L. Hogan, and Naren L. Banik Department of Neurology Medical University of South Carolina Charleston, SC 29425 *Pathology Research Service Veterans Administration Hospital Palo Alto, CA 94304

INTRODUCTION Injury to the central nervous system (CNS) [e.g., spinal cord injury (SCI) and traumatic brain injury (TBI)] is one of the main health problems in the United States as well as in the world. CNS injury is also a major killer in the United States. The majority of these injuries are caused by automobile accidents, assaults, guns, falls, sports, and other traumatic events. The extent of the loss of neurological function depends upon the severity of injury. Primary injury to the CNS causes vascular change beginning at the mechanical impact and followed by secondary pathophysiological processes. These processes eventually lead to cell demise and tissue destruction which may be devastating. The secondary injury process develops over a period of hours or days after the primary injury, i.e. initial impact to the spinal cord or brain. It is associated with synthesis or release of mediating neurochemicals which alter blood flow, ion homeostasis, and metabolism and which may also be neurodestructive agents in CNS trauma. Although the full extent and interplay of mechanisms that underlie CNS injuries are yet unknown, several factors have been implicated in the secondary injury cascade. Employing impact or compression injury models in spinal cord and the controlled cortical contusion injury model in brain, investigators have identified a number of pivotal factors, including free radicals, Ca2+ influx, proteinases and lipases, glutamate, cytokines and other mediators in the progression of secondary injury in CNS trauma1-4. The identification of these destructive agents and the timing of their pathological actions are important steps in advancing research upon CNS trauma and will ultimately lead to strategies for the treatment of CNS injury.

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One of the most devastating events in the secondary injury process of CNS trauma is the increased intracellular calcium concentrations in neurons. Calcium plays many roles in the cell, including activation of proteinases and lipases whose activities are significantly increased in CNS lesions following injury. Among these is calpain, a ubiquitous, Ca2+-activated neutral proteinase whose increased activity degrades cytoskeletal proteins in spinal cord injury, traumatic brain injury, and cerebral ischemia with concomitant loss of both cytoskeletal and myelin proteins. The loss of these proteins destablizes membrane and neuronal architecture and eventually leads to cell death. Since these proteins maintain the structure and function of neurons and their processes, it is a high priority to protect neurons by prevention of cytoskeletal protein degradation. The use of calpain inhibitors as therapeutic agents in animal trials has proven useful for this and several recent studies of treatment of injured animals with calpain inhibitors alone and/or in combination with other agents, have shown neuroprotective effects3,5-7. Although this chapter has an overall concern with several proteolytic enzymes known to be altered in CNS trauma, emphasis is put upon calpain and calpain-related therapeutic strategies. Readers should consult the literature for reviews on the role of other proteinases in CNS injury.

PROTEINS AND PROTEINASES INVOLVED IN BRAIN AND SPINAL CORD INJURY Proteins It is not possible to review the vast literature on brain and spinal cord proteins in this short chapter. Instead, we have focussed upon the examination of the finite number of CNS proteins which are endogenous substrates of the proteases in brain and spinal cord activated following injury. The degradation of cytoskeletal, axonal and myelin proteins will be taken as the index of proteolytic activity in CNS injury because these are the essential framework elements in CNS. The loss of these proteins alters the structural integrity of cells and accompanies the axonal degeneration and myelin vesiculation observed following trauma. Comprehensive pictures of brain and spinal cord proteins are in recent review articles. The majority of studies of lesions in both spinal cord injury (SCI) and traumatic brain injury (TBI) have examined endogenous cytoskeletal and axonal proteins [including microtubule associated proteins (MAP1, MAP2), fodrin (D-spectrin), neurofilament proteins (NFPs; 68kD, 150kD and 200kD)] and myelin proteins [including myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG), and the enzyme protein, 2’,3’-cyclic nucleotide 3’phosphohydrolase (CNPase)]. Degradation of cytoskeletal proteins which maintain the integrity and architecture of cells and their processes, axons, and dendrites, has been correlated with post-traumatic morphological alterations in cells and processes. Loss of neurofilament proteins in SCI and TBI is associated with axonal degeneration6,8,9. Other proteins such as MAP1 and MAP2 are also readily degraded after CNS injury. The degradation of 230kD fodrin is widely used to assess the proteolytic activities of calpain and caspase-3 which cleave fodrin at different sites producing two different peptides, a calpain specific 150kD fragment and a caspase-3 specific 120kD fragment10,11. Degradation of myelin proteins accompanies a splitting and vesiculation of myelin lamellae. The two major proteins of myelin, proteolipid protein (PLP) and myelin basic protein (MBP), constitute about 50% and 30% of the total myelin

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protein, respectively. The molecular weight of PLP is approximately 24kD while that of MBP ranges from 17.5-21kD. In rodents, there is also a small MBP with a molecular weight of 14kD. The major MBP (18kD) is the most susceptible to degradations. PLP is masked by being tightly bound (covalently) to or complexed with lipids and resists proteolysis, though it can be degraded in the presence of detergent12,l3. Other minor proteins of the myelin sheath include MOG (myelin oligodendrocyte-specific glycoprotein), MOBP (myelin oligodendrocyte-specific basic protein) DM-20 of the PLP family of proteins, CNPase, and other enzymes14.

Proteinases Proteolytic enzymes which are found in the liver and other organs are also present in brain15. Activities of brain proteinases were first demonstrated in the 1930s by Krebs16 and Kerekes et al.17 and in the 1940s by Kies and Schwimmer18. Acid proteinase was the first such activity found in brain, and then other lysosomal proteinases, cathepsins A, B, and D were isolated and purified from brain19,20. These cathepsins degrade MBP and NFPs. Cathepsin D specifically hydrolyzes the PhePhe linkage in MBP14. Acid proteinase activity has also been found in neurons21. By contrast to acid proteinases, the non-lysosomal neutral proteinases are unstable and labile, difficult to purify and assay. However, with development of purification and assay methods, Ansell and Richter19 and Marks and Lajtha20 were able to determine neutral proteinase activity in brain and spinal cord. Subsequently, several neutral proteinases have been identified and purified from spinal cord and brain, including a very high molecular weight roteosome (750kD) [multicatalytic proteinase complex (MPC)], calpain (Ca2+-activated neutral proteinase)22, metalloproteinase23 and matrix metalloproteinases24,25. MPC consists of 7- 13 subunits26,27 while calpain exists as two isoforms, µcalpain and mcalpain, each with two subunits, an 80kD catalytic and 30kD regulatory subunit. Both isoforms are absolutely dependent on calcium for activity requiring µM and mM calcium concentrations, respectively. Calpain and the metalloproteinases are also associated with myelin22,23 and degrade MBP as substrate and with enzyme activity being increased in pathophysiology in trauma and diseases6,28,29. For detailed information on CNS proteinases readers are directed to relevant recent sources4,30.

MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN SPINAL CORD INJURY Spinal cord injury, depending upon the severity, disrupts the functional axonmyelin structural unit and this leads to paralysis and other neurological deficits. The primary injury to the spinal cord disrupts blood vessels and initiates many devastating secondary pathoph siological alterations in the lesion which lead to cell death and tissue destruction8,31,23. There are extensive morphological changes, similar to those found in brain trauma, including progressive granular degeneration of axons, accumulation of hydroxyapatite-calcium crystals, vesicular degeneration of myelin, and phagocytosis by infiltrating macrophages8,31-37. Early studies of SCI lesions at the light microscopic level showed necrosis of gray and central white matter with damage to surrounding areas of white matter. Features of the lesions include edema, inflammation, and hemorrhage with infiltration of inflammatory cells (neutrophils) at 6 to 8 hours after trauma. The extent of damage to the cord depends upon the severity of injury as well as the time following trauma8,31,32,38 . Electron microscopic studies in experimental SCI have revealed progressive ultrastructural

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changes in the lesion. As early as 15 minutes after injury axons are undergoing granular degeneration and there is loosening of the myelin lamellae concomitant with significant axonal protein degradation in the lesion39. At longer intervals there is granular degeneration followed by vesiculation of the myelin sheath and inflammatory cell-mediated phagocytosis8,31,32. A most striking change in the SCI lesion is the accumulation of calcium crystals in axons as well as mitochondrial calcification8,31. This important finding first suggested that this increased calcium is responsible for the activation of calcium-activated lipases and proteinase8,39. Studies in numerous laboratories have correlated the ultrastructural changes observed in the SCI lesion at intervals following the injury with biochemical alterations. Changes have been described in lipids, cytoskeletal and myelin proteins, and lipolytic and proteolytic enzymes in the lesion and compared to sham (uninjured) controls at different times following the injury8,31,32,38,40-45. An early but significant loss of axonal and myelin proteins, particularly neurofilament proteins (NFPs), microtubule associated protein (MAP2) and myelin basic protein (MBP) is evident at 15 to 30 minutes and progresses with time following trauma8,34,39,46,47. All three major classes of NFPs (200kD, 150kD, 68kD) are progressively degraded and they are completely broken down in the SCI lesion at 6 to 72 hours after trauma. Among the myelin proteins, MBP is more susceptible to degradation in the lesion than proteolipid protein (PLP), the major protein of CNS myelin. A substantial (3040%) loss of MBP is evident at 1 hour after trauma while the loss of this protein at 24 hours following injury amounted to 90% and more. In comparison to MBP, only 40% of PLP is lost in the lesion at 24 hours after trauma. The myelin associated glycoprotein (MAG) also has been found to be extensively degraded in the lesioned cord compared to control. This time-dependent degradation of both cytoskeletal and myelin proteins in the lesion correlates very well with the ultrastructural degeneration of the axon-myelin structural unit39,48. The loss of these proteins in the lesion indicates the crucial involvement of proteolytic enzymes in the demise of cells and destruction of lesioned cord following injury. The ultrastructural or morphological changes that occur in brain following TBI and ischemia are not discussed in detail in this chapter. Nonetheless, like SCI, damage to cells and myelinated axons and axon shearing has been reported by many laboratories49-52. Diffuse axonal injury is a common feature in white matter of brain and optic nerve due to TBI, anoxia and ischemia. These changes also correlate with alterations in proteins and roteinases. For further information, consult articles cited above or related reviews53-55.

