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

细胞内运输在神经元回路形成过程中调节轴突引导受体的作用

基本信息

批准号：
9910542
负责人：
Kelly Gale Sullivan
金额：
$ 4.5万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9910542
关键词：
Adaptor Signaling Protein Address Animal Model Architecture Arginine Axon Behavior Bilateral Binding Binding Sites Biochemical Genetics Cell Adhesion Cell Culture Techniques Cell surface Cells Co-Immunoprecipitations Cognition Disorders Cognitive deficits Complex Contralateral Cues Cytoplasmic Tail Data Defect Destinations Drosophila genus Equilibrium Family Genetic Genetic Transcription Growth Cones Immunoprecipitation In Vitro Insecta Lead Learning Left Ligands Ligase Lysine Lysosomes Mediating Membrane Morphology Motor Mutate Nerve Nervous system structure Neuraxis Neurodevelopmental Disorder Neuroglia Neurons Organism Population Process Proteins RNA Interference Regulation Research Role Shunt Device Side Sorting - Cell Movement Specific qualifier value Surface Synapses System Testing Transfection Travel Ubiquitin Ubiquitination Western Blotting Work analog axon guidance axonal guidance cell behavior cognitive function fly in vivo insight knock-down late endosome member motor disorder mutant nervous system development neural circuit neurodevelopment neuronal circuitry premature prevent receptor scaffold trafficking trans-Golgi Network ubiquitin isopeptidase ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

项目摘要为了形成功能性神经系统，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，在各种分泌物和细胞的指导下寻找目标。这些配体通过与受体相互作用来引导轴突生长。视锥细胞并通过细胞骨架结构、细胞粘附和转录的变化来改变细胞行为。轴突到达目的地的复杂轨迹通常被分成更小的部分，在双边生物体中，中线是至关重要的，中间被中间目标或“选择点”打断。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左右半部之间的信息对于正常的运动和运动是必要的。这一重要过程的认知功能和缺陷可能导致多种神经发育障碍。中线交叉是由中线神经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 Netrin 等有吸引力的线索敏感，这些线索将它们吸引到中线。然而，在穿越之后，它们会对诸如狭缝之类的令人厌恶的暗示做出反应，这有助于从狭缝中退出。中线并防止重新交叉预交叉连合神经元必须防止过早反应。果蝇的 Slit 受体 Roundabout (Robo) 会受到这些令人厌恶的暗示。通过转运受体 Commissureless (Comm) 下调 Robo 表面表达，该受体分流新合成的 Robo 直接从跨高尔基体网络进入内溶酶体系统，并减少 Comm 细胞质尾部的两个 PY 基序对于其执行能力是必需的。由于 PY 基序是 Nedd4 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来下调 Robo 的精确机制。然而，Comm 和泛素连接酶之间的关系尚未得到详细探讨。提案中，我们试图测试 Comm 作为接头蛋白将 E3 连接酶引入的假设与 Robo 非常接近，并且 Robo 的泛素化将其靶向内溶酶体系统进行降解。在目标一中，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，并促进中线交叉在目标二中，我们将研究 Comm 与三种果蝇 Nedd4 家族 HECT 连接酶之间的生化和遗传相互作用（Nedd4、Su(dx) 和 Smurf）总之，拟议的工作将提供对这些机制的更深入的了解。在神经回路发育过程中调节轴突引导受体的表面表达。