DOCK7 Signaling in Neuronal Development

描述(申请人提供)：这项研究的长期目标是了解轴突发育和神经元极性建立的分子和细胞机制。神经细胞的极化能力对神经系统的组织是必不可少的，并具有深刻的功能含义。通常，神经元会极化以形成一个长轴突和多个较短的树突，以传输和接收信息，并建立对正常认知功能至关重要的电路。此外，大量证据表明，神经元极性的丧失与许多神经退行性疾病有关。因此，了解神经元极化的机制对人类健康至关重要。建立极化神经元的最初事件是单个轴突的发育。我们最近发现DOCK7是一种新的RAC GTP酶激活剂，并证明该蛋白在轴突发育的早期阶段起着至关重要的作用。此外，我们的数据揭示了DOCK7和微管调节蛋白stathmin之间的联系，并强调了微管网络调节对轴突发育的贡献。这些发现为获得对轴突发育的分子和细胞基础的新见解提供了一个独特的框架。这项申请旨在进一步阐明DOCK7功能在这一重要发育步骤中的作用和机制。为了实现这些目标，第一个特定的目标将是表征DOCK7对Stathmin磷酸化和轴突发育的影响的信号通路。具体目的2集中于DOCK7的调节，包括定义DOCK7如何被激活并选择性地定位于新生轴突的机制。将使用分子、生物化学和细胞生物学方法来解决这些目标。第三个具体目标将通过使用生化纯化技术和质谱学以及酵母双杂交方法来鉴定DOCK7相互作用的蛋白质，这些蛋白质对其在神经元极化中的功能至关重要。最后，《特定目标4》仔细研究了DOCK7在脑片迁移的皮质神经元极化中的作用。为此，将采用宫内电穿孔技术和双光子显微镜。从这些研究中获得的信息将极大地有助于理解轴突发育和神经元极化的机制。因此，它们将对理解生物医学相关过程具有重要意义，包括神经元发育、相关认知功能、神经再生和神经退行性疾病。与公共卫生相关：神经细胞的极化能力--即形成一个轴突和多个较短的树突--对神经系统的组织至关重要，并对正常认知功能和神经细胞的再生具有深远的功能影响。拟议的研究旨在了解轴突发育和神经元极化背后的分子和细胞机制。因此，它们将对许多生物医学相关过程产生重大影响，包括神经元发育、相关的认知功能、神经再生和神经退行性疾病，并可能为开发分别用于新轴突和受损轴突生成和再生的治疗剂提供基础。

DESCRIPTION (provided by applicant): The long-term objective of this study is to understand the molecular and cellular mechanisms that underlie axon development and the establishment of neuronal polarity. The ability of neuronal cells to polarize is essential for the organization of the nervous system and has profound functional implications. Typically, a neuron polarizes to elaborate one long axon and multiple, shorter dendrites so as to transmit and receive information and establish the circuitry that is critical for normal cognitive functions. Moreover, abundant evidence indicates that loss of neuronal polarity is associated with numerous neurodegenerative diseases. Thus understanding the machineries involved in neuronal polarization is of utmost importance to human health. The initial event in establishing a polarized neuron is the development of a single axon. We have recently identified DOCK7 as a novel activator of Rac GTPases, and demonstrated that the protein plays a crucial role in early steps of axon development. Moreover, our data have unveiled a link between DOCK7 and the microtubule regulatory protein, stathmin, and highlight the contribution of microtubule network regulation to axon development. These findings provide a unique framework for obtaining novel insight into the molecular and cellular underpinnings of axon development. This application aims to delineate further the role and mechanism of DOCK7 function in this important developmental step. Towards these goals, the first specific aim will characterize the signaling pathway that mediates the effect of DOCK7 on stathmin phosphorylation and axon development. Specific aim 2 focuses on the regulation of DOCK7, including the mechanisms that define how DOCK7 becomes activated and selectively localized in the nascent axon. Molecular, biochemical, and cell biological approaches will be used to address these objectives. The third specific aim will identify DOCK7-interacting proteins important for its function in neuronal polarization, by using biochemical purification techniques and mass spectrometry, and the yeast two-hybrid method. Finally, specific aim 4 scrutinizes the role of DOCK7 in the polarization of migrating cortical neurons in brain slices. To this end, in utero electroporation technology and two-photon microscopy will be employed. Information gained from these studies will greatly contribute to understanding the mechanisms that underlie axon development and neuronal polarization. As such, they will have significant implications for understanding biomedically relevant processes, including neuronal development, associated cognitive function, nerve regeneration and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: The ability of neuronal cells to polarize - i.e. to elaborate one axon and multiple, shorter dendrites - is essential for the organization of the nervous system and has profound functional ramifications for normal cognitive functions and the regeneration of nerve cells. The proposed studies are aimed at understanding the molecular and cellular mechanisms that underlie axon development and neuronal polarization. As such, they will have significant implications for numerous biomedically relevant processes, including neuronal development, associated cognitive function, nerve regeneration and neurodegenerative disease, and may provide the basis for developing therapeutic agents for the generation and regeneration of new and damaged axons, respectively.