Fascin1 in Growth Cone Motility and Guidance

项目概要： 大脑复杂的神经网络的形成始于胚胎发育。轴突引导是一个关键的 中枢神经系统精确布线的发育阶段，当轴突纤维 与特定的靶细胞连接。轴突引导的错误会导致与神经元损伤相关的神经连接缺陷。 神经系统疾病，包括癫痫。轴突的顶端有一种叫做生长锥的高度能动的结构 它可以感知并响应细胞外的引导信号来引导轴突迁移。生长锥取决于 肌动蛋白为基础的结构称为丝状伪足，以感知周围环境和检测外部指导 线索丝状伪足的形成依赖于肌动蛋白交联使平行的肌动蛋白丝成束 包括Fascin 1在内的蛋白质。在引导反应过程中，生长锥丝状伪足经历重塑， 在吸引线索的方向上，在排斥线索的方向上，我假设失去了 发育中的海马神经元中的Fascin 1将由于不能调节而导致错误的轴突引导。 丝状伪足重塑。这里提出的研究将利用分子、细胞生物学和成像技术 用体外培养的大鼠海马神经元和海马神经元研究Fascin 1在轴突生长锥中的作用和调节。 体内果蝇模型。 在目标1.1中，通过CRISPR-Cas9编辑的Fascin 1消耗对丝状伪足延伸，持久性， 和收缩将被研究。将使用三种不同的轴突引导测定来确定Fascin 1 耗尽改变了生长锥经受引导响应的能力。目标1.2将研究 Fascin 1在体外培养大鼠海马神经元生长锥中的时空动态变化 迁移和蛋白激酶C（PKC）对神经元Fascin 1的调节。以前的工作确定， Fascin 1的Ser-39被PKC磷酸化，从而消除了Fascin 1的纤维束能力，我推测 PKC通过这种作用调节轴突导向过程中生长锥丝状伪足的稳定性。目标2使用果蝇- 的方法来研究Fascin 1在体内轴突导向中的作用。果蝇表达一个单一的直系同源物 我的初步研究表明，需要Singed的存在， 蘑菇体是一种依赖轴突引导形成的神经元结构。 本文提出的研究将进一步了解轴突导向过程中的细胞骨架调节， 由于轴突引导中的错误与神经系统疾病（包括癫痫）的关联，因此具有重要性。

PROJECT SUMMARY: The formation of the brain’s intricate neural network begins in embryogenesis. Axon guidance is a critical developmental stage in which precise wiring of the central nervous system is achieved when axonal fibers connect with specific target cells. Errors in axon guidance can result in wiring defects that are associated with neurological disorders including epilepsy. The tips of axons have highly motile structures called growth cones that can sense and respond to extracellular guidance cues to direct axon migration. Growth cones depend on actin-based structures called filopodia to sense their surrounding environment and detect external guidance cues. The formation of filopodia is dependent on the bundling of parallel actin filaments by actin cross-linking proteins including Fascin1. During the guidance response, growth cone filopodia undergo remodeling, stabilizing in the direction of attractive cues and collapsing in response to repulsive cues. I hypothesize that the loss of Fascin1 in developing hippocampal neurons will result in erroneous axon guidance due to an inability to regulate filopodia remodeling. The studies proposed here will utilize molecular, cellular biology, and imaging techniques to study the role and regulation of Fascin1 in axon growth cones using cultured rat hippocampal neurons and an in vivo Drosophila model. In Aim 1.1, the effects of Fascin1 depletion via CRISPR-Cas9 editing on filopodia extension, persistence, and retraction will be studied. Three different axon guidance assays will be utilized to determine if Fascin1 depletion alters the ability of growth cones to undergo the guidance response. Aim 1.2 will study the spatiotemporal dynamics of Fascin1 in growth cones of cultured rat hippocampal neurons undergoing guided migration and the regulation of neuronal Fascin1 by protein kinase C (PKC). Previous work established that phosphorylation of Ser-39 of Fascin1 by PKC abrogates Fascin1’s filament bundling ability and I hypothesize that PKC regulates growth cone filopodia stability during axon guidance via this role. Aim 2 uses a Drosophila- based approach to study the role of Fascin1 in axon guidance in vivo. Drosophila express an single ortholog of mammalian Fascin1 called Singed and my preliminary studies indicate that the presence of Singed is required for proper formation of the mushroom body, a neuronal structure that is dependent on axon guidance to form. The studies proposed here will further the knowledge of cytoskeletal regulation during axon guidance which is of importance due to the association of errors in axon guidance with neurological disorders, including epilepsy.