Molecular Mechanisms Underlying Axon Pathfinding

描述(申请人提供)：轴突用来确保大脑中正确连接的分子机制涉及多个配体-受体相互作用，因为细胞外引导信号，如netrins和RGM(排斥性引导分子)，在轴突路径寻找过程中至关重要。参与轴突路径寻找的netrins受体包括DCC(在结直肠癌中缺失)和新生素，这是一种含有免疫球蛋白结构域的蛋白家族。新生素也是RGMS的受体。我们工作的长期目标是阐明DCC和新生素参与启动的细胞内信号机制。在这方面，PI的实验室已经确定了DCC和新生素的结合伙伴，它们可能参与netrin-1/RGM诱导的细胞内信号转导，包括PITPalpha(磷脂酰肌醇转移蛋白α)，PITPalpha(磷脂酰肌醇转移蛋白α)，(磷脂酰肌醇信号转导)，和肌球蛋白X(Myo X)，一种非传统的肌球蛋白，参与细胞黏附和丝裂原延长。事实上，初步研究表明，这些蛋白质确实对netrin-1触发的细胞内信号和调节其功能起重要作用，因为抑制PITPalpha或Myo X可以减弱netrin-1诱导的突起生长和/或生长锥体转向。Myo X似乎也参与了将DCC运送到细胞外围或突起的过程。因此，所提出的研究是基于这样的假设：PITPalpha和Myo X参与启动DCC或新生素的信号和功能。具体地说，我们将：1)分析PITPalpha在Netrin-1诱导的磷脂酰肌醇信号转导和轴突生长中的作用；2)研究PITPalpha在RGMa诱导的神经元存活中的作用；3)确定Myo X在调节DCC分布和功能中的作用。我们将利用PITPalpha或Myo X表达miRNA的神经元，神经元特异性Myo X条件性敲除小鼠，和/或通过宫内/卵内电穿孔其miRNAs在小鼠/鸡胚胎中击倒PITPalpha/Myo X表达的神经元来评估这些问题。拟议的研究结果将为控制大脑连接的细胞内机制提供洞察力。这一过程的缺陷是所有年龄段的神经障碍的基础，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。与公共健康相关：这项提议的长期目标是了解轴突寻路的分子机制，这是一个对大脑连接和功能至关重要的过程。这一过程中的缺陷会导致早期胚胎死亡、智力低下和脊髓损伤的恢复受阻。

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.