In addition to their role in the growth, development and maintenance of normal brain structure and function, neurotrophic growth factors have been implicated in neurodegenerative disorders. The overall goal of the proposed study is to gain an understanding of the molecular mechanisms by which neurotrophic growth factors act to regulate gene expression during neural development, degeneration and regeneration. These investigations have been facilitated through studies of the effects of NGF on PC12 pheochromocytoma cells, a cell line of adrenal origin that responds to NGF, but not EGF, by extending neurities and becoming electrically excitable. Using subtractive screening methods, we have cloned and characterized an mRNA that is rapidly, transiently and selectively regulated in PC12 cells by the neurotrophic growth factors NGF and basic fibroblast growth factor (bFGF), but not by EGF and insulin. This clone, NGF33.1, corresponds to an mRNA (VGF) that is nervous system-specific with peak levels found in developing and adult central and peripheral nervous tissues. VGF is therefore the most rapidly and selectively regulated neural mRNA yet identified; in addition, VGF mRNA is reinduced following readdition of NGF to neuronally-differentiated PC12 cells that were previously washed and deprived of NGF. We propose to use the VGF gene as a model to investigate the early events and the selective actions of NGF and bFGF in neural differentiation and in regeneration following neuronal injury. We have cloned and fully sequenced the VGF gene; transcriptional and post- transcriptional regulation of VGF gene expression will be investigated by transfection analysis and studies of VGF mRNA stability. The neural- specific expression of VGF mRNA during development will be further characterized using in situ hybridization, more extensive RNase protection analysis, and through the production of transgenic mice. We propose to study the function of the polypeptide encoded by the VGF gene by transfecting eukaryotic expression vectors, containing the VGF coding sequence in sense and anti-sense orientations, into PC12 cells, and by characterizing the effect(s) of antisera, generated against the encoded VGF polypeptide, and anti-sense oligonucleotides on PC12 cells and primary neural cultures. Further characterization of the VGF gene and encoded polypeptide offers a unique opportunity to assess the rapid but selective mechanism(s) by which NGF and bFGF and possibly other neurotrophic growth factors trigger neural differentiation, support neuronal function in the CNS and PNS, and modulate neuronal regeneration after injury.
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