Fig 2: Morphological, biochemical, and structural analysis in mouse tissues ARepresentative hematoxylin and eosin staining of skeletal muscle in 3-month-old individuals.BHistochemical reaction specific to COX and SDH in skeletal muscle, cerebellar cortex, and kidney of the same individuals.CCOX (CIV) enzymatic activity normalized to the activity of citrate synthase (CS) in five animals per genotype as indicated at 3 and 12 months of age. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Three months: Muscle (SM)—***P = 0.0001 (WT vs. KO), kidney (K)—***P = 0.0002 (WT vs. KO), heart (H)—***P = 0.0001 (WT vs. KO), brain (B) and liver (L)—****P < 0.0001 (WT vs. KO). Twelve months: SM and L—****P < 0.0001, B—***P = 0.0008 (WT vs. KO).DWestern blot analysis of SDS–PAGE of total lysates from liver, skeletal muscle, and brain from the indicated genotypes, each lane showing the results for one animal. The graph shows the densitometric quantification of the signals obtained in the liver. Data are presented as mean ± SEM (n = 2 WT; n = 2 het; n = 3 KO). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons: ***P = 0.0002 and ****P < 0.0001 (WT vs. KO).EWestern blot analysis of 1D-BNGE of mitochondria from skeletal muscle from the indicated genotypes, each lane showing the results from one animal.FWestern blot analysis of 2D-BNGE of mitochondria from skeletal muscle from one individual of the indicated genotype. Red arrows indicate the accumulation of COX assembly intermediates in Apopt1 -/- samples. Source data are available online for this figure.

Fig 3: Imbalances in OXPHOS complexes. (a) The steady-state levels of five OXPHOS complexes by Blue Native gel electrophoresis. Fifteen µg of mitochondrial proteins from mutant and control cell lines was electrophoresed through a Blue Native gel, electroblotted and hybridized with antibodies for NDUFA9, SDHA, UQCRC2, COX5A, ATP5C (complexes I, II, III, IV, and V), and VDAC as a loading control. (b) Quantification of levels of complexes I, II, III, IV, and V in two mutant cell lines and two control cybrid cell lines was determined. The error bars indicate two standard errors of the means. *P < 0.05 and **P < 0.01 indicate the significance, according to the t-test, of the differences between mutant and control cybrid cell lines.

Fig 4: S70pBcl2 modulates mitochondrial COX activity, respiration rate and ROS production. (A) Fold change in COX activity of Jurkat cells following treatment with DDC (400 µM) for 4 h. N = 3. (B) Fold change in COX activity of Jurkat cells following treatment with two independent siSOD1 (150 nM) for 48 h. N = 3. (C) Fold change in COX activity of Jurkat cells transiently transfected with 2 µg pcDNA3.1, WT BCL2, S70A or S70E for 30 h. N = 3. (D, E) One representative line graph showing the kinetics of OCR in Jurkat cells following treatment with DDC (400 µM) or increasing doses of etoposide for 4 h. Fold change of OCR taken at point 3 ~20 min. * indicates oligomycin, FCCP or rotenone and antimycin-A injections in a timely order. N = 4. (F) One representative line graph showing the kinetic of OCR in Jurkat cells transiently transfected with 2 µg pcDNA3.1, WT BCL2, S70A or S70E for 30 h. Fold change of OCR taken at point 3 ~20 min. * indicates oligomycin, FCCP or rotenone and antimycin-A injections in a timely order. N = 6. (G, H) Fold change of Mitosox staining levels in Jurkat cells following treatment of siSOD1 for 48 h or transiently transfected with 2 µg pcDNA3.1, WT BCL2, S70A or S70E for 30 h. N = 3 and 4 respectively. Bar graphs showing mean and SD. Two-tailed t-test was performed for A, D and G. One-way ANOVA and Sidak's multiple comparison were performed unless otherwise specified. * P = 0.05, ** P = 0.01, *** P = 0.001, **** P = 0.0001.

Fig 5: Time-chart of HD phenotype development in TgHD minipig model. Results from present publication are colored in black; previously published (or unpublished) data are indicted in gray. Displayed biochemical mitochondrial parameters are dependent exclusively on HD disease (except proteins marked by #) and not on age or gender, so we propose these as biomarkers of HD development. 1Baxa et al., 2013; 2Macakova et al., 2016; 3Krizova et al., 2017; 4Vidinska et al., 2018; 5Data described in present publication; 6Askeland et al., 2018b; 7See Movie 1; 8Taras Ardan, Z.E. et al., Institute of Animal Physiology and Genetics AS CR, Czech Republic, unpublished data. Enzymatic activity: COX, cytochrome c oxidase (RCCIV); CS, citrate synthase; NCCR, NADH-cytochrome c oxidase (RCCI-III); SQR/CS, ratio of succinate dehydrogenase (RCCII) to citrate synthase activity. Respiration parameters: GM, glutamate-malate (RCCI-dependent respiration); CII, RCCII-dependent respiration; CIV, RCCIV-dependent respiration; CI/CIV, ratio of RCCI- to RCCIV-dependent respiration. Proteins: SDH30, subunit of complex II; OSCP, oligomycin-sensitivity conferring protein subunit of ATPase (RCCV); OPA1, optic atrophy 1 protein; DRP1, dynamin-related protein 1.