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对环境做出反应。这些高度互联的 网络在发育过程中出现，因为神经元将投影延伸到指定的会议地点，确定合作伙伴， 并开始突触形成。尽管以后可能会修改初始连接，但连接的总体模式 是可预测的，因此表明伴侣的选择可以由基因组编码。它遵循的是基因 通过识别具有改变模式的突变体，分析可以有力地用于发现突触特异性基因 互联互通。为了识别新的、保守的靶标选择蛋白，我将筛选突变 这会扰乱线虫依赖神经元特异性突触的独特行为。这项工作的重点是 PVD感觉神经元及其突触靶标Pvc和Ava。PVD刺激激活其主要成分聚氯乙烯 伙伴，并触发向前移动。然而，如果移除了PVC连接，则改为激活AVA， 导致了反向运动。因此，选择性破坏pvd→pvc或pvd→ava的突变体 连接可以从容易区分的行为(例如，向前和向后移动)中识别。使用它的 线虫的生命周期短，遗传工具强大，尤其适用于无偏见的遗传筛选。在AIM 1我将使用光遗传策略在正向遗传EMS突变筛查中激活PVD，该突变筛查使用 高通量视频记录系统(WormLab)，以识别具有选择性改变运动的突变。 具有这些特定运动表型的行为突变体将通过GFP(GFP)进行筛选 跨突触伙伴重建)标记，以确认PVD→Pvc或Pvd→AVA突触 在突触发生过程中被破坏。将使用分子克隆方法来识别受影响的突触 特异性基因。目标2采用了一种独立的方法，这源于对突触 PVD中的特异性基因应由细胞自主转录因子(TF)调控。我的战略利用了 先前从RNA-Seq分析中获得的35个富含PVD的TF的列表。我将使用RNAi和可用的基因 GRASH标记试验中的突变体，以测试这些TF中的每一个在pvd→pvc或 PVD→AVA突触发生。这种转铁蛋白筛查的优点是可以失调多个靶基因。 同时获得强健的突触特异性表型。我将使用PVD特定的RNA-Seq来识别目标 然后分别测试它们在PVD突触特异性中的作用 行为测试和抓取标记物。总之，线虫的这些方法有望揭示关键 可在更复杂的神经系统中测试保守作用的突触特异性决定因素 以及与突触发生改变相关的神经障碍的链接，如自闭症谱系障碍 (ASD)。