PROTEINASES IN CNS TRAUMA In the mid 1970s and early 1980s the activities of various hydrolytic enzymes were determined in the lesions of spinal cord following injury. Changes in the activities of several other hydrolases, including N+-K+-ATPases, acetylcholinesterase, cytochrome reductase, and lysosomal hydrolases (e.g., acid phosphatase) have also been determined in the lesion. The activity of these enzymes 44 were altered in the lesion when compared to control . However, the increase in the activities of lysosomal enzymes in the lesion appears to contribute to tissue degeneration only at longer times after injury and not in the secondary injury cascade of the first day. Similar hydrolytic enzymes, including phospholipases and others were studied in TBI and their activities were also found to be altered in brain following injury44,56.

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Detailed studies on the kind of proteolytic enzymes that are involved in tissue destruction in CNS trauma were first examined in the lesion of experimental SCI in the early 1980s. Cytoskeletal and myelin proteins were found to be progressively degraded with time following injury in SCI lesions, which suggested that proteolytic enzymes may be one of the mediators of secondary injury in tissue destruction. Since various proteinases may be involved in this process, the activities of both extralysosomal neutral and lysosomal acidic proteinases were determined. Soluble fractions from lesioned and control spinal cords (autologous and sham) were isolated and incubated at different pHs (cathepsin D-like activity, pH 3.0; cathepsin B-like, pH 6.0; and uncharacterized neutral proteinase, pH 7.4) using purified MBP as a substrate. Using an indirect assay method, the activities of different enzymes were determined by assessing the extent of loss of MBP using SDS-PAGE. The degradation of MBP by neutral proteinases in the lesion was progressive, usually with concomitant production of MBP-breakdown products43. In comparison to neutral proteinase, MBP breakdown by cathepsin B and cathepsin D-like proteases was negligible. In addition, the specific inhibitor of cathepsin D, pepstatin, did not inhibit MBP degradation while this breakdown of MBP was significantly prevented by leupeptin, a neutral proteinase and cathepsin B inhibitor. Leupeptin, on the other hand, did not prevent MBP breakdown by cathepsin B (at pH 6.0). These studies revealed that in the lesion, the neutral proteinase activity was much greater than that of the cathepsins and was primarily responsible for MBP degradation. In addition, neutral proteinase activity progressively increased with time following injury leading to a 300% increase in activity in the lesion at longer intervals (24 hours) following injury compared to autologous and sham controls43. In CNS injury there is infiltration of inflammatory/immune cells which secrete many proteinases, including matrix metalloproteinase (MMPs). Activities and expression of these enzymes increases in a number of CNS disorders, including multiple sclerosis (MS), Alzheimer’s disease, ischemia, and glioblastoma24,57-61. Since inflammation is common to CNS injury and MMPs are associated with inflammation, activities of these enzymes were examined in SCI and TBI. Activity of gelatinase B (MMP-9), an enzyme involved in the opening of the blood-brain barrier (BBB)62, was increased in the SCI lesion as well as in experimental brain trauma. Increased hippocampal expression of MMPs, including MMP-3, MMP-9, and gelatinase B are found in percussion TBI during functional recovery58,59. Upregulation in the expression of MMP genes has been reported in cerebral ischemia and intracerebral hemorrhage and stroke63,64. While MMPs are being increasingly implicated in the pathogenesis of several CNS diseases, MMPs also have been shown to facilitate recovery from CNS injury65. Readers can pursue the role of MMPs in different neurodegenerative disorders in the recent literature. As in the studies on cathepsins carried out in spinal cord injury, little has been reported on lysosomal proteinases in TBI. NFPs are known to be degraded by both calpain and cathepsins suggesting that the loss of these proteins in TBI may not be due to calpain alone. Other proteinases such as proteosome (multicatalytic proteinase complex) may also be involved.

CALPAIN HYPOTHESIS The calpain hypothesis of tissue destruction in CNS trauma was first developed as a result of studies conducted on spinal cord following injury. The findings of substantially greater neutral proteinase than acid proteinase activity in the lesion suggested a pivotal role for neutral proteinases (present in endogenous glial cells) in

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mediating tissue destruction in SCI before the infiltration of neutrophils/macrophages occurs41,42. The appearance of macrophages/inflammatory cells in the lesion occurs about 6 to 8 hours after injury, suggesting that cell death and tissue destruction occumng soon after injury is carried out by neutral proteinases while acid proteinases emanating from infiltrating lymphocytes contribute to this destructive process at later times. Taken together, the loss of cytoskeletal (NFPs) and myelin proteins (MBP), substantially greater neutral proteinase activity, increased calcium levels, granular degeneration of axon, vesiculation of myelin, and findings of similar changes in the CaC12-induced myelopathy model led investigators to implicate a role for calpain in tissue destruction associated with SCI33,39,42,44,66,67. This hypothesis derived from a host of studies was further supported by the demonstration that axonal (e.g., NFP, MAP2) and myelin proteins (MBP, MAG) were excellent calpain substrates. A direct role for calpain in tissue destruction was subsequently demonstrated by findings of increased calpain activity and translational expression by immunocytochemical technique in the lesion following trauma47,68-71. Our current studies further define a crucial role for calpain in cell injury/death not only in the lesion, but also in areas remote from the lesion epicenter. As in the SCI lesion, calpain activity and expression are progressively increased in the penumbra following trauma and the increase is greater caudal than rostral to the impact site72. Findings of increased intracellular calcium levels in regions of brain and spinal cord following trauma, axonal injury, and ischemia also implicated its role in calpain activation73-75. Subsequent studies demonstrated extensive loss of cytoskeletal proteins in TBI and ischemia3,6. These findings also implicated the involvement of a calpain-like protease in tissue damage related to brain trauma and ischemia5,76,77.

CHARACTERISTICS OF CALPAIN Calcium activated neutral proteinases (calpain), also known as cysteine endopeptidases (EC 3.4.22.17), are subclassified as ubiquitous and tissue specific. Ubiquitous calpain exists as microcalpain (µcalpain ) and millicalpain (mcalpain) isoforms requiring µM and mM calcium concentratiohs for activation, respectively. Both calpain isoforms consists of an 80kD catalytic and a 30kD regulatory subunit. At least 95% of calpain in the central nervous system (CNS) is present as the mcalpain isoform. µCalpain is largely associated with neurons while mcalpain is predominantly glial78,79. The mcalpain isoform in the CNS is present in cytosol as well as associated with myelin22,80,81 and the oligodendroglial cell body and processes82. Calpains are inactive in the cytosol and µcalpain is activated by autolysis of the 80kD catalytic subunit into the 76kD form and the 30kD regulatory subunit into the 19kD form in the presence of increased Ca2+ concentrations11,83. Both the µ and m calpain interact with membrane lipids (e.g., phospholipids, glycolipids) to increase the Ca2+ sensitivity for their activation84-89. Both µ and mcalpains are associated with the endogenous inhibitor, calpastatin, in the cytosol. Calpastatin is also degraded by activated calpain when the calpain:calpastatin ratio is increased. Calpain digests many proteins, including cytoskeletal (NFP), myelin (MBP, MAG), myofibrillar, enzymatic (phospholipase C, protein kinase C), histones, transcriptional factors (Fos, Jun), hormones, and others. In spite of its involvement in the degradation of many cellular proteins and processing of enzymes and hormones, the physiological function of calpain is unclear. Nevertheless, a large number of studies recently have implicated calpain as a primary mediator in the pathophysiology of many diseases. Its role is delineated in demyelinating diseases (e.g., MS) and degenerating diseases such as Parkinson’s disease, cerebral ischemia,

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Alzheimer's disease, and in rodent cataract formation 83,90-94. These findings also indicate an important role for calpain inhibitors as therapeutic agents for the treatment of a variety of diseases. To this end, the recent determination of the three dimensional structure of calpain will facilitate the development of specific inhibitors as agents for treatment95,96. In order to obtain a more detailed understanding of the biochemical and molecular pro erties of calpain, readers can consult several excellent reviews on related areas27,97,98.

ROLE OF CALCIUM IN SPINAL CORD INJURY One of the most important findings in SCI research was the demonstration of hydroxyapatite crystallites in both the axoplasm and mitochondria in the cord lesion as early as 15 to 30 minutes following injury. There was mitochondrial calcification and granular chan es in axonal filament in the SCI lesion at longer intervals following trauma8,38,99. The level of total calcium was determined in the lesion and found to be increased progressively following injury33,35,36. These findings suggested the involvement of increased calcium in tissue destruction in SCI. Subsequent studies provided strong support for a direct role for calcium in the mediation of tissue destruction in SCI with a CaC12-induced myelopathy model. This model, like SCI, showed granular degeneration of axons and vesiculation of the myelin sheath concomitant with the accumulation of calcium h droxyapatite crystallites66 and degradation of cytoskeletal and myelin proteins33,39,67. In contrast, other divalent or monovalent cations (e.g., Mg2+, K+, Na+, Cl-) did not induce any significant morphological or biochemical changes66. In both SCI and the CaC12-induced myelopathy models, the observed progressive granular degeneration of axon and vesiculation of myelin most likely resulted from the loss of structural proteins such as NFPs, MAPs, MBP, and PLP. Since these proteins are also calpain substrates, it is likely that increased calpain activation in SCI may be associated with higher levels of calcium in the lesion as well as in the penumbra to the lesion. Thus, elevated intracellular calcium levels have been implicated in cell death and axonal degeneration not only in SCI, but also in axonal injury in optic nerve and axotomyinduced axonal degeneration75,100-105. Morphological and biochemical changes similar to those in SCI were found in organotypic embryonic mouse spinal cord cultures as well as in spinal cord segments incubated with calcium106. The hypothesis that an increased intracellular calcium concentration causes cell death has been demonstrated in vivo in the CNS following injury and other systems, including ischemia57,107, muscular dystrophy108,109, toxic liver injury110, glutamate neurotoxicity, cataract formation111,112, and optic nerve degeneration113. Calcium-mediated cell injury/toxicity was partially inhibited by calcium channel blockers and calpain inhibitors114-117.

