The PRRT2 knockout mouse recapitulates the neurological diseases associated with PRRT2 mutations

Abstract

Heterozygous and rare homozygous mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia episodic ataxia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function. Recently, an important role for PRTT2 in the neurotransmitter release machinery, brain development and synapse formation has been uncovered. In this work, we have characterized the phenotype of a mouse in which the PRRT2 gene has been constitutively inactivated (PRRT2 KO). β-galactosidase staining allowed to map the regional expression of PRRT2 that was more intense in the cerebellum, hindbrain and spinal cord, while it was localized to restricted areas in the forebrain. PRRT2 KO mice are normal at birth, but display paroxysmal movements at the onset of locomotion that persist in the adulthood. In addition, adult PRRT2 KO mice present abnormal motor behaviors characterized by wild running and jumping in response to audiogenic stimuli that are ineffective in wild type mice and an increased sensitivity to the convulsive effects of pentylentetrazol. Patch-clamp electrophysiology in hippocampal and cerebellar slices revealed specific effects in the cerebellum, where PRRT2 is highly expressed, consisting in a higher excitatory strength at parallel fiber-Purkinje cell synapses during high frequency stimulation. The results show that the PRRT2 KO mouse reproduces the motor paroxysms present in the human PRRT2-linked pathology and can be proposed as an experimental model for the study of the pathogenesis of the disease as well as for testing personalized therapeutic approaches.

Keywords: Audiogenic seizures; Cerebellum; Hippocampus; Knockout mouse; Motor paroxysms; Proline-rich transmembrane protein 2; Synaptic transmission.

Fig. 1

Somatic growth during postnatal development. A. Representative picture of WT, HET and KO PRRT2 mouse pups at P4 and P16. B–E. Analysis of the markers of somatic growth revealed no differences between WT, HET and KO mice during development on body weight (B), body temperature (C), body length (D) and tail length (E). Data (means ± SEM) were obtained from cohort 1 (10 litters): N = 15 WT, 43 HET and 17 PRRT2 KO. Two-way ANOVA and post-hoc Fisher PLSD test.

Fig. 2

Gross brain histology of cerebral cortex and corpus callosum. Representative confocal images of NeuN staining (green) in the motor (A), somatosensory (B) and visual (C) cortices of WT and PRRT2 KO mice and the respective morphometric analysis of the thickness of both cortex and corpus callosum. Data are expressed as means ± SEM; **p < 0.01, ***p < 0.001; two-tailed unpaired Student's t-test. N = 3 for both WT and KO mice. Scale bar, 100 μm.

Fig. 3

β-gal activity in the CNS of adult PRRT2 KO mice. A. Representative picture of β-gal staining in isolated adult brains from WT, HET and KO PRRT2 mice. A strong expression of β-gal was evident in the cerebellum of HET and KO mice, while WT mice were negative. B. PRRT2 was expressed in layer VIb of the neocortex (B1), septal nuclei (B2) and claustrum (B3). C, D. PRRT2 was expressed in hippocampal regions, particularly DG, hilus and CA3 (C1, D1, D2). Thalamic regions, amygdala (C2), periaqueductal grey, superior colliculus and neocortical layer VIb were also positive. E. A strong expression of PRRT2 was present in the cerebellum, particularly in the granular cell layer, while Purkinje cells were negative (E1). In the molecular layer, only sparse interneurons were positive (E2). F. An intense PRRT2 expression was present in the substantia gelatinosa and the dorsal horn of the spinal cord (F1-F2). Scale bars: 2 mm (B-D), 1 mm (E, F) and 0.1 mm (insets). N = 3 PRRT2 KO mice. G. Quantitative analysis of PRRT2 (left) and LacZ (right) mRNA levels (means ± SEM) in the cortex (CX), hippocampus (HIP) and cerebellum (CB) of WT, HET and KO mice by qRT-PCR. *p < 0.05, ***p < 0.001, one-way ANOVA and post-hoc Bonferroni's test. Samples were run in triplicate from N = 3 for both WT and KO mice.

Fig. 4

Motor behavioral patterns shown by PRRT2 mouse line during postnatal development and at adulthood. A–D. A higher frequency of bouncing (A), loss of balance (B), back walking (C) and a longer duration of grooming (D) were observed in KO mouse pups as compared with HET and WT littermates during 3-min maternal separation at P4, P8, P12 and P16. E. The panel represents the percentage of time spent by WT, HET and KO pups performing the various behaviors analyzed at P4, P8, P12 and P16. F. Righting reflex latencies measured at the end of the maternal separation test. N = 14 WT, 43 HET and 17 KO mouse pups. G–H. A higher frequency of loss of balance (G) and back walking (H) was still present in adult PRRT2 KO mice. N = 8 WT, 14 HET and 13 KO male mice. All data are expressed as means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA and post-hoc Fisher PLSD test.

