Haploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy

ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of mitochondrial DNA (mtDNA). Mutations in the ACO2 gene were identified in patients suffering from a broad range of symptoms, including optic nerve atrophy, cortical atrophy, cerebellar atrophy, hypotonia, seizures and intellectual disabilities. In the present study, we identified a heterozygous 51 bp deletion (c.1699_1749del51) in ACO2 in a family with autosomal dominant inherited isolated optic atrophy. A complementation assay using aco1-deficient yeast revealed a growth defect for the mutant ACO2 variant substantiating a pathogenic effect of the deletion. We used patient-derived fibroblasts to characterize cellular phenotypes and found a decrease of ACO2 protein levels, while ACO2 enzyme activity was not affected compared to two age- and gender-matched control lines. Several parameters of mitochondrial function, including mitochondrial morphology, mitochondrial membrane potential or mitochondrial superoxide production, were not changed under baseline conditions. However, basal respiration, maximal respiration, and spare respiratory capacity were reduced in mutant cells. Furthermore, we observed a reduction of mtDNA copy number and reduced mtDNA transcription levels in ACO2-mutant fibroblasts. Inducing oxidative stress led to an increased susceptibility for cell death in ACO2-mutant fibroblasts compared to controls. Our study reveals that a monoallelic mutation in ACO2 is sufficient to promote mitochondrial dysfunction and increased vulnerability to oxidative stress as main drivers of cell death related to optic nerve atrophy.


Results
Identification of the deletion c.1699_1749del51 in the ACO2 gene in a patient with dominant inherited optic nerve atrophy. We screened 9 unrelated German patients suffering from autosomal dominant optic nerve atrophy for mutations using whole exome sequencing (WES). For the selection of putative disease-causing genes, we considered a sub panel of genes associated with optic atrophy, including OPA1, OPA3, TMEM126A, WFS1, MFN2, SPG7, ACO2, RTN4IP1 and AFG3L2, and identified one male individual with the heterozygous deletion c.1699_1749del51 in the ACO2 gene (NCBI reference NM_001098.3; hereafter referred to as ACO2-mutant or ACO2.mut) ( Fig. 1a; individual III.4). The deletion was subsequently validated in genomic DNA of the index patient by Sanger sequencing (Fig. 1b). The same deletion in ACO2 was found in all three other clinically affected family members, including the index patient's son (individual IV.2), his first-degree female cousin (individual III.2) and her son (individual IV.1). The index patient's daughter (individual IV.3) also carried the mutation but did not show clinical signs of disease when examined at the age of 16 years (Fig. 1a). From the WES data, we could exclude mutations in OPA1, OPA3, TMEM126A, WFS1, MFN2, SPG7, RTN4IP1 and AFG3L2 and the common mtDNA mutations associated with LHON at positions 11778/ND4, 3460/ND1 and 14484/ND6 in the index patient.
The patient was 26 years old at diagnosis. Clinical examination of the patient was performed at the age of 55 years at the Institute for Ophthalmic Research and the Section for Clinical Neurogenetics of the University Clinic of Tübingen, Germany. The optic nerve atrophy manifested with a decreased visual acuity on the right eye (0.7 decimal, corresponding to approx. LogMAR 0.2), while the left eye was amblyopic in the course of esotropia with an acuity of 0.08 (decimal, LogMAR 2.1). During 30 years of disease progression, the best corrected visual acuity decreased to 0.2 (decimal, LogMAR 0.7) on the right eye, while remaining constant on the left eye. Visual field examination revealed central scotomas on both eyes (Fig. 1c).
Ophthalmological examination showed a distinct temporal pallor of the optic nerve on funduscopy, indicating an atrophy of the optic nerve, with otherwise normal morphological findings of the anterior and posterior eye segment (Fig. 1d). Examination of the retinal nerve fiber layer via spectral domain optical coherence tomography (SD-OCT) shows thinning of the optical nerve fibers in the temporal regions (Fig. 1e).
