PRRT2 Regulates Synaptic Fusion by Directly Modulating SNARE Complex Assembly

Abstract

SummaryMutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed to regulate neurotransmitter release. However, the mechanism of action is unclear. Here, we use reconstituted single vesicle and bulk fusion assays, combined with live cell imaging of single exocytotic events in PC12 cells and biophysical analysis, to delineate the physiological role of PRRT2. We report that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N-terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrate that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficiencies corresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders.

A PRRT2 variant in a Chinese family with paroxysmal kinesigenic dyskinesia and benign familial infantile seizures results in loss of interaction with STX1B

“…However, the precise regulatory mechanism underlying the interaction between PRRT2 and STX1B remains unclear. Recently, PRRT2 has been reported to regulate the assembly of the SNARE complex, synaptic vesicle docking, and fusion . Considering that cerebellar synaptic dysfunction due to PRRT2 deficiency is closely related to the occurrence of PKD, it would be interesting to determine whether and how PRRT2 modulates the formation of SNARE complexes containing STX1B in the cerebellum.…”

Paroxysmal and cognitive phenotypes in Prrt2 mutant mice

“…TEM analysis of synapses in wild type (WT) and KO cortex showed no significant difference in synapse density (P = 0.1824; Figure S2C) or active zone length (P = 0.0781; Figure S2D). (15,20 and 25 cm/s) was significantly lower in KO compared with WT in both fore (P = 0.0016) and hind limbs (P = 0.0004). C,D, Prrt2 KO mice showed shorter stride length than WT in both fore (P = 0.0051) and hind limbs (P = 0.0019) across the three speeds.…”

“…In vitro knockdown in primary neurons results in synaptic defects; namely decreased synaptic contacts, reduced synchronous neurotransmitter release and an insensitivity to extracellular Ca 2+ . A role for PRRT2 in inhibition of vesicle fusion at the synapse has also been described . Silencing of PRRT2 in vivo causes delayed neuronal migration during development and decreased dendritic spine density …”

“…18 A role for PRRT2 in inhibition of vesicle fusion at the synapse has also been described. 20 Silencing of PRRT2 in vivo causes delayed neuronal migration during development and decreased dendritic spine density. 21 Recently, two groups have described mouse models lacking PRRT2 protein.…”

Activity‐dependent cleavage of dyskinesia‐related proline‐rich transmembrane protein 2 (PRRT2) by calpain in mouse primary cortical neurons

“…Amino acids used in calpain-resistant substitution mutants are highlighted with yellow. C, Lysates of Neuro2A cells transfected with PRRT2wt-EGFP cDNA (lanes 1-3), PRRT2 LSRHP243_247FIDDD-EGFP cDNA (244-uncleaved mutant; lanes 4-6), PRRT2 HSPP201_204IDDD-EGFP cDNA (201-mut; lanes 7-9), PRRT2 LAGPG251_255FIDDD-EGFP cDNA (252-mut; lanes 10-12) or PRRT2 LQQLV219_223FIDDD-EGFP cDNA (220-mut; lanes 13-15) were treated with 4 mM CaCl 2 and 16.8 ng/µL native calpain-1 at 30°C for 0 hr (lanes 1, 4, 7, 10, 13, 16), 0.5 hr (lanes 2, 5, 8, 11, 14, 17), or 2 hr (lanes 3,6,9,12,15), and then immunoblotted with anti-GFP antibody. A black arrowhead and arrow indicate the full-length PRRT2-EGFP (lanes 1, 4, 7, 10, 13) and 12K-CTF-EGFP (lanes 2, 3, 8, 9, 11, 12), respectively.…”