Fascin1 in Growth Cone Motility and Guidance

Fascin1 在生长锥运动和指导中的作用

基本信息

批准号：
10606165
负责人：
Katherine Rebecca Hardin
金额：
$ 4.77万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-15 至 2025-05-14
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10606165
关键词：
Actins Adult Axon Biological Assay Biological Models Brain Brain Diseases Bundling CRISPR/Cas technology Cell Culture Techniques Cells Central Nervous System Cues Cultured Cells Cytoskeleton Data Defect Development Developmental Process Drosophila genus Embryonic Development Environment Epilepsy F-Actin Fiber Filament Filopodia Fingers Genes Genetic Growth Cones Hippocampus Image Imaging Techniques Knock-out Knowledge Label Link Lobe Membrane Memory Microfilaments Modeling Molecular Molecular Biology Molecular and Cellular Biology Morphology Movement Mushroom Bodies Neurons Olfactory Learning Orthologous Gene Phosphorylation Process Protein Kinase C Proteins RNA Interference Rattus Regulation Role Sampling Sensory Side Signal Transduction Structure System Testing Traction Training Work autism spectrum disorder axon growth axon guidance axonal pathfinding cell motility extracellular fascin fly in vivo insight migration mutant nervous system disorder neural network neuronal circuitry pharmacologic protein crosslink receptor response spatiotemporal training opportunity

项目摘要

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.

项目概要：大脑复杂的神经网络的形成始于胚胎发生，轴突的引导至关重要。当轴突纤维形成时，中枢神经系统的精确布线得以实现的发育阶段与特定目标细胞的连接错误可能会导致与相关的接线缺陷。包括癫痫在内的神经系统疾病。轴突的尖端具有高度活动的结构，称为生长锥。可以感知并响应细胞外引导信号以指导轴突迁移。基于肌动蛋白的结构，称为丝状伪足，可以感知周围环境并检测外部引导丝状伪足的形成依赖于肌动蛋白交联的平行肌动蛋白丝的捆绑。包括 Fascin1 在内的蛋白质在引导反应期间，生长锥丝状伪足经历重塑、稳定。朝着吸引性线索的方向前进，并根据令人厌恶的线索而崩溃。发育中的海马神经元中的 Fascin1 将因无法调节而导致轴突引导错误这里提出的研究将利用分子、细胞生物学和成像技术。使用培养的大鼠海马神经元和神经元研究 Fascin1 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 编辑消除 Fascin1 对丝状伪足延伸、持久性、将研究三种不同的轴突引导测定法来确定 Fascin1 是否有效。耗尽会改变生长锥进行指导反应的能力，目标 1.2 将研究培养大鼠海马神经元生长锥中 Fascin1 的时空动态先前的工作证实了蛋白激酶 C (PKC) 对神经元 Fascin1 的迁移和调节。 PKC 对 Fascin1 Ser-39 的磷酸化消除了 Fascin1 的丝束能力，我勇敢地 PKC 通过这一作用在轴突引导过程中调节生长锥丝状伪足的稳定性。基于方法研究 Fascin1 在体内轴突引导中的作用。哺乳动物 Fascin1 称为 Singed，我的初步研究表明 Singed 的存在是必需的为了正确形成蘑菇体，蘑菇体是一种依赖轴突引导形成的神经结构。这里提出的研究将进一步加深对轴突引导过程中细胞骨架调节的了解。由于轴突引导错误与神经系统疾病（包括癫痫）之间的关联，这一点很重要。