Impaired presynaptic Ca2+ influx in PRRT2- deficient neurons

PRRT2 is a single causative gene for paroxysmal kinesigenic dyskinesia disease. Several studies have identified an array of heterozygous loss-of-function nonsense/frameshift and missense mutations in the gene encoding this protein that through non-sense mediated RNA decay and/or degradation of truncated/misfolded protein cause a condition of haploinsufficiency. These mutations are described in a large number of patients affected by paroxysmal disorders such as benign familial infantile seizures, infantile convulsion choreoathetosis, migraine, hemiplegic migraine, paroxysmal kinesigenic dyskinesia/choreoathetosis, benign familial infantile seizures/epilepsy, and episodic ataxia. PRRT2 is a small gene located on chromosome 16p11.2 already expressed at early postnatal stages, and its expression increases till reaching a plateau at 1 month of life; high levels of PRRT2 mRNA were detected in many tissues of the nervous system, especially in the extrapyramidal system. We have previously shown that PRRT2 is a key component of regulated exocytosis and its silencing dramatically impairs neurotransmitter release by markedly reducing release probability. Moreover, we demonstrated that PRRT2 interacts with the fast Ca2+ sensors synaptotagmin 1/2 and endows the SNARE complex with Ca2+ sensitivity. Here, we investigated the altered Ca2+ dependence of glutamatergic synaptic transmission as a possible causative role in the pathogenesis of PRRT2-linked paroxysmal diseases by focusing on voltage-gated Ca2+channels (VGCaCh) and presynaptic Ca2+ influx. Thanks to the use of multiple experimental approaches, including electrophysiology, immunocytochemistry, biochemistry and live Ca2+ imaging, we investigated the changes of somatic and presynaptic VGCaCh subtypes in PRRT2-deficient neurons but also the altered Ca2+ dependence of the synaptic transmission. We observed that PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory synaptic currents (eESPCs) recorded at increasing concentrations of extracellular Ca2+. This effect was associated with a reduction of P/Q- and N-type VGCa currents paralleled by a decrease in the amplitude of N- and P/Q-type sensitive eESPCs. Using surface biotinylation, we observed that PRRT2 deletion impaired the P/Q-type VGCaChs trafficking to the membrane. Double immunostainings revealed that in presynaptic terminals lacking PRRT2, the P/Q-type VGCaChs diffused in the presynaptic membrane and reduce their clustering at the active zone. In parallel, using SyGCaMP-6s, an ultra-sensitive fluorescent Ca2+ indicator selectively expressed at the presynapse, we investigated presynaptic Ca2+ influx and observed a significant decrease of the P/Q-dependent presynaptic Ca2+ signal in PRRT2-deficient synapses. Our results strongly suggest that PRRT2 deletion causes mistargeting of P/Q-type VGCaChs far away from the active zone, reducing the availability of VGCaCh strictly associated with the release machinery and thereby the glutamate release probability. Altogether our data highlights the central role of PRRT2 in the release machinery, indeed, the lack of this protein impaired the N- and P/Q-type channels trafficking from the cytoplasm to the membrane causing of a reduction of intracellular Ca2+ in consequence of a reduction of release probability.