calpain and Multiple-Sclerosis

Communication in the central nervous system (CNS) occurs through initiation and propagation of action potentials at excitable domains along axons. Action potentials generated at the axon initial segment (AIS) are regenerated at nodes of Ranvier through the process of saltatory conduction. Proper formation and maintenance of the molecular structure at the AIS and nodes are required for sustaining conduction fidelity. In myelinated CNS axons, paranodal junctions between the axolemma and myelinating oligodendrocytes delineate nodes of Ranvier and regulate the distribution and localization of specialized functional elements, such as voltage-gated sodium channels and mitochondria. Disruption of excitable domains and altered distribution of functional elements in CNS axons is associated with demyelinating diseases such as multiple sclerosis, and is likely a mechanism common to other neurological disorders. This review will provide a brief overview of the molecular structure of the AIS and nodes of Ranvier, as well as the distribution of mitochondria in myelinated axons. In addition, this review highlights important structural and functional changes within myelinated CNS axons that are associated with neurological dysfunction.

Topics: Action Potentials; Alzheimer Disease; Animals; Axons; Brain Injuries; Calpain; Central Nervous System; Humans; Mitochondria; Multiple Sclerosis; Myelin Sheath; Ranvier's Nodes

Experimental autoimmune encephalomyelitis (EAE), a widely recognized animal model of multiple sclerosis (MS), is highly useful for studying inflammation, demyelination, and neurodegeneration in the central nervous system (CNS). EAE exhibits many similarities with MS, which is a chronic inflammatory disease affecting CNS white matter in humans. Various studies have indicated that EAE is a particularly useful animal model for understanding both the mechanisms of immune-mediated CNS pathology and also the progressive clinical course of MS. Demyelination and axonal dysfunction have previously been shown in MS and EAE but current evidences indicate that axonal damage and neuron death also occur, demonstrating that these diseases harbor a neurodegenerative component. Recent studies also have shown that the activation of calpain and caspase pathways contribute to the apoptotic death of oligodendrocytes and neurons, promoting the pathological events leading to neurological deficits. Apoptosis is involved in the disease-regulating as well as in the disease-promoting processes in EAE. This review discusses the major involvement of calpain and caspase pathways in causing demyelination and neurodegeneration in EAE animals.

Topics: Animals; Calpain; Demyelinating Diseases; Disease Models, Animal; Humans; Multiple Sclerosis; Nerve Degeneration; Signal Transduction

Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function. However, alterations in calcium homeostasis lead to persistent, pathologic activation of calpain in a number of neurodegenerative diseases. Pathologic activation of calpain results in the cleavage of a number of neuronal substrates that negatively affect neuronal structure and function, leading to inhibition of essential neuronal survival mechanisms. In this review, we examine the mechanistic underpinnings of calcium dysregulation resulting in calpain activation in the acute neurodegenerative diseases such as cerebral ischemia and in the chronic neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, prion-related encephalopathy, and amylotrophic lateral sclerosis. The premise of this paper is that analysis of the signaling and transcriptional consequences of calpain-mediated cleavage of its various substrates for any neurodegenerative disease can be extrapolated to all of the neurodegenerative diseases vulnerable to calcium dysregulation.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Animals; Brain Ischemia; Calpain; Humans; Huntington Disease; Multiple Sclerosis; Neurodegenerative Diseases; Parkinson Disease; Prion Diseases; Signal Transduction; Trauma, Nervous System

Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as μ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

Topics: Animals; Calpain; Carbamates; Cell Survival; Cysteine Proteinase Inhibitors; Dipeptides; Enzyme Activation; Gene Knockdown Techniques; Humans; Inflammation; Microglia; Motor Neurons; Multiple Sclerosis; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Th1 Cells; Th17 Cells

