signature=13050112fc99b889380a50a1d8da0626,The Metabolomic Bioenergetic Signature of

Chemicals and reagents

Methanol (MeOH), water, formic acid (Optima LC/MS grade) and glutamine were purchased from Thermo-Fisher Scientific (Illkirch, France). Isotope metabolite standards including 17α-Hydroxyprogesterone-d8 (2,2,4,6,6,21,21,21-d8), L-Thyroxine-13C6, Succinic acid-2,2,3,3-d4, Pyruvic acid-1-13C and DL-Alanine-15N with >98% purity were acquired from Sigma Aldrich (St. Quentin Fallavier, France) as well as oligomycin, carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A and aspartate. All antibodies (ab42364, EP1332Y, ab186695 and ab186696), the NAD/NADH and ATP Assay Kit (ab65348 and ab83355) were obtained from Abcam (Paris, France) and the Mitotracker® green from Molecular Probes (Oregon, USA). Tris-Glycine Gel was purchased from Life Technologies (Illkirch, France) and DMEM-F12 from Jacques Boy Institute of Biotechnology (Reims, France). The DMEM medium supplemented with FBS (fetal bovine serum) was acquired from PAN-biotech (Wimborne, UK) and the Seahorse XFe Base Medium from Agilent Technologies (Santa Clara, CA, USA).

Cell cultures

Immortalized mouse embryonic fibroblasts (MEFs) from Opa1−/− knockout C57BL/6 mice and Opa1+/+ wild-type controls were cultivated in a medium consisting of Dulbecco’s modified Eagle medium with nutrient mixture F12 (DMEM-F12) supplemented with 10% FBS at 37 °C, 5% CO2. Both cell lines shared the same passage numbers. The analyses of Opa1−/− and Opa1+/+ MEF cell lines were all performed within the same exponential growth stage. When needed, cell culture medium was supplemented with 20 mM aspartate at pH 7.4 (±0.2) for 48 h.

Western blot analysis

Eight million cells of each MEF cell line were collected before confluence (n: 5). Ice-cold radio-immunoprecipitation assay (RIPA) buffer was utilized to lyse samples for 15 min at 4 °C followed by centrifugation at 20,000 g at 4 °C for 20 min. Protein extracts (30 μg) were separated on 4–20% Tris-Glycine Gel and electron-transferred to nitrocellulose membranes according to standard procedures. After blocking the free binding sites with 5% BSA (bovine serum albumin) reconstituted in phosphate-buffered saline with 0.2% Tween-20, the membranes were probed with anti-Opa1 (ab42364) and anti-tubulin-α (EP1332Y) antibodies. Anti-mouse and anti-rabbit fluorescence (1:10,000) (ab186695 and ab186696, respectively) were used as secondary antibodies. The images were quantitatively acquired using Image Studio 2.1 software (Li-Cor, Lincoln, NE, USA).

Mitochondrial oxygen consumption

Cells were seeded in XF Cell Culture Microplates (Seahorse, Agilent Technologies, Santa Clara, CA, USA) at a concentration of 30,000 cells/well in 100 μL DMEM 4.5 g/L medium supplemented with 10% FBS and 1 mM glutamine and incubated for 6 h at 37 °C in 5% CO2 atmosphere. Before the experiments, the culture medium was removed from each well and replaced by 100 μl of Seahorse XFe Base Medium pre-warmed at 37 °C and supplemented with 1 mM glutamine, pH 7.4 (±0.4). Cells were incubated in a CO2-free incubator at 37 °C for 1 h. Before the measurements, the XFe Extracellular Flux Analyzer (Seahorse, Agilent Technologies, Santa Clara, CA, USA) automatically mixed the assay media in each well for 10 min to allow the oxygen partial pressure to reach equilibrium and three baseline measurements were taken before the addition of any of the compounds loaded in the cartridge. Injection ports on the sensor cartridge were loaded with oligomycin (2 μg/mL), a titration of carbonyl cyanide 4‐ (trifluoromethoxy) phenylhydrazone (FCCP) ranging from 0.25 μM to 3 μM, and antimycin A (2 μg/mL). OCR (oxygen consumption rate) values refer to the average oxygen consumption rates during the measurement cycles, which in this case consisted of a 3 min wait, 3 response measurements before and after the addition of the compounds. OCR values were normalised to the number of cells/well.

NAD/NADH and ATP measurement

Total cellular NAD, NADH and ATP was carried out using the NAD/NADH and ATP assay kit according to the manufacturer’s instructions.

