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

描述（由申请人提供）：胶质细胞和内皮细胞在神经系统细胞外环境的稳态调节中发挥核心作用。离子组成和总的细胞外容积由这些细胞主动调节。在许多病理条件下，稳态平衡丧失，导致细胞外空间肿胀，神经元功能障碍和细胞凋亡。尽管神经胶质功能障碍的严重影响，仍然有很多了解的分子机制，支配这种重要形式的生理调节。该研究的重点是由果蝇ser/thr激酶Fray介导的信号级联。Fray调节其下游效应子的活性，阳离子-氯化物协同转运蛋白NCC 69，在血神经屏障的胶质细胞中。该级联参与外周神经中细胞外体积的调节：在该信号级联中有缺陷的神经具有广泛的肿胀。这种肿胀也发生在神经元电活动增加的高活性突变体中。我们已经发现果蝇蛋白质和它们的人类对应物SPAK和NKCC 1之间具有广泛的分子和功能同源性。有充分的证据表明，哺乳动物的NKCC 1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反应，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外周神经活动依赖性肿胀的分子元件。在第二个具体目标中，我们将使用敲低RNAi筛选来鉴定大量先前未鉴定的分子参与者，这些分子参与者在血神经屏障处起作用以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鉴定参与胶质细胞稳态的关键分子组分的有力策略。通过对它们的鉴定，R21的提议将为后续研究奠定基础，以更好地解决胶质细胞如何调节其细胞外环境。 公共卫生相关性：调节细胞外环境的分子信号对正常神经系统功能至关重要。了解这一点与广泛的病理条件有关，包括缺血，创伤性脑损伤和中风。我们将确定参与这种重要形式的生理调节的关键分子，有助于这些神经系统疾病的合理分析。

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. PUBLIC HEALTH RELEVANCE: The molecular signals that regulate the extracellular environment are of critical importance for normal nervous system function. Understanding this is relevant to a wide range of pathological conditions, including ischemia, traumatic brain injury, and stroke. We will identify the key molecular players involved in this critical form of physiological regulation, contributing to a rational analysis of these disorders of the nervous system.