Subunit-Specific Regulation Of Glutamate Receptors

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

My laboratory studies the regulation of glutamate receptor trafficking and localization using a combination of biochemical and molecular techniques. Glutamate receptors are the major excitatory neurotransmitter receptors in the mammalian brain and are a diverse family with many different subtypes. The ionotropic glutamate receptors include AMPA, NMDA, and kainate receptor subtypes, each of which are formed from a variety of subunits. The metabotropic glutamate receptors (mGluR1-8) are G protein-coupled receptors (GPCRs), which are assembled as homodimers. We focus on defining subunit-specific mechanisms that regulate the synaptic localization and functional regulation of glutamate receptors as well as synaptic scaffolding proteins. These mechanisms include posttranslational modifications such as phosphorylation, as well as protein-protein interactions. Currently, my research program is focused specifically of the molecular mechanisms regulating NMDA receptors. The overall goal is to better understand NMDA receptor function under normal circumstances and the specific dysfunction underlying some neurodevelopmental disorders. NMDA receptors are multi-subunit complexes (tetramers) composed of homologous subunits (GluN1; GluN2A-D; GluN3A-B). We have made significant progress in the detailed characterization of the synaptic expression of NMDARs and the role of GluN2A and GluN2B in receptor trafficking and synaptic expression. We primarily focus on GluN2A and GluN2B because these subunits are highly expressed in hippocampus and cortex and are known to undergo activity- and developmentally-regulated trafficking events. Over the last decade, we have shifted to a new approach to studying structure/function of NMDARs using human genetics to inform our research. We began with this "bedside-to-bench" strategy to help guide us in testing receptor domains that are important for synaptic function. We used information from published papers and public databases that report variants identified by deep sequencing of patients with neurological or psychiatric disorders. We then began conducting experiments on missense variants identified in GRIN genes (that encode NMDA receptor subunits. Specifically, we are examining variants causing mutations in the intracellular C-terminal domain of the GluN2 NMDAR subunits (GluN2A and GluN2B). As the human genetics data have accumulated, it has become clear that many de novo mutations in NMDA receptor subunits are highly associated with neurodevelopmental disorders including autism spectrum disorder, intellectual disability and epilepsy. Therefore, our goal is to better understand the synaptic dysfunction caused by these disease-associated rare variants with an eye towards developing therapeutics. Because of our expertise in studying receptor trafficking and protein interactions, we primarily focus on rare variants identified in the intracellular C-termini of NMDA receptor subunits, although we are also embarking on studies using mice with GRIN2B haploinsufficiency. In a recently published study, we characterized a rare variant in GluN2A (S1459G) identified in an epilepsy cohort (Mota Vieira et al., 2020). The patient also was diagnosed with intellectual disability. This de novo mutation is within the extreme C-terminal domain near the PDZ ligand. We found that this serine is a CaMKII site and phosphorylation of this residue dictates the receptor interactions with PSD-95 and sorting nexin 27 (SNX27). Thus, we identified a regulatory site that determines the trafficking and synaptic expression of GluN2A-containing NMDA receptors. In a related study, we have characterized a GRIN2A rare variant (identified in a patient with neurodevelopmental disorders) encoding a frameshift that results in a truncation of half of the C-terminal domain as well as encoding a unique stretch of amino acids. We find that the receptor is mistargeted and displays increased surface expression, but specifically enriched at extrasynaptic sites. There are defects in protein interactions, spine density, and synapse number. We collaborate with clinicians (NINDS and NIMH) to collect patient data and better understand the clinical presentation so we can compare with genotype and synaptic function. Again, our approach reveals new findings in NMDA receptor structure/function. We hope to use the information from the analyses of different pathogenic rare variants to better understand NMDA receptor trafficking and localization and help test for more precise therapeutics. In addition, we have ongoing studies of rare variants in GluN2B that result in extensions of the subunit. Thus far we find that these de novo mutations disrupt binding to scaffolding proteins, reduce receptor surface expression. These studies are ongoing. Over the last year, we have put intense effort into the development of reliable protocol to differentiate excitatory neurons expressing NMDA receptors from iPSCs. This requires a multi-step protocol because the most common single step differentiation method does not yield neurons with reliable robust expression of NMDARs. We have made significant progress and are currently collaborating to use CRISPR to introduce patient identified rare variants.