The proposed research concentrates on three aspects of axonal regeneration in the nervous system. The first specific aim addresses the investigator's hypothesis that the repellent Sema3A might interfere with the regeneration of peripheral sensory and motor axons were it not for the presence of Sema3B and Sema3C in the peripheral nerve. He proposes that these two related semaphorins directly antagonize Sema3A repellent activity by competing for a receptor component, neuropilin-1. Sema3B or C would be overexpressed in transgenic mice either in Schwann cells using the P0 promoter or in epidermal cells using a keratin promoter. Alternatively, a soluble form of NP-2 that should bind and inactivate Sema3B and C, but not A, could be expressed in the same way. If overexpression of Sema3B or C increases regeneration, and overexpression of soluble NP-2 decreases regeneration, the hypothesis would be supported. The second specific aim is directed towards an analysis of the molecule recently purified by the Schwab group, NI250, which is purported to be a general inhibitor of regeneration in the CNS. The Schwab group published microsequence data from the protein they have spent the last 10 years characterizing and the investigator has used this information to identify a full-length EST clone. Preliminary studies suggest that a recombinant version of the very small extracellular portion of this protein can inhibit axon outgrowth. The investigator proposes to determine where NI250 is expressed, to see if any of the proteins most related to NI250 have similar outgrowth inhibiting activities, to define those portions and residues within the extracellular moiety that are required for inhibitory activity, and to generate an alkaline phosphatase (AP)-tagged version of this extracellular moiety to search for a receptor. Finally, the investigator has identified 5 stem cell lines in the Omnibank database in which the NI250 gene is likely to be disrupted. He proposes to analyze the resulting KO mice for normal myelin function and their capacity for regeneration. If the mice develop normally, and if NI250 is a major impediment to regeneration in the CNS, then regeneration may be improved in the KO mice. The third specific aim is focused on the further characterization of three molecules that have been identified with microarray techniques to be upregulated after crushing the sciatic nerve, and which may therefore be associated with regenerative events. It is proposed to further characterize their normal distributions and regulation during regeneration, and to determine if their overexpression (with a herpes virus expression system) in cultured DRG or retinal neurons affects various parameters of axon outgrowth. Similar assays will be performed while reducing the expression of these proteins in cultured cells with trituration-loaded antibodies or antisense oligonucleotides.
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