Understanding the processes underlying morphological differentiation of neurons is a central issue in the study of degenerative and regenerative phenomena in the human nervous system. Although neuritic outgrowth is closely linked to cytoskeletal reorganization, and is significantly influenced by neurotrophic molecules, little is known about the mechanisms by which neurotrophic signals mediate such changes. The overall goal of the proposed research is to examine the specific subcellular processes underlying growth cone behavior, using cultures of rat brain neurons undergoing morphological remodelling induced by neurotrophin-3 (NT-3). Specifically, effects of NT-3 on growth cone size and shape will be analyzed using light microscopy and computer-assisted morphometry, while fluorescence techniques will be employed in examining the accompanying alterations in the cytoskeleton. These observations will serve to characterize the model system for subsequent mechanistic studies testing the hypothesis that NT-3 effects on the growth cone are mediated by members of the Rho subfamily of Ras-related guanylate cyclases, which are known mediators of actin-based morphological effects. PCR will be used to verify neuronal expression of Rho proteins, and fluorescently-tagged fusion proteins will be generated and used to observe Rho protein localization in living cells. The role of Rho protein activation in NT-3 induced growth cone remodelling will be revealed via specific perturbations, including inactivation by C3 transferase of C. botulinum and expression of dominant inhibitory forms of Rho proteins in primary cultures and NT-3 responsive PC12 cells.
