Extracellular Matrix Signaling in Axon Outgrowth and Regeneration

描述（由申请人提供）：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连接。在胚胎发育和神经再生中，轴突通过整合来自引导线索的信号来定位高度结构化的细胞外环境。这些线索由邻近预期轴突路径的细胞产生，并被细胞外基质（ECM）结合或构建到细胞外基质（ECM）中，以引导轴突并建立神经肌肉突触。虽然许多研究都集中在描绘'经典的'轴突引导途径介导的线索，如Netrins，Ephrins，和裂缝，ECM组件和它们的修改直接轴突在胚胎生长和再生的机制还没有完全理解。 该提案的目标是定义ECM成分及其修饰所控制的分子和细胞过程，以在发育和再生过程中指导脊髓运动轴突。在斑马鱼中，最近的研究表明，lh 3/diwanka（一种在轴突路径边界细胞中表达的糖基转移酶）的突变导致轴突寻路错误。此外，功能丧失实验表明lh 3/diwanka通过ECM蛋白聚糖胶原蛋白XVIII（col 18 a1）提供指导。Col 18 a1是一种非典型的胶原蛋白，它含有几种对轴突寻路很重要的信号分子的蛋白质相互作用结构域。最后，ECM蛋白，包括col 18 a1，在轴突损伤部位和沿着再生轴突所采取的路径上调，与轴突再生过程中的可能功能一致。 在这个提议中，我将定义ECM成分及其修饰在发育和再生过程中用于指导脊髓运动轴突的机制。首先，我将确定ECM修改的时间要求，指导两个不同类别的脊髓运动轴突。其次，我将通过定义正确的运动轴突指导所必需的蛋白质相互作用结构域来确定运动轴突指导中的col 18 a1信号传导机制。最后，我将通过比较野生型神经再生的速率和保真度与不存在这些修饰的情况下的速率和保真度来确定lh 3/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ECM介导的神经肌肉连接的发展和重建所必需的指导途径。 公共卫生相关性：脊髓损伤的受害者遭受终身的运动和感觉丧失，因为脊髓中受损的神经无法再生。与此相反，脊髓周围被切断的神经再生并重新启动感觉和运动活动。这些研究的结果将定义引导周围神经到达其目标的机制，希望这些途径将为患有神经损伤（包括脊髓损伤）的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.