ROLE OF CALCIUM IN BRAIN INJURY Altered concentrations of ions in brain injury has been found to affect brain functions. Increased release of K+ may interfere with the membrane transport system, metabolism, and synaptic functions; disrupt energy homeostasis; deprive neurons of their oxygen supply; and lead to neuronal damage after TBI118-122. Decrease in the Mg2+ concentration following TBI not only may impair energy metabolism, glucose utilization and oxidative phosphorylation, but is also known to

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regulate transport and accumulation of Ca2+ in cells. Therefore, it is possible that alterations in Mg2+ concentrations in TBI may cause Ca2+-mediated neuronal loss in brain following injury 123. This is supported by reports of elevated intracellular Ca2+ levels in brain regions after experimental TBI and ischemia73,74,124-126 . In addition, the decrease in extracellular Ca2+ levels associated with profound functional disabilities following cortical compression/contusion injury in rats was not affected by pretreatment with glutamate receptor antagonists74,127. A recent study has suggested that excessive intracellular Ca2+ resulting from TBI in rats was adsorbed on mitochondrial membranes causing inhibition of the electron transport chain and energy metabolism128. It could therefore be concluded that in the pathophysiology of degenerative diseases, the influx and/or release of calcium from intracellular storage sites may cause cell death, involving many complex pathways, particularly in the secondary injury process following TBI and SCI. One of the pathways in which Ca2+ is involved is the activation of enzymes such as phospholipases and proteinases. An increased Ca2+ level also activates calpain, a Ca2+-dependent cysteine protease which mediates cytoskeletal protein degradation and neurodegeneration in human and experimental animal models of ischemia, SCI, and TBI3,5,,76,129-131. Diffuse axonal injury in white matter also has been implicated due to loss of cytoskeletal proteins and may be mediated by calpain132-134. In fact, the activation of calpain in the pathophysiology of CNS injury, ischemia, and other neurodegenerative diseases has now been firmly establised135-139.

CALPAIN ACTIVITY IN SPINAL CORD INJURY Since small samples of lesioned spinal cord are problematic, commonly used radioactive methods utilizing labeled substrates (e.g., casein) for assaying calpain activity are not reliable. These methods also require partial purification of the enzyme and removal of the endogenous inhibitor, calpastatin. In light of this problem, indirect methods for determination of calpain activity in limited amounts of tissue samples have been used39,47,70,76,77,140 . Several approaches have been taken to evaluate in vivo calpain activity in the lesion of spinal cord including (1) the degradation of endogenous proteins which are known calpain substrates; (2) the production of calpain-cleaved spectrin fragments; and (3) the formation or appearance of the active form (76kD) of µcalpain. Many endogenous proteins, including NFPs, MAP2, MBP, MAG, and spectrin, have now been identified as excellent substrates of calpain22,46,118,141-144. Some of these proteins are also degraded by other proteinases such as the cathepsins, matrix metalloproteinases, and caspase-343,145,146 whose cleavage sites and degradation products may be different as well as identical in some cases. For example, calpain and caspase-3 produce fragments of different sizes from spectrin. However, both in vivo and in vitro studies suggest a crucial role for calpain in neurofilament and spectrin degradation since the loss of 68kD NFP and spectrin is prevented (6 months), stefin B-deficient mice developed ataxia and myoclonic seizures, similar to symptoms seen in the human disease. In addition, a large number of cerebellar granule cells are lost in the mutant mice by apoptosis, indicating that stefin B has a role in preventing cerebellar apoptosis by an as yet unknown mechanism.

Hereditary Cystatin C Amyloid Angiopathy (HCCAA) HCCAA is an autosomal dominant disorder characterized by deposition of amyloid primarily in the central nervous system, although amyloid deposits have also been detected in other organs. Clinically, this deposition results in brain haemorrhages in young individuals (25-35 years old), which are the major cause of death within couple of years from the first stroke104,105. The main component of the amyloid deposits was identified by protein sequencing as a variant of cystatin C. The amyloid cystatin C differs from normal cystatin C by an amino acid substitution of Leu68Gln resultin from a single T A mutation in the codon for Leu68 in exon 2 of the cystatin C gene106,107. As a consequence of this mutation the Alu I restriction site found in the normal gene is lost, resulting in an extra ~630 base pair Alu I fragment, which also enables diagnosis of the disease. Furthermore, the concentration of cystatin C in the cerebrospinal fluid of patients with HCCAA is ~3-fold decreased compared to healthy individuals108, suggesting that this could be another causative event. In vitro studies indicated that the Leu68Gln cystatin C variant is substantially less stable than the wild type protein and more prone to dimerization38,109. Moreover, increased accumulation was observed in NIH/3T3 cells expressing Leu68Gln cystatin C compared with same cells expressing wild-type cystatin C. The intracellularly accumulating mutant cystatin C was located mainly in the endoplasmic reticulum as insoluble protein suggesting that this is an important event in the molecular pathophysiology of HCCAA110.

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81. E. Bednarski, J.C. Lauterborn, C.M. Gall, and G. Lynch, Lysosomal dysfunction reduces brain-derived neurotrophic factor expression, Exp. Neurol. 150:128 (1998). 82. E. Bednarski and G. Lynch, Selective suppression of cathepsin L results from elevations in lysosomal pH and is followed by proteolysis of tau protein, Neuroreporf 9:2089 (1998). 83. T. Yamashima, Y. Kohda, K. Tsuchiya, T. Ueno, J. Yamashita, T. Yoshioka, and E. Kominami, Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis', Eur. J. Neurosci. 10:1723 (1998). 84. LE. Hill, E. Preston, R. Monette, and J.P. MacManus, A comparison of cathepsin B processing and distribution during neuronal death in rats following global ischemia or decapitation necrosis, Brain Res. 751:206 (1997). 85. K. Tsuchiya, Y. Kohda, M. Yoshida, L. Zhao, T. Ueno, J. Yamashita, T. Yoshioka, E. Kominami, and T. Yamashima, Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors, Exp. Neurol. 155:187 (1998). 86. T. Nitatori, N. Sato, S. Waguri, Y. Karasawa, H. Araki, K. Shibanai, E. Kominami and Y. Uchiyama Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis, J Neurosci. 15:1001 (1995). 87. L.A. Pennacchio, D.M. Bouley, K.M. Higgins, M.P. Scott, J.L. Noebels, and R.M. Myers, Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice, Nature Genet. 20:25 1 (1998). 88. G.J. Pilkington, Tumor cell migration in the central nervous system, Brain Pathol. 4:157 (1994). 89. H. Graeff, N. Harbeck, L. Pache, 0. Wilhem, F. Janicke, and M. Schmitt, Prognostic impact and clinical relevance of tumor associated proteases in breast cancer, Fibrinolysis 6:45 (1992). 90. D. McCormick, Secretion of cathepsin B by human gliomas in vitro, Neuropathol. Appl. Neurobiol. 19:146 (1993). 91. M. Sivaparvathi, R. Sawaya, S.W. Wang, A. Rayford, M. Yamamoto, L.A. Liotta, G.L. Nicolson, and J.S. Rao, Overexpression and localization of cathepsin B during the progression of human gliomas, Clin. Exp. Metastasis 13:49 (1995). 92. L.L. Demchik, M. Sameni, K. Nelson, T. Mikkelson, and B.F. Sloane, Cathepsin B and glioma invasion, Int. J. Dev. Neurosci. 17:483 (1999). 93. T. Mikkelsen, P.S. Yan, K.L. Ho, M. Sameni, B.F. Sloane, and M.L. Rosenblum, Immunolocalization of cathepsin B in human glioma: implications for tumor invasion and angiogenesis, J. Neurosurg. 83:285 (1995). 94. M. Sivaparvathi, M. Yamamoto, G.L. Nicolson, Z.L. Gokaslan, G.N. Fuller, L.A. Liotta, R. Sawaya, J.S. Rao, Expression and immunohistochemical localization of cathepsin L during the progression of human gliomas, Clin. Exp. Metastasis 14:27 (1996). 95. M. Sivaparvathi, R. Sawaya, Z.L. Gokaslan, K.S. Chintala, and J.S. Rao, Expression and the role of cathepsin H in human glioma progression and invasion, Cancer Left. 104:121 (1996). 96. J. Kos and T.T. Lah, Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer, Oncology Reports 5:1349 (1998). 97. T. Strojnik, J. Kos, B. Zidanek, R. Golouh, and T. Lah, Cathepsin B immunohistochemical staining in tumor and endothelial cells is a new prognostic factor for survival in patients with brain tumors, Clin. Cancer Res. 5:559 (1999). 98. J.M. Serratosa, R.M. Gardiner, A.E. Lehesjoki, and L. Pennacchio, The molecular genetic bases of the progressive myoclonus epilepsies, Adv. Neurol. 79:383 (1999). 99. L.A. Pennacchio, A.E. Lehesjoki, N.E. Stone, V.L. Willour, K. Virtaneva, J. Miao, E. D'Amato, L. Ramirez, M. Faham, M. Koskiniemi, J.A. Warrington, R. Norio, A. de la Chapelle, D.R. Cox, and R.M. Myers, Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1), Science 271:1731 (1996). 100. M.D. Lalioti, H.S. Scott, C. Buresi, C. Rossier, A. Bottani, M.A. Morris, A. Malafosse, and S.E. Antonarakis, Dodecamer repeat expansion in cystatin B gene in progressive myoclonus epilepsy, Nature 386:847 (1997). 101. R.G. Lafreniere, D.L. Rochefort, N. Chretien, J.M. Rommens, R. Kalviainen, U. Nousiainen, G. Patry, K. Farrell, H. Soderfeld, B.R. Hale, O.H. Cossio, T. Sorosen, M.A. Pouliot, T. Kmiec, M.R. Pranzatelli, F. Andermann, E. Andermann, and G. Rouleau, Unstable insertion in the 5' flanking region of the cystatin B gene is the most common mutation in progressive myoclonus epilepsy type 1, EPM1, Nat. Genet. 15:298 (1997). 102. M. Lalioti, M. Mirotsou, C. Buresi, M.C. Peitsch, C. Rossier, M. Baldy-Moulinier, A. Bottani, A. Malafosse, and S.E. Antonarakis, Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPMl), Am. J. Hum. Genet. 60:342 (1997).

