R-Ras3 and Cell Survival in the CNS

R-Ras3 和 CNS 中的细胞存活

• 批准号: 6829094
• 负责人: ANDREW M CHAN
• 金额: $ 29.66万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829094
• 关键词: G protein; PC12 cells; biological signal transduction; cell differentiation; enzyme activity; epidermal growth factor; fibroblast growth factor; fluorescence resonance energy transfer; genetically modified animals; guanine nucleotide binding protein; guanosinetriphosphatases; hippocampus; immunocytochemistry; laboratory mouse; laboratory rat; nerve growth factors; neurogenesis; neurons; neurotrophic factors; receptor binding; terminal nick end labeling; tissue /cell culture; tissue mosaicism

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.

描述(由申请人提供)：支配神经发生的细胞内信号事件没有很好的定义。本研究旨在阐明Ras相关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前体细胞的增殖，介导其向成熟神经元的转化，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。在目标1中，将研究R-Ras3对神经生长因子和碱性成纤维细胞生长因子的选择性反应的分子机制，而不是EGF。使用PC12和H19-7神经细胞系，比较在EGF、bFGF和NGF刺激下RRas3、H-RAS和Rap1a的激活动力学。TrkA/FGFR对接蛋白SNT在介导R-Ras3选择性激活方面的能力将被检测。将使用一组R-Ras3/H-RAS嵌合体来定位R-Ras3初级序列中介导对bFGF和NGF的选择性反应的结构域。在目标2中，将研究R-RAS3的下游信令事件。R-Ras3刺激Ras相关GTPase Rap1a的GTP负载的能力将通过亲和下拉试验来检测。将测试Rap1A的显性负突变体Rap1AN17阻断R-Ras3生物学功能和信号转导的能力。R-Ras3、H-Ras和Rap1A在营养因子刺激下的时空激活将通过荧光共振能量转移(FRET)技术进行监测。在目标3中，将使用原代海马神经元和R-Ras3缺失的小鼠来描述R-Ras3在神经发生中的作用。R-Ras3、H-Ras和Rap1A改变神经前体细胞的增殖能力或原代海马神经元的分化和存活计划的能力将被检测。这些是通过使用溴脱氧尿嘧啶核苷掺入试验来定量有丝分裂，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，并通过TUNEL试验来定量凋亡细胞来实现的。R-Ras3基因缺陷的小鼠转基因品系将被分析与神经元相关的缺陷。将对胎儿和出生后的中枢神经系统进行详细的组织学和免疫组织化学检查。然后将进行行为测试，以测试R-Ras3缺陷动物是否表现出任何运动和学习障碍。