Axon guidance through the bifunctional cue WFIKKN2 and its receptors

项目摘要/摘要 神经元之间的连接作为神经网络的一部分，控制着神经系统中的信息流 并因此塑造行为输出。轴突向正确靶点的引导是神经回路中的关键步骤 在胚胎发育过程中进行组装，了解这一过程是至关重要的，就像神经元连接错误一样 导致电路功能障碍和疾病。我们报告了一种新的轴突引导线索WFIKKN2，它是一种 几个DCC家族受体的配基，并建议研究这些信号的生化性质 复合体及其在神经元连接中的功能。DCC是吸引人的轴突引导信号Netrin的受体。 1，这种配体-受体对塑造了从线虫到人类的各种有机体中的神经元连接。 在脊椎动物中，DCC家族由五个成员组成，Netrins可以结合其中的两个-DCC和新生素。 另外三个家庭成员--朋克、诺普和原生长素--在这一过程中出现了相当大的分歧 在进化中，网状蛋白并不结合，它们在轴突引导中的功能仍然难以捉摸。我们发现， 分泌的多结构域蛋白WFIKKN2可与Punc、Nope和Protogenin结合，但不能与DCC和negenin结合。我们 为发育中的感觉神经元和运动神经元表达这些新发现的受体提供证据 WFIKKN_2，而WFIKKN_2在身体周围的多个组织中表达。其他初步结果 表明WFIKKN2作为运动轴突的引诱剂和感觉轴突的排斥剂，以及表型 对WFIKKN2基因敲除小鼠的分析有力地支持了WFIKKN2介导的排斥力有助于感觉的观点 轴突导航到它们的外周目标。我们提出了一种多学科的方法来研究WFIKKN2及其 受体进一步验证关于它们在神经回路形成中作用的假说。我们将使用蛋白质 相互作用分析绘制介导配体-受体相互作用的结构域并确定配体如何结合 特异性出现在DCC家族中。我们还将结合使用培养神经元的轴突引导分析 功能操作分析Punc、Nope和Protogenin对WFIKKN_2依赖的作用 轴突的吸引和排斥，我们将使用生化方法来确定 WFIKKN2信令。为了阐明WFIKKN2及其受体在体内的轴突引导功能，我们将 使用老鼠遗传学和最先进的神经解剖学方法。我们的工作将揭示 运动和感觉轴突引导，它扩展了已知的神经元连接分子工具包，通过发现 一种新型的双功能轴突引导线索及其受体。我们的研究进一步有可能发现新的 引导信号的细胞内介体。从长远来看，我们的工作可以帮助阐明 轴突引导受体基因家族的进化多样化可能允许更复杂的连接 神经系统。总体而言，通过提供对神经元连接机制的重要见解，这项研究将 有助于我们理解神经发育疾病的病因学，并有助于 各种疾病状态下神经回路修复和神经再生的新治疗方法。

PROJECT SUMMARY/ABSTRACT Connectivity between neurons as part of neural networks governs information flow through the nervous system and therefore shapes behavioral output. Guidance of axon to their correct targets is a key step in neural circuit assembly during embryonic development, and understanding this process is critical, as neuronal miswiring can cause circuit dysfunction and disease. We report identification of a novel axon guidance cue, WFIKKN2, as a ligand for several DCC family receptors and propose to investigate the biochemical nature of these signaling complexes and their functions in neuronal wiring. DCC is a receptor for the attractive axon guidance cue Netrin- 1, and this ligand-receptor pair shapes neuronal connectivity in organisms as diverse as nematodes and humans. In vertebrates, the DCC family comprises five members, and Netrins can bind two of these – DCC and Neogenin. Three additional family members – Punc, Nope, and Protogenin – have diverged considerable over the course of evolution, do not bind Netrins, and their functions in axon guidance have remained elusive. We found that the secreted multi-domain protein WFIKKN2 binds Punc, Nope, and Protogenin, but not DCC and Neogenin. We provide evidence that developing sensory and motor neurons express these newly identified receptors for WFIKKN2, while WFIKKN2 is expressed in multiple tissues in the body periphery. Additional preliminary results show that WFIKKN2 acts as an attractant for motor axons and a repellant for sensory axons, and phenotype analysis of WFIKKN2 knockout mice strongly supports the idea that WFIKKN2-mediated repulsion helps sensory axons navigate to their peripheral targets. We propose a multidisciplinary approach to study WFIKKN2 and its receptors further and test hypotheses about their functions in neural circuit formation. We will use protein interaction assays to map the domains mediating ligand-receptor interactions and determine how ligand binding specificity arises within the DCC family. We will also combine axon guidance assays using cultured neurons with functional manipulations to dissect the contributions of Punc, Nope, and Protogenin to WFIKKN2-dependent axon attraction and repulsion, and we will use biochemical approaches to identify downstream mediators of WFIKKN2 signaling. To elucidate the axon guidance functions of WFIKKN2 and its receptors in vivo, we will employ mouse genetics and state-of-the-art neuroanatomical methods. Our work will uncover mechanisms of motor and sensory axon guidance, and it expands the known molecular toolkit for neuronal wiring by discovering a novel, bifunctional axon guidance cue and its receptors. Our studies further have the potential to identify new intracellular mediators of guidance cue signaling. In the long term, our work can help elucidate how the evolutionary diversification of axon guidance receptor gene families may allow the wiring of more complex nervous systems. Overall, by providing important insights into mechanisms of neuronal wiring, this research will contribute to our understanding of neurodevelopmental disease etiology, and it can aid in the development of novel therapeutic approaches for neural circuit repair and nerve regeneration in various disease states.