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103. M. Lalioti, H.S. Scott, P. Genton, D. Grid, R. Ouazzani, A. M’Rabet, S. Ibrahim, R. Gouider, C. Dravet, T. Chkili, A. Bottani, C. Buresi, A. Malafosse, and S.E. Antonarakis, A PCR amplification method reveals instability of the dodecamer repeat in progressive myoclonus epilepsy (EPM1) and no correlation between the size ofthe repeat and age at onset, Am. J. Hum. Genet. 62:842 (1998). 104. G. Gudmundsson, J. Hallgrimsson, T.A. Jonasson, and O. Bjarnson, Hereditary cerebral haemorrhage with amyloidosis, Brain 95:387 (1972). 105. O. Jensson, G. Gudmundsson, A. Amason, H. Blöndal, I. Petursdottir, L. Thorsteinsson, A. Grubb, H. Löfberg, D. Cohen, and B. Frangione, Hereditary cystatin C (gamma-trace) amyloid angiopathy of the CNS causing cerebral hemorrhage, Acta Neurol. Scand. 76:102 (1987). 106. J. Ghiso, O. Jensson, and B. Frangione, Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of gamma-trace basic protein (cystatin C), Proc. Acad. Sci. USA 83:2974 (1986). 107. A. Palsdottir, M. Abrahamson, L. Thorsteinsson, A. Amason, I. Olafsson, A. Grubb, and O. Jensson, Mutation in cystatin C gene causes hereditary brain haemorrhage, Lancet 861 1:603 (1988). 108. A. Grubb, O. Jensson, G. Gudmundsson, A. Amason, H. Lofberg, and J. Malm, Abnormal metabolism of gamma-trace alkaline microprotein. The basic defect in hereditary cerebral hemorrhage with amyloidosis, N. Engl. J. Med. 311:1547 (1984). 109. I. Ekiel and M. Abrahamson, Folding-related dimerization of human cystatin C, J. Biol. Chem. 271:1314 (1996). 110. M. Bjarnadottir, B.S. Wulff, M. Sameni, B.F. Sloane, D. Keppler, A. Grubb, M. Abrahamson, Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells, Mol. Pathol. 51:317 (1998). 111. G. Guncar, G. Pungercic, I. Klemencic, V. Turk, and D. Turk, Crystal structure of MHC class IIassociated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S, EMBO J. 18:793 (1999).

PROTEASES AND THEIR INHIBITORS IN GLIOMAS

Peter A. Forsyth1, Dylan R. Edwards2, Marc A. LaFleur2 and V. W. Yong1 Oncology & Clinical Neurosciences, University of Calgary and Department of Medicine, Tom Baker Cancer Centre Calgary, Alberta T2N 1N4, Canada 2 School of Biological Sciences, University of East Anglia Norwich, Norfolk NR4 7TJ, England 1

INTRODUCTION Extracellular proteases encompass several large families of molecules with a broad range of functions. These enzymes have been studied largely in the traditional context of their ability to degrade or cleave certain specific substrates, but it is becoming clear that they have important functions in diverse cellular activities such as angiogenesis, apoptosis and cell signaling that are not completely understood. Understanding the multifunctional nature of these molecules and their ability to affect a number of cellular control systems is very important for several reasons. First, knowledge of protease action will contribute to the unraveling of pathobiological mechanisms underlying a number of diseases. Second, these are important therapeutic targets and several clinical trials of protease inhibitors are already in progress. Third, since proteases are involved in a number of cellular processes, some beneficial and some deleterious, they must be clearly understood so that their manipulation is as selective as possible and the full therapeutic benefit maximized. In this chapter we outline the role of proteases and their inhibitors in gliomas. We highlight matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) along with the serine proteases and cathepsins since these are the most extensively studied proteases in gliomas. In addition, their potential as novel glioma treatments is the most advanced with MMPs; two clinical trials of synthetic MMP inhibitors in glioma patients are currently underway. Little is known about the large number of other proteases that have not been studied in gliomas.

GLIOMAS & THE BRAIN'S EXTRACELLULAR MATRIX Primary brain tumors are a heterogeneous group of tumors which arise from the tissues of the brain (glia, neurons, ependyma, choroid plexus etc.) or its coverings (the meninges). The two most common primary brain tumors are meningiomas and gliomas. The majority of meningiomas are cured by surgery and further treatment is generally not needed. In contrast, malignant gliomas are uniformly fatal in spite of aggressive surgical resection, radiotherapy and chemotherapy. Since gliomas are the major clinical challenge in neurooncology they are the focus of this review. Gliomas affect 25,000 Americans and Canadians a year and malignant gliomas (MGs) are the most common type. These are fatal tumors and the median survival of these MG patients is only about 12 months; only 2% of patients with the most malignant type Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenurn Publishers, New York, 2001.

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survive > 3 years1-3. Gliomas are important for three reasons: 1) they are the most common solid tumors in children, 2) they are becoming more common, and 3) their prognosis has not changed significantly in the past 20 years. Clearly, better treatments are desperately needed and inhibition of proteases may be an important novel glioma treatment when used in conjunction with conventional treatments. Proteases have been implicated in the two cardinal features of malignant gliomas: their marked invasiveness and vascularity. Gliomas extend tendrils of tumor cells into the surrounding brain. Isolated glioma cells migrate and invade several centimeters beyond the main tumor mass and render these surgically incurable. Similarly the most malignant of these tumors, glioblastoma multiforme (GBM) are among the most vascular tumors known. Both their invasiveness and vascularity remain major barriers to their effective treatment and are not targeted by our available treatments. Glioma invasion occurs predominantly along myelinated white matter tracts and blood vessels4. It occurs, with little or no destruction of the surrounding neuronal structures, at least when only a few cells are involved. As a consequence of its predilection for white matter tracts distant spread occurs along the optic radiations, corpus collosum or anterior commissure. Generally invasion is conceived of as a three step process: 1) receptor mediated adhesion of tumor cells to matrix proteins in the ECM, 2) degradation of the ECM by proteases creating a space and environment for the glioma cells to move into, and 3) active movement that requires receptor turnover, membrane synthesis and rearrangement of cytoskeletal elements. The second process is the focus of this review and can not be appreciated without some understanding of the brain's ECM4. There is very little ECM in the brain in contrast to other organs. The brain's ECM is mostly composed of proteoglycans and glycoproteins. The ECM's major components are collagen (particularly type IV), chondroitin sulphate, laminin, elastin, fibronectin, vitronectin, entactin, tenascin, heparan sulfate proteoglycan and hyaluronic acid. Classical ECM components (laminin, collagen type IV, fibronectin and vitronectin) are limited to vascular basement membranes (which glioma cells invade along but do not usually invade through and the glia limitans externa; the latter is another true basement membrane that covers the cortical surface. All of these aforementioned ECM components are considered as ligands that participate in glioma adhesion, ligand-receptor and signal transduction-messenger interactions that allow tumor invasion to occur. Many of these ECM components are also synthesized and deposited by glioma cells themselves creating a microenvironment that presumably facilitates their invasiveness, survival or proliferation. ECM macromolecules thought to be secreted by gliomas include tenascin, vitronectin, collagen types I, III, IV and VI, fibronectin, laminin, hyaluronan, chondroitin sulfate and heparin sulfate proteoglycans. Several families of cell surface receptors (e.g. integrins) have been identified that interact with specific domains of ECM proteins. These interactions trigger diverse cellular events such as cell attachment, adhesion, changes in cell morphology and activation of second messenger pathways. Several proteases have been implicated in the pathophysiology of gliomas, including the matrix metalloproteinases (MMPs), serine proteases (urokinase and tissue plasminogen activators; uPA and tPA), cysteine proteases (cathepsin B and S) and aspartic proteases (cathepsin D). In addition glycosidases are also important factors, but they will not be considered in detail here.

