Regulation of Neuronal Survival by Ras-like GTPase

描述（由申请人提供）：异常细胞凋亡导致多种神经退行性疾病。因此，阐明调节神经元存活的信号转导机制对于理解基础生物学和治疗干预都很重要。神经营养因子通过激活小的gtp结合蛋白Ras来刺激神经元存活，Ras通过翻译和引导神经营养因子启动的信号进入多种信号通路发挥作用。最近的数据表明，ras依赖的信号通路PI-3激酶/Akt和MEK/ERK，以及ras独立的MEK5/ERK5信号级联是神经营养因子依赖生存的主要介质。我们发现了一组新的，进化上保守的ras相关蛋白。人类至少有两个高度相关的基因（Rit和Rin），而果蝇只表达一个家族成员（Ric）。这些蛋白质代表了一个基因家族，其功能是调节从果蝇到人类的信号网络。嗜铬细胞瘤细胞中组成型活性突变体Rit的过表达通过激活mek依赖但PI-3激酶/ akt独立的信号通路诱导神经突生长和存活。此外，原代神经元中激活的Rit表达可抑制营养因子戒断诱导的细胞凋亡并促进轴突生长。因此，Rit以一种不同于Ras的方式控制生存通路，可能控制MEK5/ERK5信号通路，或调节一种激活ERK激酶的新信号通路。我们假设Rit的功能是翻译和引导神经营养因子启动的信号，以一种不同于Ras的方式促进生存信号级联。本文描述了评估这一假设的三个具体目标。特异性目的1将确定Rit信号通路的激活是否促进原代培养神经元的存活，以及Rit功能是否对神经元细胞存活至关重要。特异性目标2将通过神经营养因子相关或神经营养因子独立的途径研究调节Rit功能的细胞外刺激。特异性Aim 3将检查rit介导的ERK激活机制，并确定已建立的ERK依赖性促存活信号通路对rit介导的神经元存活是否重要。初步研究表明，果蝇对Ric活性的改变很敏感，从而导致翅膀和眼睛的发育缺陷。我们将采用遗传筛选方法来鉴定与Ric相互作用的基因，并分析脊椎动物同源物在神经细胞中对Rit功能的贡献能力。通过生物化学、分子生物学和遗传学的综合应用，这些研究将为理解这种独特的神经元存活调节剂的功能奠定基础。

DESCRIPTION (provided by applicant): Aberrant apoptosis contributes to various neurodegenerative disorders. Elaborating the signal transduction mechanisms that regulate neuronal survival is thus important for understanding both basic biology and for therapeutic intervention. Neurotrophins potently stimulate neuronal survival in part by activating the small GTP-binding protein Ras, which functions by translating and directing neurotrophin-initiated signals into multiple signaling pathways. Recent data indicate that the Ras-dependent signaling pathways, PI-3 kinase/Akt and MEK/ERK, and the Ras-independent MEK5/ERK5 signaling cascade, are the primary mediators of neurotrophin-dependent survival. We have discovered a novel, evolutionarily conserved group of Ras-related proteins. There are at least two highly related human genes (Rit and Rin), while Drosophila express only one family member (Ric). These proteins represent a gene family that functions to regulate signaling networks that have been conserved from flies to man. Overexpression of a constitutively active mutant of Rit in pheochromocytoma cells induces neurite outgrowth and survival by activating a MEK-dependent, but PI-3 kinase/Akt-independent, signaling pathway. In addition, activated Rit expression in primary neurons inhibits trophic factor-withdrawal induced apoptosis and promotes axonal outgrowth. Thus, Rit controls survival pathway(s) in a manner distinct from that of Ras, likely functioning to control MEK5/ERK5 signaling, or regulating a novel signaling pathway, which activates ERK kinases. We hypothesize that Rit functions to translate and direct neurotrophin-initiated signals to pro-survival signaling cascades in a manner distinct from Ras. Three specific aims are described to evaluate this hypothesis. Specific Aim 1 will determine if activation of Rit signaling pathways promotes the survival of primary cultured neurons and if Rit function is essential for neuronal cell survival. Specific Aim 2 will examine the extracellular stimuli that regulate Rit function, via neurotrophin-associated or neurotrophin-independent pathways. Specific Aim 3 will examine the mechanism of Rit-mediated ERK activation and determine if established ERK-dependent pro-survival signaling pathways are important for Rit-mediated neuronal survival. Preliminary studies have demonstrated that Drosophila is sensitive to altered Ric activity, resulting in developmental defects in the wing and eye. We will undertake a genetic screening approach to identify genes that interact with Ric, and vertebrate homologues will be analyzed for their ability to contribute to Rit function in neuronal cells. Through the combined use of biochemistry, molecular biology, and genetics, these studies will form the foundation for understanding the function of this unique regulator of neuronal survival.