The focus of this grant is the assembly of the plasma membrane proteolipid protein (PMPLP) in the synaptic plasma membrane and in myelin. This project will utilize a series of cell biologic techniques to follow this protein from its cite of synthesis in neurons and oligodendrocytes to the respective target membranes, eg. synaptic plasma membrane and myelin. Determination will be made of processing of PMPLP during the membrane assembly focusing on the acylation of the protein. The relative rate of assembly of this protein into myelin will be determined and compared to other myelin proteins such as the major myelin proteolipid and the myelin associated glycoprotein. Coated vesicles will be isolated from white matter and the presence of PMPLP and other myelin proteins determined and studies will be carried out to determine if these proteins have been newly synthesized. We will use immunologic methods to try to select as well as visualize subpopulations of coated vesicles that may transport the PMPLP or other myelin proteins. The final localization of PMPLP in the assembled myelin membrane of rat spinal cord will be examined using cryo- ultramicrotomy and immunoelectron microscopy. Studies will be carried out with anti PMPLP and visualized with colloidal gold conjugated second antibody. The assembly of the plasma membrane proteolipid into synaptic plasma membranes will be studied. Using immunological techniques we will examine if, in grey matter derived coated vesicles, PMPLP defines a specific sub class of vesicles and whether the sub class can be distinguished from those carrying synaptic vesicle proteins. The localization of PMPLP to specific membrane domains at the synapse will be determined using cryo- immunoelectron microscopy on ultra thin frozen sections of the rat dentate gyrus. The presence of PMPLP at the synapse will be analyzed in animals at different ages when a marked synaptogenesis and maturation of synapses is taking place in this brain region. The association of synaptic dysfunction with mental retardation and epilepsy suggests that this major synaptic protein could be involved in both the physiology and pathophysiology of the brain. Moreover, its associated with the myelin sheath suggest that not only may it be involved in the integrity of myelin but the abnormalities in the assembly of this protein could give rise to a constellation brain dysfunction associated with demyelinating and hypomyelinating diseases.
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