MATRIX METALLOPROTEINASES (MMPs) AND TISSUE INHIBITORS OF MMPs (TIMPs) Matrix Metalloproteinases (MMPs) The ability to breach tissue boundaries by active destruction of extracellular matrix (ECM) is the common denominator of tumor invasion, angiogenesis and metastasis. Tumor cells use a variety of degradative enzymes to destroy basement membranes and interstitial stroma5-8 endothelial cells may use the same machinery to form new blood vessels. The MMPs are the principal secreted proteinases required for ECM degradation in a variety of physiological and pathological tissue remodelling processes6-10. These are a family of zinc binding, calcium dependent endopeptidases. Twenty-two have been described11,12 (TABLE 1) and are subdivided principally by structure or by substrate preference into

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collagenases, gelatinases, stromelysins, and the membrane-type MMPs (MT-MMPs). There is a wealth of evidence for an association between deregulated production/activation of MMPs and aggressive behaviour in human cancers13-16; particularly for MMP-2 (gelatinaseA; 72 kDa) and MMP-9 (gelatinase-B; 92 kDa)5,6,17. A schema for the relationship between MMPs, tumor cells, the surrounding stroma, other proteases and the ECM is shown in FIGURE 1. Table 1: The family of vertebrate matrix metalloproteinases (MMPs) Group

Members

MMP numbers*

Collagenases

Interstitial collagenase (fibroblast-type)

MMP-I

Fibrillar collagens

Neutrophil collagenase Collagenase-3 Collagenase-4§

MMP-8 MMP-1 3 MMP-I 8

Fibrillar collagens Fibrillar collagens Unknown

Stromelysin-1

MMP-3

Stromelysin-2

MMP-IO

Laminin, non-fibrillar collagens, fibronectin Laminin, non-fibrillar collagens, fibronectin

Stromelysins

Main substrate**

Gelatinases

Gelatinase B MMP-2 (72 kDa type IV collagenase) MMP-9 Gelatinase B (92 kDa type IV collagenase)

Gelatin, Types IV and V collagens, fibronectin Gelatin, Types IV and V collagens, fibronectin

Membrane-type MMPs

MTl-MMP

MMP- 14

MT2-MMP

MMP- 15

MT3-MMP

MMP- 16

MT4-MMP MT5-MMP MT6-MMP

MMP- 17 MMP-24 MMP-25

Pro-MMP-2, collagens, gelatin Pro-MMP-2, collagens, gelatin Pro-MMP-2, collagens, gelatin Fibrinogen, pro-TNFa Pro-MMP-2 Pro-MMP-2

Matrilysin

MMP-7

Stromelysin-3

MMP-11

Metalloelastase No trivial name

MMP-12 MMP-1 9

Enamelysin Xenopus MMP CMMP Femalysin

MMP-20 MMP-2 1 MMP-21/22 MMP-23

Others

Laminin, non-fibrillar collagens, fibronectin D 1 proteinase inhibitor (serpin) Elastin Laminin, non-fibrillar collagens, fibronectin Not known Not known Not known

*MMP-4, -5 and -6 were found to be identical to other MMP family members and these designations are no longer in use. ** Although the principal substrates are listed, there is a great deal of substrate overlap. MMP = matrix metalloproteinase; MT-MMP = membrane-type matrix metalloproteinase; ? = numerical designation not yet assigned/clear. § Xenopus gene only at present.

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FIGURE 1. Representation of the relationship between tumor cells, the extracellular environment, various proteases and signalling molecules. The MMP/TIMP and uPA/uPAR systems are highlighted. The relationships are complex and the regulation of these proteases is interdependent. For example proteolysis of plasminogen (Pl’ogen) to plasmin by uPA when bound to uPAR on the cell surface also activates several MMPs which in turn contribute to proteolysis of the ECM. Furthermore, a number of membrane associated proteins, such as the MT-MMPs and uPAR activate proteases and serve to localize proteolysis to the invading edge of the tumor cell. The interaction of integrins, ECM molecules (e.g. vitronectin) with both MMPs and serine proteases is complex and further serves to localise regulation of proteolysis to the cell surface. Finally, once activated proteases influence tumor growth by liberating growth factors which in turn produce mitogenic signals through their receptors. Note that this figure applies to systemic cancers where, somewhat surprisingly, the surrounding stroma make most of the proteases. In gliomas, the tumor cells themselves may be the source of most of the MMPs. For example, IGFs are bound to IGFBP and not “free” to interact with their receptors but are liberated from IGFBP by MMPs. Abbreviations used: IGF = insulin-like growth factor; IGFBP = IGF binding protein; IGF1-R = IGF 1 receptor; M6P-R = nannose 6-phosphate receptor, also called IGF2-receptor; MMP = matrix metalloproteinase; MTMMP = membrane-type MMP; PAI-1 = plasminogen activator inhibitor type 1; Pl’ogen = plasminogen; TCF = T cell factor (also known as LeF), TIMP = tissue inhibitor of MMP; uPA = urokinase plasminogen activator; uPAR = uPA receptor.

All MMPs contain a signal peptide, a propeptide region, an n-terminal catalytic domain and a C-terminal hemopexin-like domain (with the exception of MMP-7 which lacks the C-terminus region). Intervening sequences further characterise certain MMPs such as fibronectin repeats in the gelatinases and the transmembrane region in the MT-MMPs. The propeptide is important for the control of MMP latency and activation. In the inactive zymogen, a cysteine residue within the Pro-Arg-Cys-Gly-X-Pro motif, the propeptide coordinates with the zinc ion in the active site disabling its proteolytic activity18. Activation of the MMPs requires proteolysis of the propeptide which exposes the active site. The catalytic centre consists of 3 conserved His residues and co-ordinate Zn2+ and is responsible for substrate and autolytic cleavage19. The C-terminal domain is similar in sequence to members of the hemopexin family. This domain appears to mediate substrate binding although both

Proteases and their Inhibitors in Gliomas

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N-terminal and C-terminal domains are required for proper binding and cleavage. The Nterminal and C-terminal domains are linked by a flexible linker peptide20. Both MMP-2 and -9 have an additional fibronectin-like domain within the catalytic domain, consisting of 3 tandem repeats of a fibronectin type II-like module. This domain binds to denatured collagen type IV and V, elastin, and denatured and native type I collagen21,22. MMP-9 also has a collagen domain with sequence homology to the D2 chain of type IV collagen which is also thought to be involved in substrate binding23. The activities are tightly controlled at six levels: 1) gene transcription, 2) zymogen activation by proteolysis, 3) inhibition of active forms by the TIMPs, 4) mRNA stability, 5) translational control, and, 6) storage in secretory granules (as for MMP-8)6,23-26. The latter three of these are the least characterised mechanisms of MMP control. The MMPs are functionally related but differ in their expression and association with TIMPs. For example MMP-2 is widely expressed constitutively27, whereas MMP-9 has restricted expression and is inducible27-31. Gene transcription occurs in response to a variety of factors such as cytokines, angiogenic factors and hormones. MMP genes targeted for induction contain unique DNA sequences in their promoter regions which bind specific transcription factors and increase transcription. Several MMP genes (such as MMP-1, -3, -7, -9,-11,-13) contain an AP-1 (phorbol ester responsive element) site that can be induced by the fos/jun transcription factors in response to stimuli such as EGF. In contrast, the promoter region of MMP-2 lacks AP1 sites and contains GC-rich boxed more typical of housekeeping genes. However, the MMP-2 gene regulatory region contains AP2 and YB-1 sites that control it’s expression in a tissuespecific fashion in vivo32,33. Zymogen activation occurs in MMPs when there is a disruption of the interaction between the unpaired cysteine residue in the propeptide and the34 zinc atom in the active site (FIGURE 2). This has been called the “cysteine switch” . Once this interaction is disturbed a conformational change in the enzyme results in autocatalytic or proteolytic cleavage of the remainder of the propeptide giving rise to the mature catalytically competent enzyme. These proteolytic events are often the result of a complex proteinase cascade. Some MMPs, such as MT-MMPs and MMP-11 have a furin-like recognition sequence in their propeptide and thus can be activated intracellularly (in the trans-Golgi network) by the calcium dependent transmembrane serine proteinases of the subtilisin group (furin/PACE). Other MMPs, such as MMP-1, -3 and –9 can be cleaved in their propeptide via the serine proteases such as the uPA-plasmin system, elastase and trypsin35. Subsequently, some of these activated MMPs can also activate other proMMPs as is the case with MMP-3 (stromelysin- 1) which activates proMMP-1 and proMMP-97. MMP-9 is also activated by MMP-219 .

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FIGURE 2: All MMPs possess a pro-peptide which is responsible for maintaining the enzyme in a latent state. This is due to the interaction of a cysteine residue (Cys) in the pro-peptide with a zinc ion in the active site cleft. Once the pro-peptide has been cleaved N-terminally to the cysteine residue, the interaction between the cysteine and the zinc ion is destabilized, allowing further proteolysis to occur, possibly via inter/intramolecular proteolysis. The fully active zymogen is then generated once the pro-peptide has been completely cleaved.

The activation of pro-MMP-2 is unique in this family and thought to result from a cellsurface mediated mechanism involving associations between MMP-2, MT- 1, 2,3,5,6 MMP and TIMP-236. MT-1 MMP is present on the cell surface (as all other MT-MMPs) and can be inhibited by TIMP-2 which binds via its N-terminal domain to the active site of MT-MMP37. (FIGURE 3). This binary complex then acts as a receptor for pro-MMP-2 whose C-terminal domain binds to the TIMP-2 C-terminal domain. A second MT-MMP molecule in close proximity then cleaves the proMMP-2 and activates it. Activation of MMP-2 in this model is only possible if TIMP-2 concentrations are low. High levels of TIMP-2 will inhibit both MMP-2 and MT-MMP19 (FIGURE 3B) This mechanism localizes the proteolytic activity of MMPs to the cell surface where proteolysis and invasion occur. Some researchers have reported that MMP-2 activation occurs via the uPA-plasmin system though this is still controversial38.

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FIGURE 3: MT1-MMP is found on the cell surface of many cells in an active form and has been shown to activate pro-MMP-2. This activation mechanism is dependent on MT1-MMP-TIMP-2-pro-MMP-2 interactions on the cell surface. TIMP-2 can bind both pro-MMP-2 and MTI-MMP via its C-terminal domain and Nterminal domain respectively, forming a tri-molecular complex which localizes pro-MMP-2 to the cell surface. In the presence of low levels of TIMP-2 within the pericellular environment (A), other TIMP-2-free MT1-MMP molecules within close proximity to this tri-molecular complex can cleave the pro-peptide of the 72kDa proMMP-2, generating an intermediate 64kDa form. It is then thought that further autocatalytic cleavage occurs to generate the fully mature 62kDa form. However, if there are large amounts of TIMP-2 present within this system (B), all the MTI-MMP molecules will be bound and inhibited by TIMP-2 and would therefore not be able to cleave pro-MMP-2.