The outer retinal function for the central visual field assessed by multifocal electroretinogram, as well as by the full-field electroretinogramm, was normal. The outer retina of the macular area on OCT was normal.
Neurological examination revealed normal findings apart from visual deficits described above. Especially, there were no signs of cerebellar ataxia or cognitive deficits reported before in carriers of biallelic ACO2 mutations 5,[9][10][11]13,14 . Laboratory tests found increased levels of ferritin (79 µg/dl; norm:  with normal levels of iron and transferrin. Quantitative cDNA analysis with RNA from whole blood of the index patient revealed no evidence of impaired transcript splicing (data not shown). Translation of mutant transcripts thus results in an in-frame deletion of 17 amino acids (p.567_583del17) in the ACO2 protein (XP_024308018.1) in a region which is conserved in eukaryotic mitochondrial aconitases.
The deletion c.1699_1749del51 in the ACO2 gene leads to a growth defect of a Δaco1 yeast strain. Aconitase is highly conserved across species and sequence alignment of human ACO2 (NP_001089.1) 29 and S. cerevisiae ACO1 (NP_013407.1) generated by MUSCLE 30 reveals a high similarity of both amino acid sequences (see Supplementary Fig. S1) of 66.41% (identity matrix created by Clusta12.1). We investigated the pathogenic potential of the ACO2 mutations by complementation analysis in S. cerevisiae with deletion of the yeast aco1 gene (Δaco1). Yeast cells were grown on galactose (Fig. 2a) or on ethanol medium (Fig. 2b) at different dilutions. When grown on galactose medium, yeast cells are forced to use mitochondrial respiration to maintain their energy supply 31,32 . Yeast devoid of aco1, expressing only the empty Yep51 vector showed a slight growth defect at 30 °C, while growth at 35 °C was severely affected. Expression of the human wildtype ACO2 or ACO2-S112R 9 missense mutant rescued the growth defect of Δaco1. In contrast, expression of the p.567_583del17 mutant ACO2 (ACO2.mut) was not sufficient to rescue the growth defect of Δaco1, indicating that mitochondrial respiration was impaired by this mutation (Fig. 2a).
The growth of yeast cells on a two-carbon substrate like ethanol requires a functional TCA 32 . Δaco1 yeast and Δaco1 yeast expressing the p.567_583del17 mutant ACO2 were not able to grow on ethanol substrate, while Δaco1 yeast expressing ACO2-WT or ACO2-S112R 9 showed similar growth on ethanol substrate at 30 °C and 35 °C (Fig. 2b).
Overall, these results suggest that loss of aco1 and the p.567_583del17 mutant ACO2 cause defects of the TCA and the mitochondrial respiration.
Disease-associated ACO2 mutations cause impaired mtDNA maintenance. We obtained fibroblasts from a skin biopsy of the patient and of healthy, age-and gender-matched control individuals. Western blot analyses revealed a significant decrease of ACO2 protein levels in ACO2-mutant fibroblast, compared to control fibroblasts (Fig. 3a,b).
As ACO2 is a mitochondrial protein, we also assessed the abundance of the mitochondrial marker protein Tom20 by Western blot (Fig. 3c). Here, we did not observe changes of protein levels between the fibroblast lines ( Fig. 3d).
Given the reduced ACO2 protein levels in ACO2-mutant fibroblasts, we further determined ACO2 enzyme activity using a spectrophotometric assay. In order to avoid distortion of the assay by also measuring the enzyme activity of the cytosolic isoform Aconitase 1 (ACO1), we used mitochondrial fractions. Our results revealed that despite reduced protein levels, ACO2-mutant fibroblasts showed no alteration of overall ACO2 enzyme activity, using NADP+ as a substrate (Fig. 3e).
Together, our results suggest that the heterozygous 17 amino acid deletion in ACO2 likely leads to a reduction of ACO2 protein level, probably by increased degradation of the mutant protein. But apparently, the wild type proportion of ACO2 protein is sufficient to maintain the overall enzyme activity in patient-derived fibroblasts under baseline conditions.
