R-Ras3 and Cell Survival in the Central Nervous System

描述（由申请人提供）：控制神经发生的细胞内信号传导事件尚未明确定义。本申请旨在阐明Ras相关的GT3，R-Ras 3（M-Ras）在神经发生过程中的细胞命运决定中的作用。许多营养因子参与促进神经祖细胞的增殖，介导其向成熟神经元的转变，并积极维持其随后的存活。这些反应的缺陷可能是一系列神经系统疾病的潜在机制，包括阿尔茨海默氏症、帕金森氏症、中风、遗传性视网膜营养不良和肌萎缩性侧索硬化症。我们的主要工作假设是，R-Ras 3是神经营养因子的关键信号分子，在神经元细胞中传播分化和存活信号，但不是增殖信号。在目的1中，将研究R-Ras 3对NGF和bFGF而不是EGF的选择性反应性的分子机制。使用PC 12和H19-7神经元细胞系，比较用EGF、bFGF和NGF刺激时RRas 3、H-Ras和Rap 1A的活化动力学。将检查TrkA/FGFR对接蛋白SNT介导R-Ras 3的选择性活化的能力。将使用一组R-Ras 3/H-Ras嵌合体对R-Ras 3一级序列内介导对bFGF和NGF的选择性反应性的结构域进行作图。在目标2中，将研究R-Ras 3的下游信号传导事件。R-Ras 3刺激Ras相关GTP酶Rap 1A的GTP负载的能力将通过亲和下拉测定来检查。将测试Rap 1A的显性失活突变体Rap 1A N17阻断R-Ras 3生物学功能和信号传导的能力。通过荧光共振能量转移（FRET）技术监测营养因子刺激后R-Ras 3、H-Ras和Rap 1A的空间和时间激活。在目标3中，将使用原代海马神经元和R-Ras 3缺失小鼠两者来描绘R-Ras 3在神经发生中的作用。将检查R-Ras 3、H-Ras和Rap 1A改变神经祖细胞的增殖能力或原代海马神经元的分化和存活程序的能力。这些是通过使用溴脱氧尿苷掺入测定来量化有丝分裂，用一组神经元特异性标记物免疫染色来检测分化状态的变化，以及TUNEL测定来量化凋亡细胞来实现的。将分析R-Ras 3缺陷的小鼠转基因系的神经元相关缺陷。将根据组织学和免疫组织化学标准对胎儿和出生后CNS进行详细检查。然后进行行为测试，以测试R-Ras 3缺陷动物是否表现出任何运动和学习功能障碍。

DESCRIPTION (provided by applicant): 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.