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. 2016 Jun 28;7(26):39184-39196.
doi: 10.18632/oncotarget.9258.

PRRT2 mutations lead to neuronal dysfunction and neurodevelopmental defects

Yo-Tsen Liu  1   2 , Fang-Shin Nian  3   4 , Wan-Ju Chou  4 , Chin-Yin Tai  5 , Shang-Yeong Kwan  1   2 , Chien Chen  1   2 , Pei-Wen Kuo  4 , Po-Hsi Lin  2 , Chin-Yi Chen  6 , Chia-Wei Huang  4 , Yi-Chung Lee  1   2   7 , Bing-Wen Soong  1   2   7 , Jin-Wu Tsai  4   7   8
Affiliations

PRRT2 mutations lead to neuronal dysfunction and neurodevelopmental defects

Yo-Tsen Liu et al. Oncotarget. .

Abstract

Mutations in the proline-rich transmembrane protein 2 (PRRT2) gene cause a wide spectrum of neurological diseases, ranging from paroxysmal kinesigenic dyskinesia (PKD) to mental retardation and epilepsy. Previously, seven PKD-related PRRT2 heterozygous mutations were identified in the Taiwanese population: P91QfsX, E199X, S202HfsX, R217PfsX, R217EfsX, R240X and R308C. This study aimed to investigate the disease-causing mechanisms of these PRRT2 mutations. We first documented that Prrt2 was localized at the pre- and post-synaptic membranes with a close spatial association with SNAP25 by synaptic membrane fractionation and immunostaining of the rat neurons. Our results then revealed that the six truncating Prrt2 mutants were accumulated in the cytoplasm and thus failed to target to the cell membrane; the R308C missense mutant had significantly reduced protein expression, suggesting loss-of function effects generated by these mutations. Using in utero electroporation of shRNA into cortical neurons, we further found that knocking down Prrt2 expression in vivo resulted in a delay in neuronal migration during embryonic development and a marked decrease in synaptic density after birth. These pathologic effects and novel disease-causing mechanisms may contribute to the severe clinical symptoms in PRRT2-related diseases.

Keywords: PRRT2; Pathology Section; Taiwan; neuronal migration; paroxysmal kinesigenic dyskinesia (PKD); synaptic development.

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Conflict of interest statement

All authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Genomic organization of the human PRRT2 gene and the distribution of the PRRT2 mutations identified in Taiwanese PKD patients
The genomic organization of human PRRT2 gene and the distribution of the seven PRRT2 mutations identified in Taiwanese PKD patients are illustrated in the upper panel. The predicted domains of the PRRT2 protein are shown in the lower panel. Six of the PRRT2 truncating mutations are localized in the predicted extracellular domain (amino acids 1-268), and five are clustered within or around the proline-rich domain (the red region).
Figure 2
Figure 2. Prrt2 protein recognized by a specific polyclonal antibody
A. A polyclonal antibody against amino acids 1-268 of the bacterially-expressed Prrt2 protein (I) was generated. Prrt2 protein from adult rat brain lysates was detected using the first bleed of Prrt2 anti-serum obtained from a rabbit (II, right lane). Preimmune serum drawn from the rabbit before antigen injection was the control (II, left lane). The antibody also recognized Prrt2 in mouse brain lysates and EGFP-tagged Prrt2 transfected into COS7 cells (III). B. Subcellular localization of Prrt2 protein by immunoflourescence staining with the polyclonal antibody. Endogenous Prrt2 protein (green) localized predominantly to cell membranes in cultured cortical neurons 3 and 16 days in vitro (DIV) (left panel). The distribution of EGFP-tagged Prrt2 also localized to the cell membrane in HEK293T cells transfected with EGFP-Prrt2, as revealed by the EGFP signal (green) and Prrt2 immuostaining (red). The distribution of Prrt2 based on EGFP signal and antibody staining was highly concordant and resembled the pattern of endogenous Prrt2 in cultured neurons. All cells were counterstained with DAPI staining (blue) to show the nuclei. Scale bar = 10 μm. C. Prrt2 protein level revealed by immunostaining with polyclonal antibody in cultured neurons transfected with Prrt2 shRNA. Upper panel: Prrt2 expression was knocked down by cotransfecting short hairpin RNA (shRNA) with Prrt2 into cultured cells. Two shRNA plasmids targeting different Prrt2 mRNA regions showed 65% (shPrrt2 #1) and 98% (shPrrt2 #2) knockdown efficiency 48 hours after transfection. Lower panel: Immunostaining in cultured neurons shows that endogenous Prrt2 signal (red) was significantly reduced in shPrrt2-transfected neurons (green, arrows) compared with the surrounding untransfected cells. All cells were counterstained with DAPI staining (blue) to show the nuclei. Scale bar = 10 μm.
Figure 3
Figure 3. Localization of Prrt2 at the pre- and post-synaptic membranes
A. Membrane fractionation assay and post-synaptic density (PSD) preparation of the protein from an adult rat brain. Prrt2 and SNAP25 were present and co-fractionated in both pre- and post-synaptic compartments. PSD95 and GluA1 are PSD proteins that were enriched in PSD fractions. Synaptotagmin I (Sygt1)-a presynaptic vesicle protein was present in the LP2 fraction, but absent from all PSD fractions. PSD95, GluA2 and N-cadherin are post-synaptic membrane proteins that were absent from the LP2 fraction. H = homogenate, S1 = low-speed supernatant, P1 = nuclear pellet and debris, S2 = cytosol plus light membrane, P2 = crude synaptosomal fraction, S3 = cytosol, P3 = light membranes, LS1 = synaptosomal cytosolic fraction, LP1 = synaptosomal membrane fraction, LS2 = luminal fraction of synaptic vesicles, LP2 = synaptic vesicle membrane fraction. PSDI, II and III represent the detergent extractability of PSD resident proteins. PSD1 represents Single Triton X-100 extraction. PSDII and PSDIII were obtained in the second round of Triton extraction and Sarkosyl extraction of PSDI, respectively. B. Immunostaining of Prrt2 and SNAP25 in GFP-expressing cultured hippocampal neurons (DIV 17). A dendritic segment with multiple spines (specialized protrusive structures) is shown in the large image. The amplified images highlight the localization of Prrt2 (red) and SNAP25 (blue), which co-localized at the tip of a spine (orange arrow) where a synapse was forming.
Figure 4
Figure 4. Subcellular localization of PKD-related PRRT2 mutations in cultured HEK293T cells
The six prematurely truncating Prrt2 mutants (P91QfsX, E199X, S202HfsX, R217EfsX, E217PfsX and R240X) were retained in the cytoplasm and failed to be properly transported to the membrane. Some faint signal from the p.P91QfsX, p.E199X and p.S202HfsX Prrt2 mutant proteins could be found in the nucleus. The missense R308C mutant protein had a membrane localization similar to the pattern for WT-Prrt2. Scale bar = 10 μm.
Figure 5
Figure 5. Reduced expression level of the R308C PRRT2 mutant
A. GFP- and HA-tagged WT-Prrt2 and the R308C mutant were transiently transfected into the HEK293T cell line. Prrt2 expression levels were determined using Western blotting and were normalized to that of WT-Prrt2. GAPDH was used as an internal control. B. A significant reduction of Prrt2 levels was observed for the R308C mutant (*: p < 0.05, **: p < 0.01). Error bars indicate SEM. Statistics were performed by a two-tailed, unpaired Student's t-test.
Figure 6
Figure 6. Delay in neuronal migration by Prrt2 knockdown during development
A. Coronal sections of the mouse brains 3 and 5 days after in utero electroporation of shControl or shPrrt2 constructs at E14.5. Three days after electroporation, cells expressing EGFP (green) and shControl had started migration, with many cells having already reached the CP. More cells were restricted to the VZ and IZ in the brains electroporated with each of the shPrrt2. This migration delay was rescued by co-transfection of shPrrt2 and Prrt2 constructs (upper panel). When brain sections were examined 5 days post electroporation, most cells in both shControl and shPrrt2 groups had reached the CP (lower panel). All sections were stained with DAPI (blue) to show the cell nuclei. Scale bars = 100 μm. B. Statistical analysis showed significant differences in cell distribution in the CP, IZ and VZ at E17.5for the four conditions. Error bars represent SEM; *: p < 0.05, ** : p < 0.01.
Figure 7
Figure 7. Decreased dendritic spine density after Prrt2 knockdown
A. Dendritic spines were imaged at P21 and P30 in brains electroporated in utero with control or Prrt2 shRNA along with EGFP constructs at E14.5. Lower spine densities for Prrt2 shRNA-expressing pyramidal neurons were observed on representative dendrites at both P21 and P30. B. Statistical analysis of spine density of dendrites at P14, P21 and P30. There was no significant difference between control and Prrt2 shRNA-expressing groups at P14. Significant decreases in spine density for Prrt2 shRNA-expressing cells were observed at P21 and P30. *: p < 0.05.
Figure 8
Figure 8. Schematic diagram showing effects of PRRT2 mutation on neuronal migration and dendritic spines in cortical neurons
A. Radial glia cells (light green) produce postmitotic neurons (dark green). B. Postmitotic neurons then migrate along the radial fiber to the CP. C. After reaching the cortical plate, the cells extend dendrites and form synaptic connections with other neurons through dendritic spines. Box shows a magnification of the pre- and post-synaptic regions. PRRT2 (pink) is localized at both the pre- and post-synaptic membranes through interactions with SNAP2518, 25, 44 and the AMPA receptor25, 33, 34. (A', B') In neurons carrying PKD-associated PRRT2 mutations, reduced expression, mislocalization and other potential defects delay the neuronal migration. (C') PRRT2 mutation also decreases the dendritic spine density and causes synaptic dysfunction, possibly through SNAP25 and/or AMPA receptor pathways.
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References

