Function and Structure Adaptations in Forebrain Development

 描述（由申请人提供）：研究人员发现，自闭症谱系障碍（ASD）和其他精神疾病的风险基因和相关分子网络参与突触发育和功能。因此，疾病风险在于神经通信的基本要素，这为在这种竞争性更新应用中提出的研究提供了一个焦点。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，我们最近发现这些伙伴包括与ASD风险有关的其他蛋白质。实验将解决一个主要的知识差距，通过确定的机制，MET及其相互作用体的成员有助于早期突触发育的电路是脆弱的ASD。这些研究建立在当前资助期内的新发现基础上：1）在啮齿动物和灵长类前脑突触发生高峰期，MET在发育中的轴突和突触中富集; 2）Met的遗传缺失破坏了树突和棘结构以及新皮层层间兴奋驱动; 3）MET的功能性启动子变体改变人类社会-情感回路激活、网络连接和选择纤维束的结构完整性; 4）MET转录受其他神经发育障碍（包括Rett综合征和语言延迟）中涉及的蛋白质调节;和5）MET与发育重要的突触蛋白（包括β-连环蛋白）直接相互作用。所提出的实验将1）通过共免疫印迹/质谱法确定MET突触蛋白相互作用组（Aim 1），2）测量与野生型小鼠相比，Met缺失小鼠的新皮质和纹状体中突触前和突触后蛋白表达的变化，包括MET相互作用配偶体，使用无偏全局同量异位素标记进行基于相对和绝对定量（iTRAQ）的蛋白质组学和靶向高分辨率/准确质量（HR/AM）蛋白质组学（目的2），和3）确定MET相互作用组，包括MET连环蛋白，对体外突触发育（目的1和2）和体内新皮层回路的功能成熟（目的3）的作用。具体而言，体外实验将使用siRNA破坏突触前和突触后MET相互作用组，并测量对突触发生和囊泡聚集的影响。体内子宫内电穿孔将操纵突触后相互作用体蛋白的表达，然后将输入功能映射到第5层皮质-纹状体神经元并测量棘大小和密度。这项研究计划提供了一个翻译的理解蛋白质参与突触发育相关的电路功能障碍。将新型蛋白质组学技术与细胞和电路功能结果相结合，以确定新皮层中典型和非典型突触发育的基础机制，这将产生很大的影响。

 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.