The long term objectives of this proposal are to learn how nerve growth factor (NGF) production is regulated and how NGF regulates neuronal survival and function.
The specific aims of this proposal are to elucidate the mechanisms that underlie basal and modulated NGF gene expression. To address regulation at the level of transcription, the rat and mouse NGF gene promoters were cloned. The promoters were linked to the human growth hormone (hGH) gene and the fusion genes shown to express at low levels in L929 cells, reflecting the normally low levels of NGF production in neuronal targets. In contrast, a NGF/hGH fusion gene was expressed at high levels in the submandibular gland of transgenic mice, reflecting the normally high levels of NGF production in this non-target tissue. These results show that the 250 to 1000-fold difference in expression is transcriptionally mediated but the mechanisms are unknown. Treatment of L929 cells with IL-1 and the phorbol ester TPA transiently increased NGF mRNA levels. However, the underlying mechanisms are presently unknown. To elucidate the mechanisms that mediate the large difference in the basal transcription of the NGF gene in neuronal targets and SMG, the basal cis elements will be mapped by deletion analyses using transient expression in L929 cells. Regions containing cis elements will be subjected to footprint analyses with nuclear extracts from SMG, L929 cells, and liver to identify differentially bound nuclear factors. To determine if footprints occur on basal cis elements, contacts between transacting factors and cis elements will be determined with methyl interference assay's; the corresponding bases mutagenized, and the effects assessed in transient expression assays. Transacting activator factors will be characterized by gel-shift analyses, Southwestern blots, and will be cloned using expression libraries from L929 cells. To study the mechanisms by which IL-1 and TPA raise NGF mRNA levels in L929 cells, the time course and cycloheximide-sensitivity of the response will be characterized. The transcriptional component will be studied using transient expression of 5' deletion mutants of NGF/hGH in IL-1 and TPA-stimulated L929 cells. Transcriptionally active segment will be subjected to footprinting before and after IL-1 or TPA treatment, and the nuclear binding proteins characterized. The post-transcriptional component will be studied by transient expression of fusion genes containing the wild-type or mutagenized 3"""""""" ends of the NGF gene. Collectively, these experiments are important steps toward a complete understanding of NGF biosynthesis and the rational design of pharmacologic agents that stimulate or inhibit NGF production in diseases that are associated with functional NGF deficiency of excess, including Alzheimer disease.
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