The regenerative capacity of the Central Nervous System (CNS) is a key factor implicated in the pathogenesis of neurodegenerative diseases. In the present study, the regenerative capacity of the CNS is considered using one of the markers of regeneration, Growth Associated Protein-43 (GAP-43) and its proteolytic fragment GAP-43-3 in the Experimental Autoimmune Encephalomyelitis (EAE) animal model of multiple sclerosis. The EAE on Wistar rats was characterized as an adequate model of multiple sclerosis, with typical clinical (pares and paralysis) and morphological (infiltration of spinal cord and deformation of motoneurons) disorders. Normally about 60% of GAP-43 is cleaved by m-calpain and stays in the form of GAP-43-3. During severe form of EAE up to 85% of GAP-43 can be found cleaved. We speculated that the cleavage of GAP-43 can play a crucial role for regenerative capacity of CNS during EAE development. Thus the distribution of GAP-43 and GAP-43-3 in the spinal cord was analyzed. The manifestation of clinical signs of EAE has been found to be in correlation with the levels of GAP-43 proteolysis both in the homogenate of the spinal cord and on the spinal cord slices. The immunoreactive staining enabled the observation of the accumulation of GAP-43-3 predominantly in microglial cells.

Topics: Animals; Calpain; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; GAP-43 Protein; Gene Expression; Male; Microglia; Motor Neurons; Multiple Sclerosis; Nerve Regeneration; Proteolysis; Rats; Rats, Wistar; Spinal Cord

Multiple sclerosis (MS) pathology is marked by the massive infiltration of myelin-specific T cells into the CNS. Hallmarks of T helper (Th) cells during active disease are pro-inflammatory Th1/Th17 cells that predominate over immunoregulatory Th2/Treg cells. Neurodegeneration, a major factor in progressive MS, is often overlooked when considering drug prescription. Here, we show that oral dosing with SNJ-1945, a novel water-soluble calpain inhibitor, reduces experimental autoimmune encephalomyelitis clinical scores in vivo and has a two pronged effect via anti-inflammation and protection against neurodegeneration. We also show that SNJ-1945 treatment down-regulates Th1/Th17 inflammatory responses, and promotes regulatory T cells (Tregs) and myeloid-derived suppressor cells in vivo, which are known to have the capacity to suppress helper as well as cytotoxic T cell functions. Through analysis of spinal cord samples, we show a reduction in calpain expression, decreased infiltration of inflammatory cells, and signs of inhibition of neurodegeneration. We also show a marked reduction in neuronal cell death in spinal cord (SC) sections. These results suggest that calpain inhibition attenuates experimental autoimmune encephalomyelitis pathology by reducing both inflammation and neurodegeneration, and could be used in clinical settings to augment the efficacy of standard immunomodulatory agents used to treat MS. Multiple sclerosis (MS) pathology is marked by inflammation and infiltration of myelin-specific T cells into the central nervous system. Inflammation leads to neurodegeneration in progressive MS which also leads to epitope spreading, feedback looping to more inflammation. Calpain can play a role in both arms of the disease. Here, oral dosing with SNJ-1945, a novel water-soluble calpain inhibitor, reduces experimental autoimmune encephalomyelitis clinical scores in vivo and has a two-pronged effect via anti-inflammation and protection against neurodegeneration.

Topics: Animals; Blotting, Western; Calpain; Carbamates; Cell Proliferation; Cell Separation; Cysteine Proteinase Inhibitors; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immunohistochemistry; Immunomodulation; In Situ Nick-End Labeling; Male; Mice; Monocytes; Multiple Sclerosis; Nerve Degeneration; Real-Time Polymerase Chain Reaction; T-Lymphocytes, Helper-Inducer

Topics: Animals; Calpain; Carbamates; Cysteine Proteinase Inhibitors; Encephalomyelitis, Autoimmune, Experimental; Immunomodulation; Male; Multiple Sclerosis; Nerve Degeneration

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) β ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm. In addition, we investigated the mechanism of action of DPN treatment-induced recovery during EAE. Given that prophylactic and therapeutic treatments with DPN during EAE improved remyelination-induced axon conduction, and that ER (α and β) and membrane (m)ERs are present on oligodendrocyte lineage cells, a direct effect of treatment on oligodendrocytes is likely. DPN treatment of EAE animals resulted in phosphorylated ERβ and activated the phosphatidylinositol 3-kinase (PI3K)/serine-threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway required for oligodendrocyte survival and axon myelination. These results, along with our previous studies of prophylactic DPN treatment, make DPN and similar ERβ ligands immediate and favorable therapeutic candidates for demyelinating disease.

