(VERBATIM FROM APPLICATION): Directed neuronal growth depends upon the dynamics of actin filaments within the highly motile growth cone. Although actin dynamics are regulated by the complex interactions of many actin-binding proteins, the high turnover rate of actin filaments in vivo is dependent upon proteins in the actin depolymerization factor (ADF)/cofilin (AC) family. AC proteins are essential in all eukaryotes and are enriched within neuronal growth cones. Furthermore, overexpression of ADF in neurons leads to an enhanced rate of outgrowth that is sustained for days. AC proteins are phosphorylated on a single regulatory site. Their phosphorylation and dephosphorylation are common targets of signaling pathways linking external signals to actin cytoskeletal reorganization. Enhanced dynamics of this phosphocycle, without a net change in the phosphorylation state of the AC proteins, often accompanies induction of ruffling membrane. Vertebrate AC proteins are phosphorylated by LIM kinases for which they are the only known substrates. Many growth factors and guidance cues signal in part through the rho family GTPases, each of which has specific targets for actin organization, but all of which target at least one member of the LIM kinase family. Hemizygosity of LIM kinase I causes Williams syndrome, a visuo-spatial cognitive defect resulting from aberrant neuronal migration in the human brain. Thus we hypothesize that the response of growth cones to various guidance cues is modulated by the regulation of AC activity, which is likely to be the final integrator of multiple signaling pathways. We propose to use a combination of molecular, cellular, immunological, and ultrastructural approaches to test the hypothesis that phosphorylation regulation of ADF activity is required for initiation of neuritic outgrowth cone pathfinding in response to attractive or repulsive guidance cues. Tropomyosin isoforms, which may compete with AC for actin filament binding, and Arp2/3, which caps pointed ends of actin and may regulate the ability of AC to depolymerize the filaments, will be localized along with AC and phosphorylated AC in growth cones undergoing a turning response. We will also test the hypothesis that guidance cues signal via bifurcating pathways that regulate both AC phosphorylation and dephosphorylation through activation of PI-3 kinase, and we will isolate and characterize the phosphatase involved. Adenoviral mediated gene transfer is a major tool to be used in sorting out the signal transduction pathways in regulating AC. These studies will advance our understanding of nerve growth cone guidance and the design of agents that allow neurons to grow and regenerated in a normally non-permissive environment.
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