The overall aim of the proposed research is to achieve an understanding of the molecular basis of neuronal growth factor actions. These actions include the establishment, maintenance and regeneration of neuronal properties including morphology (axons and dendrites), the action potential mechanism, neurotransmitter storage and release, and synaptogenesis. One such growth factor, the Nerve Growth Factor (NGF), is the one best established to have such roles in vivo. NGF is required for the survival and differentiation of the major sympathetic and sensory peripheral neurons. Including those affected in familial dysautonomia, as well as of central nervous system neurons including those cholinergic neurons affected in Alzheimer's disease. Based on previous results with the neuron-like cell line, PC12, we have established a model for the signal transduction of NGF and the second messengers which generate the growth factor actions. In the model, the cellular photo-oncogenes src and ras, transduce the NGF signal into the stimulation of two second messenger systems, cAMP and phosphatidylinositol turnover, and of their associated protein kinases. The present research will test the model directly by specific inhibition of the second messengers and of the protein kinases followed by an assessment of the resulting inhibition of their predicted functional roles in NGF actions. One set of assays involves the analysis of tyrosine hydroxylase phosphorylation (by phosphopeptide mapping) and activation (by an in situ assay), an event which results in the enhancement of catecholamine neurotransmitter biosynthesis. Another assay involves analysis of transcriptional activation of another photo-oncogene, fos, believed to provide one link, through changes in gene expression, between short and long term NGF actions. The generation and growth of neural processes, caused by NGF, will be addressed in order to examine the role of the various second messengers in this important event. The specific roles of ras and src in NGF signal transduction will also be determined through their specific inhibition and by the introduction (by gene transfection) of their oncogenic """"""""activated"""""""" forms into cells, followed by assessment of NGF actions as described above. These approaches are expected to provide insights into the possible causes of the loss of critical neural properties as well as rational approaches to treatment in Parkinson's, Alzheimer's, familial dysautonomic and other related central and peripheral degenerating diseases.
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