Molecular Regulators of Synaptic Specificity

突触特异性的分子调节剂

• 批准号: 10533260
• 负责人: Tyler J. Kennedy
• 金额: $ 6.65万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2023-08-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10533260
• 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

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).

大脑中的特定回路决定了我们如何感知和应对环境。这些高度连接的网络在发育过程中出现，因为神经元将投射延伸到定义的会议地点，识别合作伙伴，并开始突触发生。虽然最初的连接可能会被修改后，整体模式的连接是可预测的，从而表明，合作伙伴的选择可以编码的基因组。因此，遗传分析可以通过识别具有改变的连接模式的突变体来有力地发现突触特异性基因。以鉴定新的、保守的靶选择蛋白为目标，我将筛选破坏依赖于C中神经元特异性突触的独特行为的突变。优雅的这项工作的重点是PVD感觉神经元及其突触靶点，PVC和AVA。PVD刺激激活PVC，它的主要伙伴，并触发向前运动。然而，如果PVC连接被移除，AVA反而被激活，导致反向运动。因此，选择性破坏PVD→PVC或PVD→AVA连接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）。由于其短的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。在目标1中，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跨突触伴侣重构）标记物筛选具有这些特定运动表型的行为突变体，以确认PVD→PVC或PVD→AVA突触在突触发生期间被破坏。分子克隆方法将用于鉴定受影响的突触特异性基因。目的2采用一种独立的方法，源于PVD中突触特异性基因应受细胞自主转录因子（TF）调控的预期。我的策略利用了之前从RNA-Seq分析中获得的35个PVD富集的TF。我将在GRASP标记物测定中使用RNAi和可用的遗传突变体来测试这些TF中的每一个在PVD→PVC或PVD→AVA突触发生中的潜在作用。该TF筛选具有同时失调多个靶基因以获得稳健的突触特异性表型的优点。我将使用PVD特异性RNA-Seq来识别突触特异性TF的靶点，然后使用行为测定和GRASP标记物单独测试它们在PVD突触特异性中的作用。总之，这些方法在C。elegans有望揭示突触特异性的关键决定因素，这些决定因素可用于测试更复杂神经系统中的保守作用，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））的联系。