R-Ras3 and Cell Survival in the CNS

描述（由申请人提供）：控制神经发生的细胞内信号事件尚未明确定义。本应用旨在阐明ras相关的GTPase R-Ras3 （M-Ras）在神经发生过程中决定细胞命运的作用。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，包括阿尔茨海默病、帕金森病、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-Ras3是神经营养因子的关键信号分子，在神经细胞中传播分化和存活，但不是增殖信号。在Aim 1中，将研究R-Ras3对NGF和bFGF而非EGF选择性反应的分子机制。利用PC12和H19-7神经元细胞系，比较EGF、bFGF和NGF刺激RRas3、H-Ras和Rap1A的激活动力学。TrkA/FGFR对接蛋白SNT介导R-Ras3选择性激活的能力将被研究。R-Ras3初级序列中介导对bFGF和NGF选择性反应的结构域将使用R-Ras3/H-Ras嵌合体面板绘制。在Aim 2中，我们将研究R-Ras3的下游信号事件。R-Ras3刺激ras相关GTPases Rap1A的gtp负载的能力将通过亲和下拉试验进行检验。Rap1A显性负突变体Rap1A N17阻断R-Ras3生物学功能和信号转导的能力将被测试。在营养因子刺激下，R-Ras3、H-Ras和Rap1A的时空激活将通过荧光共振能量转移（FRET）技术进行监测。在Aim 3中，R-Ras3在神经发生中的作用将通过海马原代神经元和R-Ras3缺失小鼠来描述。我们将研究R-Ras3、H-Ras和Rap1A对神经祖细胞增殖能力或海马初级神经元分化和存活程序的影响。这些是通过使用溴脱氧尿苷掺入法来量化有丝分裂，用一组神经元特异性标记物进行免疫染色来检测分化状态的变化，以及TUNEL法来量化凋亡细胞来实现的。我们将分析一个缺乏R-Ras3基因的小鼠转基因系是否存在神经元相关缺陷。将根据组织学和免疫组织化学标准对胎儿和出生后的中枢神经系统进行详细检查。然后进行行为测试，以测试r - ras3缺陷的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.