We obtained lymphoblastoid cell lines (LCLs) derived from SCA28 patients carrying the p.Gly671Arg (n = 3), p.Met666Arg (n = 1), p.Met666Val (n = 3), p.Met666Thr (n = 2), and p.Thr654Ile (n = 1) mutations. Clinical features of these patients have been previously described  and are summarized in [Additional file 1]. Fourteen control LCLs were obtained from the Human Genetics Foundation of Torino (HuGeF).
LCLs were grown at 37°C and 5% CO2 in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin.
Whole genome RNA expression profiling
Total RNA was extracted from 5 × 106 LCLs using the Qiaquick RNA Easy plus extraction kit (Qiagen, Mannheim, Germany) according to manufacturer's instructions. A sample of the total RNA was visualized in a 0.6% TBE 1X/agarose gel and its quality assessed using the Agilent BioAnalyser 2100 (Agilent, Palo Alto, CA). RNA was then quantified with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Barrington IL, USA).
Whole-genome RNA expression study was performed on a subgroup of four SCA28 patients, each carrying a different AFG3L2 mutation (p.Thr654Ile, p.Met666Val, p. Met666Thr, and p.Gly671Arg). Six unrelated healthy subjects matched for sex and age at sampling were used as controls.
For each sample, total RNA (6 μg) was labeled according to the standard one-cycle amplification and labeling protocol developed by Affymetrix (Santa Clara, CA, USA). Labeled cRNA was hybridized on Affymetrix GeneChip Human Genome U133A 2.0 Arrays containing probes for over 18,400 transcripts. Hybridized arrays were stained and washed on a GeneChip Fluidics Station 450 and then scanned on a GeneChip Scanner 3000 7G (Affymetrix). Cell intensity values and probe detection calls were computed from the raw array data using the Affymetrix GeneChip Operating Software (GCOS). Further data processing was performed in the R computing environment using specific packages from the BioConductor software project . Robust Multi-Array Average (RMA) normalization was applied. Normalized data were filtered based on the Affymetrix detection call, so that only probes that had a Present call in at least one of the arrays were retained. Probes with low intensity values (less than 100) in all arrays were also excluded from statistical analysis.
Data were imported into the MultiExperiment Viewer (MeV) software , and statistical analysis was performed to detect significantly differentially expressed genes in SCA28 patients versus healthy controls, using two different tests as implemented within the MeV: Rank Product (RP)  and Significance Analysis of Microarrays (SAM) . RP was run using 100 permutations and a False Discovery Rate (FDR) of 0.005%. The SAM test was run using 1000 permutations and a FDR of 3%. Differentially expressed genes were then specifically examined based on their Gene Ontology annotations  and through the use of the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources [23–25].
Validation of data by real-time RT-PCR
Genes to be validated were selected on the basis of three criteria: (i) an expression level higher than 7, (ii) fold changes higher than 1.5 or lower than 0.7 in patients vs. controls, and (iii) the potential interest of the gene in SCA28 pathogenesis as inferred by its function, the pathway in which it was involved and/or the functional similarity with known ataxia-related genes.
Quantitative real time PCR was used to validate array findings for 11 genes in the same LCLs batches used for microarray expression profiling (see above). The validated genes were then tested using a different RNA extraction from the same subjects (4 patients vs. 6 controls) to obtain a technical replicate. In addition, a third real-time PCR experiment on the same genes was performed on LCLs generated from four new different SCA28 patients obtained later [mutations p.Met666Arg, p.Met666Thr and p.Met666Val (n = 2)], as biological replicate.
Total RNA was extracted from eight SCA28 patients and eight healthy controls as described above, and 1 μg was retrotranscribed using the Transcription First Strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). Quantitative real-time RT-PCRs were carried out on an ABI-Prism7500 Fast instrument (Applied Biosystems) using the Taqman Gene Expression Master Mix (Applied Biosystems), Universal Probe Library (UPL) technology (Roche) and according to the manufacturer’s protocol. Primers and UPL probes used for real time PCR validation are listed in Additional file 2.
Experimental Ct values were normalized to TATA-binding protein, used as endogenous control (VIC labeled pre-designed TaqMan gene expression assays, TBP
, Hs00427620_m1, Applied Biosystems). Gene expression was calculated in each sample relative to the mean of controls, using the delta-delta Ct method as described elsewhere . Each sample was examined in triplicate and differences in gene expression of patients relative to controls were statistically evaluated by the Student’s t-test for at least three independent experiments.
Cell growth analysis
Cell growth was determined using the MTT test [Carbonyl cyanide p‒(trifluoromethoxy) phenylhydrazone, 3‒(4,5‒dimethylthiazol) 2,5‒ diphenyl-tetrazolium bromide, Sigma-Aldrich] . The growth of LCLs from six patients carrying the p.Thr654Ile (n = 1), p.Met666Thr (n = 1), p.Met666Val (n = 2), p.Met666Arg (n = 1) and p.Gly671Arg (n = 1) mutations was compared to the growth of seven LCLs from healthy controls, performing six replicates for each of the five time-points analyzed (0, 3, 6, 24, and 48 hours). After overnight incubation in RPMI with 0.5% FCS, 3 × 104 cells for each time-point were washed twice in PBS solution, the pellet was resuspended in 1 ml of RPMI supplemented with 10% FCS and seeded onto 96‒well plates (150 μl/well). At each time-point, 10 μl of a 5 mg/ml MTT solution were added to each well. A negative control was included in each experiment to exclude microbial contamination.
