Extracellular Matrix Signaling in Axon Outgrowth and Regeneration

描述(申请人提供)：在脊椎动物中，运动需要脊髓运动神经元与目标肌肉形成精确的连接。在胚胎发育和神经再生中，轴突通过整合来自引导信号的信号来导航高度结构化的细胞外环境来找到它们的目标。这些信号是由预期轴突路径附近的细胞产生的，并被细胞外基质(ECM)结合或内置于细胞外基质中，以引导轴突和建立神经肌肉突触。虽然许多研究都集中在描绘由netrins、eparin和Slits等信号介导的经典轴突引导通路，但ECM成分及其修饰在胚胎生长和再生过程中指导轴突的机制尚不完全清楚。这项建议的目的是确定ECM成分及其修饰所支配的分子和细胞过程，以指导脊髓运动轴突在发育和再生过程中的作用。在斑马鱼中，最近的研究表明，LH3/diwanka突变会导致轴突寻路错误。lh3/diwanka是一种糖基转移酶，表达在与轴突路径相邻的细胞中。此外，功能丧失实验表明，lh3/diwanka通过ECM蛋白多糖胶原XVIII(Col18a1)提供指导。Col18a1是一种非典型的胶原蛋白，它含有几个信号分子的蛋白质相互作用结构域，这些信号分子对轴突的发现很重要。最后，包括col18a1在内的ECM蛋白在轴突损伤部位和再生轴突所走的路径上表达上调，这与轴突再生过程中可能的功能一致。在这项提案中，我将定义ECM组件及其修饰在发育和再生过程中指导脊髓运动轴突的机制。首先，我将确定在引导两种不同类型的脊髓运动神经轴突时，ECM改变的时间要求。其次，我将通过定义正确的运动轴突引导所必需的蛋白质相互作用结构域来确定COL18A1信号机制(S)在运动轴突引导中的作用。最后，我将通过比较野生型神经再生的速率和保真度与没有这些修饰的情况来确定lh3/diwanka介导的ECM修饰在神经再生中的作用。这些实验将共同定义神经肌肉连接发展和重建所必需的细胞外基质介导的引导通路。 公共卫生相关性：脊髓损伤的受害者终身失去运动和感觉，因为脊髓中受损的神经无法再生。相比之下，脊髓周围被剪断的神经会再生，并重新启动感觉和运动活动。这些研究的结果将确定引导周围神经到达目标的机制，希望这些途径将为包括脊髓损伤在内的神经损伤患者提供新的治疗方法。

DESCRIPTION (provided by applicant): In vertebrates, movement requires that spinal motor neurons form precise connections with target muscles. In both embryonic development and nerve regeneration, axons find their targets by integrating signals from guidance cues to navigate a highly structured extracellular environment. These cues are generated by cells neighboring the prospective axonal path and are bound by or built into the extracellular matrix (ECM) to guide axons and establish neuromuscular synapses. Although much research has focused on delineating 'classical' axon guidance pathways mediated by cues like Netrins, Ephrins, and Slits, the mechanisms by which ECM components and their modifications direct axons during embryonic outgrowth and regeneration are not fully understood. The goal of this proposal is to define the molecular and cellular processes governed by ECM components and their modifications to guide spinal motor axons during development and regeneration. In zebrafish, recent studies demonstrate that mutations in lh3/diwanka, a glycosyltransferase that is expressed in cells bordering the axonal path, lead to axon pathfinding errors. Moreover, loss-of-function experiments suggest that lh3/diwanka provides guidance through the ECM proteoglycan Collagen XVIII (col18a1). Col18a1 is an atypical collagen that contains protein interaction domains for several signaling molecules important for axon pathfinding. Finally, ECM proteins, including col18a1, are upregulated at axon injury sites and along the path taken by regenerating axons, consistent with a possible function during axonal regeneration. In this proposal, I will define the mechanisms that ECM components and their modifications utilize to direct spinal motor axons during development and regeneration. First I will determine the temporal requirements of ECM modifications in guiding two distinct classes of spinal motor axons. Secondly, I will determine the col18a1 signaling mechanism(s) in motor axon guidance by defining the protein interaction domains that are necessary for proper motor axon guidance. Lastly, I will determine the role of lh3/diwanka- mediated ECM modifications in nerve regeneration by comparing the rate and fidelity of wild type nerve regrowth to that in the absence of these modifications. Together these experiments will define ECM mediated guidance pathways necessary for the development and reconstitution of neuromuscular connections. PUBLIC HEALTH RELEVANCE: Victims of spinal cord injury suffer from lifelong loss of movement and sensation because damaged nerves in the spinal cord fail to regenerate. In contrast, sheared nerves peripheral to the spinal cord regenerate and reinitiate sensory and motor activity. Results from these studies will define mechanisms that guide peripheral nerves to their targets with the hope that these pathways will provide avenues to new therapeutic treatment for patients suffering from nerve damage, including spinal cord injury.