Multimodal synaptic profiling of patient-derived neuronal samples for the discovery of ASD therapeutics

Synaptic genes are centrally implicated in autism spectrum disorder (ASD), including synaptic adhesion molecules, sub-membranous scaffolding molecules, ion channels, receptors, mRNA-binding proteins, and transcription factors that regulate these genes. Because synaptic proteins play central roles in neuronal functions including neurodevelopment, homeostasis, plasticity, and signal transmission, understanding how genetic variation impacts synaptic protein and mRNA expression, localization, regulation, and their interplay with neuronal function is needed to understand the cellular basis of ASD. Identification of potentially convergent pathways resulting from penetrant polygenic variations would be particularly valuable towards resolving the physiological basis of disease. Such knowledge could facilitate the discovery of new lead therapeutic compounds that may restore normal brain function in patients. Historically, yeast two-hybrid and bulk synaptosome methods including co-immunoprecipitation (co-IP) have been immensely valuable to identify synaptic protein–protein and protein–mRNA interaction networks that regulate synapse formation, homeostasis, plasticity, and function. At the single-synapse and single-cell levels, however, protein/mRNA expression and localization, as well as calcium and neurotransmitter fluxes are highly heterogeneous. Consequently, distributions of synapse protein/mRNA compositions requires single-synapse-level in situ characterization. Combined in situ cell-based fluorescence imaging of multiple proteins, mRNA translation events, and calcium and glutamate fluxes allow us to understand how regulatory interactions at the synapse between protein localization and synthesis, and synaptic activity, are perturbed in ASD. Neurons differentiated from patient derived iPSCs (hiPNs), with new differentiation protocols that yield fully mature and functional synapse populations, offer an ideal system to perform these measurements with direct access to patient genotype in high-throughput. This system is superior to post-mortem human tissue that is less accessible, does not allow for live-cell functional measurements, and depends sensitively on processing conditions. This cellular imaging platform may eventually be applied to identify lead therapeutic compounds that restore neurotypical synaptic phenotypes in patient-derived neuronal samples. Finally, the platform can in principle incorporate clinical patient data to stratify patient sub-populations examined using hiPNs to empower patient-specific therapeutic discovery.