R-RAS3 AND CELL SURVIVAL IN THE CENTRAL NERVOUS SYSTEM

该应用旨在揭示一种新的ras相关g蛋白R-ras3在控制神经元细胞存活和分化中的机制。R-ras3是一种脑特异性G-蛋白，其mRNA在整个海马和小脑浦肯野细胞层中含量丰富。生物学上，R- ras3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-ras3在发育中的中枢神经系统中是介导生存和分化刺激的关键信号传感器。事实上，中枢神经系统细胞自然死亡或分化程序的失败是神经退行性疾病和恶性肿瘤最常见的潜在原因。在特异性目的1中，将检查R-ras3在成年和发育中的大鼠脑中的区域定位。原位杂交和免疫组织化学将分别使用[α - 35s UTP]标记的R-ras3互补RNA探针或特异性抗血清进行。这些研究将为神经系统中R-ras3的位置和阶段特异性表达提供重要信息。在特异性靶2中，R-ras3对两种神经细胞系PC12和H19-7细胞存活和分化的影响；在初级海马神经元中进行检查。将尝试将R- ras3诱导的生物学功能与下游信号激酶的激活联系起来。这些研究将深入了解R-ras3的生物学作用，并描绘负责神经元存活和分化的新信号通路。在Specific Aim 3中，将研究R-ras3上游信号的性质。可溶性因子和脑特异性鸟嘌呤核苷酸交换因子GRP刺激R-ras3 gtp负载的能力将被检查。最后，利用R-ras3的显性抑制突变体，研究内源性R-ras3在调节神经元细胞功能中的作用。总之，神经前体细胞从原始阶段到有丝分裂后成熟阶段的进展需要一系列可溶性因子。R-ras3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换能器。

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.