Multiple Sclerosis (MS) is a debilitating autoimmune demyelinating disease, which leads to paralysis and other functional disabilities. The factors responsible for myelin destruction in MS are not clearly understood. Thus, a pathophysiological/therapeutic study to minimize or prevent myelin damage and maximize functional recovery is warranted. Findings from animals with experimental allergic encephalomyelitis (EAE), an animal model for MS, demonstrate increased calpain activity and expression concomitant with myelin protein degradation in the CNS signifying a pivotal role for calpain in the mechanism of myelin breakdown. Using calpain inhibitors and methylprednisolone (MP) alone and in combination as therapeutic agents could inhibit myelin protein degradation, protect oligodendrocytes, and delay or prevent development of the disease. In this project, we will focus our studies on the following specific aims: 1. Define the time-course of calpain expression and activity in the spinal cord and spleen/lymph nodes in animals with EAE. 2. Examine the effects of calpain inhibitors and anti-inflammatory agents affecting myelin protein degradation which could delay or prevent the development of EAE. 3. Study the role of calpain in the induction of apoptosis in vitro in neural cells and how calpain inhibitors affect apoptotic death in glial/inflammatory cells in EAE. To support these specific aims, the following experiments are proposed: 1) Determine calpain/calpapstatin activity, expression (mRNA, protein), and the specific cellular localization (by double immunofluorescence staining) in spinal cord and spleen/lymph nodes at various times after challenge; 2) Examine the effects of cell-permeable calpain inhibitors alone and in combination with methylprednisolone in EAE development; 3) Determine calpain/calpapstatin activity, expression, and cellular localization in treated verses untreated animals and correlate these findings with the extent and type of cell death in the CNS; 4) Determine the effects of cytokines on intracellular calcium levels, calpain activation, and apoptosis in glial cell cultures; 5) Examine calpain expression, activity, and secretion from activated MPP-specific T cells and determine immunogenicity of MBP peptides generated by these cells.
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