Axon guidance through the bifunctional cue WFIKKN2 and its receptors

通过双功能信号 WFIKKN2 及其受体进行轴突引导

• 批准号: 10625432
• 负责人: Alexander Jaworski
• 金额: $ 43.52万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625432
• 关键词: Afferent Neurons; Animals; Architecture; Area; Axon; Behavioral; Binding; Binding Proteins; Biochemical; Biological Assay; Complex; Cues; Data; Defect; Development; Disease; Embryonic Development; Etiology; Evolution; Family; Family member; Functional disorder; Gene Family; Growth Cones; Human; In Vitro; Individual; Injury; Invaded; Knockout Mice; Ligand Binding; Ligand Binding Domain; Ligands; Limb Bud; Logic; Mediating; Mediator; Mesenchymal; Mesenchyme; Methods; Molecular; Motor; Motor Neurons; Mus; NTN1 gene; Nature; Nematoda; Nerve; Nerve Regeneration; Nervous System; Neural tube; Neuroanatomy; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Organism; Orphan; Output; Pattern; Peripheral; Phenotype; Process; Proteins; Receptor Gene; Reporting; Research; Role; Sensory; Sensory Ganglia; Series; Shapes; Signal Pathway; Signal Transduction; Specificity; Spinal; Spinal Ganglia; Subgroup; Tertiary Protein Structure; Testing; Therapeutic; Tissues; Vertebral column; Vertebrates; Work; axon growth; axon guidance; axonal pathfinding; domain mapping; experimental study; fly; gain of function; improved; in vivo; insight; interdisciplinary approach; knockout animal; loss of function; member; mouse genetics; neogenin; neural circuit; neural network; neurotransmission; novel; novel therapeutic intervention; paralogous gene; pharmacologic; receptor; repaired; response

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.

项目概要/摘要 作为神经网络一部分的神经元之间的连接控制着通过神经系统的信息流 从而塑造行为输出。轴突引导至正确目标是神经回路的关键步骤 胚胎发育过程中的组装，理解这个过程至关重要，因为神经元的错误接线可能会导致 导致电路功能障碍和疾病。我们报告了一种新型轴突引导线索 WFIKKN2 的鉴定，作为 几种 DCC 家族受体的配体，并建议研究这些信号传导的生化性质 复合物及其在神经元布线中的功能。 DCC 是有吸引力的轴突导向信号 Netrin 的受体 1，这个配体-受体对塑造了线虫和人类等多种生物体的神经元连接。 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成员——DCC 和 Neogenin。 另外三个家庭成员——Punc、Nope 和 Protogenin——在整个过程中出现了相当大的分歧 进化的过程中，不结合 Netrins，并且它们在轴突引导中的功能仍然难以捉摸。我们发现 分泌型多结构域蛋白 WFIKKN2 结合 Punc、Nope 和 Protogenin，但不结合 DCC 和 Neogenin。我们 提供证据表明发育中的感觉和运动神经元表达这些新识别的受体 WFIKKN2，而WFIKKN2在身体外周的多个组织中表达。其他初步结果 显示 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 敲除小鼠的分析有力地支持了 WFIKKN2 介导的排斥有助于感觉的观点 轴突导航到其外围目标。我们提出了一种多学科方法来研究 WFIKKN2 及其 进一步研究受体并测试有关它们在神经回路形成中的功能的假设。我们将使用蛋白质 相互作用测定来绘制介导配体-受体相互作用的域并确定配体如何结合 DCC 家族内部出现了特异性。我们还将使用培养的神经元进行轴突引导测定与 功能操作来剖析 Punc、Nope 和 Protogenin 对 WFIKKN2 依赖的贡献 轴突的吸引力和排斥力，我们将使用生化方法来识别轴突的下游介质 WFIKKN2 信号传导。为了阐明 WFIKKN2 及其受体在体内的轴突引导功能，我们将 采用小鼠遗传学和最先进的神经解剖学方法。我们的工作将揭示以下机制 运动和感觉轴突引导，它通过发现扩展了已知的神经元布线分子工具包 一种新型双功能轴突引导信号及其受体。我们的研究进一步有可能发现新的 引导信号传导的细胞内介质。从长远来看，我们的工作可以帮助阐明如何 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。总的来说，通过提供对神经元连接机制的重要见解，这项研究将 有助于我们对神经发育疾病病因学的理解，并且可以帮助发展 各种疾病状态下神经回路修复和神经再生的新治疗方法。