Physiological role of PRRT2 and its involvement in the pathogenesis of paroxysmal disorders

Mutations in the PRoline-Rich Transmembrane protein 2 gene (PRRT2) underlie a heterogeneous group of paroxysmal disorders encompassing infantile epilepsy, paroxysmal kinesigenic dyskinesia, a combination of these phenotypes and migraine. For the majority of the pathogenic PRRT2 variants, the mutant proteins are not expressed or not correctly targeted to the plasma membrane, resulting in a loss-of function mechanism for PRRT2-related diseases. PRRT2 is a neuron-specific, type II transmembrane protein of 340 amino acids with an important functional role in synapse formation and maintenance, as well as in the regulation of fast neurotransmitter release at both glutamatergic and GABAergic terminals. The PRRT2 knock-out (PRRT2-KO) mouse, in which PRRT2 has been constitutively inactivated, displays alterations in brain structure and a sharp paroxysmal phenotype, reminiscent of the most common clinical manifestations of the human PRRT2-linked diseases. To gain further insights on the pathogenic role of PRRT2 deficiency, I used Multi-Electrode Arrays (MEAs) to characterize neuronal activity generated by primary hippocampal cultures obtained from the PRRT2-KO mouse embryos and to assess the epileptic propensity of cortico-hippocampal slices obtained from the same animal model. This experimental approach revealed a state of heightened spontaneous activity, hyper-synchronization in population bursts of action potentials (APs) and enhanced responsiveness to external stimuli in mutant networks. A complex interplay between (i) a synaptic phenotype, with weakened spontaneous transmission and increased short-term facilitation, and (ii) a marked increase in intrinsic excitability of excitatory neurons as assessed by single-cell electrophysiology, upholds this network phenotype. Furthermore, our group has generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from heterozygous and homozygous siblings carrying the most common C.649dupC mutation. Patch-clamp recordings in neurons from homozygous patients showed an increased Na+ current that was fully rescued by expression of exogenous wild-type PRRT2. A strikingly similar electrophysiological phenotype was observed in excitatory primary cortical neurons from the PRRT2-KO mouse, which was accompanied by an increased length of the axon initial segment (AIS). At the network level, mutant cortical neurons grown on MEAs also displayed a state of spontaneous and evoked hyper-excitability and elevated propensity to synchronize their activity in network bursting events.