In this application we propose a novel molecular genetic approach to the study of protein trafficking in the oligodendrocyte and to the assembly of myelin sheaths from its components. These studies are made possible by the development of transgenic mice that can carry and expression oligodendrocytes, genes encoding foreign proteins. The foreign proteins employed in these studies (the VSV G, influenza HA and VSV-M) have been well characterized and their intracellular transport extensively studied by others in polarized epithelial cells and fibroblasts. These studies have shown that the VSV G and M proteins contain """"""""address"""""""" signals that target them to the basal-lateral membrane domain, while the Flu-HA contains signals targeting it to the apical domain of polarized epithelial cells. We propose to prepare transgenic mice in which these viral genes are controlled by myelin basic protein's promoter/enhancer so that their expression will be limited to the oligodendrocyte and Schwann cells. These proteins, synthesized in situ, will be used to probe the trafficking of proteins within the oligodendrocyte and to define those features necessary for correct incorporation in myelin. Our preliminary work with transgenic mice expressing the VSV G indicates that the G enters the myelin sheaths synthesized by both Schwann cell and oligodendrocyte. We propose to further document the localization of the G protein in myelin and its relative impoverishment in cell membrane by biochemical and immunoelectron microscopic methods. We will create transgenic mice expressing the Flu-HA and study the localization of this protein. If the targeting of this protein differs from that of VSV G, we will prepare transgenic mice expressing VSV G/Flu-HA fusion proteins to identify the region of carrying the important """"""""address"""""""" signals, as was done by others using polarized epithelial cells. The transport and targeting of a natural myelin transmembrane protein, PLP, will be studied in cultured epithelial cells and in transgenic mice. The location of address signals of this protein will be probed by examining the transport and sorting of PLP deletion mutants in both cultured epithelial cells and in the oligodendrocytes of transgenic mice. Extrinsic myelin membrane proteins which do not pass through the Golgi network presumably are targeted and transported by a separate mechanism from that transporting proteins matured in the Golgi. We will examine whether the oligodendrocyte system for sorting extrinsic proteins is related to that which targets viral M proteins to specific membrane domains in epithelial cells by preparing transgenic mice that express the VSV, measles and Flu M (membrane) proteins. Finally, we will examine whether foreign extrinsic membrane proteins which are targeted to myelin can substitute for myelin basic protein by crossing the appropriate transgene onto a shiverer mouse background.
