Evaluating the protein homeostasis of N-methyl-D-aspartate receptors containing pathogenic GluN2B subunits

Project Summary 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.