Fascin1 in Growth Cone Motility and Guidance

Fascin1 在生长锥运动和指导中的作用

• 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
• 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

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.

项目总结： 大脑复杂神经网络的形成始于胚胎发育。轴突引导是一个关键 当轴突纤维达到中枢神经系统的精确连接的发育阶段 与特定的目标单元连接。轴突引导中的错误可能导致与以下相关的配线缺陷 包括癫痫在内的神经系统疾病。轴突的顶端有称为生长锥体的高度活动的结构。 它可以感知并对细胞外引导信号做出反应，从而直接进行轴突迁移。生长锥体依赖于 基于肌动蛋白的结构称为丝状足，用于感知周围环境和检测外部指导 暗示。丝状伪足的形成依赖于肌动蛋白交联后平行肌动蛋白细丝的捆绑。 包括Fascin1在内的蛋白质。在引导反应期间，生长锥丝状足突经历了重塑、稳定 在吸引人的线索的方向上，并对排斥的线索做出反应而崩溃。我假设失去的是 发育中的海马神经元中的Fascin1会因为无法调节而导致错误的轴突引导 丝状足部重塑。这里提出的研究将利用分子、细胞生物学和成像技术。 用培养的大鼠海马神经元和AN研究Fascin1在轴突生长锥体中的作用和调节 体内果蝇模型。 在Aim 1.1中，通过CRISPR-Cas9编辑来耗尽Fascin1对丝状伪足的延伸、持久性、 并将对撤回进行研究。将利用三种不同的轴突引导分析来确定Fascin1 耗竭改变了生长锥体进行导向反应的能力。AIM 1.2将研究 体外培养大鼠海马神经元生长锥体中Fascin1的时空动态变化 蛋白激酶C(PKC)对神经元Fascin1的迁移和调控先前的工作证实了 蛋白激酶C对Fascin1Ser-39的磷酸化取消Fascin1‘S丝束能力和I假说 在轴突引导过程中，PKC通过这一作用调节生长锥丝状足突的稳定性。《目标2》使用了一只果蝇- 目的：研究Fascin1在体内轴突引导中的作用。果蝇表达一个单一的同源基因 哺乳动物Fascin1被称为烧灼，我的初步研究表明，灼烧的存在是必需的 为了正确地形成蘑菇体，一种依赖于轴突引导形成的神经元结构。 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 由于轴突指导中的错误与包括癫痫在内的神经疾病有关，这一研究具有重要意义。