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.
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