Immunoblot analysis Cells were treated with DCA as described above. After 24 h of treatment, proteins were extracted from cells in 1% SDS supplemented with 2 mM sodium orthovanadate and Cømplete protease inhibitor cocktail (Roche). Lysates were resolved by electrophoresis on 12% SDS-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane (PVDF; Thermo). The membrane was blocked for 1 h at room temperature in 5% skimmed milk in TBS-T (Tris-buffered saline with 0.1% Tween). The membrane was then probed overnight with primary antibodies in 1% skimmed milk in TBS-T at 4uC, washed in TBS-T, and then probed with the appropriate horseradish peroxidase (HRP)- conjugated secondary antibody for 60 min at room temperature. Primary antibodies were rabbit polyclonal phosphodetect anti- PDH-E1a(pSer293) 1:500 (AP1062; Calbiochem) and mouse monoclonal anti-PDHE1a 1:750 (#456600, Invitrogen). Secondary antibodies were anti-rabbit (1:2500) and anti-mouse (1:5000) HRP conjugates (Thermo). Proteins were visualized with ECL western blotting substrate (Pierce Biotechnology). b-actin was used as a loading control. Densitometric analysis was performed using ImageJ software. The relative levels of pSer293 E1a and total E1a were quantified. The pSer293 to total E1a ratio was calculated and normalized against vehicle-treated non Tg mice. Histological analysis and immunofluorescence Transgenic and non-transgenic mice groups (n = 3 per group) were treated as described above, from 70 days of age. After 20 days, mice were transcardially perfused with 4% paraformaldehyde fixative in DPBS under deep anesthesia (pentobarbital, 50 mg/kg i.p.). The lumbar spinal cords were postfixed and embedded in paraplast. 5 mm-thick, serial sections were stained with toluidine blue or processed for immunofluorescence. For GFAP immunode- tection, sections were permeabilized (0.2% Triton X-100 in PBS) and unspecific binding blocked (10% goat serum, 2% BSA, 0.2% Triton X-100 in DPBS), incubated with primary antibody (mouse monoclonal Cy-3 conjugated anti-GFAP; 1:600, Sigma) overnight, and mounted with glycerol. Images were obtained using an Olympus IX81 epifluorescence microscope. Assessment of motor neuron number and astrogliosis in the lumbar spinal cord The number of motor neurons was assessed by counting every cell on lamina IX of Rexed displaying motor neuron morphology with nucleus and nucleolus on every fifth toluidine blue stained 5 mm section (at least 25 sections per animal) through the lumbar spinal cord. Quantification of astrogliosis was performed on images obtained from every fifth GFAP immunostained section (20 sections from each group) using ImageJ software (NIH). Ventral horn area occupied by GFAP immunofluorescence was measured and expressed as a percentage of total ventral horn area in each section. Oxygen consumption Oxygen consumption studies were performed either on spinal cord tissue or in astrocyte monolayers. Tissue or cell respiration was evaluated using Oxygraph 2 K (Oroboros Instruments Corp). Oxygen consumption was recorded at 37uC in intact cells or spinal cord tissue. The rate of oxygen consumption was calculated by means of the equipment software (DataLab) and was expressed as pmol of O2?s21?ml21. For cell respiration, astrocyte monolayers were treated with DCA (0.5 and 5 mM) or vehicle for 24 h. Then, astrocytes were scraped and resuspended at 26106 cells/ml in culture medium. DCA Effects in ALS Models Mitochondrial oxygen consumption and RCR (respiratory control ratio) was calculated as: RCR = maximum uncoupled flux (FCCP)2(antimycin A-inhibited flux)/(oligomycin-inhibited flux)2(antimycin A-inhibited flux) of intact cells respiring, oxygen consumption after addition of 2 mg/ml oligomycin, 0.5 mM steps of FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone and followed by 2.5 mM antimycin A respectively as described [54]. For spinal cord studies 70 day-old mice were treated with DCA or vehicle for 20 days (n = 3 per group). Then, immediately following sacrifice, lumbar spinal cords were dissected. Spinal cord samples were immediately rinsed in respiration medium (Sucrose 110 mM, Mops 60 mM, EGTA 0.5 mM, BSA 1 g/l, MgCL2 3 mM, KH2PO4 10 mM, HEPES 20 mM, pH 7,1) and set in the Oroboros oxygraph for high resolution respirometry. Mitochon- drial oxygen consumption was measured as indicated in cell studies. Grip strength measurements Motor function was tested with a Grip Strength Meter (San Diego Instruments, San Diego CA). Tests were performed by allowing the animals to grasp the platform with both hind limbs, followed by pulling the animals until they released the platform. The force measurement was recorded twice a week from week 6 (baseline) until death in four separate trials. Neuromuscular junction measurements The extensor digitorum longus (EDL) and the soleus muscles from the same animals used for oxygen consumption studies were dissected immediately after sacrifice. Muscles were immersed for 60 minutes in 0.5% paraformaldehyde in DPBS, rinsed with DPBS (three times, 15 minutes each) and mechanically dissociated into small bundles of fibers using fine forceps under a stereomicroscope. The teased fibers were incubated with a blocking buffer containing 50 mM glycine, 1% BSA and 0.5% Triton X-100 for at least 3 hours and then in tetramethylrhoda- mine-conjugated a-bungarotoxin (TMR-BgTx) (T0195 Sigma; 1:1500 in blocking buffer) overnight (ON) at 4uC. After washing (three times, 20 minutes each) under agitation with PBS, fibers were left ON at 4uC in glycerol-Tris pH 8.8 (4:1) which was used as mounting medium for all the preparations. Image acquisition and quantitative image processing: the teased fiber preparations were observed by epifluorescence using an Olympus IX81 microscope. For each EDL muscle the images of 15–35 neuromuscular junctions were taken and stored for later analysis using Adobe Photoshop software. The total area, defined as the area delimited by the external outline of the TMR-BgTx - stained endplate marked with the Lasso tool, including both stained and non-stained areas was measured. The resulting numbers of selected pixels were counted with the Histogram tool. Data were expressed as percentage of neuromuscular junction total area from non transgenic control animals. Statistics Survival curves were compared by Kaplan-Meier analysis with the Log-rank test using Sigmaplot 12 (Systat software). All culture assays were performed in duplicate and each experiment was repeated at least three times. Quantitative data were expressed as mean 6 SEM and ANOVA and Student’s t test were used for statistical analysis, with p,0.05 considered significant. When the normality test failed, comparison of the means was performed by one-way ANOVA on ranks followed by the Kruskal–Wallis test. Data from GFAP were analyzed using a one-way ANOVA and compared by all pairwise multiple comparison procedures (Holm- Sidak method). All statistics computations were performed using PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34776