Netrin signaling during neuronal development

描述（由申请人提供）：在发育中的神经系统中，神经细胞和轴突在到达最终目的地的途中对各种吸引和排斥的引导信号作出反应。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，在其他情况下将他们的反应转换为一个特定的双功能线索，在短期和长期范围内行动。为了更好地理解这些事件背后的分子机制，一个重要的步骤是识别将引导线索与定向引导结合在一起的受体复合物。揭示细胞外信号转化为生长锥细胞骨架变化的分子机制，可能有助于更好地理解某些发育障碍，也有助于开发治疗干预措施，以改善损伤后的神经再生。本研究的中心目的是表征发育中的神经系统中介导生长锥对双功能引导线索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家族的网蛋白受体参与介导吸引和排斥；相反，UNC-5家族的成员单独或与DCC联合参与信号排斥。UNC5如何独立于DCC发出排斥信号尚不清楚。在这里，我们假设UNC5通过UNC5A的同聚化和UNC5B-D与孤儿受体PUNC的异聚化发出两种不依赖dc的排斥力信号。我们决心阐明Netrin-1如何介导轴突排斥。我们将(1)在体外研究脊椎动物脊髓神经突中DCC不依赖斥力的特征，(2)通过结合生化和遗传方法定义介导DCC不依赖斥力的受体复合物，以及(3)通过利用原代神经元培养的定向和生长反应，结合功能丧失和功能获得方法，评估这些受体在体外和体内Netrin-1信号传导中的功能。这项工作将为理解神经元如何将双向提示（如Netrin-1）转化为定向指导提供基础。此外，它在提供对双功能线索在神经发育过程中引导轴突所使用的潜在受体机制的见解方面应该具有更普遍的重要性。

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.