RABPHILIN3A: FROM NMDA RECEPTOR MEMBRANE DYNAMICS TO NEURODEVELOPMENTAL DISORDERS

Rabphilin3A (Rph3A) is a synaptic protein essential for regulating exo- and endocytosis processes at presynaptic terminals and dendritic spines. It promotes the stabilization of the NMDA-type glutamate receptors (NMDARs) at the cell surface, forming a ternary complex with the PSD-95 and GluN2A subunit of the receptor. This complex is needed for long-term potentiation (LTP) induction and NMDAR-dependent hippocampal behaviors, such as spatial learning. Under physiological conditions, Rph3A is present in approximately 50% of hippocampal spines. Notably, Rph3A positive (Rph3A+) spines exhibit enlarged spine head and postsynaptic density size, indicating enhanced synaptic stability. LTP induction increases both the number of Rph3A+ spines and NMDAR synaptic retention, thus indicating that plasticity promotes the postsynaptic localization of the protein. Importantly, the disruption of Rph3A/NMDAR complex impairs GluN2A accumulation at postsynaptic membrane, preventing LTP induction. Our group has also previously demonstrated that overexpression of Rph3A in primary hippocampal neurons is sufficient to occlude the LTP-induced formation of new dendritic spines, confirming a key role for the Rph3A/GluN2A pathway in these processes. Given these premises, the first aim of my thesis was to determine whether Rph3A serves as a molecular marker for potentiated synapses. Using calcium imaging in primary hippocampal neurons, I found that overexpression of Rph3A leads to significantly increased postsynaptic calcium influx. To assess the role of Rph3A at the level of individual synapses, I performed single-spine glutamate uncaging in live neurons. Spines overexpressing Rph3A failed to undergo further potentiation upon stimulation, suggesting that they were already in a potentiated state. Additionally, I investigated how Rph3A influences the dynamics and synaptic localization of NMDARs. In the second part of my thesis, I explored the pathological consequences associated with RPH3A gene. By combining trio-based exome sequencing, GeneMatcher data, and analysis of the 100,000 Genomes Project, our collaborators identified six heterozygous missense variants in RPH3A gene. Four cases had a neurodevelopmental disorder (NDD) with untreatable epileptic seizures [p.(Gln73His)dn; p.(Thr450Ser)dn; p.(Arg209Lys); p.(Gln508His)], and two cases [p.(Asn618Ser)dn; p.(Arg235Ser)] showed high functioning autism spectrum disorder (ASD). Using primary hippocampal neurons, I demonstrated that four of these variants (p.(Arg209Lys), p.(Thr450Ser), p.(Gln508His), and p.(Asn618Ser)) disrupted the synaptic co-localization of PSD-95 and GluN2A and led to increased surface levels of GluN2A at extrasynaptic sites. In addition, RPH3A mutants induce defects in NMDAR activity at synaptic sites, affecting postsynaptic calcium and glutamate influx at dendritic level. Collectively, this PhD work identifies Rph3A as a molecular marker of potentiated synapses and a critical regulator of synaptic NMDAR localization. Moreover, it provides the first evidence that missense gain-of-function variants in RPH3A can disrupt synaptic function and contribute to a spectrum of neurodevelopmental disorders, from untreatable epilepsy to autism spectrum disorder.