Compound heterozygous mutations in ACO2 were previously shown to cause a ~ 50% reduction of mtDNA copy number 10 , leading us to further investigate mtDNA maintenance in ACO2-mutant fibroblasts by RT-PCR. Our analysis revealed that mtDNA copy number (Fig. 3f) and mtDNA transcription (Fig. 3g) were significantly reduced in ACO2-mutant fibroblasts compared to control cells, while we did not observe changes of the amount of deletions in the major arc of the mitochondrial genome (Fig. 3h).

ACO2-mutant fibroblasts display reduced mitochondrial respiratory function. ACO2 is an
important enzyme of the TCA and is therefore critically involved in mitochondrial metabolism, which was assessed by measurement of the oxygen consumption rate (OCR) in whole fibroblasts (Fig. 4a) www.nature.com/scientificreports/ ( Fig. 4b), maximal respiration ( Fig. 4c), spare respiratory capacity (Fig. 4d) and proton leak ( Fig. 4e) were significantly reduced in ACO2-mutant fibroblasts, compared to control fibroblasts, whereas ATP production ( Fig. 4f) and the coupling efficiency ( Fig. 4g) were not changed. These results suggest that mitochondrial respiration is impaired in fibroblasts under baseline conditions. The assessment of mitochondrial respiration was further completed by measurements of the extracellular acidification rate (ECAR; Fig. 4h), which allows conclusions on glycolysis. Results show that neither glycolysis ( Fig. 4i), glycolytic capacity (Fig. 4j), glycolytic reserve (Fig. 4k), nor non-glycolytic acidification (Fig. 4l) were changed in ACO2-mutant fibroblasts.
From these results, we conclude that ACO2-mutant fibroblasts are able to maintain their energy supply by glycolysis under normal cell culture conditions. By contrast, the observed reduction of mitochondrial respiration in the presence of mutant ACO2 suggests that patient cells may not be able to adapt their metabolism to challenging conditions. ACO2-mutant fibroblasts are more susceptible to oxidative stress. The observed alterations of mitochondrial respiration in ACO2-mutant fibroblasts led us to investigate the consequence of mutant ACO2 on mitochondrial integrity. First, we analyzed mitochondrial morphology in fibroblasts under baseline conditions (Fig. 5a) and found no changes of mitochondrial aspect ratio, indicative for length (Fig. 5b), or mitochondrial form factor, indicating branching (Fig. 5c), in ACO2-mutant fibroblasts compared to control cells.
Also, we did not observe alterations of mitochondrial membrane potential in ACO2-mutant fibroblasts using TMRE staining, neither under baseline conditions, nor under Valinomycin treatment (Fig. 5d). Furthermore, we did not observe changes of mitochondrial superoxide production in ACO2-mutant fibroblasts, compared to control cells, when using MitoSOX staining under baseline conditions and under complex I inhibition with Piericidin A (Fig. 5e).
Finally, we investigated the effect of oxidative stress on cell viability by using the LDH assay. Cell viability in ACO2-mutant fibroblasts was comparable to control fibroblasts under baseline conditions, however, after oxidative challenge by treatment with H 2 O 2 , we observed a significant increase of cell death in ACO2-mutant fibroblasts compared to control cells (Fig. 5f). www.nature.com/scientificreports/ Together, the results suggest that mutations in ACO2 lead to an increased susceptibility to oxidative stress.
