Opa1 relies on cristae preservation and ATP synthase to curtail reactive oxygen species accumulation in mitochondria

Abstract

Reactive oxygen species (ROS) are a common product of active mitochondrial respiration carried in mitochondrial cristae, but whether cristae shape influences ROS levels is unclear. Here we report that the mitochondrial fusion and cristae shape protein Opa1 requires mitochondrial ATP synthase oligomers to reduce ROS accumulation. In cells fueled with galactose to force ATP production by mitochondria, cristae are enlarged, ATP synthase oligomers destabilized, and ROS accumulate. Opa1 prevents both cristae remodeling and ROS generation, without impinging on levels of mitochondrial antioxidant defense enzymes that are unaffected by Opa1 overexpression. Genetic and pharmacologic experiments indicate that Opa1 requires ATP synthase oligomerization and activity to reduce ROS levels upon a blockage of the electron transport chain. Our results indicate that the converging effect of Opa1 and mitochondrial ATP synthase on mitochondrial ultrastructure regulate ROS abundance to sustain cell viability.

Keywords: Bioenergetics; F(1)F(O)-ATP synthase; Mitochondrial cristae; Opa1; ROS; Ultrastructure.

Graphical abstract

Fig. 1

OPA1 prevents mitochondrial dysfunction and cell death under bioenergetic stress. a, b) Cell death of MAFs of the indicated genotype, incubated for the indicated time in medium supplemented with the indicated monosaccharide. Data are mean ± SEM of four independent experiments. c) Gem-color coded images of mtSypHer fluorescence ratios (em: 535 nm) after alternate excitation (exc: 500/430 nm) of WT or Opa1^tg MAFs incubated for 24 h in complete DMEM supplemented with the indicated monosaccharide. The gem color scale is shown. Scale bar, 20 μm d) Quantitative analysis of experiments performed as in c. mtSypHer ratios are shown as mean excitation 500/430 nm ratios. e) Representative pseudocolor-coded FRET ratio images of mtATeam matrix ATP reporter in cells incubated for 24 h in the indicated medium. The pseudocolor scale is shown. Scale bar, 20 μm. f) FRET fluorescence ratios of WT or Opa1^tg MAFs, as in (e). Results show the average emission ratio (525/475 nm). g) Mitochondrial redox levels quantified from ratiometric excitation ratio (400/480 nm) image analysis mt-roGFP1 fluorescence, in cells grown for 24 h in medium supplemented with either glucose or galactose. In box plots, boxes represent SEM, middle line mean, whiskers 5^th-95^th percentile, dots the individual values of the independent experiments. *p < 0.05 in a two-way ANOVA versus control.

Fig. 2

Opa1 prevents ROS accumulation in cells relying on mitochondrial metabolism. a) Mitochondrial redox levels quantified from ratiometric excitation ratio (400/480 nm) image analysis mt-roGFP1 fluorescence in WT or Opa1^tg MAFs challenged for 2 h with vehicle or 5 μM AA before recordings. Results are expressed as mean ± SEM of three independent experiments. *p < 0.05 in an unpaired two-sample Student's t-test. b) Fold increase in MitoSOX fluorescence after treatment with AA (10 μM, 15 min) in cells of the indicated genotype grown for 24 h in the indicated media. Data are mean ± SEM of four independent experiments. *p < 0.05 in a two-way ANOVA versus control (a, b) or paired two-sample Student's t-test versus control (c) c) Cell death determined by flow cytometry as the percentage of annexin-V, PI double positive events from WT and Opa1^tg MAFs MAFs, incubated for 48 h in the presence of the indicated monosaccharides and exposed to 5 μM AA for 4 h. In box plots, boxes represent SEM, middle line mean, whiskers 5^th-95^th percentile, dots the individual values of the independent experiments.

Fig. 3

Opa1 requires ATP synthase activity to prevent mitochondrial ROS accumulation. a) Protein lysates from MAFs of the indicated genotype and incubated for 24 h in medium supplemented with the indicated monosaccharide, were separated by SDS-PAGE and immunoblotted with the indicated antibodies. b) WT cells transfected with empty (EV) or mitochondrial-targeted catalase (mtCAT) were lysed after 24 h equal amounts of protein were separated by SDS-PAGE and immunoblotted using the indicated antibodies. c) MitoSOX fluorescence values of WT and Opa1^Tg cells transfected as indicated and incubated in galactose medium for 24 h; a.u.: arbitrary units. d) Analysis of TMRM fluorescence in cells transfected as indicated, incubated in galactose medium for 24 h and treated where indicated with 10 μM AA and 2 μM FCCP. Data are mean ± SEM of four independent experiments. e) Cell death after 48 h incubation in galactose medium in cells transfected with the indicated plasmids and treated with AA (5 μM, 4 h). f) MitoSOX mean fold changes from four experiments performed with cells grown in galactose supplemented medium for 24 h and challenged with AA (10 μM, 15 min). Where indicated, cells were pre-incubated with oligomycin (1 μM, 5 min). In box plots, boxes represent SEM, middle line mean, whiskers 5^th-95^th percentile, dots the individual values of the independent experiments. *p < 0.05 in a two-way ANOVA.

Fig. 4

Opa1 promotes ATP synthase monomers assembly and oligomers stability. a) Complexes from 40 μg mitochondrial extracts from cells of the indicated genotypes, incubated for 24 h in the indicated media, were separated by Blue-native PAGE (BNGE) and analyzed for ATP synthase in-gel activity. b) Densitometric analysis of experiments as in (a). c, d) BNGE (c) and densitometric analysis (d) of immunoblotted mitochondrial extracts from WT or Opa1^tg cells incubated for 24 h as indicated from at least four independent experiments as in a). Ratios between the bands corresponding to oligomeric (oligomers, Vo + dimers, Vd) and the sum of all conformations (Vo + Vd + Vm + F₁) dimeric are shown. e, f) BNGE blot (e) and densitometric analysis (f) of OPA1 High Molecular Weight (HMW) complexes of protein extracts (40 μg) from MAFs of the indicated genotype incubated for 24 h in the presence of the referenced media. In box plots, boxes represent SEM, middle line mean, whiskers 5^th-95^th percentile, dots the individual values of the independent experiments. *p < 0.05 in a two-way ANOVA.

Fig. 5

Opa1 requires the ATP synthase dimerization subunit e to prevent ROS accumulation. a, b) Representative electronic micrographs (a) and cristae profile (b) of mitochondria from MAFs of the indicated genotype transfected with the indicated shRNA for 72 h. The cristae profiles illustrate cristae and their width along the overall mitochondrial length (μm). Arrowheads indicate intercristal matrix space. c) Analysis of mitochondrial matrix redox status in cells co-transfected with mt-roGFP1 and the indicated shRNAs. Data represent mean ± SEM of 400/492 nm fluorescence ratio. Where indicated, cells were treated for 2 h with 5 μM AA. d) Spontaneous death of cells of the indicated genotype transfected with the indicated shRNA after 72 h incubation in glucose or galactose medium. In box plots, boxes represent SEM, middle line mean, whiskers 5^th-95^th percentile, dots the individual values of the independent experiments. *p < 0.05 in a two-way ANOVA.