Topics: Animals; Blotting, Western; Brain-Derived Neurotrophic Factor; Calpain; Caspase 3; Corpus Callosum; Electrophysiological Phenomena; Encephalomyelitis, Autoimmune, Experimental; Estrogen Receptor beta; Female; Immunohistochemistry; Ligands; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron; Multiple Sclerosis; Myelin Sheath; Nitriles; Oligodendroglia; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Postural Balance; Selective Estrogen Receptor Modulators; Signal Transduction; Spinal Cord; TOR Serine-Threonine Kinases

Chromogranin B (CGB) is a high capacity, low affinity calcium binding protein in the endoplasmic reticulum (ER) that binds to the inositol 1,4,5 trisphosphate receptor (InsP3R) and amplifies calcium release from ER stores. Recently, it was discovered that levels of CGB-derived peptides are decreased in the cerebrospinal fluid of multiple sclerosis (MS) patients. One of the mechanisms by which neurodegeneration in MS is thought to occur is through increased levels of intra-axonal calcium. The combination of excess intracellular calcium and dysregulated levels of CGB in MS led us to hypothesize that CGB may be involved in MS pathophysiology. Here, we show in a mouse model of MS that CGB levels are elevated in neurons prior to onset of symptoms. Once symptoms develop, CGB protein levels increase with disease severity. Additionally, we show that elevated levels of CGB may have a role in the pathophysiology of MS and suggest that the initial elevation of CGB, prior to symptom onset, is due to inflammatory processes. Upon development of symptoms, CGB accumulation in neurons results from decreased ubiquitination and decreased secretion. Furthermore, we show that calpain activity is increased and levels of InsP3R are decreased. From these results, we suggest that the elevated levels of CGB and altered InsP3R levels may contribute to the axonal/neuronal damage and dysregulated calcium homeostasis observed in MS. Additionally, we propose that CGB can be a biomarker that predicts the onset and severity of disease in patients with MS.

Topics: Animals; Calpain; Chromogranin B; Encephalomyelitis, Autoimmune, Experimental; Exocytosis; Inositol 1,4,5-Trisphosphate Receptors; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Neurons; RNA, Messenger; Spinal Cord; Ubiquitination

While multiple molecular mechanisms contribute to midbrain nigrostriatal dopaminergic degeneration in Parkinson's disease (PD), the mechanism of damage in non-dopaminergic sites within the central nervous system, including the spinal cord, is not well-understood. Thus, to understand the comprehensive pathophysiology underlying this devastating disease, postmortem spinal cord tissue samples (cervical, thoracic, and lumbar segments) from patients with PD were analyzed compared to age-matched normal subjects or Alzheimer's disease for selective molecular markers of neurodegeneration and inflammation. Distal axonal degeneration, relative abundance of both sensory and motor neuron death, selective loss of ChAT(+) motoneurons, reactive astrogliosis, microgliosis, increased cycloxygenase-2 (Cox-2) expression, and infiltration of T cells were observed in spinal cord of PD patients compared to normal subjects. Biochemical analyses of spinal cord tissues revealed associated inflammatory and proteolytic events (elevated levels of Cox-2, expression and activity of μ- and m-calpain, degradation of axonal neurofilament protein, and concomitantly low levels of endogenous inhibitor - calpastatin) in spinal cord of PD patients. Thus, pathologically upregulated calpain activity in spinal cords of patients with PD may contribute to inflammatory response-mediated neuronal death, leading to motor dysfunction. We proposed calpain over-activation and calpain-calpastatin dysregulation driving in a cascade of inflammatory responses (microglial activation and T cell infiltration) and degenerative pathways culminating in axonal degeneration and neuronal death in spinal cord of Parkinson's disease patients. This may be one of the crucial mechanisms in the degenerative process.

Topics: Alzheimer Disease; Axons; Calcium-Binding Proteins; Calpain; Case-Control Studies; Cell Death; Cytoskeletal Proteins; Gliosis; Humans; Huntington Disease; Inflammation; Multiple Sclerosis; Nerve Degeneration; Neurons; Parkinson Disease; Spinal Cord; T-Lymphocytes

Multiple sclerosis (MS) pathology is marked by the massive infiltration of myelin-specific T cells into the central nervous system (CNS). During active disease, pro-inflammatory Th1/Th17 cells predominate over immunoregulatory Th2/Treg cells. Here, we show that calpain inhibition downregulates Th1/Th17 inflammatory cytokines and mRNA in MS patient peripheral blood mononuclear cells (PBMCs) activated with anti-CD3/28 or MBP. Interestingly, calpain inhibition elevated IDO gene expression in MS PBMCs, which was markedly decreased in calpain expressing cells. Functional assay showed that incubation of MS patient PBMCs with calpain inhibitor or recombinant IDO attenuates T cell proliferation. These results suggest that calpain inhibition may attenuate MS pathology and augment the efficacy of standard immunomodulatory agents used to treat this disease.