ACO2 enzyme activity correlates with the severity of the clinical phenotype, but not with the protein amount of ACO2. A number of studies reported cases of patients carrying mutations in the ACO2 gene, including analyses in patient-derived fibroblasts. From the available literature, we noticed that the enzymatic activity of mutant ACO2 does not correlate with the protein level, fitting to our observation of reduced ACO2 protein level (Fig. 3a,b) in spite of unchanged enzyme activity (Fig. 3e). Metodiev and colleagues described clinical presentation and cellular phenotypes in families with different mutations in ACO2 and found that the compound heterozygous mutations c.220 C>G (p.Leu74Val)/c.1981 G>A (p.Gly661Arg) results in 20% protein level and 66% enzyme activity, while the homozygous mutation c.776 G>A (p.Gly259Asp) does not affect protein levels, but reduced enzyme activity to 5% 5 . Protein levels were reduced to 20% and enzyme activity to 31% (f) mtDNA copy number was analyzed by RT-PCR and indicated as ratio of the copy numbers of the mitochondrial gene ND1 to the nuclear encoded gene B2M. Significance was calculated by Mann-Whitney test (n = 9). (g) mtDNA transcription was analyzed by RT-PCR and indicated as ratio of the copy numbers of the D-Loop to the mitochondrial gene ND1. Significance was calculated by Mann-Whitney test (n = 3). (h) Major arc deletions in the mitochondrial genome were analyzed by RT-PCR and indicated as the ratio of the copy numbers of ND4, which is located on the minor arc, to ND1, which is located on the major arc of the mtDNA. Significance was calculated by Mann-Whitney test (n = 3). All data were indicated as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. www.nature.com/scientificreports/ in a compound heterozygous carrier of the mutations c.2208 G>C (p.Lys736Asp) 5 . Other studies also reported unchanged ACO2 protein levels, while the enzyme activity was reduced to 20% (compound heterozygous mutations c.2135 C>T; p.Pro712Leu 10 ), or 25% (homozygous c.1240T>G; p.Phe414Val 33 ), respectively (Fig. 6). We blotted the ACO2 enzyme activity against the protein levels reported in several studies and noticed that the level of ACO2 enzyme activity seems to correlate with the severity of the clinical phenotype (Fig. 6). Patients with higher enzymatic activity display milder clinical symptoms, e.g. a patient carrying compound heterozygous mutations c.220 C>G (p.Leu74Val)/c.1981 G>A (p.Gly661Arg) showed 66% enzyme activity in fibroblasts and displayed optic atrophy 5 , while the homozygous mutation c.776 G>A (p.Gly259Asp) was associated with 5% enzyme activity and severe symptoms including optic atrophy, ataxia, cerebellar atrophy and seizures 5 . Fitting to this observation, the patient described in the present study presented with an isolated optic atrophy (Fig. 1) and an unaffected ACO2 enzyme activity in patient-derived fibroblasts (Figs. 3e, 6).

Discussion
In the present study, we provide first evidence for haploinsufficiency of the ACO2 gene as a cause of autosomal dominant isolated optic atrophy. We used WES to identify potential disease-causing mutations and found a novel heterozygous deletion, c.1699-1749del51, in the ACO2 gene, resulting in a shortened ACO2 protein with an internal 17 amino acid deletion.
Mutations in ACO2 have been described in the context of early fatal or neurodegenerative disease in homozygous or compound heterozygous state. The clinical symptoms range from mildly affected patients, suffering from isolated optic atrophy 9 , to severely diseased patients, presenting with cortical or cerebellar atrophy, hypotonia, seizures or intellectual disabilities starting in early infancy 5,[9][10][11][12][13] . Based on the genetic results, we reason that the identified heterozygous c.1699_1749del51 ACO2 mutation causes an autosomal dominant inherited isolated optic atrophy with reduced penetrance in the herein described family.
The pathogenic nature of the ACO2 deletion c.1699_1749del51 identified in our study was further substantiated by performing a yeast complementation assay. The deletion of the mitochondrial aco1 gene caused a severe growth defect on galactose medium as well as on ethanol medium. Complementation with a vector expressing the ACO2-mutant variant (ACO2 c.1699_1749del51) was not sufficient to rescue this phenotype, indicating that both, mitochondrial respiration and the TCA cycle were affected. Similar phenotypes of aco1-deficient yeast complemented with different ACO2 variants were reported in previous studies 5,9,25 .
