Netrin signaling during neuronal development

描述（由申请人提供）：在发育中的神经系统中，神经细胞和轴突在通往其最终目的地的途中对各种吸引和排斥的引导线索作出反应。在他们的旅程中，他们遵循一个复杂的轨迹，在某些情况下使用多个线索，在其他情况下切换他们对特定双功能线索的反应，在短距离和长距离上采取行动。在获得更好地理解这些事件背后的分子机制的一个重要步骤是确定受体复合物，耦合的指导线索，定向指导。揭示细胞外信号转化为生长锥细胞骨架变化的分子机制可能会导致更好地了解某些发育障碍，并有助于开发治疗干预措施，以改善损伤后的神经再生。该建议的中心目的是表征在发育中的神经系统中介导生长锥对双功能导向因子Netrin-1的反应的受体复合物。Netrin-1吸引几类轴突，同时排斥其他轴突。DCC家族的Netrin受体涉及介导吸引和排斥两者;相反，DCC-5家族的成员涉及单独或与DCC组合的信号排斥。UNC 5如何独立于DCC发出排斥信号尚不清楚。在这里，我们假设，UNC 5信号两种类型的DCC-独立的排斥，通过同源多聚化UNC 5A和通过异源多聚化UNC 5 B-D与孤儿受体PALORA。我们决心阐明Netrin-1如何介导轴突排斥。我们将（I）在体外研究脊椎动物脊髓神经突的DCC-独立排斥，（II）通过使用生物化学和遗传学方法的组合来定义介导DCC独立排斥的受体复合物，和（III）通过利用原代神经元培养物的定向和生长反应与神经元的缺失和获得相结合，在体外和体内评估这些受体在Netrin-1信号传导中的功能。功能接近。这项工作将为理解神经元如何将双向线索（如Netrin-1）转化为定向指导提供基础。此外，它应该是更普遍的重要性，在神经发育过程中提供的双功能线索，以指导轴突的潜在受体机制的见解。

DESCRIPTION (provided by applicant): In the developing nervous system, nerve cells and axons respond to a variety of attractive and repulsive guidance cues en route to their final destination. On their journey they follow a complicated trajectory using in some circumstances multiple cues, in others switching their responsiveness to a particular bifunctional cue, acting over short and long range. An essential step in obtaining a better understanding of the molecular mechanisms that underlie these events is to identify receptor complexes that couple guidance cues to directed guidance. Uncovering the molecular mechanisms that translate an extracellular cue into a change in the growth cone cytoskeleton is likely to lead to a better understanding of certain developmental disorders, and also to contribute towards development of therapeutic interventions to improve nerve regeneration following injury. The central aim of this proposal is to characterize receptor complexes that mediate responses of growth cones to the bifunctional guidance cue Netrin-1 in the developing nervous system.Netrin-1 attracts several classes of axons, while repelling others. Netrin receptors of the DCC family are implicated in mediating both attraction and repulsion; in contrast members of the UNC-5 family are implicated in signaling repulsion alone or in combination with DCC. How UNC5 signals repulsion independent of DCC is not known. Here we hypothezise that UNC5 signals two types of DCC-independent repulsion, through homomultimerization of UNC5A and through heteromultimerization of UNC5B-D with the orphan receptor PUNC. We are determined to elucidate how Netrin-1 mediates axonal repulsion. We will (I) characterize DCC- independent repulsion in vertebrates spinal neurites in vitro, (II) define the receptor complexes mediating DCC independent repulsion by using a combination of biochemical and genetic approaches and (III) evaluate the function of these receptors in Netrin-1 signaling in vitro and in vivo by utilizing directional and outgrowth responses of primary neuronal cultures in combination with loss- and gain-of-function approaches. This work will provide the foundation for understanding how neurons translate a bidirectional cue, such as Netrin-1, into directed guidance. In addition, it should be of more general importance in providing insights into the underlying receptor mechanism used by bifunctional cues to guide axons during neurodevelopment.