Regulation of Neuronal Survival by Ras-like GTPase

描述(由申请人提供)：异常的细胞凋亡导致各种神经退行性疾病。因此，阐述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是重要的。神经营养因子部分通过激活小的GTP结合蛋白RAS而有效地刺激神经元存活，RAS通过翻译和引导神经营养因子启动的信号进入多个信号通路发挥作用。最近的研究表明，依赖RAS的信号通路，即PI-3激酶/Akt和MEK/ERK，以及不依赖RAS的MEK5/ERK5信号通路，是神经营养素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。至少有两个高度相关的人类基因(Rit和Rin)，而果蝇只表达一个家庭成员(Ric)。这些蛋白质代表了一个基因家族，其功能是调节从苍蝇到人的保守的信号网络。在嗜铬细胞瘤细胞中过表达RIT的结构活性突变体通过激活依赖于MEK但不依赖于PI-3激酶/Akt的信号通路来诱导轴突生长和存活。此外，原代神经元中活化的RIT表达抑制了营养因子撤除诱导的细胞凋亡，促进了轴突的生长。因此，RIT以一种不同于RAS的方式控制生存途径(S)，可能起到控制MEK5/ERK5信号的作用，或者调节一条新的信号通路，激活ERK激酶。我们假设RIT的功能是将神经营养素启动的信号转译并引导至促生存信号级联，其方式与RAS不同。描述了三个具体目标来评估这一假说。具体目标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功能是否对神经细胞生存至关重要。《特定目标2》将通过神经营养素相关或非神经营养素途径，研究调节RIT功能的细胞外刺激。具体目标3将研究RIT介导的ERK激活的机制，并确定已建立的ERK依赖的促生存信号通路是否对RIT介导的神经元存活重要。初步研究表明，果蝇对Ric活性的改变很敏感，导致翅膀和眼睛的发育缺陷。我们将采用基因筛选的方法来识别与Ric相互作用的基因，并将分析脊椎动物同源物对神经细胞RIT功能的贡献。通过生物化学、分子生物学和遗传学的结合使用，这些研究将为理解这种独特的神经元生存调节因子的功能奠定基础。

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.