Topics: Calpain; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Leukocytes, Mononuclear; Male; Middle Aged; Multiple Sclerosis; Reverse Transcriptase Polymerase Chain Reaction; Th1 Cells; Th17 Cells

The lack of disease-modifying pharmacological agents for effective treatment of multiple sclerosis (MS) still represents a large and urgent unmet medical need. Our previous studies showed that ligands to type 2 imidazoline receptors (I(2)R) were effective in protecting spinal cord injury caused by experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this study, we further examined the protective property of a very selective ligand of I(2)R, 2-(2-benzofuranyl) 2-imidazoline (2-BFI) against EAE. Importantly, a mechanism of 2-BFI-mediated protection was investigated which possibly involves an I(2)R binding protein, brain-creatine kinase (B-CK), as well as CaATPase and calpain. The enzymatic activity of B-CK and CaATPase was significantly reduced in EAE injured spinal cord. Reduction of B-CK activity in EAE spinal cord may lead to energy reduction and dysfunction in cellular calcium homeostasis. Increased intracellular calcium evokes elevation of calpain activity occurring in EAE spinal cord which causes further tissue damage. Indeed, EAE injured spinal cord showed significant reduction in CaATPase and increase calpain activities. Remarkably, spinal cord tissue from mice treated daily with 2-BFI during the progression of EAE significantly restored B-CK and CaATPase enzymatic activities and showed no induction in calpain activity. Moreover, EAE spinal cord from 2-BFI treated mice also demonstrated better preservation of myelin; reduced axonal injury, as evidenced by the lower level of β-APP expression, and above all, highly improved neurobehavioral scores (p<0.01; n=10). These findings suggest that 2-BFI can be further developed as a therapeutic drug for MS treatment.

Topics: Animals; Axons; Benzofurans; Calcium-Transporting ATPases; Calpain; Creatine Kinase, BB Form; Encephalomyelitis, Autoimmune, Experimental; Female; Imidazoles; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Spinal Cord Injuries

Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose dependently with the calpain inhibitor calpeptin (50-250 microg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased, with down-regulation of proapoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of proapoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS.

Topics: Animals; Axons; Blotting, Western; Calpain; Cell Death; Cysteine Proteinase Inhibitors; Dipeptides; Down-Regulation; Encephalomyelitis, Autoimmune, Experimental; Fluorescent Antibody Technique; Gliosis; In Situ Nick-End Labeling; Inflammation; Male; Multiple Sclerosis; Nerve Tissue Proteins; Oligodendroglia; Rats; Rats, Inbred Lew; Spinal Cord; Tissue Embedding

Multiple sclerosis (MS) is a devastating autoimmune demyelinating disease of the central nervous system (CNS). This study investigated whether expression and activity of the calcium-activated protease calpain correlated with Th1/Th2 dysregulation in MS patients during states of relapse and remission. Calpain expression and activity were significantly increased in peripheral blood mononuclear cells (PBMCs) from MS patients, compared to controls, with the highest expression and activity noted during relapse. Th1 cytokines were highest and Th2 cytokines were lowest in MS patients during relapse. Treatment with calpain inhibitor, calpeptin, decreased Th1 cytokines in PBMCs from MS patients. Calpain inhibitor also reduced degradation of myelin basic protein (MBP) by inhibiting the calpain secreted from MBP-specific T cells. Taken together, these results suggested calpain involvement in Th1/Th2 dysregulation in MS patients.

