Molecular Regulators of Synaptic Specificity

突触特异性的分子调节剂

• 批准号: 10581824
• 负责人: Tyler J. Kennedy
• 金额: $ 0.25万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-02 至 2023-08-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581824
• 关键词: Adopted; Affect; Afferent Neurons; Animal Model; Architecture; Axon; Behavior; Behavioral; Behavioral Assay; Biological Assay; Brain; Caenorhabditis elegans; Cells; Cloning; Complex; Computer software; Databases; Defect; Dendrites; Development; Electronic Medical Records and Genomics Network; Environment; Excision; Gene Expression; Genes; Genetic; Genetic Screening; Genome; Goals; Individual; Instinct; Label; Life Cycle Stages; Link; Locomotion; Maintenance; Mammals; Maps; Methods; Modeling; Molecular; Molecular Analysis; Molecular Cloning; Morphogenesis; Movement; Mutagenesis; Mutation; Nervous system structure; Neurodevelopmental Disorder; Neurons; Nociception; Nociceptors; Oranges; Output; Pathway interactions; Pattern; Phenotype; Process; Proteins; RNA Interference; Reproducibility; Research; Role; Site; Specificity; Synapses; Testing; To specify; Video Recording; Work; autism spectrum disorder; behavior test; design; expectation; genetic analysis; grasp; in vivo; live cell imaging; meetings; mutant; mutation screening; nervous system disorder; new therapeutic target; novel; optogenetics; postsynaptic; programs; reconstitution; response; stem; synaptogenesis; tool; transcription factor; transcriptome sequencing

Project Summary Specific circuits in the brain determine how we sense and respond to our environment. These highly connected networks emerge during development as neurons extend projections to defined meeting sites, identify partners, and begin synaptogenesis. Although initial connections may be modified later, the overall pattern of connectivity is predictable thus suggesting that partner selection can be encoded by the genome. It follows that genetic analysis can be powerfully employed to find synaptic specificity genes by identifying mutants with altered patterns of connectivity. With the goal of identifying novel, conserved target selection proteins, I will screen for mutations that disrupt distinctive behaviors that depend on neuron-specific synapses in C. elegans. This work focuses on the PVD sensory neuron and its synaptic targets, PVC and AVA. PVD stimulation activates PVC, its dominant partner, and triggers forward movement. If the PVC connection is removed, however, AVA is activated instead, resulting in reverse locomotion. Thus, mutants that selectively disrupt either PVD→PVC or PVD→AVA connections can be identified from readily distinguished behaviors (e.g., forward vs reverse movement). With its short life cycle and powerful genetic tools, C. elegans is especially useful for unbiased genetic screens. In Aim 1 I will use an optogenetic strategy to activate PVD in a forward genetic EMS mutagenesis screen that uses a high-throughput video recording system (WormLab) to identify mutants with selectively altered locomotion. Behavioral mutants with these specific locomotory phenotypes will be screened with GRASP (GFP Reconstitution Across Synaptic Partners) markers to confirm that either PVD→PVC or PVD→AVA synapses are disrupted during synaptogenesis. Molecular cloning methods will be used to identify the affected synaptic specificity genes. Aim 2 adopts an independent approach that stems from the expectation that synaptic specificity genes in PVD should be regulated by cell autonomous transcription factors (TFs). My strategy exploits a list of 35 PVD-enriched TFs previously derived from RNA-Seq profiling. I will use RNAi and available genetic mutants in the GRASP marker assay to test each of these TFs for potential roles in either PVD→PVC or PVD→AVA synaptogenesis. This TF screen has the advantage of dysregulating multiple target genes simultaneously for a robust synaptic specificity phenotype. I will use PVD-specific RNA-Seq to identify the targets of the synapse-specific TFs and then test them individually for roles in PVD synaptic specificity using the behavioral assay and GRASP markers. Together, these approaches in C. elegans are expected to reveal key determinants of synaptic specificity that can be tested for conserved roles in more complex nervous systems and for links to neurological disorders associated with altered synaptogenesis such as Autism Spectrum Disorder (ASD).

项目摘要 大脑中的特定回路决定了我们如何感知和应对环境。这些高度关联的 在发育过程中，神经元将投射扩展到定义的会议地点，识别合作伙伴， 并开始突触形成。虽然初始连接可能会在以后修改，但连接的总体模式 是可预测的，因此表明伴侣选择可以由基因组编码。由此可见，遗传 通过鉴定突变体的模式改变，分析可以有效地发现突触特异性基因 连接性。以鉴定新的、保守的靶选择蛋白为目标，我将筛选突变 破坏了依赖于C.优美的这个工作主要集中在 PVD感觉神经元及其突触靶点PVC和AVA。PVD刺激激活PVC，其主导 合作伙伴，并触发向前移动。然而，如果PVC连接被移除，则AVA被激活， 导致反向运动。因此，选择性破坏PVD→PVC或PVD→AVA的突变体 可以从容易区分的行为（例如，正向运动与反向运动）。与其 短生命周期和强大的遗传工具，C. elegans对于无偏遗传筛选特别有用。在Aim中 1我将使用光遗传学策略在正向遗传EMS诱变筛选中激活PVD，该筛选使用 高通量视频记录系统（WormLab）来鉴定具有选择性改变的运动的突变体。 将用GRASP（GFP）筛选具有这些特定运动表型的行为突变体 在突触伙伴之间的重建）标记，以确认PVD→PVC或PVD→AVA突触是 在突触形成过程中被破坏分子克隆方法将用于鉴定受影响的突触 特异性基因AIM 2采用了一种独立的方法，这种方法源于对Synaptic PVD中特异性基因的表达受细胞自主转录因子（TF）的调控。我的策略利用了 先前从RNA-Seq分析得到的35种PVD富集的TF的列表。我将使用RNAi和可用的基因 突变体，以测试这些TF中的每一种在PVD→PVC或 PVD→AVA突触发生。这种TF筛选具有多个靶基因失调的优点 同时用于强大的突触特异性表型。我将使用PVD特异性RNA-Seq来识别目标 的突触特异性的TF，然后测试他们单独的PVD突触特异性的作用，使用 行为测定和GRASP标记物。总之，这些方法在C。秀丽线虫有望揭示 突触特异性的决定因素，可以在更复杂的神经系统中测试保守的作用 以及与突触发生改变相关的神经系统疾病，如自闭症谱系障碍， （ASD）中指定的值。