Molecular genetic analysis of ion regulation by glia at the blood-nerve barrier

描述(由申请人提供)：神经胶质细胞和内皮细胞在神经系统细胞外环境的动态平衡调节中起着核心作用。离子组成和细胞外总体积由这些细胞主动调节。在许多病理情况下，动态平衡失衡，导致细胞外间隙肿胀、神经元功能障碍和细胞凋亡。尽管神经胶质功能障碍有着严重的影响，但关于支配这一关键形式的生理调节的分子机制，仍有许多有待研究。研究的重点是由果蝇丝氨酸/Thr激酶Fray介导的信号级联反应。FRAY调节其下游效应物--阳离子-氯代转运体NCC69在血-神经屏障的神经胶质细胞中的活性。这一级联反应参与了周围神经细胞外体积的调节：在这一信号级联反应中有缺陷的神经有广泛的肿胀。这种肿胀也发生在多动突变体中，这些突变体的神经元电活动增加。我们已经发现果蝇蛋白与其人类同源蛋白Spak和NKCC1之间存在广泛的分子和功能同源性。有很好的证据表明，哺乳动物NKCC1功能障碍与中风有关，并导致神经系统水肿。在这个项目中，我们将详细研究分子级联，目的是识别新的成分，并更好地了解神经活动如何影响神经胶质功能。在具体目标1中，我们使用遗传学方法来表征参与周围神经活动依赖性肿胀的分子元件。在第二个具体目标中，我们将使用敲击式RNAi筛选来识别大量以前未识别的分子成员，它们在血神经屏障发挥作用，调节神经系统的细胞外环境。使用具有广泛哺乳动物同源性的遗传模型系统是快速识别参与神经胶质细胞内稳态的关键分子成分的有效策略。有了他们的鉴定，这项R21提案将为后续研究更好地解决胶质细胞如何调节细胞外环境奠定基础。

DESCRIPTION (provided by applicant): Glia and endothelial cells play a central role in the homeostatic regulation of the extracellular environment of the nervous system. The ionic composition and overall extracellular volume is actively regulated by these cells. In many pathological conditions homeostatic balance is lost, leading to swelling of the extracellular space, neuronal dysfunction, and apoptosis. Despite the severe impact of glial dysfunction, much remains to the learned about the molecular mechanisms that govern this critical form of physiological regulation. The focus of the study is a signaling cascade mediated by the Drosophila ser/thr kinase Fray. Fray regulates the activity of its downstream effector, the cation-chloride cotransporter NCC69, in glial cells of the blood-nerve barrier. This cascade is involved in the regulation of extracellular volume in peripheral nerves: nerves defective in this signaling cascade have extensive swelling. Such swelling also occurs in hyperactie mutants that have increased neuronal electrical activity. We have found extensive molecular and functional homologies between the Drosophila proteins and their human counterparts, SPAK and NKCC1. There is good evidence that dysfunction of NKCC1 in mammals is associated with stroke, and leads to edema in the nervous system. In this project we will examine the molecular cascade in detail, with the goal of identifying novel components, and to better understand how neural activity affects glial function. In specific aim 1 we use a genetic approaches to characterize the molecular elements that are involved in activity-dependent swelling in peripheral nerves. In the second specific aim we will use a knock-down RNAi screen to identify a substantial number of previously unidentified molecular players that function at the blood-nerve barrier to regulate the extracellular environment of the nervous system. The use of a genetic model system with extensive mammalian homology is a powerful strategy to rapidly identify the key molecular components involved in glial homeostasis. With their identification, this R21 proposal will set the stage for follow-on studies to better resolve how glia regulate their extracellular environment.