3D fluorescence imaging

Cells were seeded on coverslips in AQ4 DMEM-F12, 1% FCS, in a humidified atmosphere (95% air, 5% CO2) at 37 °C. Cells were then incubated for 15 min with 100 nM Mitotracker® green to stain the mitochondrial network. Images were acquired with an inverted wide-field microscope ECLIPSE Ti-E (Nikon, Amsterdam, Netherlands) equipped with a 100x oil-immersion objective (Nikon Plan Apo100x, N.A. 1.45) and an Andor NEO sCOMS camera controlled by Metamorph® 7.7 software (Molecular Devices, Sunnyvale, CA, USA). Twenty-one image planes were acquired along the Z-axis at 0.2 μm increments. Following image acquisition, images were first iteratively deconvolved using Huygens Essential® software (Scientific Volume Imaging, Hilversum, The Netherlands), with the maximum iteration scored at 50 and a quality threshold at 0.01, followed by 3D processing and morphometric analysis with Imaris 8.0® software (Bitplane, Zurich, Switzerland). Thirty cells of each MEF cell line were analysed for the quantitative analysis of mitochondrial shapes.

Metabolomics analysis

Samples (n = 10 for each MEF cell line) were randomly prepared as follows. After removal of the medium, the cellular monolayer was rinsed twice with an aqueous solution containing 0.22% NaCl before being quenched with cold MeOH. The cell suspension (estimated at 4 million cells), obtained after mechanical scraping, was then collected and stored at −80 °C until analysis in an aliquot of 106 cells. Test samples of each cell line (n = 3 samples/cell line) were pooled from various aliquots to validate the statistical model. Internal quality controls (QCs) were generated by mixing all the samples together.

A non-targeted reverse phase (RP) metabolomics method was validated for cell culture samples (Supplementary Table S2). Briefly, a mixture of H2O/MeOH was added to 1 million cells, which were initially fortified with the isotope metabolite standards mixture (10 μg/mL in MeOH), in order to achieve a final volume of 600 µL containing 20% water and 80% MeOH. After centrifugation, supernatants were evaporated to dryness. Samples were then reconstituted with an aqueous solution (2% MeOH) before the UHPLC-HRMS (Ultra High Pressure Liquid Chromatography-High Resolution Mass Spectrometry) analysis.

A Thermo Scientific Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), equipped with a heated electrospray ionization source, was used for this study in positive and negative modes. Ionisation conditions and MS parameters were identical to those previously described

Metabolite identification was facilitated using the MSMLSTM (Mass Spectrometry Metabolite Library of Standards) molecule library (IROA Technologies, Bolton, MA, U.S.A.) for mass spectrometry metabolomics. These commercial standards were injected in the same analytical conditions in order to create an in-house database (a final collection of 500 accurately identified carboxylic acids, amino acids, nucleotides, saccharides, fatty acids, lipids and hormones). Then, a TraceFinder 4.1 processing method was designed to allow a comparison of the Retention Times (RT), the MS/MS fragmentation spectra, the m/z and the isotopic patterns of metabolites. Ions integrated with a CV (Coefficient of Variation) higher than 30% in QC, a RT drift greater than 10 sec, and a linearity of dilution with an r² lower than 0.7 were immediately discarded. The identification criteria included accurate measurement of the m/z ratio (better than 5 ppm), a perfect isotopic pattern, an RT drift lesser than 5 s, and/or the presence of two identical fragments (level of metabolite identifications: 1, identified compounds)

Statistical analyses

Before performing statistical analyses, data were normalised by the total ion current (TIC) of each sample using Microsoft Excel software. In addition, the dataset was log10 transformed, mean-centred and scaled by the square root of the standard deviation of each variable (Pareto scaling) to reduce the contribution of the most intense ions. Statistical analyses were performed following the workflow chart shown in Fig. 3.

Multivariate analysis was carried out with Simca-P + v 14.0 (Umetrics, Umea, Sweden). Principal Component Analysis (PCA), an unsupervised method, was used to investigate the population structure and to emphasize spontaneous clustering or separation of samples on the basis of their global metabolite profiles. To highlight molecules implicated in the metabolomic signature, Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), a supervised analysis, was carried out, retaining only the metabolites that showed a strong power of discrimination and a high statistical reliability in the model. This means that variables were gradually excluded according to the results obtained from the different plots, i.e. the S-plot (visualization of intensity and reliability), the loading column plot with jack-knife confidence intervals, the coefficient plot, and finally, the Variable Importance in the Projection (VIP) plot. The purpose was to minimize the risk of over-fitting and to reduce the variability of prediction, thus simplifying interpretation of the molecular signature. OPLS-DA models were cross-validated by leaving out one-third of the samples, and this process was replicated three times. The qualities and performances of the models were evaluated using the Q²Ycum (goodness of prediction), the R²Ycum (goodness of fit) values, the cross validation-analysis of variance (CV-ANOVA), the permutation test (evaluation of the risk of over-fitting) and the prediction of a test set (6 test samples prepared and analysed like the other samples but not used for the development of the statistical model). Finally, only metabolites with a VIP value greater than 1 were considered as “highly relevant” for the metabolomic foot-printing.

Univariate analysis was performed with MetaboAnalyst 3.5

Statistical analysis of the western blots, mitochondrial respiration, and quantitative mitochondrial imaging was done with GraphPad statistical software (San Diego, CA, USA). The non-parametric Mann-Whitney test was used for western blot analysis and Student’s unpaired t-test was used for imaging and respiration analysis. For all analyses, a p-value