Pooled Optical Imaging, Neurite Tracing, and Morphometry Across Perturbations (POINT-MAP).

项目摘要 大规模的外显子组范围的关联研究确信与300多个基因有关 在神经发育障碍(NDDS)的风险中蕴藏着罕见的突变，但特定的 每种药物对分子、细胞和功能的影响大多是未知的。我们的长期目标是 实施高通量功能基因组学方法来阐明这些机制 基因及其变种。神经元延伸长而精细的分支投射并连接 沿着这些突起在突触上相互连接。大量的研究表明，改变 NDDS和其他复杂脑部疾病的神经元形态和突触。整体而言 拟议研究的目标是结合一个强大的人类神经元扰动平台 以创新的蜂窝条形码策略开发一种新技术，使高 NDD危险基因对神经元形态和功能影响的吞吐量评估 突触发生。我们的中心假设是许多新城疫风险基因在 神经元和突触功能的失调，我们可以通过 测量遗传干扰危险基因后的神经元形态。我们的目标是开发一种 用于池光学成像、轴突跟踪和形态测量的新型可扩展平台 微扰(点地图)。为此，我们将开发一种新型的光学蜂窝条形码 与CRISPR干扰有关，是神经细胞填充，可通过免疫荧光识别。 我们将建立一条自动图像分析管道，用于条形码检索、神经元追踪、 以及神经元形态和突触标志物的分析。我们将在中演示点地图 人诱导的多能干细胞来源的神经元同时评估功能 40个顶级NDD危险基因对神经元和突触形态的影响。

Project Summary Large scale exome-wide association studies have confidently implicated over 300 genes harboring rare mutations in the risk of neurodevelopmental disorders (NDDs), yet the specific molecular, cellular, and functional impacts of each are mostly unknown. Our long-term goal is to implement high-throughput functional genomics approaches to elucidate mechanisms for these genes and their variants. Neurons extend long and elaborately branched projections and connect to one another at synapses along these projections. A wealth of studies has implicated altered neuronal morphology and synapses in NDDs and other complex brain disorders. The overall objective of the proposed research is to combine a powerful human neuronal perturbation platform with an innovative cellular barcoding strategy to develop a novel technology enabling high throughput evaluation of the contributions of NDD risk genes on neuronal morphology and synaptogenesis. Our central hypothesis is that many NDD risk genes converge at the dysregulation of neuronal and synaptic function, and that we can identify such aberrations by measuring neuronal morphology after genetically perturbing risk genes. We aim to develop a novel scalable platform for Pooled Optical Imaging, Neurite Tracing, and Morphometry Across Perturbations (POINT-MAP). To do so, we will develop a novel optical cellular barcode that is linked to CRISPR perturbations, is neuronal cell-filling, and is identifiable by immunofluorescence. We will establish an automated image-analysis pipeline for barcode retrieval, neuronal tracing, and analysis of neuronal morphology and synaptic markers. We will demonstrate POINT-MAP in human induced pluripotent stem cell derived neurons to simultaneously assess the functional impact of 40 top NDD risk genes on neuronal and synaptic morphology.