Simultaneous profiling of neuronal synapse activities, proteins, and messenger RNAs at the single-cell level

PROJECT SUMMARY Autism spectrum disorder (ASD) is a debilitating neurodevelopmental disease that severely affects cognition and communication in patients. Genome-wide association studies of ASD have revealed complex polygenic variation in synapse-associated proteins. The normal development and function of neuronal circuits rely critically on the spatially controlled expression and regulation of synaptic proteins, their messenger RNAs (mRNAs), and their interactions. Thus, understanding how penetrant genetic variations associated with neurodevelopmental diseases such as ASD impact neuronal synapse development, homeostasis, plasticity, and activity is crucial for the identification of new therapeutics to treat patients. Toward this end, single-cell fluorescence imaging of neuronal synapse activity integrated with in situ measurement of synapse protein and mRNA levels and localizations offers the potential to identify convergent synaptic phenotypes in ASD. The present project builds on prior research in which we applied highly multiplexed fluorescence imaging of synaptic proteins to fixed neuronal samples to identify convergent synaptic phenotypes associated with ASD and SCZ risk genes. Specifically, we build on this technique by integrating high-throughput molecular characterization of mRNA expression levels and localizations in intact neurons together with characterization of neuronal activity in the same cells. Live-cell imaging of glutamate and calcium levels is performed followed by fixation and multiplexed fluorescence imaging of both multi-protein and mRNA levels and localizations including mRNA translational state. CRISPRi will be used to characterize how high confidence ASD-associated gene deletions affect neuronal synapse activities and their multiprotein-mRNA interaction networks. Bayesian network analysis will identify convergent networks and pathways by which ASD-associated genetic variations impact neuronal function both in rodent and human iPSC-derived neuronal cultures, in collaboration with the Stanley Center at the Broad Institute of MIT and Harvard. Development of this multi-modal synapse characterization approach will pave the way towards the discovery of new therapeutics for ASD, as well as other neurodevelopmental disorders such as SCZ using patient samples in future work.