Regulation of axon guidance receptor trafficking in the developing mammalian central nervous system

发育中哺乳动物中枢神经系统轴突引导受体运输的调节

基本信息

批准号：
10677668
负责人：
Greg J. Bashaw
金额：
$ 45.49万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-09 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10677668
关键词：
Adaptor Signaling Protein Animals Antibodies Axon Bilateral Binding Biochemical Biological Biological Assay Brain Cell Line Cells Central Nervous System Cues Data Defect Development Disease Dissociation Dorsal Drosophila genus Drosophila sli protein Dyes Elements Embryo Endosomes Exhibits Family Floor Genetic Growth Cones Human Immunofluorescence Immunologic In Situ In Vitro Injury Ligands Ligase Link Malignant Neoplasms Mammals Mediating Messenger RNA Molecular Monitor Morphogenesis Mus Nerve Regeneration Nervous System Neurons Pathway interactions Pattern Play Process Proteins Proteomics Receptor Signaling Regulation Reporter Research Role Signal Pathway Signal Transduction Signaling Molecule Spinal Spinal Cord Stains Surface Synapses System Techniques Testing Therapeutic Tissues Transcriptional Regulation Vertebral column Vertebrates Work axon guidance axon regeneration conditional knockout conditional mutant developmental disease experimental study extracellular fly gene function hindbrain in vivo insight late endosome lipophilicity motor behavior mouse genetics mutant nervous system development nervous system disorder neural circuit novel premature prevent protein expression protein transport receptor recruit repaired response screening trafficking ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY Determining how neurons are assembled into functional circuits will provide insight into developmental disorders of the nervous system and may suggest therapeutic approaches to promote nerve regeneration. To navigate to their correct targets, axons must modulate their responses to extracellular cues, and regulated intracellular protein trafficking plays a pivotal role in this process. For example, commissural axons cross the midline despite the presence of repellant ligands in order to establish connections that are essential for coordinated motor behavior. In Drosophila, the endosomal protein Commissureless (Comm) prevents commissural axons from prematurely responding to the repellant Slit, by inhibiting surface expression of the Slit receptor Roundabout1 (Robo1). In mammals, Robo receptors are also negatively regulated in commissural axons prior to midline crossing, but the mechanisms are unknown. Unlike Slit and Robo, comm is not conserved in vertebrates; however, our preliminary data indicate that the vertebrate Nedd-4 interacting proteins (Ndfip1 and Ndfip2) can act analogously to Comm to regulate the trafficking and stability of human Robo receptors in vitro, and that loss of Ndfip1or Ndfip2 function in vivo in mice results in increased expression of Robo receptors and defects in axon guidance. We will test the hypothesis that Ndfip proteins control axon guidance in the developing brain and spinal cord by recruiting Robo receptors to endosomes and triggering their degradation through interactions with Nedd-4 E3 ubiquitin ligases. In aim 1, we will use molecular, cell biological and biochemical approaches to: 1) determine whether Ndfip proteins exhibit differential effects on intracellular trafficking of Robo receptors or other axon guidance receptors, 2) delimit the sequences that are necessary and sufficient to mediate interactions between Ndfip proteins and Robo family receptors, 3) characterize the role of HECT E3 ligase activity on receptor trafficking and 4) identify the specific Nedd4 family ligase(s) that is required for Robo receptor regulation. Aim 2 will explore the embryonic expression patterns and in vivo requirements for Ndfip proteins during commissural axon guidance by examining the trajectory of commissural axons in Ndfip1 and Ndfip2 single and double mutants, using 1) immunofluorescence for pre and post-crossing commissural axon markers, and 2) unilateral lipophilic dye tracing experiments. In addition, we will generate conditional knockouts of Ndfip1, Nedd4-1 and Nedd4-2 using Cre-lines specific for commissural neurons to investigate requirements for Nedd4-1 and Nedd4-2 in spinal commissural axon guidance. Aim 3 will assess the in vivo links between Ndfip proteins and Robo receptors by 1) testing whether neurons cultured from Ndfip mutants exhibit altered repulsive responses to exogenously added Slit proteins and 2) examining genetic interactions between Ndfip and Robo mutants. Finally, a biochemical screen will be conducted to identify novel substrates of Ndfip proteins.

项目摘要确定神经元如何组装到功能电路中将提供有关发展的洞察力神经系统的疾病，并可能提出促进神经再生的治疗方法。到导航到其正确的目标，轴突必须调节其对细胞外提示的反应，并调节细胞内蛋白运输在此过程中起关键作用。例如，连合轴突越过中线尽管存在驱虫配体，以建立对协调的运动行为。在果蝇中，内体蛋白连续性（COMM）阻止通过抑制表面表达的表达狭缝受体rondabout1（robo1）。在哺乳动物中，机器人受体在合理中也受到负调节中线交叉之前的轴突，但是机制尚不清楚。与缝隙和机器人不同，通信不是在脊椎动物中保守；但是，我们的初步数据表明脊椎动物NEDD-4相互作用蛋白（NDFIP1和NDFIP2）可以类似地采取行动以调节人类机器人的贩运和稳定性体外受体，NDFIP1OR NDFIP2在小鼠体内功能的丧失导致表达增加轴突引导中的机器人受体和缺陷。我们将测试NDFIP蛋白控制轴突的假设通过招募机器人受体并触发机器人，在发育中的大脑和脊髓引导它们通过与NEDD-4 E3泛素连接酶相互作用而降解。在AIM 1中，我们将使用分子细胞生物学和生化方法：1）确定NDFIP蛋白是否对机器人受体或其他轴突引导受体的细胞内运输，2）划定序列必要且足以介导NDFIP蛋白与Robo家族受体之间的相互作用，3）表征E3连接酶活性在受体运输中的作用，4）确定特定的NEDD4家族机器人调节所需的连接酶。 AIM 2将探索胚胎表达模式在合并轴突指导期间NDFIP蛋白的体内需求通过检查的轨迹 NDFIP1和NDFIP2单一和双重突变体中的轴突，使用1）免疫荧光用于PRE和交叉后轴突标记和2）单侧亲脂性染料跟踪实验。另外，我们将使用特定于合理的Cre-line产生NDFIP1，NEDD4-1和NEDD4-2的有条件敲除神经元研究NEDD4-1和NEDD4-2在脊柱轴突指导中的需求。目标3意志通过1）测试神经元是否培养，评估NDFIP蛋白与机器人受体之间的体内链接从NDFIP突变体表现出对外源添加的缝隙蛋白的排斥反应的改变，2）检查 NDFIP和Robo突变体之间的遗传相互作用。最后，将进行生化屏幕识别NDFIP蛋白的新型底物。