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

项目概要 确定神经元如何组装成功能回路将有助于深入了解发育 神经系统疾病，并可能建议促进神经再生的治疗方法。至 为了导航到正确的目标，轴突必须调节它们对细胞外信号的反应，并受到调节 细胞内蛋白质运输在此过程中发挥着关键作用。例如，连合轴突穿过 尽管存在排斥配体，但仍处于中线，以建立必要的连接 协调的运动行为。在果蝇中，内体蛋白 Commissureless (Comm) 可防止 通过抑制连合轴突的表面表达，避免对排斥性狭缝过早做出反应 狭缝受体 Roundabout1 (Robo1)。在哺乳动物中，Robo 受体在连合神经中也受到负调节 轴突先于中线交叉，但机制尚不清楚。与 Slit 和 Robo 不同，comm 不是 在脊椎动物中保守；然而，我们的初步数据表明脊椎动物 Nedd-4 相互作用蛋白 （Ndfip1和Ndfip2）可以类似于Comm来规范人类Robo的贩运和稳定性 体外受体，小鼠体内 Ndfip1 或 Ndfip2 功能丧失会导致 Ndfip1 或 Ndfip2 表达增加 机器人受体和轴突引导缺陷。我们将检验 Ndfip 蛋白控制轴突的假设 通过将 Robo 受体招募到内体并触发来指导大脑和脊髓的发育 它们通过与 Nedd-4 E3 泛素连接酶相互作用而降解。在目标1中，我们将使用分子、细胞 生物和生化方法：1) 确定 Ndfip 蛋白是否对 Robo 受体或其他轴突导向受体的细胞内运输，2) 界定序列 介导 Ndfip 蛋白和 Robo 家族受体之间的相互作用是必要且充分的，3) 描述 HECT E3 连接酶活性对受体运输的作用，并 4) 识别特定的 Nedd4 家族 Robo 受体调节所需的连接酶。目标 2 将探索胚胎表达模式 通过检查连合轴突引导过程中 Ndfip 蛋白的体内需求 Ndfip1 和 Ndfip2 单突变体和双突变体中的连合轴突，使用 1) 免疫荧光进行前和 交叉后连合轴突标记，以及2)单侧亲脂性染料示踪实验。此外，我们 将使用特定于连合的 Cre 系产生 Ndfip1、Nedd4-1 和 Nedd4-2 的条件敲除 神经元来研究 Nedd4-1 和 Nedd4-2 在脊髓连合轴突引导中的要求。目标3将 通过 1) 测试是否培养神经元来评估 Ndfip 蛋白和 Robo 受体之间的体内联系 来自 Ndfip 突变体的 Ndfip 突变体对外源添加的 Slit 蛋白表现出改变的排斥反应，2) 检查 Ndfip 和 Robo 突变体之间的遗传相互作用。最后，将进行生化筛选 鉴定 Ndfip 蛋白的新底物。

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.