Aberrant transcriptional networks in step-wise neurogenesis of paroxysmal kinesigenic dyskinesia-induced pluripotent stem cells

Paroxysmal kinesigenic dyskinesia (PKD) is an episodic movement disorder with autosomal-dominant inheritance and marked variability in clinical manifestations. Proline-rich transmembrane protein 2 (PRRT2) has been identified as a causative gene of PKD, but the molecular mechanism underlying the pathogenesis of PKD still remains a mystery. The phenotypes and transcriptional patterns of the PKD disease need further clarification. Here, we report the generation and neural differentiation of iPSC lines from two familial PKD patients with c.487C>T (p. Gln163X) and c.573dupT (p. Gly192Trpfs*8) PRRT2 mutations, respectively. Notably, an extremely lower efficiency in neural conversion from PKD-iPSCs than control-iPSCs is observed by a step-wise neural differentiation method of dual inhibition of SMAD signaling. Moreover, we show the high expression level of PRRT2 throughout the human brain and the expression pattern of PRRT2 in other human tissues for the first time. To gain molecular insight into the development of the disease, we conduct global gene expression profiling of PKD cells at four different stages of neural induction and identify altered gene expression patterns, which peculiarly reflect dysregulated neural transcriptome signatures and a differentiation tendency to mesodermal development, in comparison to control-iPSCs. Additionally, functional and signaling pathway analyses indicate significantly different cell fate determination between PKD-iPSCs and control-iPSCs. Together, the establishment of PKD-specific in vitro models and the illustration of transcriptome features in PKD cells would certainly help us with better understanding of the defects in neural conversion as well as further investigations in the pathogenesis of the PKD disease.


Alkaline phosphatase (AKP) staining
iPSC colonies were fixed with 4% PFA (Sigma) and permeabilized by 0.2% Triton X-100 (Sigma). The alkaline phosphatase activity was detected using an AKP staining solution as instructed by the manual of Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Inc).

RNA isolation, reverse transcription and quantitative PCR
Total cellular RNA was extracted by the Trizol Reagent (Invitrogen) and cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System according to the manufacturer's guidelines. End-point RT-PCR was carried out using 0.2-1.0 μg of total RNA and oligo-d (T) primers in the PCR reaction systems (Life Technologies) reported previously [2]. Primer sequences for end-point RT-PCR are listed in Supplementary Table S1. Quantitative PCR was performed using the SYBR Premix Ex Taq (Takara Bio, Shiga, Japan) on Applied Biosystems 7900HT Fast Real-Time PCR System. Quantitative levels for all genes were normalized to the housekeeping gene GAPDH and assessed relative to the relevant control samples. Some sequences of the primers for PCR (pMXs-OCT4, pMXs-SOX2, pMXs-KLF4 and pMXs-C-MYC) have been reported previously [1, 3], other primer sequences for quantitative RT-PCR are listed in Supplementary Table S2.

Bisulfite sequencing PCR
Bisulfite genomic sequencing was carried out using the EZ DNA Methylation-Direct Kit (Zymo Research) as previously described [3]. Two μg of genomic DNA was treated according to the procedures of manufacture Active Motif. Primers specific to the human OCT4 promoter were used to amplify genomic DNA sequences with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and PCR products were cloned into the pGEM-T easy vector (Promega). Individual clones were sequenced in the Invitrogen Company (Shanghai, China). Bisulfite primer information is presented in Supplementary Table S2.

Teratoma formation
About 5x10 6 iPSCs were cultured in the presence of 10 μM Y27632 (Calbiochem) overnight. Then the cells were collected and injected intramuscularly into SCID mice anesthetized with isoflurane. At 8-10 weeks post-injection, teratomas were dissected, fixed overnight in 10% buffered formalin phosphate and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) for further analysis. Experiments complied with all relevant institutional and national animal welfare laws, guidelines and policies.

Cell growth detection
The growth curve was detected using the Cell Counting Kit-8 (CCK-8, Dojindo) according to the manufacturer's recommended protocol. Cells were plated onto 96-well plate in a density of 1X10 5 cells per well, then cultured by treating with WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], which could produce a water-soluble formazan dye upon reduction in the presence of an electron carrier, 10 μl of the CCK-8 solution was added to each well of the plate on day 1-10 and incubated for 4 hours in the incubator. The absorbance was measured at 450 nm and 600nm using the enzyme-labeled instrument (Promega).

Cell apoptosis detection by flow cytometry analysis
iPSCs cultured under the condition of step-wise neural induction were separately collected on days 0, 5 and 10. Assays were carried out using the Annexin V-PE/7-AAD Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's manual. iPSC colonies were digested into single cells and stained with Annexin V-PE and 7-AAD for 15 minutes. Levels of cell apoptosis were analyzed using a BD Accuri C6 flow cytometer (BD Accuri Cytometers, Ann Arbor, MI, USA).

Immunofluorescence staining of the human fetal brain
A human fetal brain was obtained from an aborted fetal at autopsy within 3 hours of spontaneous abortion. The brain was examined with the informed consent of the donor and in accordance with the Tongji Hospital guidelines. Brain tissues were fixed in 4% PFA at 4 °C for 1 week and then cryoprotected in a sucrose gradient (15%-20%-30%) for 72 hours at 4 °C. Then samples were frozen in OCT on dry ice.
Immunofluorescence staining of the human fetal brain was performed on 10-20 μm serial coronal sections on glass slides. Some sections were subjected to an antigen retrieval protocol. Subsequently, cryosections were blocked for 1-2 hours in Tris-buffered saline with 2% Triton X-100 and 10% donkey serum. Cryosections were incubated with the primary antibodies (anti-PRRT2 and anti-NEUN) for 24 hours at 4 °C. Secondary antibodies against the appropriate species were incubated for 2-3 hours at room temperature, counterstained with DAPI for 15 minutes and coverslipped with the Organo/Limonene Mount (Sigma).

Western blot analysis
Human fetal tissues were isolated, then suspended in a lysis buffer supplemented with 1% protease inhibitor cocktail (Sigma), rotated and triturated at 4 °C. 10 to 20 μg of protein was separated on the 10% SDS-polyacrylamide gel, then transferred to a nitrocellulose membrane and probed with a primary antibody against PRRT2 overnight at 4 °C, followed by the incubation of horseradishperoxidase-conjugated secondary antibody (Promega) at room temperature for 1-2 hours. Protein bands were visualized using ECL chemiluminescence analyzer (Thermo Scientific Pierce). GAPDH (Proteintech) were used as the loading control.