Missense variants in RPH3A cause defects in excitatory synaptic function and are associated with a clinically variable neurodevelopmental disorder.

PURPOSE: RPH3A encodes a protein involved in the stabilization of GluN2A subunit of N-methyl-D-aspartate (NMDA)-type glutamate receptors at the cell surface, forming a complex essential for synaptic plasticity and cognition. We investigated the effect of variants in RPH3A in patients with neurodevelopmental disorders. METHODS: By using trio-based exome sequencing, GeneMatcher, and screening of 100,000 Genomes Project data, we identified 6 heterozygous variants in RPH3A. In silico and in vitro models, including rat hippocampal neuronal cultures, have been used to characterize the effect of the variants. RESULTS: Four cases had a neurodevelopmental disorder with untreatable epileptic seizures [p.(Gln73His)dn; p.(Arg209Lys); p.(Thr450Ser)dn; p.(Gln508His)], and 2 cases [p.(Arg235Ser); p.(Asn618Ser)dn] showed high-functioning autism spectrum disorder. Using neuronal cultures, we demonstrated that p.(Thr450Ser) and p.(Asn618Ser) reduce the synaptic localization of GluN2A; p.(Thr450Ser) also increased the surface levels of GluN2A. Electrophysiological recordings showed increased GluN2A-dependent NMDA ionotropic glutamate receptor currents for both variants and alteration of postsynaptic calcium levels. Finally, expression of the Rph3AThr450Ser variant in neurons affected dendritic spine morphology. CONCLUSION: Overall, we provide evidence that missense gain-of-function variants in RPH3A increase GluN2A-containing NMDA ionotropic glutamate receptors at extrasynaptic sites, altering synaptic function and leading to a clinically variable neurodevelopmental presentation ranging from untreatable epilepsy to autism spectrum disorder.

Rabphilin-3A Drives Structural Modifications of Dendritic Spines Induced by Long-Term Potentiation.

The interaction of Rabphilin-3A (Rph3A) with the NMDA receptor (NMDAR) in hippocampal neurons plays a pivotal role in the synaptic retention of this receptor. The formation of a Rph3A/NMDAR complex is needed for the induction of long-term potentiation and NMDAR-dependent hippocampal behaviors, such as spatial learning. Moreover, Rph3A can also interact with AMPA receptors (AMPARs) through the formation of a complex with myosin Va. Here, we used a confocal imaging approach to show that Rph3A overexpression in primary hippocampal neuronal cultures is sufficient to promote increased dendritic spine density. This morphological event is correlated with an increase in GluN2A-containing NMDARs at synaptic membranes and a decrease in the surface levels of GluA1-containing AMPARs. These molecular and morphological modifications of dendritic spines are sufficient to occlude the spine formation induced by long-term potentiation, but do not prevent the spine loss induced by long-term depression. Overall, our results demonstrate a key role for Rph3A in the modulation of structural synaptic plasticity at hippocampal synapses that correlates with its interactions with both NMDARs and AMPARs.