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会对各种吸引人的和令人厌恶的指导信号做出反应。在它们的旅程中，它们遵循一条复杂的轨迹，在某些情况下使用多个线索，在另一些情况下，将它们的反应切换到特定的双功能线索，在短距离和长距离上采取行动。要更好地了解这些事件背后的分子机制，关键的一步是识别将指导信号与定向指导相结合的受体复合体。揭示细胞外线索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，并有助于开发治疗干预措施，以改善损伤后的神经再生。这项建议的中心目标是表征在发育中的神经系统中介导生长锥对双功能指导线索Netrin-1的反应的受体复合体。Netrin-1吸引几种类型的轴突，而排斥其他类型的轴突。DCC家族的Netrin受体参与调节吸引和排斥；相比之下，UNC-5家族的成员参与单独或与DCC联合传递排斥信号。目前尚不清楚在不依赖于DCC的情况下，unc5如何发出斥力信号。在这里，我们假设，通过Unc5A的同聚和Unc5b-D与孤儿受体PUNC的异构化，UNC5发出两种类型的非DCC非依赖性排斥信号。我们决心阐明Netrin-1是如何介导轴突排斥的。我们将(I)在体外表征脊椎动物脊椎神经突起中DCC非依赖性排斥，(Ii)结合生物化学和遗传学方法确定介导DCC非依赖性排斥的受体复合体，以及(Iii)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的方法来评估这些受体在体外和体内Netrin-1信号转导中的功能。这项工作将为理解神经元如何将双向线索(如Netrin-1)转化为定向指导奠定基础。此外，在深入了解双功能线索在神经发育过程中引导轴突的潜在受体机制方面，它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.