Mechanisms of Axonal Pathfinding Controlled by Netrin-1

在发育过程中，未成熟大脑中的分子引导轴突到达它们的目标区域， 复杂的路径。正确的寻路是建立正确的突触连接所必需的， 而轴突寻路过程中的复杂调节过程的紊乱被认为会引起各种各样的神经元损伤。 神经发育障碍Netrin-1是一种分泌的、有吸引力的轴突导向因子， 神经系统的正常形成。netrin-1介导轴突生长的机制 促进作用在很大程度上是未知的，迄今为止确定的途径似乎不一致：cAMP/PKA 信号传导、轴突内蛋白质合成和降解以及Rho家族活性的调节 GTP酶，在轴突细胞骨架的调节中具有重要作用的蛋白质。这个应用程序的目标是 为了理解这些不同的信号机制是如何整合的，从而导致特征性的轴突 生长和生长锥转向反应表征netrin-1信号传导。根据初步 研究中，我提出netrin-1通过信号机制调节轴突生长和寻路， 需要形成由PARS、PAR 6、cdc 42和aPKC β组成的信号传导复合物。在这 复合物中，PAR 6募集泛素E3连接酶Smurfl，导致RhoA降解。我进一步 假设这种复合体的形成是由PARS的本地翻译触发的，而不是 从细胞体募集PARS。 该提案的具体目的是验证该模型，并确定在体内模型中局部PARS翻译的重要性。作为理解netrin-1等分子如何调节轴突生长和寻路的长期努力的一部分，本提案的目的是：（I）证明RhoA的Smurfl依赖性泛素化在netrin-1信号传导中的作用，（II）建立局部泛素化的作用。 在netrin-1信号转导中PARS的翻译，以及（III）从遗传学角度剖析轴突定位的PARS的作用 mRNA在体内轴突寻路中的作用。从这些实验中获得的结果将为 一个新的和统一的框架来理解netrin-1信号。

During development, molecules in the immature brain steer axons to their target areas, following long and complex pathways. Proper pathfinding is required for the establishment of correct synaptic connections, and disturbances in the intricately regulated process of axonal pathfinding are thought to cause a variety of neurodevelopmental disorders. Netrin-1 is a secreted, attractive axon guidance cue that is crucial for the proper formation of the nervous system. The mechanisms by which netrin-1 mediates its axon outgrowth promoting effects are largely unknown, and the pathways identified thus far seem incongruent: cAMP/PKA signaling, intra-axonal protein synthesis and degradation, and regulation of the activity of Rho family GTPases, proteins with l<ey roles in the regulation of the axonal cytoskeleton. The goal of this application is to understand how these disparate signaling mechanisms are integrated to result in the characteristic axonal outgrowth and growth cone turning response that characterize netrin-1 signaling. Based on preliminary studies, I propose that netrin-1 regulates axon growth and pathfinding through a signaling mechanism that requires the formation of a signaling complex consisting of PARS, PAR6, cdc42, and aPKC^. In this complex, PAR6 recruits the ubiquitin E3 ligase Smurfl leading to degradation of RhoA. I further hypothesize that the formation of this complex is triggered by the local translation of PARS rather than recruitment of PARS from the cell body. The specific aims of this proposal are designed to validate this model and to determine the importance of local PARS translation in an in vivo model. As part of a long-term effort to understand how molecules such as netrin-1 regulate axonal outgrowth and pathfinding, the aims of this proposal are: (I) To demonstrate the role of Smurfl-dependent ubiquitination of RhoA in netrin-1 signaling, (II) to establish the role of local translation of PARS in netrin-1 signaling, and (III) to genetically dissect the role of axonally localized PARS mRNA in axon pathfinding in vivo. The results obtained from these experiments will create the foundation for a novel and unified framework for understanding netrin-1 signaling.