Fig. 5

Paroxysmal phenotype of PRRT2 KO mice. A. Motor paroxysms in response to audiogenic stimuli. A1. Representative trackings of WT, HET and KO mice during 1-min exposure to an audiogenic stimulus (white noise, 120 days; A1). A2. EEG recorded in WT and PRRT2 KO mice during the resting adaptation period. A3. Representative EEG traces of PRRT2 KO mice during the audiogenic response before and during wild running. A4–A7. The plots show the percentage of animals responding with wild running to the audiogenic stimulus (A4), the mean duration of wild running (A5), the percentage of animals performing jumps (A6) and the frequency of back walking performed during the audiogenic response (A7) as means ± SEM. N = 6 WT, 7 HET and 6 KO male mice. B. Pentylenetetrazol-induced seizures. A 10-s representative EEG trace of a PRRT2 KO mouse recorded during a PTZ-induced tonic-clonic seizure is shown (B1). The plots (means ± SEM) depict: the total distance covered (B2) and the duration of the immobility/freezing episodes (B3) collectively recorded at subthreshold PTZ doses (10, 20 and 30 mg/kg); the threshold for provocation of the first tonic-clonic seizure (B4); the seizure latency from the administration of the threshold doses (B5); the duration of the first tonic-clonic seizure (B6); and the compound seizure propensity index (B7) calculated as described in the Materials and Methods. Statistical analysis was performed using the Fisher exact test (A4, A6) and two-way ANOVA and post-hoc Fisher PLSD test (A5, A7, B2–B7). **p < 0.01; ***p < 0.001. N = 6 WT, 7 HET and 6 KO (A); 7 WT and 6 KO (B).

Fig. 6

Expression of synaptic markers in PRRT2 KO mice. Representative confocal images of the hilus of the DG (A) and of the granule cell (upper panel) and molecular (lower panel) layers of the cerebellum (B) of WT and PRRT2-KO mice stained with DAPI (blue), VGAT (green), VGLUT1 (red) and their merge. Histograms on the right show the mean intensity VGAT and VGLUT1 immunoreactivities and the number of VGAT- and VGLUT1-positive puncta. (ML: molecular layer; GL: granule cell layer; H: Hilus; PL: Purkinje cell layer). All data are expressed as means ± SEM; *p < 0.05, **p < 0.01; two-tailed unpaired Student's t-test. N = 3 for both WT and KO. Scale bar, 150 μm.

Fig. 7

Electrophysiology of PRRT2 KO hippocampal slices. A. Representative mEPSC traces recorded in DG GCs from WT and PRRT2 KO mice in the presence of TTX. The mean (± SEM) amplitude and frequency of mEPSCs are reported on the right for WT (open bars) and PRRT2 KO (black bars) mice. *p < 0.05, two-tailed unpaired Student's t-test. N = 18 and 12 neurons from WT (3 mice) and PRRT2 KO (3 mice), respectively. B. Left: Representative traces of eEPSCs from WT (black traces) and KO (red traces) DG GCs in response to single (top) or paired (bottom) stimuli. Right: Mean (± SEM) eEPSC amplitude (top) and paired-pulse ratio (PPR) plotted as a function of the inter-stimulus interval (ISI: 10 ms-4 s) for WT (open bar/symbol) and PRRT2 KO (black bar/symbol) mice. N = 14 and 13 neurons from WT (4 mice) and PRRT2 KO (4 mice), respectively. C. Representative mIPSC traces recorded in GCs from WT and PRRT2 KO mice in the presence of TTX. The mean (± SEM) amplitude and frequency of mIPSCs are reported on the right for WT (open bars) and PRRT2 KO (black bars) mice. N = 20 and 17 neurons from WT (3 mice) and PRRT2 KO (3 mice), respectively. D. Left: Representative traces of eIPSCs from WT (black traces) and KO (red traces) DG GCs in response to single (top) or paired (bottom) stimuli. Right: Mean (± SEM) eIPSC amplitude (top) and paired-pulse ratio (PPR) plotted as a function of the ISI (10 ms-4 s) for WT (open bar/symbol) and PRRT2 KO (black bar/symbol) mice. *p < 0.05, two-tailed unpaired Student's t-test. N = 13 and 12 neurons from WT (4 mice) and PRRT2 KO (4 mice), respectively.

Fig. 8

Electrophysiology of PRRT2 KO cerebellar slices. A. Representative mEPSC traces recorded in PCs from WT and PRRT2 KO mice in the presence of TTX. The mean (± SEM) amplitude and frequency of mEPSCs are reported on the right for WT (open bars) and PRRT2 KO (black bars) mice. N = 9 and 11 neurons from WT (3 mice) and PRRT2 KO (3 mice), respectively. B. Left: Representative traces of eEPSCs from WT (black traces) and KO (red traces) mice in response to single (top) or paired (bottom) stimuli. Right: Mean (± SEM) eEPSC amplitude (top) and paired-pulse ratio (PPR) plotted as a function of the inter-stimulus interval (ISI: 10 ms-4 s) for WT (open bar/symbol) and PRRT2 KO (black bar/symbol) mice. C. PRRT2 knockdown enhances synaptic facilitation during high frequency trains. Left: Mean (± SEM) normalized values of eEPSC amplitude showing the time course of synaptic facilitation and depression in PCs subjected to 2 s high-frequency stimulation at 10 (top), 20 (middle) and 40 (bottom) Hz. In the insets, representative traces showing synchronous EPSCs evoked by the tetanic stimulation. Right: Cumulative plots of normalized EPSC amplitudes during trains at 10 (top), 20 (middle) and 40 (bottom) Hz plotted as means (± SEM) against the stimulus number. Individual slopes were calculated from the last 10 responses in the train and statistically compared (10 Hz: 0.006 ± 0.001 and 0.010 ± 0.001, p < 0.01; 20 Hz: 0.011 ± 0.002 and 0.021 ± 0.003, p < 0.05; 40 Hz: 0.017 ± 0.004 and 0.054 ± 0.010, p < 0.05; for WT and PRRT2 KO neurons, respectively). *p < 0.05; **p < 0.01, two-tailed unpaired Student's t-test. N = 11 and 18 neurons from WT (3 mice) and PRRT2 KO (3 mice), respectively.