Regulation of Nervous System Wiring by the Robo3 Axon Guidance Receptor and its Ligand NELL2

Robo3 轴突引导受体及其配体 NELL2 对神经系统布线的调节

• 批准号: 10175061
• 负责人: Alexander Jaworski
• 金额: $ 44.75万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10175061
• 关键词: Address; Axon; Binding; Biochemical; Biological Assay; Brain; Chemosensitization; Cues; Data; Dependence; Development; Disease; Embryo; Embryonic Development; Embryonic Nervous System; Etiology; Family; Family member; Follow-Up Studies; Genetic; Goals; Growth Cones; Image; Immunohistochemistry; In Situ Hybridization; In Vitro; Injury; Knockout Mice; Ligands; Maps; Mediating; Mediator of activation protein; Methods; Molecular; Mus; Mutant Strains Mice; NTN1 gene; Nervous system structure; Neurodegenerative Disorders; Neurons; Optic Chiasm; Orphan; Pattern; Population; Proteins; Regulation; Reporter; Research; Retina; Retinal Ganglion Cells; Role; Signal Pathway; Signal Transduction; Site; Spinal Cord; Structure; Testing; Therapeutic; Visual system structure; Work; axon growth; axon guidance; axon injury; axonal pathfinding; base; experimental study; expression cloning; improved; in vitro Assay; in vivo; insight; interest; loss of function; mutant; nervous system development; netrin receptor; neural circuit; neurodevelopment; neuron development; novel; receptor; response; superior colliculus Corpora quadrigemina

PROJECT SUMMARY The guidance of axons to their correct targets is an important step in nervous system wiring, but the molecular and cellular mechanisms of axon guidance are still not completely understood. The goal of the proposed research is to elucidate the functions of a new axon guidance cue, NELL2, and its receptor Robo3 in neural development and to define the molecular mechanisms mediating NELL2-Robo3 signaling. While the receptors Robo1 and Robo2 mediate axon repulsion by guidance molecules of the Slit family, the divergent Robo family member Robo3 does not bind Slits but instead silences Slit-Robo1/2 signaling. Furthermore, Robo3 indirectly potentiates axonal attraction by Netrin-1, mediated by the Netrin receptor DCC. We recently identified a novel guidance cue, NELL2, as a repulsive ligand for the previously orphan receptor Robo3. We generated NELL2 knockout mice and found that NELL2/Robo3-mediated repulsion guides commissural axons in the embryonic spinal cord. Our findings define a novel NELL2-Robo3 signaling pathway for axon guidance and raise the possibility that it wires neural circuits throughout the nervous system. To test this idea, we will analyze the expression of NELL2 and Robo3 during neural development by several means, including genetic reporter lines. Neuronal populations that express Robo3 or project axons close to sites of NELL2 expression will be examined for NELL2 responses in vitro. In these axon guidance assays, Robo3-dependence will be tested using neurons from Robo3 mutant mice. To determine the in vivo roles of NELL2 and Robo3 in wiring neuronal populations of interest, axonal trajectories in NELL2- and Robo3-deficient mice will be analyzed by in toto imaging of the embryonic nervous system and by several complementary approaches. Based on preliminary expression data, initial studies will focus on retinal ganglion cell axon guidance. Our results from commissural neurons show that Robo3 function in axon guidance is three- fold, as it inhibits Slit-induced repulsion through Robo1/2, potentiates Netrin-1 attraction through DCC, and mediates repulsion from its own ligand NELL2. It is unclear which intracellular signaling pathways are engaged downstream of NELL2 and how Robo3 is able to perform its multiple functions simultaneously. To address these questions, we will test if Slit silencing, Netrin-1 attraction, and NELL2 repulsion can be separated in Robo3 structure-function analyses in vitro and in vivo. Using candidate and unbiased biochemical approaches, we will identify novel NELL2 receptors and downstream mediators of Robo3 signaling and determine their roles in axon guidance in vitro and neural circuit formation in vivo. Our work is expected to provide important insights into the functions of NELL2 and Robo3 in brain wiring. It will also help elucidate how different axon guidance signaling pathways interact within the same neuron, a fundamental question in neural development that is still poorly understood. By defining mechanisms of axon pathfinding, the proposed work has the potential to inform therapeutic approaches aimed at restoring damaged axonal connections after physical injury or onset of neurodegenerative disease. Furthermore, it can help define the mechanisms underlying the etiology of diseases that result from nervous system mis-wiring.

项目摘要 引导轴突到达其正确的目标是神经系统布线的重要步骤，但分子和细胞的 轴突导向的机制仍然没有完全理解。这项研究的目的是阐明 一种新的轴突导向因子NELL 2及其受体Robo 3在神经发育中的功能，并确定其分子机制。 调节NELL 2-Robo 3信号传导的机制。虽然受体Robo 1和Robo 2通过以下方式介导轴突排斥： 作为Slit家族的引导分子，分歧的Robo家族成员Robo 3不结合Slit，而是沉默 Slit-Robo 1/2信号。此外，Robo 3通过Netrin-1间接增强轴突吸引力，Netrin-1介导了Robo 3的作用。 受体DCC。我们最近发现了一种新的引导因子NELL 2，作为先前孤儿的排斥配体。 受体Robo 3。我们产生了NELL 2基因敲除小鼠，发现NELL 2/Robo 3介导的排斥引导蛋白， 胚胎脊髓中的连合轴突。我们的发现定义了一种新的NELL 2-Robo 3轴突信号通路 引导并提高它在整个神经系统中连接神经回路的可能性。为了验证这个想法，我们将 通过多种方法分析NELL 2和Robo 3在神经发育过程中的表达，包括遗传报告基因 线将检查表达Robo 3或投射轴突接近NELL 2表达位点的神经元群体的 体外NELL 2应答。在这些轴突引导测定中，将使用来自Robo 3的神经元测试Robo 3依赖性。 突变小鼠为了确定NELL 2和Robo 3在连接感兴趣的神经元群体中的体内作用， NELL 2和Robo 3缺陷小鼠的轨迹将通过胚胎神经系统的整体成像来分析 以及几种互补的方法。基于初步的表达数据，初步的研究将集中在视网膜 神经节细胞轴突引导。我们对连合神经元的研究结果表明，Robo 3在轴突引导中的功能是三个- 折叠，因为它通过Robo 1/2抑制狭缝诱导的排斥，通过DCC增强Netrin-1吸引，并介导 与自身配体NELL 2的排斥。目前还不清楚哪些细胞内信号通路参与下游的 NELL 2以及Robo 3如何能够同时执行其多种功能。为了解决这些问题，我们将测试 如果Slit沉默、Netrin-1吸引和NELL 2排斥可以在Robo 3体外结构-功能分析中分离， 和体内。使用候选和无偏见的生物化学方法，我们将确定新的NELL 2受体， Robo 3信号传导的下游介质，并确定它们在体外轴突引导和神经回路形成中的作用 in vivo.我们的工作有望为NELL 2和Robo 3在大脑布线中的功能提供重要的见解。它将 还有助于阐明不同的轴突引导信号通路如何在同一神经元内相互作用，这是一个基本的 神经发育中的一个问题，仍然知之甚少。通过定义轴突寻路机制， 这项工作有可能为旨在恢复物理损伤后受损轴突连接的治疗方法提供信息。 损伤或神经变性疾病发作。此外，它可以帮助确定潜在的病因机制， 神经系统错误连接导致的疾病。