    1. Chen WJ, Lin Y, Xiong ZQ, Wei W, Ni W, Tan GH, Guo SL, He J, Chen YF, Zhang QJ, Li HF, Murong SX, Xu J, Wang N, Wu ZY. Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia. Nature genetics. 2011;43:1252–1255. - PubMed
    1. Cloarec R, Bruneau N, Rudolf G, Massacrier A, Salmi M, Bataillard M, Boulay C, Caraballo R, Fejerman N, Genton P, Hirsch E, Hunter A, Lesca G, Motte J, Roubertie A, Sanlaville D, et al. PRRT2 links infantile convulsions and paroxysmal dyskinesia with migraine. Neurology. 2012;79:2097–2103. - PMC - PubMed
    1. Dale RC, Gardiner A, Antony J, Houlden H. Familial PRRT2 mutation with heterogeneous paroxysmal disorders including paroxysmal torticollis and hemiplegic migraine. Dev Med Child Neurol. 2012;54:958–960. - PubMed
    1. Gardiner AR, Bhatia KP, Stamelou M, Dale RC, Kurian MA, Schneider SA, Wali GM, Counihan T, Schapira AH, Spacey SD, Valente EM, Silveira-Moriyama L, Teive HA, Raskin S, Sander JW, Lees A, et al. PRRT2 gene mutations: from paroxysmal dyskinesia to episodic ataxia and hemiplegic migraine. Neurology. 2012;79:2115–2121. - PMC - PubMed
    1. Marini C, Conti V, Mei D, Battaglia D, Lettori D, Losito E, Bruccini G, Tortorella G, Guerrini R. PRRT2 mutations in familial infantile seizures, paroxysmal dyskinesia, and hemiplegic migraine. Neurology. 2012;79(21):2109–2114. - PMC - PubMed

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