R-RAS3 AND CELL SURVIVAL IN THE CENTRAL NERVOUS SYSTEM

本申请旨在阐明一种新的Ras相关G蛋白R-ras 3在控制神经细胞存活和分化中的机制。 R-ras 3是脑特异性G蛋白，其mRNA在整个海马和小脑的浦肯野细胞层中高度丰富。 在生物学上，R-ras 3促进培养细胞中的神经元分化和存活。 我们的工作假设是，R-ras 3是一个关键的信号转导介导的生存和分化的刺激在发展中的中枢神经系统。 事实上，不能在CNS中执行自然细胞死亡或分化程序是神经退行性疾病和恶性肿瘤的最常见的根本原因。 在特定目标1中，将检查R-ras 3在成年和发育中的大鼠脑中的区域定位。分别使用[α-35S UTP]标记的R-ras 3互补RNA探针或特异性抗血清进行原位杂交和免疫组织化学。 这些研究将为R-ras 3在神经系统中的位点和阶段特异性表达提供重要信息。 在具体目标二中，将检查R-ras 3对两种神经元细胞系PC 12和H19-7中的细胞存活和分化的影响;以及对原代海马神经元的影响。 将尝试将R-ras 3诱导的生物学功能与下游信号传导激酶的激活相关联。 这些研究将提供深入了解R-ras 3的生物学作用和描绘负责神经元存活和分化的新信号通路。 在具体目标3中，将研究R-ras 3上游信号的性质。 将检查可溶性因子和脑特异性鸟嘌呤核苷酸交换因子GRP刺激R-ras 3 GTP负载的能力。 最后，通过使用R-ras 3的显性抑制突变体，将研究内源性R-ras 3在调节神经元细胞功能中的作用。 总之，神经元前体细胞从原始到有丝分裂后成熟阶段的进展需要一系列可溶性因子。 R-ras 3很可能是神经发生过程中耦合分化和存活事件的关键信号转导子。

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.