Investigation of the Synaptic Molecular Network using Multiplexed Imaging

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PROJECT SUMMARY The synaptic molecular network is a complex, tightly interacting system of hundreds of proteins that forms the basis for learning, memory, and other brain functions. It is a disrupted locus of many neurological, neurodegenerative, and psychiatric disorders, and is a focal point of action for small molecule psychiatric treatments. Understanding this network and the rules that govern it is necessary for understanding the molecular etiology of brain diseases, and for the rational design of psychiatric drugs. To achieve this, we propose to apply PRISM, a recently developed multiplexed imaging tool which allows in-situ measurements of many proteins at single-synapse resolution, to construct and validate a causal, predictive model of interdependencies among proteins of the glutamatergic synapse, their subunit composition, and activation state. We will expand the repertoire of targets available for PRISM and measure these targets in a large population of synapses across a variety of chemical environments. This will serve two purposes: measurement of many ‘snapshots’ of the synaptic molecular network pulled in different directions, which is necessary for subsequent model learning, and in-depth characterization of the downstream synaptic biochemical effects of perturbations that include antidepressants of different classes. We will also combine sensitive live calcium imaging of synapse activity with subsequent PRISM measurements of the same synapses. We will then use these data to construct a Bayesian network model of causal dependencies between the probability distributions of these measurements. This Bayesian network will yield predictions about causal connections between nodes and downstream effects of perturbing certain targets, which we will subsequently test. The result of this study will be a powerful, predictive model connecting up to 30 measures of protein levels, subunit compositions, phosphorylation states, and synapse activity. This model will provide a unifying context to integrate mechanistic details of interactions between specific synaptic actors into a holistic understanding of the synapse as a whole. It will also provide predictions about system-level effects of chemical perturbations, potentially paving the way for an entirely novel modality for in vitro screening of psychiatric treatments.