Cytoskeletal Regulation During Growth Cone Migration and Axon Guidance

描述（由申请人提供）：损伤或疾病对成人中枢神经系统（CNS）连接的损害通常是不可修复的。设计修复中枢神经系统损伤的疗法需要对中枢神经系统发育的细胞机制有详细的了解。随着大脑的成熟，神经元迁移到大脑中适当的位置，并精心设计过程，引导它们到达目标，形成适当的连接。生长锥尖神经突的初始形成和轴突生长锥随后的定向运动都需要f -肌动蛋白和微管（MT）动力学。F-actin:MT相互作用可能在这两个过程中起关键作用。然而，介导这种相互作用的分子机制，以及这些相互作用如何驱动神经生成和轴突引导，尚不清楚。Ena/VASP蛋白通过控制肌动蛋白细胞骨架动力学来引导生长锥。结合小鼠遗传学、原代细胞培养、活细胞成像和电子显微镜，我的实验室发现Ena/ vasp缺陷的皮质神经元不能形成丝状足，这是一种由捆绑的f -肌动蛋白组成的手指状突起。此外，我们发现缺乏丝状足的皮质神经元不能形成神经突，并表现出改变的微管动力学。Ena/VASP突变的皮质神经元丝状伪足的恢复也挽救了神经突的形成。初步数据表明，缺乏Ena/ vasp的感觉神经元形成轴突，但表现出明显的引导缺陷。我们将使用来自Ena/VASP缺陷动物的感觉神经元制备来验证我们的工作假设，即Ena/VASP依赖性丝状足的形成使mt和肌动蛋白束之间的相互作用成为可能，而肌动蛋白束需要同时接收吸引和排斥信号。额外的新数据表明，Ena/VASP蛋白可能协调F-actin:MT相互作用，并且TRIM9蛋白与Ena/VASP和微管相互作用；TRIM9也参与轴突导航的控制。此外，包括Ena/VASP在内的蛋白质网络可能受到Lamellipodin的调节，Lamellipodin是一种整合细胞表面受体为轴突引导因子产生的信号的分子。总的来说，这些数据使我们假设Ena/VASP蛋白参与在F-actin: MTs相互作用中起关键作用的蛋白质网络，并反过来与引导受体控制的信号通路相关。我们的长期目标是了解神经元如何整合环境线索来协调其形态和运动的变化，这是建立功能性神经系统所必需的。更好地理解神经突形成和轴突引导的机制基础将为神经系统中的连接如何建立以及它们在可塑性过程中如何重塑提供基本的见解。我们研究计划的结果应该对疾病或损伤后修复这些连接的治疗方法的发展具有重要价值。公共卫生相关性：损伤或疾病对成人大脑和脊柱内形成连接的神经造成的损害往往是不可修复的。我们试图全面了解神经纤维是如何形成的，并在胎儿发育初期被引导到它们的目标，期望这将有助于我们学习修复大脑损伤。

DESCRIPTION (provided by applicant): Damage to connections within the adult Central Nervous System (CNS) by injury or disease is often irreparable. To design therapies to repair CNS damage requires a detailed understanding of the cellular mechanisms underlying CNS development. As the brain matures, neurons migrate to their proper positions within the brain and elaborate processes that are guided to their targets to form proper connections. Both initial formation of growth cone-tipped neurites, and subsequent guided locomotion of axonal growth cones, require F-actin and microtubule (MT) dynamics. F-actin:MT interactions likely play key roles in both of these processes. However, the molecular mechanisms that mediate such interactions, and how these interactions drive neuritogenesis and axon guidance, are not understood. Ena/VASP proteins function in growth cone guidance by controlling actin cytoskeleton dynamics. Using a combination of mouse genetics, primary cell culture, live cell imaging and electron microscopy, my lab found that Ena/VASP-deficient cortical neurons fail to form filopodia, finger-like processes comprised of bundled F-actin. Furthermore, we found that cortical neurons devoid of filopodia fail to form neurites, and exhibit altered microtubule dynamics. Restoration of filopodia in Ena/VASP mutant cortical neurons also rescues neurite initiation. Preliminary data indicate that Ena/VASP-deficient sensory neurons form axons, but exhibit striking guidance defects. We will use sensory neuron preparations from Ena/VASP deficient animals to test our working hypothesis is that Ena/VASP- dependent filopodia formation enables interactions between MTs and actin bundles that are required to receive both attractive and repulsive cues. Additional new data indicate that Ena/VASP proteins may act to coordinate F-actin:MT interactions, and that the TRIM9 protein interacts with both Ena/VASP and microtubules; TRIM9 is also implicated in the control of axon navigation. Furthermore, a network of proteins, including Ena/VASP, is likely regulated by Lamellipodin, a molecule that integrates signals generated by cell-surface receptors for axonal guidance factors. Collectively, these data lead us to hypothesize that Ena/VASP proteins participate in protein networks that play key roles in F-actin: MTs interactions, and are in turn linked to signaling pathways controlled by guidance receptors. Our long-term goal is to understand how neurons integrate environmental cues to orchestrate changes in their morphology and movement necessary to establish a functional nervous system. A better understanding of the mechanistic basis of neurite formation and axon guidance will provide fundamental insight into how connections in the nervous system are established and how they are remodeled during plasticity. The results of our research plan should be of great value to the development of therapeutic approaches to repair these connections subsequent to disease or injury. PUBLIC HEALTH RELEVANCE: Damage to the nerves that form connections within the adult brain and spinal column by injury or disease is often irreparable. We seek a comprehensive understanding of how nerve fibers form and are guided to their targets initially during fetal development expecting that this will help us learn to repair damage to the brain.