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

点击翻译按钮获取中文摘要

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.