Identifying the factors which influence the regenerative potential of neurons represents an important step in developing therapeutic strategies to promote axon regeneration in the mammalian CNS. We have amassed substantial preliminary evidence indicating that, contrary to previous assertions, alterations in the regenerative potential of peripheral sensory fibers, as reflected in a rise in the rate of regeneration in response to a test lesion (axonal injury) made several weeks after a so- called conditioning lesion, correlate with an increase in the amount of tubulin transported in the SCb component of slow axonal transport. Furthermore, we have found that this increase in the amount of tubulin transported in SCb is associated with a selective elevation in the expression and transport of the class II isotype of beta-tubulin betaII- tubulin). We postulate that level of expression of betaII-tubulin (and the amount of betaII-tubulin transported in nerve fibers) influences the rate of regeneration by determining the amount of tubulin transported in SCb. We plan to test our hypothesis by examining the relationship among the level of expression of betaII-tubulin, the amount of betaII-tubulin transported in nerve fibers, the amount of tubulin transported in SCb, and the rate of regeneration in two model systems, peripheral sensory fibers and optic nerve fibers regenerating in a peripheral nerve graft. The tubulin transported in these nerve fibers will be labeled by injecting 35S-methionine into L5 dorsal root ganglion and eye, respectively. The axonal transport of pulse-labeled betaII-, betaIII- tubulin, and total beta-tubulin will be analyzed using immunoprecipitation with isotype-specific monoclonal antibodies. Since the increased rate of regeneration after a conditioning lesion also correlates with a decrease in neurofilament (NF) expression and a reduction in axonal NF content, we will examine the-influences of NF expression and axonal NF content on both the rate of regeneration and the relative amount of tubulin transported in SCb. Finally, we will examine the specific influence of betaII-tubulin expression on the rate of neurite elongation by selectively altering the expression of specific beta-tubulin isotypes in cultured neurons using either viral vectors or antisense oligonucleotides.
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