Tissue Inhibitors of MMPs (TIMPs) TIMPs block the deleterious effects of elevated productiod/activation of all MMPs in vitro and in vivo 6,39,40. The four TIMP family members (TABLE 2)41-44 have distinct properties and functions23,45. Common features include the characteristic 6 loop structure, resulting from 12 conserved cysteine residues forming intrachain disulphide bonds. TIMPs also possess two domains; a highly conserved N-terminus that is critical for binding to, and inhibiting, MMP activity and a C-terminus, which governs TIMP-pro-MMP interactions46. Structural differences among TIMP proteins include the presence of N-linked glycosylation sites on TIMP-1 and -3, but not TIMP-2 or –4. Both TIMP-1 & -2 cDNA encode for 21 kDa proteins but TIMP-1 can be either singly (24 kDa) or doubly (28 kDa) glycosylated26; the functional significance of glycosylation is unclear. There are important differences between TIMPs in diffuseability, tissue distribution, transcriptional regulation, and specific association with latent gelatinases. TIMP-1, 2 & 4 are freely diffuseable but TIMP-3 is ECM-associated47. Differences in tissue distribution are outlined in TABLE 2. TIMP-1 might be actively transported into the nuclei of gingival fibroblasts48 but the significance is unclear. TIMP- 1 transcription is regulated by a number of cytokines, hormones and growth factors (eg. TGFE IL-1, IL-6 TNFD and retinoic acid)49,50 . TIMP-3 gene transcription is induced by TPA and TGFE147. Gene silencing by methylation has been described only for TIMP-351. Both TIMP-2 and - 4 are constitutively expressed and their promoter regions have distinctive features and lack the AP1 sites that confer inducibility on TIMP-1 and – 352-54. The binding

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of TIMP to the activated MMPs catalytic site leads to inhibition55. In addition, binding of specific TIMPs to the hemopexin-like domain of some MMPs regulates activation of their proforms. TIMP-1 binds to and slows activation of the latent proMMP-9 while TIMP-2 binds and regulates activation of pro-MMP-255-58. TIMPs can be inactivated by a variety of proteinases such as neutrophil elastase and trypsin59. Table 2: Properties of tissue inhibitors of metalloproteinases (TIMPs) TIMP-1

TIMP-2

TIMP-3

Chromosome gene location (human)

Xp11.23-11.4

17q2.3-2.5

22q 12.1-1 3.2

Protein (kDa)

28*

21

24,27*

22

Major sites

Ovary, bone, uterus

Lung, brain, testes

Kidney, decidua, brain

Brain, heart

Expression

Inducible

Largely constitutive

Inducible

Constitutive

Predominant form of expressed molecule

Secreted

Secreted

ECM-associated

Secreted

Pro-MMP complex

MMP-9

MMP-2

MMP-2

?

lnhibition of MT-MMP

No

Yes

Yes

?

Inhibition of gelatinases

Yes

Yes

Yes

Yes

Matrix bound

No

No

Yes

No

-/+1

+

?

No

No

Yes

No

TNFRI

No

Yes

?

?

TNFRI

?

?

Yes

?

TNFRII

No

Yes

?

?

L-selectin

No

No

Yes

?

HER2/ncu

Yes

No

?

?

IL6R

No

No

Yes

?

Apoptosis Inhibition of protein ectodomain shedding: proTNFD

TIMP-4 3p25

TIMP = tissue inhibitor of metalloproteinase; MMP = matrix metalloproteinase; MT-MMP = membrane-type matrix metalloproteinase; ECM = extracellular matrix; TACE = tumor necrosis factor alpha converting enzyme; - = inhibits apoptosis in some tissues; + = promotes apoptosis in some tissues; ? = unknown Apoptosis effects are likely tissue and context dependent 1 inhibits apoptosis in melanoma cells (168) but promotes it in lymphocytes * size of glycosylated protein

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As expected, manipulation of TIMPs affects invasive/metastatic behavior. TIMP-1 inversely correlates with metastatic potential60,61 and exogenous TIMP-1/ TIMP-2 proteins can inhibit invasion in vitro and invasion and metastases in vivo62-67. Antisense-mediated suppression of TIMP-1 induces malignant behavior in Swiss 3T3 cells68 and forced overexpression of TIMP- 1/TIMP-2 reduces the metastatic ability of melanoma cells65,66,69. Adenoviral delivery systems which over-express TIMP-370,71 find inhibition of invasion, increased apoptosis and reduced attachment. Little is known about TIMP-4 other than it will inhibit invasion, metastasis, tumor growth and angiogensis when transfected into a breast cancercell line72 .

The multifunctional role of MMPs and TIMPs It is critical to appreciate that MMPs/TIMPs do a great deal more than simply interact with each other and mediate the invasive process. They have dramatic and potent effects on a broad range of cellular functions such as growth and proliferation, apoptosis, and angiogenesis45,23,73. The effects of MMPs/TIMPs on these processes are poorly understood and this has important implications for developing novel glioma therapies. In terms of growth and proliferation the multifunctional nature of MMPs has been highlighted by intravital video microscopy which shows that extravasation occurs independently of MMP or TIMP expression74. Instead MMPs may be more important in creating and maintaining a favourable growth environment once extravasation has occured23,45 rather than in extravasation per se. The mechanisms are unknown but MMPs may indirectly stimulate growth in vivo by influencing growth factor bioavailability75-77 in the ECM or through G-protein-mediated proteolytic growth factor processing at the cell surface78. The inhibition of growth factors, such as heparin-binding EGF-like growth factor, has recently been shown to be a metalloproteinase-dependent process which can be inhibited with a synthetic MMP inhibitor (BP-94). Inhibiting the “shedding” of growth factors, such as EGF, which are critical to the signalling of tumors like gliomas is a novel mechanism by which MMPs affect tumor growth and proliferation. Other potential mechanisms through which MMPs/TIMPs may influence growth factor bioavailability have been described. One involves the liberation of IGF from the soluble binding proteins (IGFBPs). MMP-1, -2, and -3 can degrade IGFBP-3 and thus release active IGF75 and TIMP-1 can inhibit tumor growth by reducing IGF bioavailability. In this scenario TIMP-1 inhibits the MMP mediated degradation of IGFBP-3 resulting in elevated IGFBP-3 protein levels which in turn results in less “free” unbound IGF-II being available to stimulate tumor growth79. Also MMP-3 can cleave the membrane-anchored precursor form of heparin-binding epidermal growth factor that can act on cells in a paracrine or autocrine fashion76. MMPs can also negate mechanisms designed to moderate cytokine signals, as occurs in cleavage and release of the inactive type II cell surface “decoy” receptor for interleukin 177. Any or all of these types of events may be occurring in the tumor microenvironment but particularly at the tumor-stromal interface. These indirect mechanisms of influencing tumor growth have not yet been explored in gliomas. TIMPs also have unexpected effects that are potentially important. TIMPs may stimulate cell growth independently of MMP inhibition. For example TIMP levels are not always negatively correlated with tumor grade (expected if decreased TIMPs led to a more malignant and invasive phenotype80). Furthermore, experimental manipulation of TIMPs can inhibit tumor growth in addition to invasion and metastasis. TIMP-2 overexpression produces reduced melanoma growth but not metastases69 and transfection of TIMP-2 into transformed fibroblasts reduced tumor growth as well as metastases81. Finally, TIMP-1 &-2 are being recognized as potentially important cell signaling molecules; TIMP- 1 stimulates proliferation in erythroid precursors41,82 (possibly independent of its MMP inhibitory activity77) and other cell types in vitro in the absence of serum83,84. TIMP-1 also activates steroidogenesis in testis85 and has been described as accumulating in the nucleus in gingival fibroblasts48. Similarly TIMP-2 has growth-promoting activity in vitro at picomolar concentrations86 but inhibits bFGF-induced endothelial proliferation87 Whether TIMPs will be growth stimulators or inhibitors may be tissue specific, or (as appears to be the case with TIMP-2) co-mitogens that depend for their actions on other factors such as insulin or the IGF-1 receptor84. TIMPs may have dramatic effects on apoptosis but the mechanism is unresolved. Furthermore, presumably by regulating several levels of cellular control, individual TIMPs have dramatically different effects on apoptosis. The ECM may act as a “survival factor” and suppress apoptosis since proteolytic modification of the ECM or disruption of cell-matrix

