The long range goal of this research program is to understand the role of the proteolipid protein (PLP) in the development,structure and maintenance of CNS myelin. The immediate goal is to study the organization of PLP in myelin. We have previously proposed a model for the topography of PLP in the myelin membrane and will continue to test the model,using new approaches. 1. The orientation of PLP domains in the plasma membrane and membrane expansions of oligodendrocytes will be studied in primary cell cultures. Antibodies against synthetic peptides corresponding to sequences in key PLP domains will be visualized by fluorescence microscopy and immunogold electron microscopy (EM). Antibodies against myelin basic protein and galactocerebroside will be utilized as markers for the cytoplasmic and external faces, respectively. 2) The segregation of PLP along different morphological areas of myelin (e.g.compact, paranodal loops, etc.) will be visualized in mouse optic nerve myelin by immunogold EM, and the orientation of PLP domains will be visualized in isolated myelin vesicle preparations. 3) Myelin deficient mouse mutants will be studied to relate the effects of the genetic abnormalities in shiverer and jimpy mice to the segregation and/or orientation of PLP. Shiverer optic nerve will be examined for possible effects of the absence of myelin basic protein on PLP orientation and jimpy mice will be studied to understand the consequences of the abnormal PLP molecules on myelin structure. The multiple approaches overcome limitations inherent in a single method and should lead to in-depth characterization of the organization of PLP in myelin. Detailed knowledge of the orientation of PLP is relevant to the elucidation of factors that maintain the compact,multilamellar structure characteristic of myelin and is specifically important for understanding the pathophysiology od demyelinating disorders,particularly multiple sclerosis.
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