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

项目摘要 为了形成一个功能正常的神经系统，每个神经元必须找到并连接到适当的突触目标。 轴突必须经常长途跋涉寻找它们的目标，由各种分泌的和细胞的指导。 表面轴突引导分子。这些配体通过与受体相互作用来控制轴突的生长 视锥细胞，并通过改变细胞骨架结构、细胞粘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”打断了这些目标。在双侧生物体中，中线是至关重要的 中枢神经系统神经元子集的选择点，称为连合神经元，其轴突必须穿过它才能整合 身体的左右半部分之间的信息。中线交叉是正常运动所必需的， 认知功能和这一重要过程中的缺陷可导致各种神经发育障碍。 中线交叉是由中线胶质细胞分泌的吸引和排斥信号的组合控制的。 连合神经元最初对诸如Netrin的吸引性线索敏感，这将它们吸引到中线。 然而，在穿越之后，它们对诸如狭缝之类的排斥性线索做出反应，这些线索有助于从裂缝中退出。 中线，防止再次交叉。交叉前连合神经元必须防止过早反应 这些排斥性的线索，并做到这一点，部分，通过下调狭缝受体回旋（机器人）。果蝇 通过转运受体Commissureless（Comm）下调Robo表面表达， 新合成的Robo直接从trans-Golgi网络到内溶酶体系统， 机器人蛋白水平。Comm细胞质尾区的两个PY基序是其执行功能所必需的。 这些任务。由于PY基序是Nedd 4家族HECT连接酶的已知结合位点，这强烈表明， Comm通过与细胞内泛素化机制相互作用下调Robo。精确的机械 然而，Comm和泛素连接酶之间的关系尚未被详细研究。在这 建议，我们试图测试的假设，通信作为一个衔接蛋白，使E3连接酶进入 与Robo非常接近，并且Robo的泛素化将其靶向到内溶酶体系统进行降解。 在目标一，我们试图阐明的要求，泛素交通通讯机器人复杂， 内/溶酶体，促进Robo降解，并促进中线交叉。在目标二中，我们将调查 Comm与果蝇Nedd 4家族三种HECT连接酶之间的生化和遗传相互作用 （Nedd 4，Su（dx）和Smurf）。总之，拟议的工作将使人们更深入地了解这些机制， 在神经回路发育过程中调节轴突导向受体的表面表达。

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.