Molecular Mechanisms Underlying Axon Pathfinding

描述（由申请人提供）：轴突用于确保大脑中正确布线的分子机制涉及多种配体-受体相互作用，因为细胞外引导线索，如netrins和RGMs（排斥性引导分子），在轴突寻路过程中至关重要。参与轴突路径发现的netrins受体包括DCC（在结直肠癌中缺失）和再生蛋白（一种含有免疫球蛋白结构域的蛋白质家族）。再生蛋白也是RGMs的受体。我们工作的长期目标是阐明DCC和再生蛋白参与启动的细胞内信号传导机制。在这方面，PI的实验室已经鉴定了DCC和再生蛋白的结合配偶体，其可能参与netrin-1/RGM诱导的细胞内信号传导，包括PITPalpha（磷脂酰肌醇转移蛋白α），一种对）、）、（磷脂酰肌醇信号传导至关重要的蛋白，和肌球蛋白X（Myo X），一种涉及细胞粘附和丝状伪足伸长的非常规肌球蛋白。事实上，初步研究表明，这些蛋白质确实对netrin-1触发的细胞内信号传导和调节其功能有重要贡献，因为PITPalpha或Myo X的抑制减弱netrin-1诱导的神经突生长和/或生长锥转向。Myo X似乎也参与将DCC递送至细胞外周或神经突。因此，所提出的研究是基于PITPalpha和Myo X参与启动DCC或再生蛋白信号传导和功能的假设。具体而言，我们将：目的1）分析PITPalpha在netrin-1诱导的磷脂酰肌醇信号传导和神经突生长中的作用;目的2）研究PITPalpha在RGMa诱导的神经元存活中的作用;目的3）确定Myo X在调节DCC分布和功能中的作用。我们将利用表达PITPalpha或Myo X的miRNA的神经元、神经元特异性Myo X条件性敲除小鼠和/或通过子宫内/卵内电穿孔小鼠/鸡胚胎中的miRNA而敲低PITPalpha/Myo X表达的神经元来评估这些问题。拟议的研究结果将提供深入了解细胞内机制控制大脑布线。这一过程的缺陷是所有年龄段神经系统疾病的基础，从新生儿到老年人的神经退化过程。了解这些信号通路为未来新的治疗方法提供了可能的基础。公共卫生相关性：这项提议的长期目标是了解轴突寻路的分子机制，这是大脑布线和功能的关键过程。这个过程中的缺陷与早期胚胎死亡、智力迟钝和脊髓损伤恢复受损有关。

DESCRIPTION (provided by applicant): Molecular mechanisms utilized by axons to ensure proper wiring in the brain involve multiple ligand-receptor interactions, as extracellular guidance cues, such as netrins and RGMs (repulsive guidance molecule), are critical in the process of axon path-finding. Receptors for netrins involved in axon path-finding include DCC (deleted in colorectal cancer) and neogenin, a family of immunoglobin domain-containing proteins. Neogenin is also a receptor for RGMs. The long-term goal of our work is to elucidate intracellular signaling mechanisms initiated by DCC and neogenin engagement. In this regard, the PI's laboratory has identified binding partners for DCC and neogenin which are likely to participate in netrin-1/RGM-induced intracellular signaling, including PITPalpha (phosphatidylinositol transfer protein alpha), a protein essential for ),), ( phosphatidylinositol signaling, and myosin X (Myo X), an unconventional myosin implicated in cell adhesion and filopodial elongation. Indeed, pilot studies indicate that these proteins do contribute importantly to netrin-1-triggered intracellular signaling and regulating their functions, as inhibition of PITPalpha or Myo X attenuates netrin-1-induced neurite outgrowth and/or growth cone turning. Myo X also appears to be involved in delivering DCC to the cell periphery or neurites. Thus, the proposed studies are based on the hypothesis that PITPalpha and Myo X are involved in initiating DCC or neogenin signaling and functions. Specifically, we will: Aim 1) analyze the role of PITPalpha in netrin-1-induced phosphatidylinositol signaling and neurite outgrowth; Aim 2) investigate the role of PITPalpha in RGMa-induced neuronal survival; and, Aim 3) determine the role of Myo X in regulating DCC distribution and functions. We will take advantage of neurons expressing miRNA of PITPalpha or Myo X, neuron-specific Myo X conditional knock-out mice, and/or neurons with PITPalpha/Myo X expression knocked-down by in utero/ovo electroporation of their miRNAs in mouse/chicken embryos to assess these questions. Results of the proposed studies will provide insights into intracellular mechanisms controlling brain wiring. Defects in this process underlie neurologic disorders at all ages, from neonates to neurodegerative processes in the elderly. Understanding such signaling pathways provides a possible foundation for future novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: The long-term goal of this proposal is to understand molecular mechanisms underlying axon pathfinding, a process essential for brain wiring and functions. Defects in this process are implicated in early embryonic death, mental retardation, and impaired recovery of spinal cord injury.