Proteomic analysis of the myelin membrane and its lipid microdomains

Myelin is a physiologically active, highly polarized membrane that allows for dramatic savings in space, time and energy. The importance of myelin in humans is highlighted by the number of debilitating neurological diseases that occur when myelin is disrupted, such as leukodystrophies, central and peripheral neuropathies, and inflammatory demyelinating diseases such as multiple sclerosis. In order to begin to better treat these diseases, an improved understanding of the biological processes of myelin biogenesis, maintenance and function is needed. In particular, the identity of the proteins involved in these processes, as well as the organization of these proteins within membrane microdomains to produce a highly polarized membrane, will be invaluable information for the myelin biology community as well as for the treatment of many myelin disorders. ^ The specific aims of this thesis are to: (I) Establish a two-dimensional proteomic map of myelin proteins. This has involved the application of high-resolution two-dimensional gel electrophoresis, western blotting, mass spectrometry, and bioinformatics to identify both major and minor proteins. This approach has allowed us to map the gel coordinates of 98 myelin proteins (based on their unique isoelectric points and molecular weights), as well as identify novel myelin proteins, providing a comprehensive database for future research. (II) Analyze the protein composition of myelin glycosphingolipid microdomains. This Aim has employed selective detergent solubilization of myelin, developed through a detailed analysis of alternative extraction parameters, in order to isolate glycosphingolipid microdomains as detergent-insoluble, low-density membrane fractions. Coupled with techniques used in Aim 1, we were thus able to identify proteins specifically partitioned into glycosphingolipid microdomains. This identification is providing a more complete understanding of the organization of proteins and lipids in myelin, and in view of their role in signal transduction, provides insights into the functional biology of myelin.