Structure-guided functional analysis of GluA4-NPTX2 interaction during PVIN homeostatic scaling

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PROJECT SUMMARY AMPA-type glutamate receptors (AMPARs) are the major excitatory neurotransmitter receptors in the brain and changes in AMPAR number at synapses underlie learning and memory as well as human disease. A detailed understanding of how AMPARs are organized at synapses is critical to understand how synaptic strength is regulated and for the development of therapeutics to correct circuit imbalances in human disease. The long-term goal of this proposal is to use Cryo-EM to understand how the structural basis of AMPAR N-terminal domain interactions (NTDs) drive functional outcomes such as increased AMPAR accumulation and synaptic strength. The rationale for this approach is twofold 1) it will help resolve long-standing questions about the regulation of key neurotransmitter receptors; and 2) a detailed structural model of AMPARs participating in key regulatory complexes will guide future therapeutic approaches that seek to alter the strength of excitatory input onto neurons implicated in psychiatric illnesses like schizophrenia. The adhesion protein NPTX2 binds to AMPARs, clusters AMPARs at synapses, and is required for homeostatic scaling of interneuron-specific GluA4- containing AMPARs. Therefore, NPTX2-dependent GluA4 scaling is an ideal model for testing the hypothesis that direct extracellular interactions with AMPARs control synaptic strength. This approach is innovative because models of AMPAR plasticity have never been observed in structural detail. This research is significant because it will yield new insights into how AMPAR interactions drive plasticity and how this can be exploited for therapeutic benefit in the future. An example of such an approach would be a structure-guided therapeutic strategy for clustering GluA4 on the surface of Parvalbumin-expressing interneurons (PVINs), which exhibit lowered excitatory drive in models of schizophrenia. The long-term goal of this project will be achieved with the following two specific aims: 1) Determine the structure of the NPTX2/GluA4 complex via single particle Cryo-EM. and 2) Test whether NPTX2 drives GluA4 PVIN scaling through a direct interaction. For the first aim we will employ single-particle Cryo-EM to solve the structure of the activity-regulated synaptic adhesion molecule NPTX2 in complex with the interneuron- specific GluA4 AMPARs. For the second aim, we will employ transgenic mouse models, biochemistry, neuron culture, confocal light microscopy, and electrophysiology to test the hypothesis that direct binding of NPTX2 to the NTD of GluA4 drives homeostatic scaling in disease-associated PVINs. The applicant has proposed this work in part to further their long-term goal of establishing an independent research career connecting the structure of synaptic proteins to their synaptic function. The candidate will undertake extensive training in Cryo-EM and biophysics which will be facilitated by an expert mentoring team composed of an AMPAR Cryo-EM expert, an AMPAR plasticity expert, and an expert in NPTX2, all of whom will mentor the applicant through the transition to a tenure track academic position.