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.

项目总结/摘要 神经元之间的连接作为神经网络的一部分，控制着通过神经系统的信息流 从而塑造行为输出。引导轴突到达正确的靶点是神经回路的关键步骤 胚胎发育过程中的组装，理解这一过程是至关重要的，因为神经元的错误布线可以 导致电路功能障碍和疾病。我们报告了一种新的轴突导向因子WFIKKN 2的鉴定， 几个DCC家族受体的配体，并建议研究这些信号转导的生化性质 复合物及其在神经元布线中的功能。DCC是一种有吸引力的轴突导向因子Netrin的受体， 1，这种配体-受体对在线虫和人类等多种生物体中形成神经元连接。 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和再生蛋白。 另外三个家族成员-- Punc、Nope和Protogenin --在整个过程中出现了相当大的分歧 在进化的过程中，不结合Netrins，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WFIKKN 2结合Punc、Nope和Protogenin，但不结合DCC和再生蛋白。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发现的受体， WFIKKN 2，而WFIKKN 2在身体外周的多个组织中表达。其他初步结果 显示WFIKKN 2作为运动轴突的吸引剂和感觉轴突的排斥剂，表型 对WFIKKN 2基因敲除小鼠的分析强烈支持WFIKKN 2介导的排斥有助于感觉的观点。 轴突定位到它们的外围目标。我们提出了一个多学科的方法来研究WFIKKN 2及其 受体进一步和测试假设他们的功能在神经回路的形成。我们将使用蛋白质 相互作用测定以绘制介导配体-受体相互作用的结构域并确定配体结合如何 特异性在DCC家族中产生。我们还将使用培养的神经元结合联合收割机轴突引导试验， 功能操作，以剖析Punc，Nope和Protogenin对WFIKKN 2依赖性 轴突的吸引和排斥，我们将使用生物化学方法来确定下游介质， WFIKKN 2信令。为了阐明WFIKKN 2及其受体在体内的轴突导向功能，我们将 运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 运动和感觉轴突的指导，它扩大了已知的分子工具包神经元布线，发现 一种新的双功能轴突导向因子及其受体。我们的研究进一步有可能发现新的 细胞内介导的指导线索信号。从长远来看，我们的工作可以帮助阐明 轴突导向受体基因家族的进化多样性可能允许更复杂的连接， 神经系统总的来说，通过对神经元布线机制提供重要的见解，这项研究将 有助于我们了解神经发育疾病的病因，它可以帮助发展 用于各种疾病状态下的神经回路修复和神经再生的新治疗方法。