The biochemical analysis of mitochondrial function in patient-derived fibroblasts and Δaco1-yeast highlights the pathogenic relevance of the herein described ACO2 c.1699-1749del51 deletion. It is worth noting that expression of the S112R mutation showed at least a partial complementation of Δaco1-yeast, notwithstanding that this mutation was previously described to cause a recessively inherited syndrome of infantile cerebellarretinal degeneration 9 .
Mitochondrial aconitase is an evolutionary strongly conserved protein that is involved in the TCA cycle and in the maintenance of mitochondrial function. Therefore, we used patient-derived fibroblasts to further analyze the cellular mechanisms of the underlying pathology.
Recent studies suggested that the ACO2 enzyme activity in patient-derived cells seem to generally correlate with the severity of clinical symptoms 5,11,13 . Indeed, the ACO2 enzyme activity varied considerably in the different studies, ranging from 5 to > 60% in cells derived from patients with either homozygous or compound heterozygous mutations, and patients with higher ACO2 enzyme activity presented with milder clinical phenotypes 5,9,10,13 . This finding fits to the comparatively mild clinical phenotype of isolated optic nerve atrophy in the herein described patient and the unaffected ACO2 enzyme activity in the patient-derived fibroblasts.
In contrast, several studies showed that ACO2 enzyme activity does not correlate with ACO2 protein levels. In 2014, Metodiev and colleagues analyzed ACO2 protein levels and enzyme activity in fibroblasts from patients with ACO2 mutations. Cells with the lowest levels of ACO2 protein (~ 20%) showed the highest levels of remaining mutant ACO2 enzyme activity (~ 30% to > 60%, respectively), while fibroblasts expressing the highest levels of ACO2 protein (unchanged levels compared to control fibroblasts) had the lowest enzyme activities (~ 5%) 5 . Additionally, the studies of Sadat et al. and Bouwkamp et al. showed that ACO2 enzyme activity was reduced to 20% or 25%, respectively, while the ACO2 protein levels were unchanged in patient-derived fibroblasts 10,33 .
Despite the fact that ACO2 enzyme activity was not significantly affected in fibroblasts derived from the patient, the mitochondrial respiration was impaired. Basal respiration, maximal respiration and spare respiratory capacity were significantly decreased, suggesting that ACO2-mutant cells have a reduced capacity to cope with metabolically demanding conditions. Sadat and colleagues previously observed a similar impairment of mitochondrial respiration in ACO2-mutant patient-derived fibroblasts, accompanied with a 50% reduction of mtDNA copy number 10 .
Hence, it was not surprising to find that fibroblasts with mutations in ACO2 in this study showed a 50% reduction of mtDNA copy number, which was likely the cause of impaired mitochondrial respiration. In yeast, the mitochondrial ACO1 protein, which corresponds to human ACO2, was found to be crucial for mtDNA maintenance. A diploid yeast strain with heterozygous depletion of aco1 showed drastic reduction of mtDNA abundance 25 . This function of ACO1 was independent of the ACO1 catalytic activity and rather depending on retrograde metabolic pathways 25 . Therefore, we conclude that optic nerve atrophy may be caused by heterozygous mutations in the ACO2 gene independent of its enzymatic activity. We postulate that the reduction of ACO2 protein levels leads to defects in mtDNA maintenance, which subsequently impair mitochondrial respiratory function.
