Presynaptic PRRT2 Deficiency Causes Cerebellar Dysfunction and Paroxysmal Kinesigenic Dyskinesia

Abstract

PRRT2 loss-of-function mutations have been associated with familial paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions and choreoathetosis, and benign familial infantile seizures. Dystonia is the foremost involuntary movement disorder manifest by patients with PKD. Using a lacZ reporter and quantitative reverse-transcriptase PCR, we mapped the temporal and spatial distribution of Prrt2 in mouse brain and showed the highest levels of expression in cerebellar cortex. Further investigation into PRRT2 localization within the cerebellar cortex revealed that Prrt2 transcripts reside in granule cells but not Purkinje cells or interneurons within cerebellar cortex, and PRRT2 is presynaptically localized in the molecular layer. Analysis of synapses in the cerebellar molecular layer via electron microscopy showed that Prrt2^-/- mice have increased numbers of docked vesicles but decreased vesicle numbers overall. In addition to impaired performance on several motor tasks, approximately 5% of Prrt2^-/- mice exhibited overt PKD with clear face validity manifest as dystonia. In Prrt2 mutants, we found reduced parallel fiber facilitation at parallel fiber-Purkinje cell synapses, reduced Purkinje cell excitability, and normal cerebellar nuclear excitability, establishing a potential mechanism by which altered cerebellar activity promotes disinhibition of the cerebellar nuclei, driving motor abnormalities in PKD. Overall, our findings replicate, refine, and expand upon previous work with PRRT2 mouse models, contribute to understanding of paroxysmal disorders of the nervous system, and provide mechanistic insight into the role of cerebellar cortical dysfunction in dystonia.

Keywords: Prrt2; cerebellum; dystonia; knock-out mice; paroxysmal kinesigenic dyskinesia.

Figure 1.. Prrt2 expression throughout the developing mouse brain and spinal cord.

A. X-Gal staining of parasagittal brain section. B. Prrt2 expression levels in the cerebellum at P7, P15, P30, and P90. C. Moderate Prrt2 expression is seen in the hippocampus. D. Immunohistochemistry of cerebellar cortex suggests that PRRT2 protein is mainly located in the molecular layer. E. Developmental expression of Prrt2 in mouse relative to E15 frontal cortex (n=6; mean ± SD). Dissection of all brain regions readily identifiable in older mice (midbrain, frontal cortex, striatum, cerebellum, thalamus, hippocampus, and spinal cord) was not possible at E15 and P1. Scale bar: A = 2mm, B = 500mm, C = 200mm, D = 100mm.

Figure 2.. Characterization of a Prrt2 knockout model

A. Left: Western blot of PRRT2 in cerebellum of Prrt2^+/+, Prrt2^fl/fl, Prrt2^+/−, and Prrt2^−/− mice (top), with α-tubulin positive control. Right: Western blot of liver tissues in the same groups shows no expression of PRRT2. B. Semi-quantitative analysis of Western blot data (n=2; line = mean). C. PRRT2 immunohistochemistry in cerebellar cortex with rabbit anti-PRRT2 antibody. Scale bar = 50 μm.

Figure 3.. Phenotypic and motor deficiencies in Prrt2 knockout mice.

A. Weights of Prrt2^+/+, Prrt2^+/−, and Prrt2^−/− mice by sex (mean ± SD). B. Righting reflex impairments in Prrt2^+/− and Prrt2^−/− mice relative to their WT littermates emerge at P10 and disappear by P16 (two-way repeated measures ANOVA with Tukey’s multiple comparison test; genotype: F_(2,39) = 0.65, P = 0.53; asterisks indicate significance in Tukey’s multiple comparison test; box shows 25^th to 75^th percentiles, inner line is median, whiskers show 5^th and 95^th percentiles). C. Increased rope climbing time in Prrt2^+/− and Prrt2^−/− mice normalized for weight (one-way ANOVA with Tukey’s multiple comparison test; F_(2,39) = 12.6, P = 5.9 × 10⁻⁵; asterisks indicate significance in Tukey’s multiple comparison test; box shows 25^th to 75^th percentiles, inner line is median, whiskers show 5^th and 95^th percentiles) D. Prrt2^+/− and Prrt2^−/− mice showed decreased motor coordination on a round 9mm raised-beam task (one-way ANOVA with Tukey’s multiple comparison test; F_(2,39) = 7.3, P = 0.0021; asterisks indicate significance in Tukey’s multiple comparison test; box shows 25^th to 75^th percentiles, inner line is median, whiskers show 5^th and 95^th percentiles) E. Performance on the rotarod task was impaired in Prrt2^+/− and Prrt2^−/− mice across multiple days (two-way repeated measures ANOVA with Tukey’s multiple comparison test; genotype: F_(2,39) = 5.5, P = 0.0079; asterisks indicate significance in Tukey’s multiple comparison test; pink asterisks = Prrt2^+/+ vs Prrt2^−/−, grey asterisks = Prrt2^+/+ vs Prrt2^+/−; mean ± SEM). * = P<0.5, ** = P <0.01; *** = P <0.001; **** = P <0.0001.

