The paroxysmal disorder gene PRRT2 downregulates NaV channels and neuronal excitability in human neurons

Proline-Rich Transmembrane Protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes, including benign familial infantile seizures, paroxysmal kinesigenic dyskinesia and migraine. Most of the mutations of this gene lead to a premature stop codon, generating an unstable form of mRNA or a truncated protein that is degraded, pointing out the loss of the PRRT2 function as pathogenic mechanism of action. In this thesis, we have used different approaches to investigate the pathophysiological function of PRRT2. An important role for PRRT2 in the neurotransmitter release machinery, brain development and synapse formation has been uncovered by a previous work performed in our laboratory by acute silencing of PRRT2 expression. Here, we analyzed the phenotype of primary hippocampal neurons obtained from mouse PRRT2 knockout (KO) embryos. Analysis of synaptic function in primary neurons obtained from PRRT2-KO showed a largely similar, albeit attenuated, synaptic phenotype with respect to acute PRRT2 silencing characterized by weakened spontaneous/evoked synaptic transmission and increased facilitation at excitatory synapses. These effects were accompanied by a strengthened inhibitory transmission that, however, displayed faster synaptic depression. At the network level, these synaptic phenotypes, resulted in a state of increased spontaneous and evoked neurotransmitter release with increased excitability of excitatory neurons. To better dissect the physiological role of PRRT2, we characterized the phenotypes of neurons differentiated from Induced Pluripotent Stem Cells (iPSCs) from patients homozygous for the PRRT2 c.649dupC mutation. Hence, we observed an increased Na+ current and firing activity in iPSCs rescued with the re-expression of the human wild-type form of PRRT2. By use of heterologous expression system, we demonstrate that PRRT2 interacts with NaV1.2/NaV1.6, but not with NaV1.1 channels, modulating their membrane exposure and decreasing their conductances. In brief, our findings highlighted that PRRT2 mutations might be a negative modulator of NaV1.2/NaV1.6 channels and point out the critical role of this protein in the regulation of the neuronal network functionality.