Function and Structure Adaptations in Forebrain Development

 描述(申请人提供)：研究人员发现，自闭症谱系障碍(ASD)和其他精神障碍的风险基因和相关分子网络参与突触的发育和功能。因此，无序风险存在于神经通信的主要元素中，这为在这一竞争性更新应用中提出的研究提供了一个重点。先进的突触蛋白质组学和功能实验将带来对受体酪氨酸激酶c-MET(MET)及其相互作用伙伴的新理解，我们最近发现包括其他与ASD风险有关的蛋白质。实验将解决在确定MET及其互动体成员在ASD易受攻击的电路中促进突触早期发育的机制方面的一个主要知识缺口。拟议的研究基于本授权期内的新发现：1)Met在啮齿动物和灵长类前脑的突触发生高峰期丰富地发育着轴突和突触；2)Met的基因缺失扰乱了树突和脊柱的结构以及新皮质板层间的兴奋驱动；3)Met的功能启动子变体改变了人类社会情绪回路的激活、网络的连通性和选择纤维束的结构完整性；4)Met的转录受其他神经发育障碍的蛋白质调控，包括Rett综合征和语言延迟；以及5)Met直接与发育重要的突触蛋白相互作用，包括-Catenin。拟议的实验将1)通过共免疫沉淀/质谱学(AIM 1)确定MET突触蛋白交互作用组(AIM 1)，2)使用基于相对和绝对定量(ITRAQ)的无偏全局等压标签和靶向高分辨/准确质量(HR/AM)蛋白质组学(AIM 2)，测量MET缺失小鼠新皮质和纹状体中突触前和突触后蛋白质表达的变化，包括MET-NOT的相互作用伙伴(AIM 1和2)，以及3)确定MET相互作用组，包括连接素，在体外突触发育(AIM 1和2)和新皮质环路功能成熟(AIM 3)中的作用。具体地说，体外实验将使用siRNA来破坏突触前和突触后的MET相互作用组，并测量其对突触发生和囊泡聚集的影响。体内的宫内电穿孔将操纵突触后相互作用组蛋白的表达，随后将输入第5层皮质纹状体神经元的功能图谱以及脊柱大小和密度的测量。这项研究计划提供了对参与突触发育的与回路功能障碍相关的蛋白质的翻译理解。将新的蛋白质组学技术与细胞和电路功能结果相结合，将产生很大的影响，以确定新皮质中典型和非典型突触发育的机制。

 DESCRIPTION (provided by applicant): Researchers have discovered that risk genes and associated molecular networks for autism spectrum disorder (ASD) and other psychiatric disorders participate in synapse development and function. Disorder risk, therefore, lies in the principle elements of neural communication, providing a focus for studies proposed in this competing renewal application. Advanced synaptic proteomics and functional experiments will bring a new understanding of the receptor tyrosine kinase c-MET (MET) and its interacting partners, which we recently discovered include other proteins implicated in ASD risk. Experiments will address a major knowledge gap in determining the mechanisms through which MET and members of its interactome contribute to early synapse development in circuits that are vulnerable in ASD. The proposed studies build upon new discoveries made during the current grant period: 1) MET is enriched in developing axons and synapses during the peak of synaptogenesis in rodent and primate forebrain; 2) genetic deletion of Met disrupts dendritic and spine architecture and neocortical interlaminar excitatory drive; 3) the functional promoter variant of MET alters human social-emotional circuit activation, network connectivity and the structural integrity of select fiber tracts; 4) MET transcription is regulated by proteins implicatd in other neurodevelopmental disorders, including Rett Syndrome and language delay; and 5) MET interacts directly with developmentally important synaptic proteins, including -catenin. The proposed experiments will 1) determine the MET synaptic protein interactome by co-immunoprecipiation/mass spectrometry (Aim 1), 2) measure changes in pre- and postsynaptic protein expression, including MET-interacting partners, in neocortex and striatum in Met-null compared to wild type mice, using unbiased global isobaric tagging for relative and absolute quantitation (iTRAQ)-based proteomics and targeted high resolution/accurate mass (HR/AM) proteomics (Aim 2), and 3) determine the role of the MET interactome, including catenin, on synapse development in vitro (Aims 1 & 2) and functional maturation of neocortical circuits in vivo (Aim 3). Specifically, experiments in vitro will use siRNA to disrupt the pre- and postsynaptic MET interactome and measure the impact on synaptogenesis and vesicle clustering. In utero electroporation in vivo will manipulate expression of postsynaptic interactome proteins, followed by functional mapping of input to layer 5 cortico-striatal neurons and measures of spine size and density. This research program provides a translational understanding of the proteins involved in synapse development related to circuit dysfunction. There will be high impact by integrating novel proteomics technologies with cell and circuit functional outcomes for determining mechanisms that underlie typical and atypical synapse development in the neocortex.