Figure 4.. Localization of PRRT2 within the cerebellar circuit

A. In-situ hybridization of Prrt2 transcripts (green) shows expression exclusively in the granule cell layer in Prrt2^+/+ mouse and absent in Prrt2^−/− mouse cerebellar cortex. Scale bar = 20 μm. B. Fluorescent immunohistochemistry visualization of PRRT2 protein (green) in the cerebellar cortex. Protein is localized to the granule cell and molecular layers, with no colocalization with the Purkinje cell label, calbindin (red). Scale bar = 50 μm. C. Staining of PRRT2 (orange), GluR1 (green), and DAPI (blue). Merged image shows minimal colocalization of GluR1 and PRRT2. Scale bar = 50 μm. D. Staining of PRRT2 (green), vGlut1 (red), and DAPI (blue) showing extensive colocalization of vGlut1 and PRRT2. Scale bar = 50 μm. E. Spot analysis quantification of normalized colocalized spot number (n = 3 mice; unpaired t-test; * = P <0.05, ** = P <0.01, *** = P <0.001; mean ± SD).

Figure 5.. Cerebellar cortex vesical localization in Prrt2^+/+ and Prrt2^−/− mice.

A, B. TEM images of vesicles in the molecular layer of cerebellar cortex of Prrt2^+/+ (left) and Prrt2^−/− mice (right). White arrows denote docked vesicles, black arrows denote reserve vesicles. Scale bar = 100 nm C-E. Quantification of total vesicles, reserve vesicles, and docked vesicles in Prrt2^+/+ and Prrt2^−/− mice (n = 37 synapses each; Mann-Whitney test; *** = P <0.001, **** = P <0.0001; horizontal white line is the median, black horizontal lines are the 25^th and 75^th percentiles).

Figure 6.. Prrt2 knockout alters Purkinje cell short-term facilitation and excitability.

A. Examples of EPSCs in Purkinje cells evoked by 20-Hz parallel fiber stimulation in Prrt2^+/+ and Prrt2^−/− mice. B. Amplitude of EPSCs evoked by successive stimulation of parallel fiber axons (two-way repeated measures ANOVA with Tukey’s multiple comparison test; genotype: F_(2,29) = 8.4, P = 0.0013; asterisks indicate significance in Tukey’s multiple comparison test; pink asterisks = Prrt2^+/+ vs Prrt2^−/−, grey asterisks = Prrt2^+/+ vs Prrt2^+/−; mean ± SEM). Values on the y axis are normalized to the amplitude of the first EPSC. C. Purkinje cell current clamp recordings showing response to 100-pA current injections from Prrt2^+/+ and Prrt2^−/− mice. D. Firing rate of Purkinje cells in response to injected current steps (two-way repeated measures ANOVA with Tukey’s multiple comparison test; genotype: F_(2,50) = 5.5, P = 0.0068; asterisks indicate significance in Tukey’s multiple comparison test; pink asterisks = Prrt2^+/+ vs Prrt2^−/−, blue asterisks = Prrt2^+/− vs Prrt2^−/−; mean ± SEM). E. Summary of spike responses to injected current steps of CbN cells in current clamp mode (two-way repeated measures ANOVA with Tukey’s multiple comparison test; genotype: F_(2,27) = 0.77, P = 0.47; mean ± SEM). * = P <0.5, ** = P <0.01.