After 1 hour incubation at 37°C in the dark the MTT solution was carefully removed, and cells were dissolved in 100 μL of DMSO for 10 minutes at 37°C. Proliferation rate of cells was determined by measuring the optical density (OD) of each sample at 570 nm with an ELISA Microplate Reader Model 680 (Biorad). To confirm growth curves with a second method, independent of mitochondrial function, we used a cytoplasmic membrane-dye (Neuro-DiO, Biotium) whose fluorescence intensity decreases as a consequence of cell divisions. The design of the experiment was the same as that of the MTT assays, and fluorescence was analyzed by a FACS Calibur flow cytometer using CellQuest software (Becton Dickinson, USA).
Analysis of cell cycle
Cell cycle was analyzed by fluorescence labeling of DNA with Propidium Iodide (PI) , a stoichiometric dye, that binds in proportion to the amount of DNA present in the cell. LCLs from six healthy controls and six SCA28 patients carrying the p.Thr654Ile (n = 1), p.Met666Thr (n = 1), p.Met666Val (n = 2), p.Met666aArg (n = 1), and p.Gly671Arg (n = 1) mutations were compared at three different time-points (0, 3, and 24 h). Approximately 5 × 105 cells for each time-point were subjected to density gradient centrifugation Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ) to remove any dead cell and debris. Viable cells were collected at the interface and washed with PBS solution. Cells were synchronized by overnight incubation in RPMI containing 0.5% FCS, washed twice in PBS and seeded onto a 24-wells plate in 0.5 ml of RPMI 10% FCS at the final concentration of 5 × 105/ well. At the designed time-points (0, 3 and 24 h), each well was washed with PBS and resuspended in 1 ml of PBS with 0.1% BSA. One ml of 70% Ethanol was added to each sample for 45 minutes at 4°C, samples were then rinsed with PBS, suspended in 0.5 ml of PBS with 0.1% BSA and 5 μl of RNaseA [10 mg/ml], and incubated 30 minutes at 37°C. One μl of PI was added to each sample for 10 minutes at 4°C in the dark, then cells populations were analyzed on a FACS Calibur flow cytometer using CellQuest software (Becton Dickinson).
Analysis of apoptosis
Apoptosis was evaluated by fluorescence-activated cell sorting (FACS) after Annexin V/propidium iodide (PI) double staining (Biovision, San Francisco, CA, USA). LCLs from six healthy controls and six patients carrying the p.Thr654Ile (n = 1), p.Met666Arg (n = 1), p.Met666Thr (n = 1), p.Met666Val (n = 2), and p.Gly671Arg (n = 1) mutations were used .
Cells were washed twice with Annexin-Binding Buffer (ABB) , and incubated for 5 minutes in the dark with: 1) ABB alone; 2) Annexin V-FITC; 3) PI; 4) Annexin V-FITC and PI. Early apoptotic cells were only stained by Annexin V-FITC, whereas late apoptotic or necrotic cells were double-stained by Annexin V-FITC and PI. Cells were analyzed on a FACS Calibur flow cytometer using CellQuest software (Becton Dickinson). Cells in which apoptosis was induced by overnight incubation with 1 μM staurosporine were used as the positive control. To exclude that increasing apoptosis was due to starvation, the same experiments were performed without overnight incubation in RPMI with 0.5% FCS.
ATP synthesis and respiratory chain complex evaluation
Mitochondria were isolated from three SCA28 and three controls LCLs by differential centrifugation as described . Ten million cells were homogenized using a glass-Teflon homogenizer in an isotonic buffer [0.25 M sucrose, 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.2, 1 mM EDTA] supplemented with 0.1% BSA and 0.1 mg/ml digitonin. Cell debris and nuclei were pelleted twice by centrifugation at 2,500 g for 5 minute at 4°C. Supernatants were centrifuged at 12,000 g for 25 min at 4°C and the mitochondrial pellet was resuspended in isotonic buffer (0.5 M sucrose, 20 mM MOPS, pH 7.2, 1 mM EDTA).
