Two extreme Loss-of-Function GRIN2B-mutations are detrimental to tri-heteromeric NMDAR-function, but rescued by pregnanolone-sulfate

Abstract

Mutations within various N-methyl-D-aspartate receptor (NMDAR) subunits are tightly associated with severe pediatric neurodevelopmental disorders and encephalopathies (here denoted GRINopathies), for which there are no treatments. NMDARs are tetrameric receptors and can be found at the membrane of neurons in various compositions, namely in di- or tri-heteromeric forms. The GluN2B subunit appears very early in development and, therefore, prenatally this subunit is predominantly found within di-heteromeric receptors, exclusively composed of the GluN1 and GluN2B subunits. Postnatally, however, the GluN2A subunit undergoes rapid increase in expression, giving rise to the appearance of tri-heteromers containing the GluN1, GluN2A and GluN2B-subunits. The latter are emerging as the principal receptor-type postnatally. Despite more than a decade of research of numerous GRINopathies, not much is known regarding the effect of GRIN variants when these are assembled within tri-heteromers. Here, we have systematically examined how two de novo GRIN2B variants (G689C and G689S) affect the function of di- and tri-heteromers. We show that whereas a single mutated subunit readily instigates a dominant negative effect over glutamate affinity of tri-heteromers, it does not dominate other features of the receptor, notably potentiation by pregnanolone-sulfate (PS). This led us to explore PS as a potential treatment for these two severe loss-of-function (LoF) mutations in cultured neurons, in which case we indeed find that the neurosteroid rescues current amplitudes. Together, we present the first report to examine LoF GRIN2B mutations in the context of di- and tri-heteromeric receptors. We also provide the first demonstration of the positive outcome of the use of a GRIN2B-relevant potentiator in the context of tri-heteromers. Our results highlight the importance of examining how different mutations affect features in various receptor subtypes, as these could not have been deduced from observations performed on purely di-heteromers. Together, our study contributes to the ongoing efforts invested towards understanding the pathophysiology of GRINopathies as well as provides insights towards a potential treatment.