Title

Vesicular trafficking SNARE proteins may be utilized for myelin membrane synthesis by oligodendrocytes

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Cell|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Myelination involves the coordinated and rapid distribution of lipids and proteins into oligodendrocyte processes for axonal enwrapment. Little is known regarding the precise targeting mechanisms for myelin lipids and proteins, although certain aspects of this process must occur via a vesicular trafficking mechanism. In highly purified culture systems, oligodendrocytes differentiate to a point inclusive of myelin-like membrane synthesis, thus allowing for in vitro study of the machinery that delivers myelin membrane. The small GTP-binding class of proteins are involved in the targeted delivery of vesicles in a number of different cell types and transport pathways. Two-dimensional gel electrophoresis followed by probing with radiolabeled GTP demonstrated that a number of these proteins are developmentally up-regulated during myelin synthesis. Proteins of both the endocytic (Rab4, 5, 7) and exocytic (Rab3, 11) pathways were identified as well as Golgi (Rab6) and Endoplasmic reticulum (Rab2) localized proteins. Further characterization by PCR analyses of rab3 isoforms, a protein thought to be specific for neuroendocrine and synaptic vesicles, indicated that oligodendrocytes preferentially express rab3a. Other proteins involved in the formation of vesicle docking complexes at the plasma membrane were studied. The proteins VAMP-2/synaptobrevin-2, syntaxin-2, synapsin, synaptophysin, nsec-1, were expressed in oligodendrocytes, while the regulatory proteins synaptotagmin 1 and rabphilin-3a were not detected. In addition, the neuron specific t-SNARE syntaxin-1 was not detected. The t-SNARE SNAP-25 was detected as a rare mRNA without detection of protein.^ Overexpression of dominant negative mutants of the v-SNARE VAMP-2, induced morphological changes including a reduced number of processes and decreased myelin-like membrane formation. The results are suggestive of VAMP-2, and subsequently SNARE complexes, participating in the vesicular delivery of myelin membrane in the actively myelinating oligodendrocyte. ^