Pooled Optical Screens of Synaptic Function

突触功能的汇集光学屏幕

基本信息

批准号：
9979030
负责人：
Samouil Farhi
金额：
$ 25.65万
依托单位：
BROAD INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-15 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979030
关键词：
Address Affect Bar Codes Biological Assay Biological Models Cell Survival Cells Clustered Regularly Interspaced Short Palindromic Repeats Coupled Development Disease Electrophysiology (science)Environment Future Gene Family Genes Genetic Guide RNA Human Human Genetics Image In Situ Individual Institutes Investigation Knock-out Label Libraries Light Measurement Measures Mental disorders Methodology Methods Microscope Molecular Monitor Mus Mutation Neurobiology Neurons Optical Methods Optics Other Genetics Performance Phenotype Presynaptic Terminals Process Proteins Proteomics Protocols documentation RNA Reading Recycling Research Personnel Role Schizophrenia Side Synapses Synaptic Transmission Synaptosomes System Techniques Technology Testing Time Translating Vesicle Work base cell growth design exome sequencing experimental study follow-up gene function high throughput screening induced pluripotent stem cell innovation knockout gene method development mutant optogenetics patch clamp portability postsynaptic presynaptic screening synaptic function voltage

项目摘要

Though our understanding of the protein composition of the synapse and our recognition of the involvement of synaptic mechanisms in psychiatric disease both continue to grow, our ability to study the synaptic function of genes is still hampered by low throughput, high overhead assays. We propose to address this need by developing pooled screens of the effect of genetic perturbations on synaptic function in cultured neurons. Compared to arrayed screens, pooled screens are more robust to condition-to-condition variability and are thus better suited for studies of highly culture-dependent phenotypes such as synaptic transmission. Despite this advantage, it has been unclear how to translate pooled screening technology from studying cell survival or growth coupled phenotypes to dynamical functional phenotypes like those involved in neuronal function. We propose an innovative way around this problem by harnessing new optical methods. Our approach will be to perform barcoded genetic perturbations on a pool of cultured neurons, to perform time-resolved imaging assays of their postsynaptic or presynaptic function, and to subsequently use multiplexed RNA or protein readout methods to read out barcodes and match synaptic phenotypes to perturbations. Postsynaptic function will be read out using simultaneous voltage imaging and optogenetics, while presynaptic function will be read out by monitoring vesicle recycling with a pH sensitive fluorescent protein. The postsynaptic and presynaptic protocols are independent of each other and constitute separate specific aims. We will extensively characterize the performance of both assays and perform a pilot screen on the synaptic functions of 50 genes identified in Schizophrenia whole exome sequencing studies. Accomplishing these aims is possible by innovations in expression strategies, measurement protocols, and barcoding approaches. The chief significance of this work is to enable studies of synaptic function to occur earlier in the discovery process when studying new families of genes. In addition, the pilot screen we perform will generate valuable information about the basic neurobiological function of Schizophrenia associated genes. We will validate these results with traditional patch clamp approaches, and they will serve as the launching point for further investigations into the mechanisms of action of these genes.

尽管我们对突触蛋白质组成的了解以及对突触参与的认识精神疾病的突触机制都在不断发展，我们研究突触功能的能力基因仍然受到低通量、高开销检测的阻碍。我们建议通过以下方式解决这一需求开发遗传扰动对培养神经元突触功能影响的汇总屏幕。与阵列筛选相比，合并筛选对于条件间的变异性更加稳健，因此更适合高度文化依赖性表型的研究，例如突触传递。尽管如此优势，目前尚不清楚如何将混合筛选技术从研究细胞存活或生长中转化将表型与动态功能表型（如涉及神经元功能的表型）耦合。我们建议通过利用新的光学方法解决这个问题的创新方法。我们的方法是执行对培养的神经元池进行条形码遗传扰动，以对其进行时间分辨成像分析突触后或突触前功能，并随后使用多重 RNA 或蛋白质读出方法读出条形码并将突触表型与扰动进行匹配。突触后功能将使用以下方式读出同时进行电压成像和光遗传学，同时通过监测囊泡读出突触前功能使用 pH 敏感荧光蛋白进行回收。突触后和突触前协议独立于彼此并构成单独的具体目标。我们将广泛描述两者的性能对精神分裂症全外显子组中鉴定的 50 个基因的突触功能进行分析和初步筛选测序研究。通过表达策略、测量的创新，可以实现这些目标协议和条形码方法。这项工作的主要意义是使突触功能的研究成为可能在研究新的基因家族时，发生在发现过程的早期。此外，我们在试点屏幕上执行将产生有关精神分裂症相关的基本神经生物学功能的有价值的信息基因。我们将用传统的膜片钳方法验证这些结果，它们将作为进一步研究这些基因的作用机制的起点。