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contacts produces apoptosis88,89. Proteolytic processing of TNF-D and FAS ligand (FasL) from the surface of lymphoid cells by MMP-like activity also alters apoptosis 90,91. These effects on apoptosis may underlie some of the reported effects of TIMPs on cell growth84,85 and the apparent paradox (paradoxical if one expects high levels of TIMP-1 would inhibit invasion and metastases and produce a better prognosis) between high TIMP-1 levels in tumor specimens and the patient's poorer prognosis80,92. To outline what is known about TIMPs and apoptosis briefly. TIMP-1 protects against apoptosis in Burkitt's lymphoma cells and normal tonsillar B cells in a manner that is independent of its MMP-inhibitory activity93. This was not found with TIMP-2 or a synthetic MMP inhibitor, was reversed with TIMP-1 antibodies, and was associated with up-regulation of the anti-apoptotic protein Bc1-XL (but not Bcl-2). Since TIMP-1 is secreted, binds to the cell surface, and the effect is blocked by TIMP-1 antibodies, it may act through a receptormediated autocrine loop though a receptor has not been isolated. TIMP-1 has also been associated with a mature, activated phenotype in Burkitt's lymphoma cell lines93. Another mechanism though which TIMP-1 may affect cell signaling and hence apoptosis directly or indirectly is by inhibiting the shedding of the extracellular domain of growth factor receptors as has been found for the HER2 ectodomain in breast cancer cells94 (TABLE 2). The data for TIMP-2 are less clear and the effects of TIMPs on apoptosis are likely tissue-specific, dependent on the stage of differentiation and/or the cellular milieu. For example TIMP-2 inhibited mitomycin-induced apoptosis in melanoma cell lines95 but induces apoptosis in simulated human T lymphocytes (Dr. M. Lim personal comm.) possibly by inhibiting shedding of FasL or TNF-D The best-characterized TIMP in apoptosis is TIMP-3 which has been reported to protect TNF-α receptors from proteolysis by metalloproteinses. TIMP-3 transfectants in colon carcinoma lines inhibited tumor formation in nude mice96. TIMP-3 transfectants had fewer mitotic figures, were delayed in G1, died after serum starvation and their conditioned media caused cell death that was inhibited by anti-TNF-D antibodies. Transfectant cell lysate contained p55 TNF-α receptor while controls had p55 TNF-α receptor and p46 soluble TNFα inhibitor. Control conditioned media also had p46 while no soluble receptor was found in the transfectant condition media96. Others have found that adenoviral expression of TIMP-3 induced apoptosis in melanoma cells71, vascular smooth muscle cells and HeLa cells70. There are no published studies regarding the effects of TIMP-4 on apoptosis. TIMPs are involved in regulation of the shedding of a variety of protein ectodomains in addition to the TNF Receptors (TABLE 2). These effects may be connected with growth and apoptosis through modulation of adhesive signals (e.g. L-selectin) or signaling receptors or ligands (e.g Her2/neu94,97). Angiogenesis allows solid tumors to grow beyond a certain critical size (2-3mm3)98. The acquisition of angiogenic capabilities (referred to as the angiogenic switch) is a key event in tumour progression which is controlled by the balance between angiogenic factors and inhibitory molecules99-102. Extracellular matrix (ECM) remodeling is an important aspect of angiogenesis because of the structural barriers encountered by invading endothelial cells (ECs). The ECs must first dissolve their underlying BM as well as degrade ECM components as they invade into the surrounding perivascular stroma, forming new capillary sprouts. Angiogenesis is dependent, at least in part, on the actions of MMPs since both TIMPs and synthetic MMP inhibitors such as BB-94 (Batimastat) and AG3340 (Prinomastat) are anti-angiogenic103-106. In vivo, tumors arising from B16F10 melanoma cells overexpressing TIMP-2 show reduced angiogenesis95 . Moreover, endothelial tube formation induced by bFGF and VEGF is inhibited by TIMP-2 and TIMP-3, but not TIMP-1103. Several MMPs may be involved in the angiogenic process, but prime candidates include MMP-2 (gelatinaseA), cell surface MT-MMPs, and MMP-9 (gelatinase-B). All of these can degrade a wide range of substrates including interstitial collagens, gelatin, laminin, and fibronectin107-110. Several observations indicate a link between MMP-2, MT-MMPs and angiogenesis. First, during in vitro EC capillary-like structure formation, an increase in activated MMP-2 is observed compared to a monoculture of EC111. Second, pro-MMP-2 activation may involve an association with the D v E integrin, itself an essential function for EC adhesion to BM112,113. Third, PEX, a fragment of MMP-2 comprising of the C-terminal hemopexin-like domain blocks pro-MMP-2 activation on the chick chorioallantoic membrane where it disrupts angiogenesis and tumor growth 114. Fourth, in MMP-2 null mice, tumors generated by malignant cell lines displayed reduced tumor volumes and decreased levels of angiogenesis115. Fifth, endothelial sprout formation from a muscle explant embedded within

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a fibrin gel was found to be MT1-MMP dependent116. Finally, MTl-MMP-null mice show defects in vascular invasion of cartilage and fail to produce new blood vessels in response to the bFGF in a mouse corneal angiogenesis assay117,118. The role of MMP-9 in tumor associated angiogenesis is also important though less extensively studied than that of MMP-2; furthermore it is likely that both enzymes are important in tumor associated angiogenesis. Degradation of the type IV collagen in the basement membrane by endothelial cells can be mediated by both MMP-2 and -9. Type IV collagenase activity is very important in the early steps of endothelial cell morphogenesis and capillar formation. In addition MMP-9-null mice demonstrate very abnormal growth plate vascularization and ossification problems73. The precise mechanism by which MMP-9 regulates growth plate angiogenesis is unknown. Presumably it functions to release angiogenic factors or to inactivate angiogenic inhibitors. As yet unpublished data suggest the progression to the angiogenic switch and malignancy are markedly reduced in MMP-9-null mice. It is possible, for example, that MMP-9 releases sequestered VEGF or other angiogenic factors and contributes to the induction of angiogenesis in these tumors. MMPs/TIMPs have a complex role in angiogenesis that is not yet clear. The simple notion that excess activity of all MMPs leads to increased angiogenesis is not accurate. Rather these may have both angiogenic and angiostatic effects and the net effect may be MMP and tissue specific and depend on spatial-temporal expression. For instance, MMP-7, MMP-9 and, MMP- 12 have all been linked with the generation of angiostatin from plasminogen119 . Processing of endostatin from collagen XVIII also involves the actions of cathepsins and MMPs120. Clearly the contribution of MMPs/TIMPs to tumor associated angiogenesis needs to be better understood so these can be effectively manipulated therapeutically.

Synthetic Inhibitors of MMPs A number of low molecular weight synthetic MMP inhibitors are under various stages of development by the pharmaceutical industry (TABLE 3). In general, these have a peptide backbone similar to the cleavage site on collagen that binds the MMP, and they contain a hydroxamate group that coordinates the catalytic zinc ion in the active site form121. Several promising studies have been published that find antitumor activity of synthetic MMP inhibitors in a variety of in vivo tumor models. The most widely studied of these is batimastat (also called BB-94: British Biotech Ltd., Oxford United Kingdom), which produced prolonged survival in an ovarian tumor xenograft122; inhibited metastasis of melanoma123 , breast124 and colon cancer125 cell lines. The BB-94 also inhibited growth of a colon cancer xenograft126, a breast cancer cell line127 and a hemangioma105. Other synthetic MMP inhibitors (Celltech Therapeutics, Ltd., Slough, United Kingdom; Agouron Pharmaceuticals, San Diego, U.S.A.) inhibit the tumor growth in a variety of tumor models in vivo 128-130. Table 3: Metalloproteinase Inhibitors in Clinical Trials Company

Compound

Indication

Status

Agouron Bayer British Biotech British Biotech Chiroscience Chiroscience Chiroscience Roche Roche Biosciences

AG3340 Bay 12-9566 Marimastat BB-2516 BB-3644 D2163 D1927 D5410 Ro 32-3555 RS 130830

Cancer and Gliomas Cancer Cancer and Gliomas Multiple Sclerosis Cancer Cancer Inflamm. Bowel Arthritis Osteoarthritis

Phase II/III Suspended Phase II/III Phase I Phase I Preclinical Phase II Phase I Phase I

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MMPs In Gliomas The evidence that MMPs are important in the pathophysiology of gliomas is based on three observations: 1) A number of MMPs (mostly MMP-2 and -9)/ TIMPs are found in gliomas or their tumor vasculature, 2) Over-expression or dysregulation of some MMPs is associated with a poorer clinical outcome131-135. Synthetic MMP inhibitor inhibit glioma proliferation, tumor growth and vascularity in a glioma model in vivo136,106. The latter two observations are the strongest evidence that MMPs are a critical requirement for glioma growth and not simply associated with it. Various genetic manipulations in vivo, such as glioma formation in MMP-knock out mice or inducible overexpression systems in vivo have not been published in gliomas. MMP-1, -2, -3, -7, –9 131-146, TIMPs 1-4 135,140,143,147-150 and MT 1-6 MMPs144,151-153 are found in glioma cell lines and surgical specimens. MMP-2 and -9 are the most extensively studied and it seems likely both are involved in invasion and angiogenesis. MMP-2 may become important in glioma invasion and MMP-9 in glioma angiogenesis. Both MMP-2 and -9 are present in normal brain and are expressed in neurons and to a lesser extent in the glia and vasculature using both in situ hybridization (IS) and immunohistochemistry (IH)132,146; MMP-2 is also found in fetal astrocytes154. Levels of expression (using RT-PCR or Northerns), amount of protein and activity are higher for all gliomas, irrespective of grade, than in normal brain131,132 for both MMP-2 and -9. However, expression levels of MMP-2 remain relatively constant with increases in glioma grade whereas MMP-9 expression increases dramatically. This is probably due to the different tissue sources of MMPs in gliomas; the more malignant gliomas are very vascular tumors and MMP-9 is more closely associated with the vasculature in gliomas. MMP-2 is mostly expressed in glioma cells (using IS and IH) and to a much lesser extent in the vasculature and surrounding glia; MT1 -MP localization follows a similar pattern 138,144-146 . In contrast, MMP-9 is predoqinantly expressed in the vasculature with variable expression in glioma cells and little, if any staining in surrounding glia132,134,135,146 . In some patients MMP-9 expression is almost exclusively confined to the vasculature132 but in others high levels are also seen in the glioma tumor cells suggesting expression may be variable among patients. In the vasculature the MMP-9 expression seems to originate from the endothelial cells and perivascular cells. Two other comments regarding the localization of MMP-2 and -9 in gliomas should be made. First, we have never observed MMP expression in isolated tumor cells which were distant from the main tumor mass as one might expect if the early expression of MMPs facilitated invasion of isolated tumor cells. However the methods of IS and IH may be too insensitive to detect very small levels of expression. The failure to observe the expression does not mean it does not occur. In situ zymography may detect MMP activity in these cells but it has not been reported yet. Alternatively, our underlying hypothesis, that MMPs are critical factors in glioma invasion, may be incorrect and these function to maintain a favorable environment for glioma growth. Second, in contrast to systemic cancer where MMP expression predominantly localized to surrounding stromal cells and not found in the tumor cells, MMP-2 and -9 expression were found mostly in the glioma cells and vasculature and not in the surrounding glia or stroma. The reasons for this difference from systemic cancer are unknown. It could be related to the specialized "stroma" of the brain which largely lacks the tough basement membranes and collagen-rich tissue planes that are major barriers to the spread of tumors outside the CNS. The CNS ECM may pose less of a barrier to glioma invasion or the brain's ECM may regulate proteinase expression/activity in glioma cells. There is considerable evidence that MMP-2 plays a major role in glioma invasiveness. In vitro studies usually find that the best correlations are found between invasion and MMP-2 expression/activity8,144,155,156. In addition, there is a close correlation between MT1-MMP expression and activation of MMP-2 during the malignant progression of gliomas144. While MMP-2 is present in normal brain, it is both overexpressed and activated in malignant gliomas. One interpretation of these data from tumor specimens is that MT 1 -MMP is overexpressed in malignant gliomas where there is an accompanying activation of MMP-2; small amounts of MTI-MMP are found in glia surrounding gliomas but not in normal brain144 . So the idea is that the presence of both MT1-MMP and MMP-2 on glioma tumor cells allows for activation of MMP-2 at the glioma/stroma interface and this process facilitates glioma invasion. An alternative explanation is provided by in vitro

