Changes in the ionic and cellular environment in and around multiple sclerosis plaques affect the ability of mature OLs to maintain myelin sheaths and reform myelin after injury. Understanding the regulation of myelin assembly and maintenance in mature OLs will lead to strategies for blocking demyelination or enhancing remyelination in demyelinated areas. Previous studies from this laboratory have shown that a sustained increase in intracellular Ca++ levels in cultured OLs leads to a reversible retraction of membrane sheets. To further understand the relationship between Ca++ levels and maintenance of membrane sheets, they will examine the capacity of mature OLs to regulate calcium when exposed to ionophores, depolarization, inhibition or activation of voltage-gated Ca++ channels, or release of intracellular calcium. Morphology and maintenance of membrane sheets will be assessed by immunocytochemical staining and quantitative morphometry. Cell viability will be evaluated by visualization of nuclear lysis with propidium iodide and assay of mitochondrial function. Changes in Ca++ levels will be measured with Indo-1 and lase cytometry imaging. Agents identified as altering Ca++ levels, damaging mature OLs, or disrupting membrane sheets will be examined for their effects on the pattern of IEG and myelin protein gene expression. The IEGs to be examined are c-fos, c-jun, jun b, HLH 462 and zif 268, selected because of their known activity as transcription factors and their responsiveness to injury in OLs or other cell types. Myelin protein genes are likely to be downstream targets of IEG changes. In situ hybridization, immunocytochemistry, and Western and Northern blotting will be used to examine expression of the genes. The pattern of changes will be correlated with the ability of OLs to recover and maintain or reform membrane sheets. Another series of experiments will examine a critical end point in myelin assembly, the transport of intracellular vesicles containing PLP/DM-20 into and out of the plasma membrane of OL and associated membrane sheets. The balance between insertion and removal of PLp/DM-20 in the plasma membrane of OL cell bodies and membrane sheets will be examined by exposing cells to inhibitors of endocytosis. Effects of co-culture with neurons from dorsal root ganglia and the effects of upregulation of PLP/DM-20 synthesis with cyclic AMP will be assessed using immunocytochemistry and iodination of surface PLP. Related studies of insertion of galactocerebroside (GalC) and sulfatide into the extracellular face of the plasma membrane will be carried out in the immortalized OL cell line N20.1 which synthesizes the galactolipids but does not express them on the surface of the cell.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurological Sciences Subcommittee 1 (NLS)
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Kerza-Kwiatecki, a P
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Wayne State University
Schools of Medicine
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Benjamins, Joyce A; Nedelkoska, Liljana (2007) Cyclic GMP-dependent pathways protect differentiated oligodendrocytes from multiple types of injury. Neurochem Res 32:321-9
Boullerne, Anne I; Benjamins, Joyce A (2006) Nitric oxide synthase expression and nitric oxide toxicity in oligodendrocytes. Antioxid Redox Signal 8:967-80
Studzinski, Diane M; Benjamins, Joyce A (2005) Expression of P0 glycoprotein in CNS glia: effects of overexpression in N20.1 cells. Glia 52:234-44
Benjamins, Joyce A; Nedelkoska, Liljana; George, Edwin B (2003) Protection of mature oligodendrocytes by inhibitors of caspases and calpains. Neurochem Res 28:143-52
Studzinski, Diane M; Benjamins, Joyce A (2003) Regulation of CNS glial phenotypes in N20.1 cells. J Neurosci Res 73:31-41
Studzinski, D M; Benjamins, J A (2001) Cyclic AMP differentiation of the oligodendroglial cell line N20.1 switches staurosporine-induced cell death from necrosis to apoptosis. J Neurosci Res 66:691-7
Boullerne, A I; Nedelkoska, L; Benjamins, J A (2001) Role of calcium in nitric oxide-induced cytotoxicity: EGTA protects mouse oligodendrocytes. J Neurosci Res 63:124-35
Studzinski, D M; Callahan, R E; Benjamins, J A (1999) Increased intracellular calcium alters myelin gene expression in the N20.1 oligodendroglial cell line. J Neurosci Res 57:633-42
Boullerne, A I; Nedelkoska, L; Benjamins, J A (1999) Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1. J Neurochem 72:1050-60
Studzinski, D M; Ramaswamy, R; Benjamins, J A (1998) Effects of cyclic AMP on expression of myelin genes in the N20.1 oligodendroglial cell line. Neurochem Res 23:435-41

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