Evaluating the protein homeostasis of n-methyl-d-aspartate receptors containing pathogenic GLUN2B subunits

The National Institute of Neurological Disorders and Stroke awarded Case Western Reserve University $109,412 on January 1, 2026, under the Extramural Research Programs in the Neurosciences and Neurological Disorders program (CFDA 93.853) to evaluate the protein homeostasis of N-methyl-D-aspartate receptors containing pathogenic GluN2B subunits.

The research elucidates the proteostasis network regulating NMDA receptor folding, assembly, trafficking, and degradation, with emphasis on how disease-associated variants perturb these molecular mechanisms. Aim 1 tests whether disease-associated variants within the GluN2B subunit fail to assemble into functional receptors, bypassing endoplasmic reticulum quality control checkpoints and resulting in accumulation and activation of the unfolded protein response. Aim 2 examines clearance and degradation of disease-associated variant GluN2B subunits through lysosomal pathways. The work employs biochemical and molecular techniques to characterize how mutations in GRIN genes, identified through whole exome sequencing, result in loss of receptor function and reduced cell surface expression.

Case Western Reserve University's Office of Research Administration, operating in affiliation with University Hospitals of Cleveland, performs the research in Cleveland, Ohio. The performance period runs from January 1, 2026 through December 14, 2026.

Abstract

N-methyl-d-aspartate receptors (nmdars) are gated by the primary excitatory neurotransmitter, glutamate. They play essential roles in neuronal formation, synaptic maturation, as well as various central nervous system functions, such as learning and memory. Nmdars are heterotetrameric assemblies, typically composed of two GLUN1 subunits and two GLUN2 subunits. The grin genes that encode the glun subunits of nmdars are highly intolerant to genetic variation which indicates mutations are more likely to result in disease states. Recently, whole exome sequencing has identified a large number of mutations to the grin genes that result in a loss of receptor function and reduced expression on the cell surface. The subunit composition of nmdars is vital for their regional and functional expression on the cell surface, but receptors must first conform to the native structures within the endoplasmic reticulum (ER) before being trafficked to the plasma membrane. Despite the importance the ER has in establishing the correct folding of proteins, much remains to be understood regarding the essential steps in nmdar folding, oligomerization, and anterograde trafficking. The overarching goal of this study is to elucidate the proteostasis network regulating nmda receptor folding, assembly, trafficking, and degradation. We further seek to determine how disease-associated variants (davs) perturb these molecular mechanisms. Utilizing numerous biochemical and molecular techniques, aim 1 will test the hypothesis that davs within the GLUN2B subunit are unable to assemble into functional receptors, failing ER quality control checkpoints, resulting in their accumulation within the ER and subsequent activation of the unfolded protein response. Aim 2 will test the hypothesis that that the clearance and degradation of the DAV GLUN2B subunits occurs through the lysosome via two distinct pathways: macroautophagy and selective ER- phagy. Nmdars are anticipated to undergo degradation via autophagy under physiological and pathological conditions. However, in the case of davs, we believe selective ER-phagy is also activated to remove aberrant GLUN2B subunits from the crowded ER lumen. Additionally, aim 2 will investigate the role of the LC3B interacting region (LIR) motif present in the highly conserved c-terminal domain of the GLUN2B subunit. The proposed study will also characterize the effects of these davs in the context of cortical neurons derived from human induced pluripotent stem cells. Results from these studies will provide great insight into the molecular mechanisms and cellular pathways responsible for maintaining the homeostasis of nmdars, perhaps even defining subunit-specific mechanisms. Indeed, these results hold promise to identify novel therapeutic targets for treatment of disorders in which nmdars are dysregulated, including grin diseases, schizophrenia, autism spectrum disorder, and alzheimer's disease.