MOLECULAR BASES OF NEURONAL CONNECTIVITY

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DESCRIPTION: Although injury to the visual system in mature mammals generally results in a permanent loss of function, recent studies show that CNS neurons can be induced to regenerate their axons if exposed to appropriate cellular and molecular conditions. The first two aims of this proposal will test the hypothesis that one factor that stimulates retinal ganglion cells to regenerate their axons is a small molecule, AF-1. In goldfish, a species in which CNS neurons regenerate their axons spontaneously, we have found that glia of the optic nerve secrete two factors that induce ganglion cells to extend long axons in culture. AF-1, the more potent of the two factors, exerts an equally dramatic effect on ganglion cells of the rat. We will obtain AF-1 and AF-2 from glial cultures and purify these to homogeneity by gel filtration, reverse-phase, and hydrophilic affinity chromatography. Based upon the sequence data, we will generate synthetic peptides, test these for activity and utilize them to generate antibodies. We will utilize the predicted nucleotide sequence and/or the antibodies to isolate the fish and human genes that encode the putative precursor proteins. We will examine whether AF-1 enhances axonal outgrowth in mammalian systems ranging in complexity from isolated ganglion cells to retinas with peripheral nerve grafts in vivo. We will investigate whether AF-1 works synergistically with the defined neurotrophin, BDNF, and examine whether it stimulates expression of proteins involved in axonal regeneration in vivo, e.g., the membrane phosphoprotein GAP-43. Aim 3 will examine the hypothesis that the expression of GAP-43 is regulated in part through controlling the stability of its mRNA. We have identified regions within the 3' untranslated region of GAP-43 mRNA which serve as binding sites for proteins that may be important in regulating mRNA stability, and have identified three proteins that bind to these regions. Using PC12 cells as a model system, we have found that the binding of one of the proteins is induced by NGF and parallels the increase in GAP-43 expression. We will use a variety of molecular biological approaches to examine the importance of the identified nucleotide domains and the associated binding proteins in controlling the stability of GAP-43 mRNA. Together, these studies should add considerably to understanding molecular mechanisms that control the neuron's growth state, and may ultimately contribute to the development of methods for enhancing regeneration of injured connections in the human visual system.