Mechanisms of Axonal Pathfinding Controlled by Netrin-1

在发育过程中，未成熟大脑中的分子将轴突引导到它们的目标区域，遵循长时间和 复杂的路径。建立正确的突触连接需要正确的路径寻找， 而在错综复杂的轴突寻路过程中的干扰被认为导致了各种 神经发育障碍。Netrin-1是一种分泌的、有吸引力的轴突引导信号，对 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，到目前为止确定的途径似乎不一致：cAMP/PKA 信号转导、轴突内蛋白质合成和降解以及Rho家族活性的调节 GTP酶，与L等人合作的蛋白质，在轴突细胞骨架的调节中发挥作用。此应用程序的目标是 为了了解这些不同的信号机制是如何整合在一起导致特征性轴突的 以Netrin-1信号为特征的突起和生长锥体转向反应。基于初步的 研究表明，netrin-1通过一种信号机制调节轴突生长和寻路 需要形成一个由PARS、PAR6、CDC42和aPKC^组成的信号复合体。在这 复合体，PAR6招募泛素E3连接酶SMurfl，导致RhoA的降解。我进一步 假设这种复合体的形成是由PAR的本地翻译触发的，而不是 从细胞体中招募PAR。 这项建议的具体目的是为了验证这一模型，并确定在活体模型中局部PARS翻译的重要性。作为了解Netrin-1等分子如何调控轴突生长和寻路的长期努力的一部分，该提议的目的是：(I)证明依赖于SMurfl的RhoA泛素化在Netrin-1信号中的作用；(Ii)建立局部 翻译PARs在netrin-1信号中的作用，以及(Iii)从遗传学角度剖析轴向定位的PARs的作用 在活体轴突寻路中的mRNA.从这些实验中获得的结果将为 为理解netrin-1信号的新的和统一的框架。

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.