Patients
We enrolled two Japanese families with segregating dominant traits for cerebellar ataxia. There are 10 affected individuals in the family 1 and five in the family 2. Blood samples were obtained from eight affected individuals and three unaffected individuals in family 1, and five affected individuals and two unaffected individuals in family 2 (Fig. 1a). All patients were diagnosed with SCA by neurologists. Prior to this study, we confirmed that all affected individuals had no pathogenic mutations causing SCA1–3, 6, 8, and dentatorubral-pallidoluysian atrophy. The study was approved by the Human Subjects Committees of Hiroshima University; all subjects provided written informed consent.
Linkage analysis
The samples used for linkage analysis were 1-III-2, 1-III-4, 1-III-6, 1-III-8, 1-III-11, 1-III-13, 1-IV-2, 1-IV-3, and 1-IV-4. Because it was possible that 1-IV-3 and 1-IV-4 did not reach the appropriate age at onset, the two samples were treated as unknown in the pedigree file of linkage analysis. Genomic DNA (gDNA) was extracted from the peripheral lymphocytes of the participants according to standard protocols. We used a Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA) for genotyping of single nucleotide polymorphisms (SNPs), and linkage analysis was performed by Allegro software, estimating the dominant inheritance [11].
Exome Sequencing
Exome sequencing was carried out using three samples from 1-III-6, 1-III-11, and 1-III-13, as previously described [12]. For family 2, exome sequencing was also performed with the sample from 2-III-1. gDNA libraries were prepared using a SeqCap EZ Human Exome Library v2.0 (Roche, Basel, Switzerland). Sequencing was performed with 100-bp paired-end reads on a HiSeq2000 sequencer (Illumina, San Diego, CA, USA). We used BWA (http://bio-bwa.sourceforge.net/) [13] for alignment and mapping, Samtools (http://samtools.sourceforge.net/) [14] and Picard (http://broadinstitute.github.io/picard/) for SAM/BAM handling, GATK (http://www.broadinstitute.org/gatk/) [15] and Samtools for variant calls, and Annovar (http://annovar.openbioinformatics.org/) [16] for annotation. Functional predictions due to amino acid changes were estimated using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) [17], SIFT (http://sift.bii.a-star.edu.sg/) [18], and Mutation Taster (http://www.mutationtaster.org/index.html) [19]. Control exome sequences were obtained from Japanese patients undergoing exome analysis for diseases other than SCA. All reported genomic coordinates were in GRCh37/hg19. The identified mutations were validated with a standard polymerase chain reaction (PCR)-based amplification followed by sequence analysis with an Applied Biosystems 3130 DNA sequencer (Thermo Fisher Scientific, Waltham, MA, USA).
Expression vector
Wild-type CACNA1G (short isoform; BC110995.1, NM_198382.2) in the pCMV-SPORT6 plasmid (pCMV-SPORT6-CACNA1G) was purchased from Dharmacon (Lafayette, CO, USA). The mutation c.5075G > A corresponding to c.5144G > A in the longest isoform (NM_018896.4) was introduced by site-directed mutagenesis using QuikChange Lightning (Agilent Technologies, Santa Clara, CA, USA) and verified by bidirectional sequencing. The IRES-EGFP sequence was amplified by PCR from the pIRES-EGFP plasmid and inserted at the termination codon of the cDNA sequence in pCMV-SPORT6-CACNA1G (pCMV-SPORT6-CACNA1G-IG) using an In-Fusion HD Cloning Kit (TaKaRa Bio, Shiga, Japan).
Cell culture, transfection, and immunofluorescence
The primary antibodies used in this study were anti-CACNA1G [20], and anti-alpha 1 sodium potassium ATPase (Abcam, Cambridge, UK).
HeLa and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nakarai Tesque, Kyoto, Japan) supplemented with 10 % fetal bovine serum and penicillin/streptomycin (PS) in a 37 °C incubator with 5 % CO2. For immunofluorescence analysis, cells were grown on chamber slides (SCS-008; Matsunami, Osaka, Japan) coated with poly-l-lysine (Sigma-Aldrich, St. Louis, MO, USA), and were transiently transfected with pCMV-SPORT6-CACNA1G using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. After 48–72 h, the cells were fixed in 4 % paraformaldehyde, washed with phosphate-buffered saline (PBS), blocked, and permeabilized with 0.2 % Tween20. Cells were incubated overnight at 4 °C with anti-CACNA1G and anti-alpha 1 sodium potassium ATPase antibodies and then treated with secondary antibodies. Images were obtained using confocal microscopy (LSM510; Carl Zeiss, Jena, Germany). The nuclei were visualized using DAPI.
Cells for whole-cell patch clamping were grown in glass-bottom plates (μ-Dish 35 mm low; ibidi, Martinsried, Germany) for 24 h following transfection with SPORT6-CACNA1G-IG using Lipofectamine LTX.
Electrophysiology
Whole-cell recordings were made from GFP-expressing HEK293T cells using an upright microscope (BX51WI; Olympus, Tokyo, Japan) equipped with an IR-CCD camera system (IR-1000; DAGE-MTI, Michigan, IN, USA) at room temperature. The intracellular solution was composed of 110 mM CsCl, 20 mM TEA-Cl, 10 mM NaCl, 5 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, and 0.4 mM 2Na-GTP (pH 7.3, adjusted with CsOH). The pipette access resistance was about 2–3 MΩ. The composition of the extracellular solution was 10 mM NaCl, 105 mM TEA-Cl, 10 mM 4-AP, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 20 mM glucose, bubbled with 95 % O2 and 5 % CO2. Ionic currents were recorded with an EPC-10 (HEKA Elektronik, Lambrecht, Germany). The signals were filtered at 3 kHz and digitized at 20 kHz. On-line data acquisition and off-line data analysis were performed using PATCHMASTER software (HEKA Elektronik, Lambrecht, Germany). Relative conductance and steady-state inactivation plots were fitted by the following Boltzmann equations:
$$ \begin{array}{ll}\frac{G}{G_{max}}\hfill & =\frac{1}{1+ exp\left(\left({V}_{half}-{V}_m\right)/k\right)}\hfill \\ {}\frac{I}{I_{max}}\hfill & =\frac{1}{1+ exp\left(\left({V}_m-{V}_{half}\right)/k\right)}\hfill \end{array} $$
Derivation of patient-specific fibroblasts and generation of induced pluripotent stem cells (iPSCs)
Patient 2-III-1 harbored the CACNA1G mutation. Thus, a 3-mm punched skin biopsy was obtained from the upper arm. The skin sample was mechanically cut into small pieces and cultured in 100-mm cell culture dishes in DMEM containing 20 % fetal bovine serum. Outgrowth of dermal fibroblasts appeared after 7–14 days, and cultures were split at 1:3 after reaching 80 % confluence. Peripheral blood was obtained from a healthy donor after providing written informed consent in accordance with the institutional review board guidelines. Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation with a BD Vacutainer CPT (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions.
Human complementary DNAs for reprogramming factors were transduced into dermal fibroblasts and PBMCs with episomal vectors (pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL; Addgene, Cambridge, MA, USA) [21, 22]. The generated iPSCs were maintained on a mitomycin C-inactivated SNL feeder cell layer in DMEM/F12 containing GlutaMaxI (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20 % KnockOut Serum Replacement (KSR; Thermo Fisher Scientific, Waltham, MA, USA), 5 ng/ml recombinant human FGF basic (Wako, Osaka, Japan), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and PS under 5 % CO2.
Purkinje cell differentiation from iPSCs
The differentiation of Purkinje cells from iPSCs was performed as described previously [23, 24].