Oligodendrocytes the myelin forming cells of the CNS, develop from an identified precursor cell (OPC). OPCs are abundant in developing and adult animals suggesting that these cells carry out functions in addition to providing a pool of oligodendrocyte precursors. OPCs express on their surfaces NG2 a chondroitin sulfate proteoglycan that can inhibit axonal growth and guide axon elongation in vitro. The expression of NG2 is maximal during development and again when the brain suffers an injury. This suggests that OPCs can provide negative, growth inhibitory cues to both developing and regenerating axons and that NG2 is the biochemical basis for this contact mediated growth inhibition. The goal of this proposal is to test this hypothesis of OPC function.
Under Specific Aim 1 the complete distribution of OPCs and NG2 in the developing CNS will be mapped and compared to the distribution of newly formed axon tracts and to other putative growth inhibitory molecules. This data will provide the anatomical framework against which to evaluate the hypothesis that OPCs define areas that are non-permissive for axon growth during early CNS development.
Under Specific Aim 2 the membrane properties of OPCs will be directly evaluated using a in vitro membrane carpet assays. Fusion proteins that encode small regions of NG2 will be prepared and tested for their ability to inhibit axonal outgrowth and neutralizing monoclonal antibodies prepared against those domains of NG2 that are active in these assays. Knock-in cell lines will be created that express these growth inhibitory domains and the ability of these lines to inhibit axon growth evaluated. Time-lapse video microscopy will be used in Specific Aim 3 to determine whether the induction of growth cone collapse is a mechanism by which OPCs inhibit axonal growth.
In Specific Aim 4 the ability of membranes prepared from CNS scar tissue to promote or inhibit axonal outgrowth will be tested and the role of NG2 in any inhibition evaluated using the domain specific neutralizing anti-NG2 antibodies.
This aim also includes a detailed biochemical analysis of the temporal patterns of expression of growth promoting and growth inhibiting molecules after injury. The proposed studies will provide important information concerning the functions of a newly recognized class of glial cells during development, tissue repair and regeneration. They will also provide information concerning the functions of proteoglycans in the CNS.
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