Linear ubiquitination of the NMDA receptor GluN2A subunit facilitates the GluN2B-to-GluN2A switch and synaptic maturation

Abstract

N-methyl-D-aspartate-type glutamate receptors (NMDARs) initiate the synaptic plasticity underlying learning and memory. In forebrain excitatory neurons, NMDARs are heteromeric tetramers composed of two GluN1 subunits and two glutamate ionotropic receptor NMDA type subunit 2A (GluN2A) or GluN2B subunits. At birth, NMDARs contain primarily GluN2B, but within weeks, GluN2A-containing receptors predominate the forebrain, comprising over 65% of total NMDARs in adulthood. This rapid subunit switch is essential for neonatal cognitive development, yet mechanisms driving it remain unclear. Particularly, while GluN2B levels remain relatively constant, GluN2A increases several 100-fold, despite its mRNA rising by only ~10-fold, strongly suggesting involvement of unknown posttranslational regulation. Here, we show that in the neonatal mouse forebrain, the linear ubiquitination axis, composed of the E3 ligase complex LUBAC and the deubiquitinase OTULIN, shifts transiently toward higher activity, with HOIP upregulated and OTULIN downregulated. In neonatal mice, experimentally reducing the axis activity by OTULIN overexpression causes persistent synaptic immaturity and adult cognitive deficits. Using proteomic and biochemical assays, we identified GluN2A as a key substrate: Linear ubiquitination at six lysines in the GluN2A C-terminus stabilizes the subunit and promotes its synaptic expression, whereas disrupting this modification destabilizes GluN2A by promoting lysosomal degradation. Consistently, overexpression of wild-type GluN2A rescues OTULIN-induced synaptic immaturity, whereas the ubiquitination-deficient GluN2A-6KR mutant fails to rescue and further exacerbates this defect. OTULIN overexpression selectively promotes GluN2A degradation, thereby delaying the GluN2B-to-GluN2A switch and synaptic maturation. These findings reveal a role for the linear ubiquitination axis in selectively stabilizing GluN2A, supporting rapid synaptic and cognitive development.