In addition, we further investigated mitochondria-related phenotypes by assessment of the ECAR, which indicates glycolytic function, mitochondrial morphology, mitochondrial membrane potential and mitochondrial superoxide production, none of which showed changes in ACO2-mutant fibroblasts compared to control www.nature.com/scientificreports/ www.nature.com/scientificreports/ fibroblast lines. These results are not surprising given the fact that fibroblasts are able to maintain their energy demand by glycolysis. We finally assessed cell viability in our fibroblast lines and were not able to detect differences at baseline conditions. However, when cells were challenged with oxidative stress using H 2 O 2 treatment, ACO2-mutant fibroblasts showed a markedly increased level of cell death compared to control cells. ACO2 was previously described to be sensitive to oxidative stress 20,21 and to play a role in oxidative stress-related pathways [22][23][24] . In 2011, Cantu et al. showed that paraquat-induced inhibition of ACO2 caused an increased production of H 2 O 2 and ferrous iron accumulation with subsequent increased apoptosis of rat dopaminergic N27 cells. The authors concluded that ACO2 protects cells from apoptosis by the regulation of H 2 O 2 production and iron accumulation in a mitochondrial metabolism-dependent manner 34 .
Additionally, it was shown that overexpression of ACO2 prevents H 2 O 2 -induced mtDNA damage and the resulting mitochondrial translocation of p53 and apoptosis in alveolar epithelial cells, while knockdown of ACO2 enhanced H 2 O 2 -induced mtDNA damage and subsequent apoptosis 35 . In the light of these results, we can conclude that the observed increased susceptibility of ACO2-mutant fibroblasts to H 2 O 2 -induced oxidative stress is linked to impaired ACO2 function and subsequent mitochondria-induced cell death.
Given the insufficiency of the ACO2 mutant protein to rescue the growth defect of Δaco1-yeast, together with the phenotypes described in ACO2-mutant fibroblasts and the autosomal dominant inheritance of the heterozygous mutation, we reason that the c.1699-1749del51 deletion causes a haploinsufficiency.
It is worth noting that temporal pallor of the optic nerve is a common trait of mitochondrial defects and autosomal dominant optic atrophy (ADOA; i.e. Kjer´s syndrome) 36,37 . Up to 70% of ADOA cases are caused by mutations in the OPA1 gene 38 . Interestingly, recent studies showed that OPA1 and ACO2 are involved in similar mechanisms, thereby possibly explaining shared clinical phenotypes in patients carrying mutations in ACO2 or OPA1. In particular, OPA1 regulates mtDNA integrity by tethering nucleoids to the inner mitochondrial membrane, subsequently influencing mtDNA replication and distribution 39,40 . Consequently, knockout of OPA1 caused disorganization of the cristae and a decrease of mtDNA copy number in mouse embryonic fibroblasts 39 . Furthermore, knockdown of OPA1 induced by siRNA in murine neurons led to a decrease of baseline mitochondrial respiratory function and aconitase enzyme activity while at the same time aconitase protein levels were not affected. In addition, neurons with OPA1 knockdown were more susceptible to oxidative stress, resulting in elevated rotenone-induced cell death, likely due to an impairment of the ROS defence in these cells 41 . However, mutations in OPA1 were excluded in the herein described patient.
In summary, our study provides evidence for a novel mutation in the ACO2 gene causing haploinsufficiency in a pedigree with autosomal-dominant inherited optic nerve atrophy. Thereby, our study adds further details to the complex picture of mitochondrial defects as underlying cause of optic nerve atrophy caused by mutations in ACO2.

Structural analysis of deleted segment 571-583 in human ACO2.
To predict the effects of deleting positions 571-583 in ACO2 we used a three-dimensional structure of the analyzed protein and inspected non-covalent interactions (hydrogen bonds, salt bridges) in the structure. The structure of human ACO2 (NP_001089.1) has not been solved yet. However, its amino acid sequence is 96.5% identical to the sequence of the same enzyme from pig (pdb entry 1b0j). Therefore, we used this structure as a template to predict the structure of human ACO2 14 . The structure was predicted using the homology-modelling software MODELLER 46 . The MolProbity web-server 47 was then used to optimize side chain orientations and to add hydrogen atoms to the structure.
