MOLECULAR MECHANISMS OF NEURONAL DIFFERENTIATION

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The mechanisms by which acquisition of the neuronal phenotype is controlled in the mammalian nervous system are poorly understood. Our work is aimed at defining the signaling pathways for two prominent regulators of neuronal phenotype, neuronal growth factors and excitability. The rat pheochromocytoma cell line, PC12, is the premier cell culture model for the differentiating actions of the neurotrophin, NGF. Major actions of NGF on PC12 cells, including the extension of neuronal processes (neurites), stimulation of neurotransmitter synthesis, the induction of neural genes, and the prevention of apoptotic cell death are all mediated through a proto-oncoprotein pathway in which Ras is a central component. A series of partial loss- of-function mutants of Ras and mutants of putative Ras-effector proteins, transfected into PC12 cells, will be used to reveal the Ras downstream effector proteins that control distinct neural traits. Depolarization-induced calcium influx exerts long-term effects on neuronal phenotype. Mutant forms of Src and PYK2 protein tyrosine kinases expressed in PC12, will be used to determine the relative contributions of these kinases in causing downstream calcium-mediated phosphorylation events required for both Ras-dependent and Ras- independent pathways. The involvement of the long term tyrosine phosphorylation events in mediating neuronal phenotypic and apoptotic changes will be determined by specific manipulation of the stimulated tyrosine kinase. Rin is a novel calcium/calmodulin-activated protein that is related to Ras and present exclusively in neuronal cells. Rin and an activated form of Rin will be expressed in PC12 cells to determine the extent to which Rin can regulate distinct aspects of neuronal phenotype in response to calcium elevation, and to determine if Rin can interact with the normal Ras signaling effectors. Neuronal growth factors and calcium influx control neuronal phenotype and cell death through independent but convergent signaling pathways. Our studies are aimed at elucidating mechanisms for these controls, the disruption of which underlies a variety of degenerative nervous system disorders.