Molecular Regulators of Synaptic Specificity

突触特异性的分子调节剂

基本信息

批准号：
10187355
负责人：
Tyler J. Kennedy
金额：
$ 6.64万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
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/10187355
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 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

项目摘要

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).

大脑中的特定电路确定了我们如何对我们的环境进行响应，因为神经元的发展效果是可以预测的，因此可以预测，可以预测选择的伴侣。遗传分析可以通过与识别新颖的蛋白质识别杂交来找到突触规范的基因，我将筛选出依赖神经元特异性突触的突变。目标，PVC和AVA。它的生命周期和强大的遗传工具ETEC策略在前遗传EMS Mutajenesis屏幕中使用高通量视频记录系统（Wormlab）来识别具有选择性改变的运动的突变体（GFP重新固定在突触党中）。标记以确认pvd→AVA突触是破坏的。富含的TF源自RNA-Seq分析，用于PVD→PVC或PVD→AVA突触发生的fs。使用行为测定和喘着气的RKER在PVD突触特异性中进行单独测试。作为自闭症谱系障碍（ASD）。