Yeast drop dilution assay. The growth assay of yeast was done as described before 9,32 . Strains harbouring the appropriate plasmids were grown at 30 °C in synthetic depleted (SD) medium containing 0.67% (wt/vol) yeast nitrogen base (Difco, Detroit, MI) and 2% galactose or 3% ethanol supplemented with the appropriate amino acids (50 mg/ml) overnight, followed by drop dilution growth on the indicated media agar plates.
Fibroblast cell culture. Informed consent was obtained from all individuals included in this study prior to skin biopsy collection. Skin biopsies were obtained from one male patient with the c.1699_1749del51 deletion in ACO2. Age-and gender-matched healthy control individuals were recruited from the Luxembourg Parkinson's study and fibroblasts were provided by the Integrated Biobank Luxembourg (IBBL) within the framework of the National Centre for Excellence in Research on Parkinson's disease (NCER-PD 48 ). All fibroblasts were grown in cell culture approved flasks and plates (BD Bioscience, Heidelberg, Germany; Corning, Kaiserslautern, Germany; Greiner Bio-One GmbH, Frickenhausen, Germany; Thermo Fisher Scientific, Braunschweig, Germany) with DMEM +/+ medium (4.5 g/l Glucose + 15% FBS + 1% Pen/Strep) and were incubated at 37 °C and 5% CO 2 . Fibroblasts were splitted with trypsin-EDTA (0.25%) phenol red (Thermo Fisher Scientific, Braunschweig, Germany). Cell cultures were tested for mycoplasma contamination once per month using the Plasmo Test™ Detection Kit (InvivoGen). For all experiments, fibroblasts were used below passage 9.  www.nature.com/scientificreports/ SEY chemiluminescence 2800 Fc (Li-COR, Lincoln, USA). Three independent samples per cell line (n = 3) were assessed. Image J software was used for relative signal quantification.
Mitochondrial fractionation for measurement of Aconitase 2 activity. For measurement of ACO2 enzyme activity, mitochondrial fractions were prepared from fibroblasts as described before 49 . Fibroblasts were collected using trypsin-EDTA (0.25%) phenol red (Thermo Fisher Scientific, Braunschweig, Germany). Obtained cell pellets contained ~ 6 × 10 6 to 10 × 10 6 cells and were mixed with 240 µl homogenization buffer (10 mM Tris, pH7.4; 1 mM EDTA; 250 mM Sucrose; Complete Protease Inhibitor (Roche, Germany)) on ice. Cells were homogenized for 1 min and samples were subsequently mixed with homogenization buffer to a total volume of 4 ml. The homogenate was centrifuged for 10 min at 4 °C, 1500×g. The supernatant was centrifuged again for 10 min at 4 °C, 1500×g. The supernatant from this step was afterwards centrifuged for 10 min at 4 °C, 10,000 rpm and this step was repeated once. The resulting supernatant contained the cytoplasmic fraction and the pellet contained the crude mitochondria fraction.
Biochemical measurement of Aconitase 2 activity. The Aconitase-340 assay (Bioxytech, Foster City, USA) was used to measure enzymatic activity of ACO2, according to the manufacturer's protocol. Measurements were performed on the monochromatic spectrophotometer SPECORD 210Plus (Analytic Jena AG, Jena, Germany). Crude mitochondria fractions were used as described above. The OD values from the beginning (OD1) and the end (OD2) of the phase of linear increase was used to calculate the ACO2 enzyme activity. Absorption rate: �A 340 /min = OD2−OD1 time . Net rate was determined by subtraction of the blank rate form the samples rate: Net�A 340 /min = �A 340 /min Sample − �A 340 /min (Blank) . Aconitase activity (mU) was calculated in consideration of the molar extinction coefficient,ε , for NADPH ( ε (NADPH) = 6220 M −1 cm −1 , the temperature correction coefficient, c (c = 2.4435) and the assay dilution, d (d = 4): Aconitase activity ( mU) = NetA 340 /min c·ε · d . The enzyme activity of aconitase was corrected to the dilution and normalized to protein concentration of the sample. Sample Dilution Correction (mU 1 ) = mU Dilution . Protein Correction: mU 2 = mU 1 Protein concentration . mtDNA analysis. Mitochondrial major arc deletions, transcription-associated 7S DNA and copy number were quantified using a real-time PCR (RT-PCR) approach with TaqMan probes as previously described 50 . In brief, three probes targeting different regions within the mitochondrial genome are quantified simultaneously. A probe in the mtDNA gene ND1, which is located in the minor arc and typically spared from deletions, is measured relatively to a probe in the mtDNA gene ND4, which is located within the major arc in a region that is commonly affected by the 4977 bp deletion. In addition, with a probe targeting the mitochondrial D-loop, the abundance of 7S DNA is measured relative to ND1 to determine the proportion of mtDNA molecules currently undergoing transcription 51 . Finally, the nuclear encoded single-copy gene B2M was used as a reference to quantify the amount of wildtype mtDNA copies (ND1:B2M).
