Functional Analysis of Semaphorin 5A In Vivo

描述（由申请人提供）：正常的神经系统功能主要取决于神经元连接的复杂网络的正常发育。众所周知，由于疾病或损伤导致的神经网络组装缺陷或突触连接中断会导致严重的神经功能缺损。脑信号蛋白是一类重要的分子，已知其调节轴突生长、导向和可塑性。我们已经确定Semaphorin 5A（Sema 5A）作为一种双功能轴突导向分子，在体外以蛋白聚糖依赖的方式调节神经元的生长。具体而言，在硫酸软骨素蛋白聚糖（CSPG）的存在下，Sema 5A抑制神经突生长，而在硫酸乙酰肝素蛋白聚糖（HSPG）的存在下，SemaSA促进神经突生长。本提案中概述的研究直接建立在我们的体外研究结果基础上，旨在研究Sema 5A在体内神经系统发育过程中特定神经元群体中的功能特征。在具体的目标1，我建议有条件地消融Sema 5A在胚胎神经系统中，以研究其在发展过程中的一个主要的边缘纤维束称为反束。我建议将我们的Sema 5A条件小鼠与特定的cre驱动系杂交，以选择性地消融胚胎神经系统中的Sema 5A。在具体目标2，1中，将探索机制问题，以深入了解Sema 5A如何以蛋白聚糖依赖性方式调节神经元生长。我将使用的方法来实现我的目标，包括使用最先进的小鼠遗传学，免疫组织化学和生物化学技术。为了询问与Sema 5A功能相关的机制问题，我将采用来自野生型和Sema 5A突变动物的原代神经元进行体外膜条纹测定。总的来说，预计这些研究将为Sema 5A在特定中枢神经系统纤维束发育中的作用提供重要的见解，重要的是，揭示哪些蛋白聚糖将Sema 5A介导的生长转换为Sema 5A介导的抑制。如果成功，我们的研究将提供CSPGs如何影响神经元生长的第一个机制线索。Lay language总结：硫酸软骨素蛋白聚糖（CSPG）是一种细胞外大分子，越来越多地被认为是神经细胞生长发育过程中的重要调节因子。此外，CSPG已经涉及限制损伤后的神经系统再生。这里提出的工作是面向任务的-它的目的是了解CSPGs如何指导神经细胞不生长。我们期望发现新的生物学原理，这些原理可能与治疗发育障碍和/或促进损伤或疾病后的神经系统修复有关。

DESCRIPTION (provided by applicant): Proper nervous system functioning critically depends on the proper development of an intricate network of neuronal connectivity. It is well established that defects in neural network assembly or interruption of synaptic connections as a result of disease or injury leads to severe neurological deficits. The Semaphorins are one important class of molecules known to regulate axonal growth, guidance, and plasticity. We have identified Semaphorin 5A (Sema5A) as a bi-functional axon guidance molecule that regulates neuronal growth in a proteoglycan-dependent manner in vitro. Specifically, in the presence of chondroitin sulfate proteoglycans (CSPGs), Sema5A inhibits neurite outgrowth, whereas in the presence of heparan sulfate proteoglycans (HSPGs), SemaSA promotes neurite outgrowth. The studies outlined in this proposal directly build on our in vitro findings and are aimed at the functional characterization of Sema5A in specific neuronal populations during nervous system development in vivo. In specific Aim 1, I propose to conditionally ablate Sema5A in the embryonic nervous system in order to study its role during the development of a major limbic fiber tract called the fasciculus retroflexus. I propose to cross our Sema5A conditional mice with specific cre- driver lines to selectively ablate Sema5A in the embryonic nervous system. In specific Aim 2,1 will explore mechanistic questions to gain insights into how Sema5A regulates neuronal growth in a proteoglycan dependent manner. The methods I will use to achieve my goals include the use of state-of-the-art mouse genetics, immunohistochemical and biochemical techniques. To ask mechanistic questions related to Sema5A function, I will employ in vitro membrane stripe assays with primary neurons from wild-type and Sema5A mutant animals. As a whole, the studies proposed are anticipated to provide important insights into how Sema5A functions in the development of specific central nervous system fiber tracts, and importantly, reveal which proteoglycans switch Sema5A-mediated growth to Sema5A-mediated inhibition. If successful, our studies will provide the first mechanistic clues of how CSPGs influence neuronal growth. Lay language summary: Chondroitin sulfate proteoglycans (CSPGs) are large extracellular molecules that are increasingly being recognized as important regulators of nerve cell growth during development. In addition, CSPGs have been implicated in limiting nervous system regeneration following injury. The work ' proposed here is mission-oriented-it is aimed at understanding how CSPGs instruct nerve cells not to grow. We expect to uncover new biological principles that may be relevant for the treatment of developmental disorders and/or promote nervous system repair following injury or disease.