This application aims to unravel the mechanisms by which a novel Ras-related G-protein, R-ras3 in controling neuronal cell survival and differentiation. R-ras3 is a brain-specific G- protein and its mRNA is highly abundant in the entire hippocampus and the Purkinje cell layers of the cerebellum. Biologically, R- ras3 promotes neuronal differentiation and survival in cultured cells. Our working hypothesis is that R-ras3 is a critical signal transducer in mediating survival and differentiation stimuli in the developing CNS. Indeed, a failure to execute the natural cell death or differentiation programs in the CNS is the most common underlying cause of neurodegenerative diseases and malignancies.
In Specific Aim one, the regional localization of R-ras3 in the adult and developing rat brain will be examined. In situ hybridization and immunohistochemistry will be performed using [alpha-35S UTP]-labeled R-ras3 complementary RNA probes or specific antisera, respectively. These studies will provide vital information as to the sites and the stage-specific expression of R-ras3 in the nervous system.
In Specific Aim two, the effects of R-ras3 on cell survival and differentiation in two neuronal cell lines, PC12, and H19-7; and in primary hippocampal neurons will be examined. Attempts will be made to correlate R- ras3 induced biological functions with the activation of downstream signaling kinases. These studies will provide insights into the biological actions of R-ras3 and the delineation of novel signaling pathways responsible for neuronal survival and differentiation.
In Specific Aim 3, the nature of the upstream signals of R-ras3 will be investigated. The ability of soluble factors and a brain-specific guanine nucleotide exchange factor, GRP, to stimulate the R-ras3 GTP-loading will be examined. Finally, by using a dominant inhibitory mutant of R- ras3, the role of endogenous R-ras3 in regulating neuronal cell functions will be investigated. In summary, the progression of neuronal precursor cells from primitive to postmitotic-matured phase requires a repertoire of soluble factors. It is highly likely that R-ras3 is a critical signal transducer in coupling differentiation and survival events during neurogenesis.
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