Respiratory chain activity was evaluated using a protocol previously described  and adapted to LCLs. In brief, isolated mitochondria were incubated at 37°C for 30 min in a respiratory buffer (0.25 M sucrose, 20 mM MOPS, 1 mM EDTA, 5mM inorganic phosphate, 0.1% BSA, and 1 mM ADP, pH 7.4). The function of each respiratory chain complex was tested by providing buffers containing specific substrates and inhibitors. (1) Buffer 1 (5 mM pyruvate and 1 mM malate) and (2) buffer 2 (5 mM glutamate and 1 mM malate) were used to stimulate complex I, II, III, IV, and V-dependent ATP synthesis. (3) Buffer 3 containing a complex I inhibitor (1 mM rotenone) and 10 mM succinate, was used to measure ATP production that is dependent on complex II, III, IV, and V. Finally, (4) buffer 4 [2 mM antimycin A -inhibitor of complex III- and 0.1 mM tetramethyl-p-phenylenediamine (TMPD) / 2 mM ascorbate] was used to evaluate the ATP level resulting from the activity of complex IV and V.
ATP concentration was determined with the luciferin-luciferase method as described previously . In brief, isolated mitochondria were lysed with the ATP lysis buffer (200 mM NaOH and 500 nM EDTA), and an aliquot of the obtained extract was diluted with the ATP dilution buffer (100 mM NaOH and 500 nM EDTA). Twenty microliters of this mixture were added to 100 μl of the assay solution (250 mM glycylglycine, 2 mM EGTA, 2 mM MgCl2, 0.4 g/L BSA, 7.5 mM DTT, 15 μM luciferin, and 10 μg/ml luciferase), and the ATP content was measured using a luminometer. Three independent experiments were performed, and the significance of the means calculated using two-tailed Student’s t-test.
Quantification of total cellular ATP levels
The amount of cellular ATP was measured by using the Adenosine 5'-triphosphate (ATP) Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO, USA), as described previously . ATP content was measured in duplicate, according to manufacturer’s instructions, using an appropriate internal ATP standard for calibration. Total protein quantification was performed using classical Bradford protocol.
Thiobarbituric acid reactive substances (TBARS) measurement
Lipid peroxidation was evaluated measuring the Thiobarbituric acid reactive substances (TBARS) levels, following published methods . Cells were washed twice in PBS and the pellet resuspended in 600 μl of PBS. Five μl of Triton X-100 and 500 μl of “TBA solution” (0.375% Thiobarbituric acid and 30% Trichloroacetic Acid in 0.5 N HCl) were added to 500 μl of cells. Samples were boiled at 100°C for 20 minutes and quickly cooled by immersion in an ice bath, and centrifuged at 13000 g for 5 minutes. The absorbance of 300 μL of supernatants was read at 532 nm with a Synergy HT microplate reader (Bio-Tek Instruments, Winooski, VT), reading the absorbance at 532 nm. TBARS values were expressed as nmol/mg proteins.
Reactive Oxygen Species evaluation
Intracellular Reactive Oxygen Species (ROS) levels were determined by using the fluorescent dye, DCFH-DA, 2,7-dichlorodihydrofluorescein diacetate (5 μM). DCFH reacts with ROS to generate a new highly fluorescent compound, dichlorofluorescein, which can be analyzed by flow cytometry. Cells were incubated with DCFH-DA (5 μM) at 37°C for 30 min, washed twice with PBS, and then measured on a FACS Calibur flow cytometer using CellQuest software (Becton Dickinson). Cells treated with H2O2 (10 μM) were used as the positive control. Three independent experiments were performed.
Mitochondrial DNA analysis
Evaluation of mtDNA copy number was performed with an absolute quantitative TaqMan real-time PCR using the LightCycler480 (Roche), as previously detailed . Long-range PCR was performed to screen for the presence of mtDNA deletions or rearrangements, as described .
Total proteins were extracted from LCLs of four patients and four healthy controls, using the RIPA Lysis Buffer [TrisHCl (50 mM), NaCl (150 mM), Triton (1%)] and quantified using standard Bradford protocol. Proteins (20-40 μg) were denaturated for 5 min at 99°C in Laemmli Buffer (Biorad) and separated by SDS–PAGE (10% or 12%) and transferred to nitrocellulose membranes (Biorad) in Tris-Glycine Buffer with 20% methanol. Western Breeze Kit (Invitrogen) was used for blocking, hybridization, secondary antibodies, and detection of antibodies, following manufacturer’s instructions. Hybridizations were performed with primary antibodies against POLγ (Abcam, ab116049, 1:1000), TFAM (Abcam, ab131607, 1:1000), VDAC (Abcam, ab15895, 1:700), DRP1 (BD Biosciences, #611112, 1:1000), OPA1 (BD Biosciences, #612606, 1:1000), MFN1 (Abcam, ab57602, 1:1000), MFN2 (Abcam, ab56889, 1:1000), ATPase-α (BD Bioscience, #612516, 1:1000) and NDUFA9 (LifeSpan Bioscience, LS-C133337 1:1000). Beta-actin antibody (Abcam Ab8227, 1:2000) or Lamin B1 antibody (Abcam, ab16048, 1:6000) were used as loading controls.
Part of the Western blot assays (DRP1, OPA1, MFN1, and MFN2) were performed after synchronization of the cells, obtained through overnight incubation in RPMI with 0.5% FCS followed by serum replacement with RPMI medium supplemented with 10% FCS for 24 hours.