Functional consequences of mutations in GRIN2A and GRIN2B associated with mental disorders

Abstract

GRIN2A and GRIN2B encode the GluN2A and GluN2B subunits of the NMDA receptor, a subtype of ionotropic glutamate receptor that displays voltage-dependent block by Mg2+ and a high permeability to Ca2+. These receptors play important roles in synaptogenesis, synaptic transmission and synaptic plasticity, as well as contributing to neuronal loss and dysfunction in several neurological disorders. Recently, individuals with a range of childhood onset epilepsies, intellectual disability and other neurodevelopmental abnormalities have been found to carry heterozygous gene-disrupting or protein-altering point mutations in GRIN2A and GRIN2B. This thesis addresses the hypothesis that these point mutations cause key functional disturbances to NMDA receptor properties that contribute to neurodevelopmental disorders. To test this hypothesis, a group of related mutations were selected for functional assessment in heterologous systems: four missense mutations affecting residues in or near the subunit pore regions, all of which are associated with epilepsy and intellectual disability. To model the impact of gene disrupting mutations in GRIN2A, a preliminary analysis of the functional consequences of GluN2A haploinsufficiency in a genetically modified rat was also performed. Three of the four missense mutations were found to be associated with profound alterations in fundamental NMDA receptor properties: compared to wild type, GluN2AN615K was found to reduce Mg2+ block, GluN2BN615I and GluN2BV618G to cause potentiation by Mg2+, and GluN2AN615K and GluN2BN615I showed reduced conductance. GluN2AR586K was not found to influence the parameters assessed. When GluN2AN615K was expressed alongside wild type subunits in the same NMDA receptor, it was found to have a dominant negative effect. Finally, I established successful gene targeting in a new rat Grin2A knock-out model, and observed that heterozygous neurons had lower GluN2A protein expression and current density, making a good model to study human epilepsies associated with loss of a GRIN2A allele. This thesis provides evidence that three missense mutations in GRIN2A and GRIN2B affect physiologically important properties of the NMDA receptor, and that GluN2A haploinsufficiency influences important neural properties in vitro. This is consistent with these mutations causing disease and highlights these and related mutations as potential therapeutic targets in the future.