The proposed experiments will be concerned with the localization and function of enzymes related to myelination, transport across the myelin sheath, and the maintenance of myelin. Biochemical isolation methods and enzyme assays have been used to demonstrate carbonic anhydrase (CA) and 5'-nucleotidase activities in myelin and to quantify these enzyme activities in myelin itself and in the cells that synthesize and maintain myelin, the oligodendroglia. The next logical steps are precise immunocytochemical localization of CA and 5'-nucleotidase in the nervous system and elucidation of their function in myelin and oligodendroglia. Although the abundance of CA in oligodendroglia is now well-known, the presence of CA in astroglia and compact myelin is controversial and will be investigated by immunocytochemical methods. Immunocytochemistry with anti-CA will also be used as a tool for identifying cells in the spinal cords of dysmelinating mutant (shiverer) mice, for visualizing the processes of satellite oligodendrocytes in normal and shiverer mouse gray matter, and for characterizing developing oligodendroglia and their processes in white matter during normal myelination. A hypothetical function of CA, that of providing bicarbonate to acetyl CoA carboxylase for lipid biosynthesis, will be tested in normal white matter homogenates and oligodendroglia. Immunocytochemical localization of 5'-nucleotidase in the developing rat, normal mouse, and shiverer mouse CNS will also be undertaken, and rat sciatic nerves will be embedded in paraffin for immunocytochemical localization of both CA and 5'-nucleotidase. The hypothetical transport function of 5'-nucleotidase will be tested in vitro in myelin vesicles. The understanding of the precise locations and the functions of these enzymes in myelin, oligodendroglia and Schwann cells will represent progress toward defining the mechanisms and sources of stress (e.g., neurotoxins, infection, or the inflammatory response) that could damage myelin directly or perturb glial cell function, and toward devising ways of preventing such stress.
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