Functional Analysis of Semaphorin 5A In Vivo

描述（由申请人提供）：正常的神经系统功能严重依赖于复杂的神经元连接网络的适当发展。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可导致严重的神经功能缺损。信号蛋白是一类重要的调节轴突生长、引导和可塑性的分子。我们已经确定信号蛋白5A （Sema5A）是一种双功能轴突引导分子，在体外以蛋白聚糖依赖的方式调节神经元生长。具体来说，在硫酸软骨素蛋白聚糖（CSPGs）存在时，Sema5A抑制神经突生长，而在硫酸肝素蛋白聚糖（HSPGs）存在时，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的基础上，旨在研究Sema5A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Aim 1中，我建议有条件地切除胚胎神经系统中的Sema5A，以研究其在被称为回束的主要边缘纤维束发育过程中的作用。我建议将我们的Sema5A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Sema5A。在特定的Aim 2中，我将探讨机制问题，以深入了解Sema5A如何以蛋白聚糖依赖的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，免疫组织化学和生化技术。为了了解与Sema5A功能相关的机制问题，我将使用野生型和Sema5A突变动物的原代神经元进行体外膜条纹实验。总的来说，这些研究有望为Sema5A在特定中枢神经系统纤维束发育中的功能提供重要见解，并且重要的是，揭示哪些蛋白聚糖将Sema5A介导的生长转换为Sema5A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。摘要：硫酸软骨素蛋白多糖（CSPGs）是一种大的细胞外分子，在神经细胞发育过程中被认为是重要的调节因子。此外，CSPGs还与损伤后神经系统再生受限有关。这里提出的工作是以任务为导向的，旨在了解CSPGs如何指示神经细胞不生长。我们期望发现新的生物学原理，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.