Regulation of protein targeting in axon guidance and neuronal morphogenesis

描述(申请人提供)：发展复杂的神经元形态和准确的神经连接需要在细胞环境中严格控制轴突生长和对引导信号的反应。精确的引导线索受体的亚细胞定位和细胞骨架的定位信号对于轴突引导是必不可少的。蛋白质通过复杂的膜运输和轴突运输系统定位于特定的细胞位置。神经元由于其高度极化和复杂的结构，特别依赖于多样化和高保真的蛋白质运输。蛋白质运输和运输的缺陷是多种人类发育和神经退行性疾病的基础，包括阿尔茨海默病、夏科特-玛丽-图斯和尼曼-皮克病。尽管它们很重要，但人们对神经元中这些关键过程的调控机制知之甚少。该领域面临的一个主要挑战和该项目的长期目标是了解神经元在自然环境中发育时蛋白质定位和细胞骨架动力学是如何控制的，在自然环境中，神经元必须整合多种细胞外信号。我们建立了一个模型，在该模型中，我们可以成像完整斑马鱼胚胎中神经元货物运输、蛋白质定位和细胞骨架变化的动力学。脊椎动物的感觉神经元必须延伸不同的中枢和外周轴突，才能形成感觉回路。我们发现，这些轴突对轴突引导线索表现出不同的反应。此外，我们还发现了内体转运和动蛋白适配器Calsyntenin-1(Clstn-1)在感觉轴突引导中的作用。在目标1中，我们建议确定Clstn-1如何调节内体到不同轴突间隔的运输路线。在目标2中，我们将研究调节神经营养素-3和Semaphorin 3d受体特异性定位的机制。我们将检验这一假设，即Clstn-1和另一类动蛋白适配器--折叠蛋白反应介体蛋白(CRMPs)的功能是将受体靶向特定的轴突区段。在目标3中，我们将确定Clstn-1、CRMPs、Sema3d和神经营养因子-3如何聚合来调节局部的细胞骨架动力学。我们独特的模型允许我们将轴突运输、引导受体定位和细胞骨架变化的分子事件与特定的轴突引导决定联系起来，在它们自然发生的时间和地点。阐明调节感觉轴突生长、引导和蛋白质运输的分子信号对于了解神经退行性疾病、神经病理性疼痛障碍以及轴突损伤后再生的条件至关重要。我们的实验将揭示这种机制，从而可能有助于确定疾病治疗的分子靶点。

DESCRIPTION (provided by applicant): Development of complex neuronal morphology and accurate neural connections requires tightly controlled axon outgrowth and responses to guidance signals in the cell environment. Precise subcellular localization of guidance cue receptors and localized signaling to the cytoskeleton are essential for axon guidance. Proteins are targeted to specific cell locations by complex membrane trafficking and axonal transport systems. Neurons are particularly dependent on diverse and high fidelity protein trafficking because of their highly polarized and complex structure. Defects in protein trafficking and transport underlie multiple human developmental and neurodegenerative diseases, including Alzheimer's disease, Charcot-Marie-Tooth, and Niemann Pick disease. Despite their importance, the mechanisms regulating these critical processes in neurons are poorly understood. A major challenge to the field and the long term goal of this project is to understand how protein localization and cytoskeletal dynamics are controlled as neurons develop in their natural environment, where they must integrate multiple extracellular cues. We established a model in which we can image dynamics of neuronal cargo transport, protein localization, and cytoskeletal changes in the intact zebrafish embryo. Vertebrate sensory neurons must extend distinct central and peripheral axons to form the sensory circuit. We found that these axons show distinct responses to axon guidance cues. Moreover, we discovered roles for endosomal trafficking and the kinesin adaptor Calsyntenin-1 (Clstn-1) in sensory axon guidance. In Aim 1 we propose to determine how Clstn-1 regulates endosome transport routes to different axon compartments. In Aim 2 we will investigate mechanisms regulating specific localization of receptors for Neurotrophin-3 and Semaphorin3d. We will test the hypothesis that Clstn-1 and another class of kinesin adaptors, the Collapsin response mediator proteins (CRMPs) function to target receptors to specific axon compartments. In Aim 3 we will determine how Clstn-1, CRMPs, Sema3d and Neurotrophin-3 converge to regulate localized cytoskeletal dynamics. Our unique model allows us to connect the molecular events of axonal transport, guidance receptor localization and cytoskeletal changes to specific axon guidance decisions at the time and place they naturally occur. Elucidation of the molecular signals regulating sensory axon growth, guidance, and protein trafficking is critical for understanding neurodegenerative disorders, neuropathic pain disorders and the conditions under which regeneration after axon injury can occur. Our experiments will uncover such mechanisms and thus may help to identify molecular targets for disease treatment.