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

项目摘要 确定神经元是如何组装成功能电路的，将有助于了解发育过程。 神经系统疾病，并可能建议治疗方法，以促进神经再生。到 轴突必须调节它们对细胞外信号的反应，并调节它们对细胞外信号的反应。 细胞内蛋白质运输在该过程中起关键作用。例如，连合轴突穿过 尽管存在排斥配体，但为了建立对于 协调的运动行为在果蝇中，内体蛋白Commissureless（Comm）阻止了 连合轴突过早响应排斥性狭缝，通过抑制表面表达的 狭缝受体Roundabout 1（Robo 1）。在哺乳动物中，Robo受体也在连合中负调控， 轴突之前中线交叉，但机制是未知的。与Slit和Robo不同， 然而，我们的初步数据表明，脊椎动物Nedd-4相互作用蛋白 （Ndfip 1和Ndfip 2）可以类似于Comm来调节人类Robo的贩运和稳定性 受体，并在小鼠体内Ndfip 1或Ndfip 2功能的丧失导致表达增加， Robo受体与轴突导向缺陷。我们将测试Ndfip蛋白控制轴突的假设 通过将Robo受体募集到核内体并触发 它们通过与Nedd-4 E3泛素连接酶的相互作用而降解。在目标1中，我们将使用分子，细胞 生物学和生物化学方法，以：1）确定Ndfip蛋白是否表现出对 Robo受体或其他轴突导向受体的细胞内运输，2）界定 必要且足以介导Ndfip蛋白和Robo家族受体之间的相互作用，3） 表征HECT E3连接酶活性对受体运输的作用和4）鉴定特异性Nedd 4家族 Robo受体调节所需的连接酶。目的2将探索胚胎表达模式 以及在连合轴突引导过程中Ndfip蛋白的体内需求， Ndfip 1和Ndfip 2单突变体和双突变体中的连合轴突，使用1）免疫荧光法检测前和 交叉后连合轴突标记，和2）单侧亲脂性染料示踪实验。另外我们 将使用连合特异性的Cre-lines产生Ndfip 1、Nedd 4 -1和Nedd 4 -2的条件敲除 神经元研究Nedd 4 -1和Nedd 4 -2在脊髓连合轴突引导中的需求。目标3将 通过以下方式评估Ndfip蛋白和Robo受体之间的体内联系：1）测试培养的神经元是否 从Ndfip突变体表现出改变排斥反应外源添加的Slit蛋白和2）检查 Ndfip和Robo突变体之间的遗传相互作用。最后，将进行生化筛查， 鉴定Ndfip蛋白新底物。

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.