Fig 1.
Schematic of the components and fluxes included in the computational model, and comparison of model simulations with experimental measurements.
(A) Figure adapted from [26]. Addition of pharmacological modulators are simulated by inhibiting (red flat-headed arrows) or increasing (red arrow) the activity of the indicated model component, and arrows point to the text label of the affected model component. We assume the rapid conversion of superoxide to hydrogen peroxide (H2O2; not shown). ANT: adenosine nucleotide transferase; ΔΨm: mitochondrial membrane potential; H+: protons; H2O2: Hydrogen peroxide; IMM: inner mitochondrial membrane; IMS: inter-membrane space; OMM: outer mitochondrial membrane; Pi: Phosphate; Q: Ubiquinone; QH2: Ubiquinol. (B,C) (B) Model simulations of the mitochondrial stress test closely resembled (C) experimental measurements of the oxygen consumption rate (OCR) in primary cortical neurons. OCR in (C) was measured in wildtype primary cortical neurons at baseline and following addition of Oligomycin (Oligo, 2 μg/ml), FCCP (0.5 μM) and AntimycinA (AntiA, 1 μM), as described in [17], with nonmitochondrial respiration subtracted. Panel C reproduced from [17]. Traces represent individual simulations or wells, and the mean of all traces is shown in black. (D-G) The simulated response (Sims) of ΔΨm to (D) Rot, (E) AntiA and (F) Oligo, as well as the (G) simulated response of NAD(P)H to Rot (foldchange, FC) also resembled experimental measurements (Expt; data reproduced from [17]).
Fig 2.
Simulating impairments in model components provides mechanistic insight.
(A) Basal OCR simulated in physiological conditions (PC; no simulated impairment), in the presence of a complex I impairment (CI80%; CI activity = 80% PC activity), and in the presence of increased proton leak (Hle200%; Hle activity = 200% PC activity). (B) Mitochondrial membrane potential (ΔΨm) simulated at baseline and following the addition of Oligomycin (Oligo), in physiological conditions (left two box-plots) and with a CI impairment (CI80%; right two box-plots). (C) ΔΨm foldchange over baseline following the addition of Oligomycin, in physiological and CI impairment conditions (CI80%). Individual simulations (dots) are coloured relative to PC simulations (see Methods): White: Within ±10% of PC; Darkening shades of red indicate increase above PC, darkening shades of blue indicate decrease below PC. n = 1000 simulations for each condition.
Fig 3.
Simulating impairments of critical model components predicts their impact on bioenergetic parameters.
The activity of the indicated model components (as a % of the simulated physiological conditions (PC)) is plotted on the x-axis against basal oxygen consumption rate (OCR), maximal OCR, basal mitochondrial membrane potential (ΔΨm, normalised to a min ΔΨm of −50 mV, for visualisation), and basal concentrations of mitochondrial ATP, NADH, and cytosolic ROS (H2O2). Impairments were simulated in (A-C) ETC complexes I, III and IV [CI, CIII, CIV]; (D) the dehydrogenase flux providing substrate to the ETC [DH]; (E) the F1Fo ATP synthase [F1]; (F, I) proton leak [Hle]; (G) cytosolic ATP production [KDyn]; (H) cytosolic ATP consumption [KCons].
Fig 4.
Categorising simulated impairments enables comprehensive analysis of the effects of mitochondrial ETC impairments on key bioenergetic parameters.
(A-C) Predicted impact of reduced complex I (CI) activity on (A) mitochondrial membrane potential (ΔΨm), (B) mitochondrial ATP (ATPm) levels and (C) mitochondrial NADH (NADHm) levels at baseline and following the simulated addition of Oligomycin (Oligo; F1Fo ATP synthase inhibitor). FC: foldchange (compared to basal value). Heatmap values are the median of 50 simulations. The red box indicates F1Fo ATP synthase reversal predicted in the presence of severe CI defects, as evidenced by a decrease in ΔΨm following Oligomycin addition. (B,D,F) The foldchange (FC) response to Oligomycin is grouped into 7 categories, as described in Methods (decrease---, decrease--, decrease-, no change, increase + , increase++, increase+++ compared to simulated physiological conditions [100% CI activity]). The effect of other impairments is provided in Supp Data.
Fig 5.
