The intracellular signaling events governing neurogenesis are not well defined. This application aims to elucidate the role a Ras-related GTPase, R-Ras3 (M-Ras) in cell fate decision during neurogenesis. A host of trophic factors are involved in promoting the proliferation of neuroprogenitors, mediating their transition into matured neurons, and actively maintaining their subsequent survival. Defects in these responses may be the underlying mechanism for a range of neurological disorders including Alzheimer's, Parkinson's, stroke, hereditary retinal dystrophies and Amyotrophic Lateral Sclerosis. Our major working hypothesis is that R-Ras3 is a key signaling molecule for neurotrophic factors, propagating differentiation and survival but not proliferative signals in neuronal cells.
In Aim 1, the molecular mechanism responsible for the selective responsiveness of R-Ras3 to NGF and bFGF but not EGF will be investigated. Using PC12 and H19-7 neuronal cell lines, the kinetics of activation of RRas3, H-Ras, and Rap1A upon stimulation with EGF, bFGF, and NGF will be compared. The ability of a TrkA/FGFR docking protein SNT in mediating the selective activation of R-Ras3 will be examined. The structural domains within R-Ras3 primary sequence that mediate the selective responsiveness to bFGF and NGF will be mapped using a panel of R-Ras3/H-Ras chimeras.
In Aim 2, the downstream signaling events of R-Ras3 will be investigated. The ability of R-Ras3 to stimulate the GTP-loading of a Ras-related GTPases Rap1A will be examined by an affinity pull-down assay. The ability of a dominant negative mutant of Rap1A, Rap1A N17, to block R-Ras3 biological functions and signaling will be tested. The spatial and temporal activation of R-Ras3, H-Ras and Rap1A upon trophic factor stimulation will be monitored by the Fluorescence Resonance Energy Transfer (FRET) techniques.
In Aim 3, the role of R-Ras3 in neurogenesis will be delineated using both primary hippocampal neurons and R-Ras3-null mice. The ability of R-Ras3, H-Ras and Rap1A to alter the proliferative capacity of neuroprogenitors, or the differentiation and survival program of primary hippocampal neurons will be examined. These are achieved by using the bromodeoxyuridine incorporation assay to quantify mitogenesis, immunological staining with a panel of neuronal specific markers to detected changes in the differentiation state, and the TUNEL assay to quantify apoptotic cells. A mouse transgenic line deficient in R-Ras3 will be analyzed for neuronal-associated defects. A detailed examination of both fetal and postnatal CNS with respect to histological and immunohistochemical criteria will be performed. Behavioral tests will then be conducted to test if R-Ras3-deficient animals display any motor and learning dysfunction.
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