Arg209Lys and Gln508His missense variants in Rabphilin 3A cause pre- and post-synaptic dysfunctions at excitatory glutamatergic synapses.

The synaptic protein Rabphilin 3A (Rph3A), encoded by the RPH3A gene, is a known binding partner of the NMDA receptor (NMDAR) complex, which is essential for synaptic plasticity and cognitive functions. A recent report demonstrated a causal association between missense variants in the RPH3A gene and neurodevelopmental disorders, manifesting as either drug-resistant epilepsy with intellectual disability or as autism spectrum disorder with learning disability. In this study, we used primary hippocampal neurons to analyse synaptic effects induced by the p.(Arg209Lys) and p.(Gln508His) RPH3A variants, located in the N-terminal disordered region and the C-terminal C2A domain of Rph3A, respectively. We found that both the mutants exert effects on pre- and post-synaptic events mediated by Rph3A, despite their different positions within the Rph3A amino acid sequence. Notably, in both cases, RPH3A variants reduced presynaptic glutamate release and led to decreased synaptic retention of NMDARs containing the GluN2A subunit, a primary binding partner of Rph3A. These changes were associated with a reduced frequency of calcium events at dendritic spines, indicating an overall significant dysregulation of glutamatergic synaptic transmission.