Unsupervised clustering separates impairments according to the induced bioenergetic phenotype.
Impairments generally cluster together as they induce distinct bioenergetic phenotypes. Red/blue shading indicates the predicted change in the presence of the simulated impairment compared to physiological conditions (PC) as described in Methods. Row annotations indicate the component with the simulated impairment (CI, complex I; CIII, complex III; CIV, complex IV; F1, F1Fo ATP synthase; DH: dehydrogenase flux; Hle, proton leak; KCons, cytosolic ATP consumption; KDyn, cytoslic ATP production) and the magnitude of the defect [black-white: 100%−0% of activity in PC]. Leak = oligomycin-insensitive oxygen consumption rate (OCR).
Fig 6.
Unsupervised clustering predicts that an increased proton leak may underlie the bioenergetic phenotype measured in Parkin KO dopaminergic neurons (Giguiere et al, 2018).
(A) Experimental bioenergetic phenotype in Parkin KO dopaminergic neurons (PARK) as measured by [50] – increased basal OCR, unchanged maximal OCR, reduced mitochondrial ATP. (B, inset) The experimental phenotype clustered with a simulated increase in proton leak (Hle), equivalent to increased mitochondrial uncoupling, and large impairments in cytosolic ATP production (KDyn). Red/blue shading indicates predicted change compared to physiological conditions (PC) as described in Methods. Row annotations indicate the component with the simulated defect (CI, complex I; CIII, complex III; CIV, complex IV; F1, F1Fo ATP synthase; DH: dehydrogenase flux; Hle, proton leak; KCons, cytosolic ATP consumption; KDyn, cytosolic ATP production) and the magnitude of the defect [black-white: 100%−0% of activity in PC]. (B) A simulated increase in Hle (350% PC) reproduced the experimental observations [compare with Figs 2A and 2G from [50]].
Fig 7.
Model-guided semi-automated analysis predicts that combined defects in complex I, F1Fo ATP synthase and proton leak can mechanistically explain the bioenergetic phenotype observed in Pink1 KO neurons.
(A-C) Experiments in primary cortical neurons from Pink1 KO mice identified (A) significant reductions in basal and maximal oxygen consumption rates (OCR), **p < 0.01, post-hoc comparison; (B) reduced ΔΨm sensitivity to Antimycin A (AA; CIII inhibition), ***p < 0.001, genotype x treatment interaction (pre- vs post-drug), linear mixed effects model; and (C) no change in the ΔΨm response to Rotenone (Rot; CI inhibition; p = 0.597). Data are shown as mean + /-SEM (A) and mean + /-st. dev (B,C). (D) Unsupervised clustering of simulated and experimental phenotypes demonstrated that while defects in Hle or DH partially reproduced Pink1 KO (Pink1) experimental behaviour, no single defect accurately recapitulated all experiments. (E-G) Simulated experiments [(E) OCR, (F) ΔΨm foldchange (FC) response to Rotenone, (G) ΔΨm foldchange (FC) response to Antimycin A] demonstrated that the combined impairments of DH84% and KCons40% accurately reproduces the entire set of experiments in Pink1 KO neurons. PC; simulated physiological conditions.
Table 1.
Sensitivity analysis parameter settings: Model components varied throughout this study, and the corresponding parameter values to simulate the range of impaired/increased activity. PC: simulated Physiological Condition.
Table 2.
Categorising modelled outputs using quantile thresholds from the simulated physiological condition (PC) distribution. Thresholds were defined as ±10%, ± 25% and ±40% change from PC median, corresponding to the 40th/60th, 25th/75th, 10th/90th percentiles of the baseline PC distribution (n = 1000).
Table 3.
Summary of the predicted changes in bioenergetic parameters (rows) in the presence of severe impairments in the indicated model components (columns): These predictions agree with established bioenergetic knowledge, further highlighting the accuracy of the model. Blue = decrease, white = no change, red = increase. Abbreviations: CI, complex I; CIII, complex III; CIV, complex IV; DH: Dehydrogenase flux, F1: F1Fo ATP Synthase, Hle: Mitochondrial proton leak, KCons: Cytosolic ATP consumption, Kdyn: Cytosolic ATP production. Down arrow indicates decreased activity of the indicated component, up arrow indicates increased activity.