The long-term objective of this proposal is to study the molecular mechanism(s) of fetal neuronal development and neuronal differentiation under the effects of the female hormone, estradiol (E2). The investigators hypothesize that E2 induced nNOS is an important molecular mediator of E2 mediated sustenance of neuronal differentiation in NGF-withdrawal PCl2 cells. This hypothesis will be tested under the following specific aims: to determine the molecular mechanism by which E2 sustains neuronal differentiation and neurite outgrowth of PCl2 cells, derived originally from a rat pheochromocytoma, by determining whether E2-induced NO is required for the continued differentiation program; and to identify the cis-acting response elements in the nNOS promoter, the transcription factors that transactivate the nNOS gene, and the signaling transduction pathways required for estrogen-mediated induction of nNOS gene expression. These experiments will direct future studies critical to the understanding of E2- mediated fetal neuronal development and neuronal differentiation, that will lend insights into the approach to neurodegenerative diseases. In order to achieve these goals, the research design and methods will include the PC12 cell line, which is widely used as a model system to study NGF- mediated neuronal differentiation. The E2 mediated molecular mechanisms which participate in sustaining PCl2 cell differentiation following NGF-withdrawn are not known. Blocking production of Nitric Oxide (NO) with an inhibitor of NOS, N (omega)-nitro-L-arginine methyl ester (L-NAME), but not D-NAME, inhibits neurite extension, implicating nNOS in the differentiation process. E2, like NGF, markedly induces nNOS activity and mRNA levels in neuronal cells and anti-estrogen drugs act as strong inhibitors of purified nNOS, suggesting that E2 may sustain neuronal differentiation and neurite growth via an NO- mediated process. Based on these observations, the investigators hypothesize that E2 induced nNOS is an important molecular mediator of E2 mediated sustenance of neuronal differentiation in NGFwithdrawal PCl2 cells.
