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.

Role of Rph3A in brain injury induced by experimental cerebral ischemia-reperfusion model in rats.

AIM: The aim was to study the role of Rph3A in neuronal injury induced by cerebral ischemia-reperfusion. METHODS: The protein and mRNA levels of Rph3A in penumbra were detected by Western blot. The localization of Rph3A in different cell types in penumbra was detected by immunofluorescence. Apoptosis in the brain was detected by TUNEL staining. We tested neurobehavioral evaluation using rotarod test, adhesive-removal test, and Morris Water maze test. We examined the expression and localization of Rph3A in cultured neurons and astrocytes in vitro by Western blot and ELISA, respectively. RESULTS: The mRNA and protein levels of Rph3A had significantly increased in brain penumbra of the rat MCAO/R model. Rph3A was mainly distributed in neurons and astrocytes and was significantly increased by MCAO/R. We downregulated Rph3A and found that it further worsened the cerebral infarct, neuronal death and behavioral, cognitive, and memory impairments in rats after MCAO/R. We also found that ischemia-reperfusion upregulated the in vitro protein level and secretion of Rph3A in astrocytes but led to a decrease in the protein level of Rph3A in neurons. CONCLUSION: The increase in Rph3A in the brain penumbra may be an endogenous protective mechanism against ischemia-reperfusion injury, which is mainly dominated by astrocytes.

Presynaptic congenital myasthenic syndrome with altered synaptic vesicle homeostasis linked to compound heterozygous sequence variants in RPH3A.

BACKGROUND: Monogenic defects of synaptic vesicle (SV) homeostasis have been implicated in many neurologic diseases, including autism, epilepsy, and movement disorders. In addition, abnormal vesicle exocytosis has been associated with several endocrine dysfunctions. METHODS: We report an 11 year old girl with learning disabilities, tremors, ataxia, transient hyperglycemia, and muscle fatigability responsive to albuterol sulfate. Failure of neuromuscular transmission was confirmed by single fiber electromyography. Electron microscopy of motor nerve terminals revealed marked reduction in SV density, double-membrane-bound sacs containing SVs, abundant endosomes, and degenerative lamellar bodies. The patient underwent whole exome sequencing (WES) and relevant sequence variants were expressed and studied in a mammalian cell line. RESULTS: Chromosomal microarray studies and next generation sequencing (NGS) of mitochondrial DNA were unrevealing; however, NGS of genomic DNA showed two rare sequence variants in the gene encoding rabphilin 3a (RPH3A). The paternally inherited variant c.806 G>A (p.Arg269Gln) involves a substitution of a conserved residue in the linker region, while the maternally inherited variant c.1390 G>T (p.Val464Leu) involves a conserved amino acid substitution in the highly conserved C2A region. Expression studies revealed that p.Arg269Gln strongly impairs the binding of rabphilin 3a to 14-3-3, which is a proposed regulator of synaptic transmission and plasticity. In contrast, the binding of rabphilin 3a to 14-3-3 is only marginally impaired by p.Val464Leu; thus, the pathogenic role of p.Val464Leu remains unclear. CONCLUSION: In summary, we report a patient with a multisystem neurologic disorder and altered SV regulation attributed to defects in RPH3A, which grants further studies of this gene in human disorders of synaptic transmission.

PKN1 Directs Polarized RAB21 Vesicle Trafficking via RPH3A and Is Important for Neutrophil Adhesion and Ischemia-Reperfusion Injury.

Polarized vesicle transport plays an important role in cell polarization, but the mechanisms underlying this process and its role in innate immune responses are not well understood. Here, we describe a phosphorylation-regulated polarization mechanism that is important for neutrophil adhesion to endothelial cells during inflammatory responses. We show that the protein kinase PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to guanosine triphosphate (GTP)-bound RAB21. These interactions are important for polarized localization of RAB21 and RPH3A in neutrophils, which leads to PIP5K1C90 polarization. Consistent with the roles of PIP5K1C90 polarization, the lack of PKN1 or RPH3A impairs neutrophil integrin activation, adhesion to endothelial cells, and infiltration in inflammatory models. Furthermore, myeloid-specific loss of PKN1 decreases tissue injury in a renal ischemia-reperfusion model. Thus, this study characterizes a mechanism for protein polarization in neutrophils and identifies a potential protein kinase target for therapeutic intervention in reperfusion-related tissue injury.