PRRT2 frameshift mutation reduces its mRNA stability resulting loss of function in paroxysmal kinesigenic dyskinesia

Abstract

A heterozygous frameshift PRRT2 mutation (c.649_650InsC) has been identified as the major causative mutation in several paroxysmal disorders, including paroxysmal kinesigenic dyskinesia (PKD). Since PKD is an autosomal dominant disorder and since the frameshift mutations of PRRT2 may create a truncated protein, it remains unclear whether this mutation causes toxic gain of function or loss of function. By generating Prrt2 knock-in (KI) mice that express human PRRT2 with the c.649_650InsC mutation and by comparing the phenotypes of Prrt2 KI mice with knockout (KO) mice, we find that both KI and KO mice show the same extents of impaired rotarod and balance beam performance as well as the same sensitivity to seizure induction. Both KI and KO mice show altered formation of SNARE complex and number of synaptic vesicles. In addition, western blotting of KI mouse brain tissues could not detect truncated PRRT2 protein that might be generated by the c.649_650InsC mutation. Moreover, the level of PRRT2 mRNA in KI mice is significantly decreased, recapitulating the reduction of PRRT2 mRNA reported in PKD patients. Furthermore, mutant PRRT2 mRNA is unstable and showed shortened half-life than wild-type PRRT2 mRNA. Our studies suggest that PRRT2 frameshift mutation leads to the loss of function by affecting its mRNA stability, a mechanism that is different from haploinsufficiency due to dysfunctional protein or gain of function caused by truncated protein.

Keywords: Decreased mRNA stability; Frameshift mutation; Loss of function; Prrt2 KI mice; Truncated protein.

Fig. 1.. Generation of PRRT2 KO and KI mice.

(A) Schematic diagram of generation of mutant PRRT2 mice. (B) Genotyping of WT and mutant mice using genomic DNA from mouse tail by PCR. (C) Sequencing results of WT and Mutant mice. (D) Western blotting of the cerebellum lysates from WT, KO and KI.

Fig. 2.. Comparable PKD-like behavioral phenotypes in PRRT2 KO and KI mice.

(A) Body weight of WT, KO and KI mice from 2 to 8 weeks of age. (B) Elevated plus maze assay of WT, KO and KI mice at 4 and 8 weeks of age. (C) Motor behavior of WT, KO and KI mice at 4 and 8 weeks of age in grip strength, rotarod and beam balance. (D) Foot-printing assay on WT, KO and KI at the age of 8 weeks. FW, fore-paw width; FL, fore-paw length; HW, hind-paw width; HL, hind-paw length, were recorded. Age-matched WT mice (n=24), KO mice (n=15) and KI mice (n = 15) were examined in above assays. (E) Flurothyl-induce seizure experiments on WT, KO and KI mice at 8 weeks of age. Latency to first myoclonic jerks (MJ) and generalized tonic-clonic seizure (GTCS) were recorded. One-way ANNOVA followed with Turkey’s multiple comparisons test, *P< 0.05, n=10 per group.

Fig. 3.. Increased formation of SNARE complex and number of synaptic vesicles in PRRT2 KO and KI mice.

(A) Western blotting of adult WT, KO and KI mice brain tissues with synapse related protein. SYN1, Synapsinl; SYT, Synaptotagmin. STX1A, Syntaxin1A. One-way ANNOVA followed with Turkey’s multiple comparisons test, n=6 per group. (B) Full gel western blotting of non-boiled synaptosomes purified from the whole brain tissues of 2-month-old WT, KO and KI mice. One-way ANNOVA followed with Turkey’s multiple comparisons test, *P< 0.05, **P< 0.005, n=3 per group. (C) Representative transmission electron microscopy images of nerve terminals from 2-month-old WT, PRRT2 KO and KI mice. Scale bar, 100 nm. (D) Quantitative analysis of total synaptic vesicles (SVs), docked SVs and mean length of active zone. One-way ANNOVA followed with Tukey’s multiple comparisons test, *P< 0.05, n=3 per group.

Fig. 4.. Reduced mRNA stability of mutant PRRT2 leads to undetectable PRRT2 protein.

(A-B) Western blotting of transfected HEK293 cells and adult mouse cortex lysates. The blots were probed with anti-PRRT2 antibody (EM516) and anti-HA antibody. Vinculin served as a loading control. Unpaired-student’s t test, *** P=0.001, n=4 per group. (C-D) Western blotting of WT, KO and KI mice cerebellum injected with MG132 (C) or 3-MA (D) with the indicated concentrations and times. (E) Quantitative real-time PCR (qPCR) assay of WT and mutant PRRT2 mRNA in the cerebellum and cortex of adult WT and KI. Unpaired-student’s t test, n=4 per group, **P=0.0056 (cerebellum), **P=0.0014 (cortex). (F-G) QPCR assay on the half-life of WT and mutant PRRT2 mRNA in transfected Hek293 cells after Actinomycin D treatment at different time point. One-way ANNOVA followed with Tukey’s multiple comparisons test, ****P< 0.0001, n=6 per group.