Molecular mechanisms of neuron motility and axon guidance

大脑的功能依赖于精确而复杂的轴突连接模式。广泛的长期 这个项目的目标是了解这种轴突连接的模式是如何在发育过程中建立的。当 这种连接不能正确地形成或随后丢失，这可导致大范围的 神经发育、精神和神经变性疾病。 该提案特别关注基于RNA的调控机制。调控基因的一个关键优势 在mRNA水平上的表达是蛋白质表达可以被引导到特定的亚细胞区域， 时间和空间特异性-神经元中的一个重要优势，其具有高度的空间特异性， organization.因此，基于RNA的调节在轴突导向、神经元迁移和神经元再生中起关键作用。 突触可塑性，虽然具体机制仍然知之甚少。在这里，基于RNA的 将在微管细胞骨架（Aim 1）的调节和轴突通路中研究其机制 在复杂的选择点上进行选择（目标2）。目的1集中在微管细胞骨架，这是至关重要的 在神经元结构和运动中的作用。我们最近的工作已经确定了基于RNA的 微管的调节。具体地说，微管加端蛋白APC结合微管蛋白Tubb 2b mRNA，在2000 - 2005年之间。 轴突中Tubb 2b翻译所需的位点，生长锥中动态微管的形成，以及 体内神经元迁移。这开辟了一个新的研究领域，以RNA为基础的调控， 微管细胞骨架一个目标是研究特定微管蛋白mRNA的协调调节 其在其3 'UTR中具有APC结合位点并引起大多数人微管蛋白病。另一个目标将使用 时间推移成像，以具体了解如何基于RNA的调节控制微管动力学， 包括微管在负端起始和在正端组装的基本新模型， 端除了轴突之外，这些机制的特征在于突触棘的形成。目标2将 继续研究脊髓中线处的连合轴突引导，这是一个特征良好的模型， 发育轴突寻路已知基于RNA的调节发生在连合轴突内，包括 上调已经穿过中线中间靶的远端轴突节段中mRNA的翻译。 然而，很少有人知道的机制，包括RNA结合蛋白的参与，其 下游mRNA靶点或上游调控途径。该目的将表征特异性RNA结合 在轴突节段上显示高度选择性表达的蛋白质，在中线中具有强而独特的表型， 指导，并与中线轴突中调控的mRNA相互作用;以及上游配体和 受体与这些基于RNA的调节机制在物理上和功能上相互作用。这些研究 将提供有关轴突发育和基于RNA的调节的基本机制的新信息。

The brain relies for its function on a precise and complex pattern of axonal connections. The broad long-term goal of this project is to understand how this pattern of axon connections is set up during development. When such connections fail to form properly, or are subsequently lost, this can lead to a broad range of neurodevelopmental, psychiatric and neurodegenerative disorders. This proposal focuses particularly on RNA-based regulatory mechanisms. A key advantage of regulating gene expression at the mRNA level is that protein expression can be directed to specific subcellular regions with temporal and spatial specificity – an important advantage in neurons, which have a high degree of spatial organization. Accordingly, RNA-based regulation plays key roles in axon guidance, neuron migration and synapse plasticity, although the specific mechanisms remain poorly understood. Here, RNA-based mechanisms will be studied in regulation of the microtubule cytoskeleton (Aim 1), and in axon pathway selection at a complex choice point (Aim 2). Aim 1 focuses on the microtubule cytoskeleton, which has crucial roles in neuron structure and motility. Our recent work has now identified a mechanism for RNA-based regulation of microtubules. Specifically, microtubule plus-end protein APC binds tubulin Tubb2b mRNA, at a site required for Tubb2b translation in axons, formation of dynamic microtubules in the growth cone, and neuron migration in vivo. This opens up a new field of investigation into RNA-based regulation of the microtubule cytoskeleton. One goal will be to investigate coordinated regulation of specific tubulin mRNAs which have APC binding sites in their 3'UTR and cause most human tubulinopathies. Another objective will use time-lapse imaging to understand specifically how RNA-based regulation controls microtubule dynamics, including fundamental new models for both microtubule initiation at the minus end, and assembly at the plus- end. In addition to axons, these mechanisms will be characterized in formation of synaptic spines. Aim 2 will continue studies of commissural axon guidance at the spinal cord midline, a well-characterized model of developmental axon pathfinding. RNA-based regulation is known to occur within commissural axons, including upregulated translation of mRNAs in distal axon segments that have crossed the midline intermediate target. However, little has been known of the mechanisms, including the RNA-binding proteins involved, their downstream mRNA targets, or upstream regulatory pathways. This Aim will characterize specific RNA-binding proteins that display highly selective expression on axon segments, strong and distinct phenotypes in midline guidance, and interactions with mRNAs regulated in axons at the midline; as well as upstream ligands and receptors that interact physically and functionally with these RNA based regulatory mechanisms. These studies will provide novel information on fundamental mechanisms of axon development, and RNA-based regulation.