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experiments with the rat C6 glioma line in which the critical proteolytic enzyme was MT1MMP and not MMP-2157. These investigators found that the ability of C6 glioma cells to overcome the inhibitory properties of myelin and allow invasion along myelin occurred with MT1 -MMP expression; this invasive phenotype could be mimicked when fibroblasts were transfected with MT1-MMP. This is independent of MMP-2 activity since invasion was not inhibited with TIMP-1, a potent inhibitor of MMP-2 but not MT1-MMP. However, as inhibition was also not observed with BB-94, an effective inhibitor of MT- 1 -MMP158 there may be contributions from other metalloproteinases. Whether these results apply to humans, or in vivo, remains to be determined. The six MT-MMPs have all been found in gliomas144,152,153 . In addition to activation of MMP-2 they may also activate other MMPs such as MMP-13 (collagenase-3). Information on the MT-MMPs is at present very preliminary but there are clear differences in their expression in normal brain and gliomas. MT1-, 2- and 6-MMPs are not expressed in normal brain whereas MT3-, 4-, 5-MMP transcripts are151-153. The expression in gliomas is somewhat inconsistent but MT5-MMP may be the most commonlyoverexpressed; MT1-, 2-, 3-, 4- and 6- are over expressed in a smaller number of samples 151-153 . The cellular origin of these has been described for MT1-, 2-, and 3-MMP but not yet for MT4-, 5-, 6-MMP. MTland 2-MMP are found in malignant glioma cells and, to a lesser extent, in the endothelial cells. Little or none is found in normal brain. MT3-MMP was found in normal brain but the cellular origin was not clear151 . Clearly the importance of these enzymes which may be critical to glioma invasion needs to be better understood.

Integrins and Protease Activity Integrins are cell surface receptors that mediate the physical and functional interactions between a cell and its ECM. These are being increasingly studied in glioma migration and invasion159 . Cell surface integrins may physically "grip" the ECM proteins and simultaneously interact with cytoskeletal elements within the cell to regulate cell adhesion, shape and motility. In addition, the interaction of ECM components with integrins can affect signalling pathways and regulation of protease activity. For example, ligation of the DvE3 integrin provides a survival signal for endothelial cells in vitro and in vivo160 and disruption of this receptor inhibits angiogenesis by inducing endothelial cell apoptosis112. Similarly, vitronectin (ligates DvE 3 and DvE 5 integrins) may protect glioma cells from apoptosis161 . αvβ3 integrin may also influence glioma migration and invasion by regulating the localization and activation of tumor-derived proteases at plasma membrane of glioma cells in two ways. First the αvβ3 integrin directly binds activated MMP-2, concentrates its proteolysis at the tumor cell surface113. Second, vitronectin (the ligand of DvE3) binds, depending on its confirmation, either the protease plasminogen, its inhibitor plasminogen activator inhibitor type 1 (PAI-1), or the urokinase receptor162. These mechanisms may serve to focus both serine proteinase and metalloproteinse activities in the vicinity of the cell membrane. Clearly the complexity of αvβ3, vitronectin and plasminogen, PAI-1 or the urokinase receptor may permit integrins to regulate serine protease-mediated proteolysis. In this scenario plasminogen binding to av b3 and vitronectin would produce enhanced proteolysis and invasion whereas binding of PAI-1 may reduce proteolysis but allow a better "grip"for tumor cell locomotion162 . PAI-1 over expression, somewhat paradoxically, is correlated with increased tumorginecity in gliomas163. This tumor enhancing role of PAI-1 is endorsed by studies of PAI- 1 -/- mice, which have shown that transplanted malignant keratinocytes fail to invade and establish vascularized tumors in these animals, but angiogenesis and tumor growth could be restored upon adenoviral deliver of PAI-164.

TIMPs In Gliomas Although all TIMPs seem to block glioma invasion their effects on a variety of other cellular processes such as proliferation apoptosis or angiogenesis may be quite distinct from each other. Little is known regarding TIMPs in gliomas and inconsistencies exist. Some investigators find a reduced expression of TIMP-1 &-2165 with increasing glioma grade. This suggests a lack of TIMP contributed to the increased aggressiveness of malignant gliomas. However, we 149 and others 135,143,150 find an upregulation of TIMPs -1 &-2 with increasing grades of gliomas. Interestingly, over-expression of TIMP- 1 in a glioma cell line166produced

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the expected reduction in invasion in vitro but also decreased proliferation. This observation was not otherwise explained but is potentially important. TIMP-3 in gliomas has been reported by only three groups149,150; one51 found reduced expression by gene silencing via methylation. No study has reported TIMP-4 in gliomas. We evaluated the expression of TIMPs 1-4 in gliomas149 and found that TIMP-1 was positively, whereas TIMP-4 was negatively, correlated with glioma grade. TIMP-4 levels are initially high in low and midgrade tumors but in GBMs return to levels seen in normal brain tissue. TIMP-2 & -3 expression levels did not vary with tumor grade and are expressed at low levels. The patterns of TIMP localization (using IS and IH) were also different . TIMP-1 was expressed in the tumor vasculature > tumor cells, whereas TIMP-2 was diffusely expressed. TIMP-4 was expressed only in glioma tumor cells though we cannot rule out very low levels of expression in the tumor vasculature using these techniques. Since TIMP- 1 & -4 have dissimilar localizations and expression patterns this suggests their functions in gliomas may be very different. This is certainly not consistent with the simple notion that a loss of TIMP expression allows the malignant progression of gliomas. The increase in TIMP- 1 expression with glioma grade may stimulate glioma proliferation (e.g. via VEGF upregulation167 ), act as a growth factor or be a compensatory increase in expression. In contrast, the reduction of TIMP-4 expression in higher grade tumors may reflect enhanced tumor growth, angiogenesis or invasion or alternatively, enhanced expression of TIMP-4 may confer growth advantages early in the progression of gliomas which are less important once transformation to the highly malignant GBM has occurred. Breast cancer lines which over-express TIMP-4 have produced decreased proliferation, invasion and microvascular density168. Like TIMP-2, TIMP-4 can associate with the Cterminal hemopexin domain of pro-MMP-2169 and this raises the possibility that the TIMP-2 and -4 may have different abilities to regulate MT-MMP-mediated pro-MMP-2 activation.

Synthetic Inhibitors of MMPs In Gliomas There are only four studies of synthetic inhibitors of MMPs (SynMMPIs) in gliomas to our knowledge 106,136,170 (Penny Costello unpublished observations). In terms of in vitro studies Tonn et al. 1999170 reported that both BB-94 and BB-2516 had the expected effects on invasion using the Matrigel assay or spheroid confrontation assay. Neither compound was found to be cytotoxic, however marked inhibition of proliferation, which in some cases inhibited proliferation completely, was found using the U251 cell line. This contrasts to our own data136 using a different MMP inhibitor called AG3340. This did not inhibit proliferation or viability at concentrations 100µM. The mechanism by which BB-94 or BB2516 might affect proliferation in vitro was not explained. It should be noted that the concentration of 50µM of BB-2516 used to inhibit proliferation is approximately 1000 times higher than IC50 for collagenase, MMP-2 or MMP-9. The observation, therefore, is intriguing but unexplained. One possibility is that the BB-94 and BB-2516 (but not AG3340) inhibit a sheddase which liberates EGF from membrane-bound heparin-binding EGF and subsequently allows this liberated EGF to interact with its receptor. Alternatively BB-94 or BB-2516 may inhibit the liberation of other growth factors from the matrix or their binding proteins in serum. Further studies are needed to clarify the potential growth modifying actions of these inhibitors in vitro and in vivo. All in vivo studies are done using the U87 malignant glioma cell line implanted subcutaneously in SCID-NOD or nude mice106,136 (Costello unpublished papers). These all find a markedly reduced rate of glioma growth, proliferation, angiogenesis and invasion in vivo using either BB-94 (Penny Costello unpublished observations) or AG3340106,136 (FIGURE 4). Since AG3340 does not affect glioma viability or proliferation in vitro (at least in our hands at pharmacologically relevant concentrations) we speculated that AG3340’s effects in vivo are mediated by inhibiting angiogenesis and/or by affecting growth factor bioavailability. The effect of AG3340 on glioma vascularity is particularly striking 106. All of these SynMMPIs only slowed tumor growth in vivo and neither produced tumor “cure” or caused tumor regression. Indeed they were not designed or expected to act as cytotoxic agents. Finally, it is still unknown if SynMMPIs are effective in intracerebral glioma models. The mechanism by which synthetic MMP inhibitors reduce angiogenesis in gliomas is unknown but probably complex. As mentioned previously the simple notion that excess MMP activity leads inevitably to increased angiogenesis may not be accurate. MMPs may be involved in the generation of the endogenous angiogenesis inhibitors angiostatin and

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FIGURE 4: Effects of AG3340 on s.c.. growth of the malignant glioma cell line U87. Effects of vehicle control or AG3340 treatment (begun on day 0) on tumor length (length X width); treatment began when tumors were easily measurable and clearly growing. A, in experiment lA, a significant difference in tumor size appeared by day 21 (P < 0.01, Wilocxon test) and remained until day 31 (P