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. As whole exome and whole genome sequencing have become more accessible, many rare variants have been identified in NMDA receptor subunits genes (GRINS). The expanding list of pathogenic rare variants associated with neurodevelopmental disorders has led to the recognition of GRIN Disorders. The work in my lab is focused on understanding NMDA receptor function under normal circumstances and the specific dysfunction underlying GRIN 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. Using human genetics to inform our research, we implement a "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 revealed that many de novo mutations in NMDA receptor subunits are often pathogenic and lead to co-morbidities 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 have expanded our studies to include truncating or frameshift mutations. In addition, we are analyzing mice with GRIN2B haploinsufficiency (GluN2B knock-out heterozygous mice) to gain insight into the dysfunction underlying a significant subpopulation of people diagnosed with GRIN2B disorders. In one ongoing study, we are evaluating rare variants in GluN2B that result in extensions of the subunit. Thus far we find that these two de novo mutations disrupt binding to scaffolding proteins, reduce receptor surface expression, and destabilize the protein in a variety of expression systems. Because these two variants result in extensions and disrupt binding to MAGUK proteins, we again examined databases to look for other variants that specifically disrupt the PDZ ligand on 2B. One such variant is a disease-associated frameshift in the distal C-terminal tail of GluN2B, which also displays decreased protein expression and surface expression in heterologous cells and in neurons. These data all demonstrate that disruption of the PDZ ligand of GluN2B in humans is not tolerated and results in neurodevelopmental disorders including epilepsy. In addition to CTD missense variants, we are studying truncating mutations in 2B and 2A using mouse models. We have generated mouse lines of 2B and 2A truncations and will evaluate resulting phenotypes using biochemical, physiological, and behavioral approaches. In addition, we are collaborating to investigate tRNA strategies to rescue the phenotype. Perhaps this would be a therapeutic that could be developed in the future for nonsense variants in GRIN genes. We have also been studying the scaffolding protein CNKSR2, which is a high confidence gene associated with neurodevelopmental disorders. We find that CNKSR2 binds to NMDA receptors and forms a multimolecular complex. We have generated mouse models of KO and missense mutations of CNKSR2, which we are currently characterizing.