Seahorse-XF e 96 extracellular flux analyser. Measurement of oxygen consumption rate (OCR). We used whole cells to analyse OCR with the XF e 96 extracellular flux Analyzer (Seahorse Bioscience, USA) as previously reported 52,53 . Twenty-four hours before the measurement, 14,000 cells per well were seeded into SF96 cell culture microplates (Seahorse Bioscience, USA) and incubated over night at 37 °C. The preparation of the assay cartridge, assay media and cell culture microplate for the measurement was done according to manufacturer's protocol (Seahorse Bioscience, USA). During measurement, cells were subsequently treated with 1 µM Oligomycin, 250 nM FCCP and a 5 µM mixture of Rotenone and Antimycin A (all chemicals obtained from Sigma Aldrich, Germany). For normalization of OCR to total protein concentration per well, the cells were lysed in the well after OCR measurement, using a lysis buffer with Triton X-100 and Complete Protease Inhibitor (Roche Applied Science, Mannheim, Germany) for 10 min at room temperature. Protein concentrations were measured using Bradford solution (Bio-Rad Laboratories, Munich, Germany) according to the manufacturer's protocol. OCR raw data were normalized to total protein concentration in each well. www.nature.com/scientificreports/ Factor (FF = (perimeter 2 )/(4*π*area)) as indicator of mitochondrial branching and Aspect Ratio (AR = major axis/minor axis) as indicator of mitochondrial length. For each cell line, between 75 and 85 cells were imaged and analyzed.

Measurement of extracellular acidification rate (ECAR
Flow cytometry. Flow cytometry measurement was performed with the BD LSR Fortessa (Becton, Dickinson and Company, Ermbodegem, Belgium). At least 10,000 cells per sample were measured and analysed using the Flowjo software (Flowjo LLC, Oregon, USA). An unstained control was included in every experiment in order to determine the autofluorescence level of each fibroblast line. Fibroblasts were grown under standard conditions at a density of 200,000 cells per well in 6 well plates. For measurement of the mitochondrial membrane potential, cells were treated with 5 nM Valinomycin (Sigma Aldrich Chemie GmbH, Munich Germany) for 14 h. Fibroblasts were harvested with trypsin and stained with 20 µM TMRE (Thermo Fisher Scientific, Germany) for 20 min at 37 °C. For measurement of mitochondrial ROS, the growth medium was exchanged to low Glucose medium (DMEM 1.5 g/l Glucose + 1% Pen/Strep) without FBS 24 h prior to the experiment. Cells were subsequently treated with 20 nM Piericidin A (Santa Cruz, Dallas, Texas) for 14 h in order to inhibit complex I of the respiratory chain. Cells were harvested in trypsin and washed twice with HBSS (Thermo Fischer Scientific, Braunschweig, Germany Statistical analyses. Graph-Pad Prism 8.0 software was used to assess the statistical significance. All measurements were independently repeated three times or more, as indicated in the figure legends (n indicates the number of independent biological replicates). In order to account for small sample size, we used non-parametric tests throughout as detailed in the figure legends.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.