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 Guroff2 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 activator34, cysteine proteases5, calpain6, 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 oligoden-
Role of Proteases in the Pathophysiology of Neurodegenerative Diseases, edited by Lajtha and Banik. Kluwer Academic/Plenum Publishers, New York, 2001.
drocyte glycoprotein (MOG)10, and another basic protein, the myelin-associated oligodendrocyte 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 MS12, 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. Lampert17 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 calcium20, 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 metalloproteinases21, plasminogen activator22, calpain23, 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 MS24. 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 pep-statin, for cathepsin D25, inhibitors of plasminogen act ivator26, 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.
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This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.