The Role of Intracellular Trafficking in Regulating Axon Guidance Receptors During Neuronal Circuit Formation

项目摘要 为了形成一个有功能的神经系统，每个神经元都必须找到并连接到适当的突触靶点。 轴突必须经常长距离移动以寻找它们的目标，由各种分泌和细胞引导。 表面轴突引导分子。这些配体通过与生长过程中的受体相互作用来控制轴突。 通过改变细胞骨架结构、细胞黏附和转录来改变细胞行为。 轴突到达目的地的复杂轨迹通常被分解成更小的片段， 中间目标或“选择点”点缀其间。在双边生物体中，中线是至关重要的 中枢神经系统神经元的一个子集的选择点，称为连合神经元，其轴突必须穿过它才能整合 身体左右半部分之间的信息。中线交叉对于正常的运动和 认知功能和这一重要过程中的缺陷可导致多种神经发育障碍。 中线交叉是由中线胶质细胞分泌的吸引和排斥线索的组合控制的。 连合神经元最初对吸引人的暗示(如Netrin)很敏感，这会将它们吸引到中线。 然而，在交叉后，它们会对令人厌恶的暗示做出反应，例如狭缝，这有助于从 中线并防止再次交叉。交叉前连合神经元必须防止过早反应 并部分通过下调狭缝感受器环形交叉路口(Robo)来做到这一点。果蝇 通过贩运受体CommIsless(Comm)下调Robo表面表达，从而分流 新合成的Robo从跨高尔基网络直接进入内溶酶系统，并减少 ROBO蛋白质水平。Comm细胞质尾部的两个PY基序是其执行能力所必需的 这些任务。由于PY基序是已知的Nedd4家族Hect连接酶的结合位点，这强烈表明 COMM通过与细胞内泛素化机制相互作用来下调Robo的表达。精密机械 然而，Comm和泛素连接酶之间的关系还没有被非常详细地探讨。在这 建议，我们试图测试假设，Comm作为一个适配器蛋白，将E3连接酶带入 与Robo非常接近，Robo的泛素化将其靶向内溶体系统进行降解。 在第一个目标中，我们试图阐明泛素将Comm-Robo复合体运输到 内切/溶酶体，促进ROBO降解，促进中线交叉。在第二个目标中，我们将调查 Comm与三种果蝇Nedd4家族Hect连接酶的生化和遗传相互作用 (Nedd4、SU(DX)和蓝精灵)。总之，拟议的工作将提供对这些机制的更深层次的洞察 在神经回路发育过程中调节轴突引导受体的表面表达。

Project Abstract To form a functional nervous system, each neuron must find and connect to the appropriate synaptic targets. Axons must often travel long distances in search of their targets, directed by a variety of secreted and cell surface axon guidance molecules. These ligands steer axons by interacting with receptors on their growth cones and altering cell behavior through changes in cytoskeletal architecture, cell adhesion, and transcription. The complex trajectories that axons take to reach their destinations are often broken up into smaller segments, which are punctuated by intermediate targets or “choice points.” In bilateral organisms, the midline is a vital choice point for a subset of CNS neurons, called commissural neurons, whose axons must cross it to integrate information between the left and right halves of the body. Midline crossing is necessary for normal motor and cognitive function and defects in this vital process can result in a wide variety of neurodevelopmental disorders. Midline crossing is controlled by a combination of attractive and repulsive cues secreted by midline glia. Commissural neurons are initially sensitive to attractive cues such as Netrin, which draw them to the midline. After crossing, however, they become responsive to repulsive cues such as Slit, which facilitate exit from the midline and prevent re-crossing. Pre-crossing commissural neurons must prevent premature responsiveness to these repulsive cues and do this, in part, by downregulating the Slit receptor Roundabout (Robo). Drosophila downregulate Robo surface expression via the trafficking receptor Commissureless (Comm), which shunts newly-synthesized Robo directly from the Trans-Golgi Network to the endolysosomal system and decreases Robo protein levels. Two PY motifs in the cytoplasmic tail of Comm are necessary for its ability to perform these tasks. As PY motifs are known binding sites for Nedd4-family HECT ligases, this strongly suggests that Comm downregulates Robo by interacting with intracellular ubiquitination machinery. The precise mechanistic relationship between Comm and ubiquitin ligases, however, has not been explored in great detail. In this proposal, we seek to test the hypothesis that Comm acts as an adaptor protein that brings E3 ligases into close proximity to Robo, and that ubiquitination of Robo targets it to the endolysosomal system for degradation. In aim one, we seek to elucidate the requirement for ubiquitin to traffic the Comm-Robo complex to endo/lysosomes, facilitate Robo degradation, and promote midline crossing. In aim two, we will investigate the biochemical and genetic interactions between Comm and the three Drosophila Nedd4 family HECT ligases (Nedd4, Su(dx), and Smurf). In summary, the proposed work will provide deeper insight into the mechanisms regulating surface expression of axon guidance receptors during the development of neural circuits.