Topics: Biomarkers; Calcium; Calcium Signaling; Calpain; Cytokines; Dipeptides; Enzyme Inhibitors; Female; Humans; Male; Multiple Sclerosis; Myelin Basic Protein; Neutrophils; Recurrence; Th1 Cells; Th2 Cells; Up-Regulation

Damage to axons in acute multiple sclerosis (MS) lesions is now well established but the mechanisms of this damage remain obscure. Here we have applied a panel of antibodies that identify cell populations and proteins contained in them with a view to detecting those cells and proteins that are localised particularly closely to damaged axons in acute, sub-acute and border-active MS plaques. Results are expressed semi-quantitatively and graphs produced that show that many of the markers show enhanced expression at sites of axon damage. However, the sharpest increase in expression in relation to axon damage was seen for Calpain I (micro-calpain), inducible nitric oxide synthase and MMP-2, suggesting that these proteins may form part of a group of proteins responsible for the initiation of myelin and/or axon damage seen in MS lesions.

Topics: Amyloid beta-Peptides; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Axons; Biomarkers; Brain; Calpain; CD3 Complex; Humans; Immunohistochemistry; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Multiple Sclerosis; Nitric Oxide Synthase Type II; Osteopontin; Plaque, Amyloid; Proteins; Sialoglycoproteins; T-Lymphocytes; Tyrosine

Topics: Amyloid beta-Protein Precursor; Axons; Brain; Calpain; Humans; Macrophages; Major Histocompatibility Complex; Multiple Sclerosis; Nerve Degeneration; T-Lymphocytes, Cytotoxic

In autoimmune demyelinating diseases such as multiple sclerosis (MS), the degradation of myelin proteins results in destabilization of the myelin sheath. Thus, proteases have been implicated in myelin protein degradation, and recent studies have demonstrated increased expression and activity of a calcium-activated neutral proteinase (calpain) in experimental allergic encephalomyelitis, the corresponding animal model of MS. In the present study, calpain activity and expression (at translational and transcriptional levels) were evaluated in white matter from human patients with MS and Parkinson's and Alzheimer's diseases and compared with that of white matter from normal controls. Western blot analysis revealed that levels of the active form of calpain and calpain-specific degradation products (fodrin) were increased by 90.1% and 52.7%, respectively, in MS plaques compared with normal white matter. Calpain translational expression was up-regulated by 462.5% in MS plaques compared with controls, although levels of the specific endogenous inhibitor, calpastatin, were not altered significantly. At the transcriptional level, no significant changes in calpain or calpastatin expression were detected by reverse transcription-PCR. Using double immunofluorescent labeling, increased calpain expression was observed in reactive astrocytes, activated T cells, and activated mononuclear phagocytes in and adjacent to demyelinating lesions. Calpain activity and translational expression were not increased significantly in white matter from patients with Parkinson's or Alzheimer's diseases compared with that of normal controls. Because calpain degrades all major myelin proteins, the increased activity and expression of this proteinase may play a critical role in myelinolysis in autoimmune demyelinating diseases such as MS.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Demyelinating Diseases; Fluorescent Antibody Technique; Humans; Multiple Sclerosis; Protein Biosynthesis; Transcription, Genetic

In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.

Topics: Animals; Antibodies, Monoclonal; Calpain; Encephalomyelitis, Autoimmune, Experimental; Galactosylceramides; Glial Fibrillary Acidic Protein; Immunoenzyme Techniques; Inflammation; Multiple Sclerosis; Neuroglia; Rats; Rats, Inbred Lew; Spinal Cord

Degradation of myelin basic protein (MBP) in human man myelin was monitored by electroimmunoblotting. Problems of variation between, as well as within, electroimmunoblots were overcome by the introduction of an internal standard in each sample, thus allowing reproducible quantification of MBP. The Ca2+-dependent protease acting on MBP was active at endogenous levels of Ca2+ (congruent to 300 micrograms/g myelin) and was inhibited in the presence of Ca2+ chelators. Extensive degradation of MBP occurred rapidly in the presence of added Ca2+, reaching a plateau after a 1 h incubation (80-85% degradation). The proteolytic activity was not enhanced in the presence of 2-mercaptoethanol. It was most active at neutral pH and at temperatures approaching physiological conditions. No difference was observed between proteolytic activities of control and multiple sclerotic myelin. It is suggested that fluctuations in the accessibility of free Ca2+ to the protease may lead to the regulation of Ca2+-activated myelinolysis.

Topics: Brain; Calcium; Calpain; Edetic Acid; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Humans; Hydrogen-Ion Concentration; Immunologic Techniques; Mercaptoethanol; Multiple Sclerosis; Myelin Basic Protein; Myelin Sheath; Temperature