ETHE1 and MOCS1 deficiencies: Disruption of mitochondrial bioenergetics, dynamics, redox homeostasis and endoplasmic reticulum-mitochondria crosstalk in patient fibroblasts

Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical findings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identified in the same cells, while variable alterations in mitochondrial fusion and fission were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.

www.nature.com/scientificreports www.nature.com/scientificreports/ Mutation analysis. Genomic DNA from fibroblasts was extracted using a Quick-DNA TM Miniprep Plus Kit, Zymo Research, Irvine, CA, USA, according to manufacturer's instructions, followed by PCR to amplify desired regions on each gene. Gel extraction of the PCR product was performed with a Zymoclean TM Gel DNA recovery kit, Zymo Research, followed by Sanger sequencing.
Total RNA was isolated from fibroblasts using a Quick-RNA TM MiniPrep kit, Zymo Research, and cDNA was synthesized using SuperScript III First-Strand Synthesis System, Thermo Fisher Scientific, Waltham, MA, USA, according to manufacturer's instructions. cDNA was used for RT-PCR, and gel extraction of the PCR products was performed with the Zymoclean TM Gel DNA recovery kit, Zymo Research, followed by Sanger sequencing.
Mitochondrial respiration. The oxygen consumption rate (OCR) was measured with a Seahorse XF e 96 Extracellular Flux Analyzer, Agilent Technologies, Lexington, MA, USA. Cells were seeded in 96-well Seahorse tissue culture microplates pre-coated with poly-D-lysine (80,000 cells per well), Sigma-Aldrich Co., St. Louis, MO, USA, for cell adherence. Subsequently, the plate was incubated, prior to the assay, at 37 °C for 1 h without CO 2 in basal media containing unbuffered DMEM. The initial OCR was evaluated to establish the basal respiration, the ATP-linked respiration was determined following injection of the complex V inhibitor oligomycin and the maximal respiration following the injection of 1 µM of the uncoupling agent FCCP. All cell lines were measured with four to eight wells per cell line. After the assay, protein concentration in each well was measured using the DC ™ Protein Assay kit, Bio-Rad Laboratories, Hercules, CA, USA. Data were expressed in pmol/min/ mg of protein.
ATP production. ATP production was measured by bioluminescence using an ATP determination kit (ATPlite ™ ), PerkinElmer Inc., Waltham, MA, USA, according to the manufacturer's instructions. Luminescence was quantitated in a SpectraMax ® i3x Platform multi-mode microplate reader system, Molecular Devices LLC, Sunnyvale, CA, USA. After the assay, the protein concentration in each well was measured using the DC ™ Protein Assay kit, Bio-Rad Laboratories, Hercules, CA, USA. Data are reported in μmol of ATP produced/mg of protein.
Mitochondrial mass and superoxide production. Mitochondrial mass and superoxide production were Western blotting. Cells were grown in T175 flasks and, at 95-100% confluence, harvested by trypsinization, pelleted and stored at −80 °C. For whole cell lysate preparation, the pellets were resuspended in RIPA buffer containing a protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany. Homogenates were kept on ice for 30 min and mixed every 10 min, then centrifuged at 14,000 g for 10 min at 4 °C, and the supernatant was collected.

Statistical analysis.
Statistical analysis was performed with GraphPad 5.0 software. Student's t test or one-way ANOVA followed by Tukey multiple range test were applied for comparisons between groups. Differences were considered significant when P < 0.05.

Results
Mutation analysis and protein content of ETHE1, MOCS1 and SO. Sequencing of ETHE1, MOCS1, MOCS2 and MOCS3 from genomic DNA and cDNA was performed to confirm the previously reported mutations of fibroblasts from four patients with ETHE1 deficiency 30 and determine the mutation of a fibroblast cell line with MoCD (Supplementary Information: Table S1). Fibroblast levels of ETHE1 and MOCS1 (molybdenum cofactor biosynthesis protein 1; MOCS1A and MOCS1B; 2 enzymes encoded by MOCS1) proteins were then evaluated by western blotting (Fig. 1). Cell lines ETHE1-1, ETHE1-3 and ETHE1-4 had no immunodetectable ETHE1 protein (cell lines with exon deletion or splice site mutation) while ETHE1-2 had similar ETHE1 protein as control cells, indicating that the missense mutation in ETHE1-2 leading to the substitution of Asp165 by a Gly causes complete loss of ETHE1 activity. MOCS1 deficient cells had a reduced amount of MOCS1 (MOCS1A and MOCS1B), as well as SO.
Oxygen consumption and ATP production. As a measure of the bioenergetic state of patient fibroblasts oxygen consumption was determined using a Seahorse analyzer. Basal, maximal and ATP-linked respiration was decreased in all cell lines (Figs 2, 3 and Supplementary Fig. S1). Treatment of cells with 40 nM JP4-039 for 24 h increased these measurements in ETHE1-3 and ETHE1-4 ( Fig. 2) and MOCS1 deficient cells (Fig. 3). ATP production measured with ATPlite luminescence assay kit was also decreased in ETHE1-3 and ETHE1-4 and the MOCS1 deficient cells (Fig. 4). No alterations were observed in ETHE1-1 and ETHE1-2 cell lines. Taken together, these data show bioenergetic impairment in MOCS1 and some ETHE1 deficient fibroblasts.
Mitochondrial mass and dynamics. Mitochondrial mass is often increased in the face of respiratory chain dysfunction 40,43,44 , and so this parameter was evaluated in patient cells using the probe MitoTracker Green (Fig. 5A,B), a mitochondrial dye that localizes to mitochondria with minimal dependence on mitochondrial membrane potential. ETHE1-2, ETHE1-4 and MOCS1 deficient cells were found to have increased mitochondrial mass compared to control fibroblasts, while no differences were observed in ETHE1-1 and ETHE1-3 fibroblasts. Since alterations in mitochondrial mass might result from changes in mitochondrial dynamics, we measured the levels of the main proteins involved in mitochondrial fusion (mitofusin 1 -MFN1, mitofusin 2 -MFN2, and optic atrophy type 1 -OPA1) and fission (dynamin related protein 1 -DRP1). We also determined the phosphorylation of DRP1 on Ser637 and Ser616 as it has been reported that both total levels of DRP1 and its phosphorylation play a role in regulation of mitochondrial fission 45 . MFN1 and MFN2 content was decreased in ETHE1-1, whereas MFN1, MFN2 and OPA1 content was increased in ETHE1-3 ( Fig. 5C and Supplementary Fig. S2). DRP1 was increased in ETHE1-2 ( Fig. 5C and Supplementary Fig. S2). Furthermore, Ser637 phosphorylation of DRP1 was markedly decreased in all ETHE1 deficient cell lines, whereas Ser616 phosphorylation was mildly decreased in ETHE1-2, ETHE1-3 and ETHE1-4 ( Fig. 5C and Supplementary Fig. S2). MOCS1 deficient cells had lower levels of MFN1 and MFN2, but no alterations in OPA1 content, compared to control cells ( Fig. 5D and Supplementary  Fig. S3). DRP1 content was increased and Ser637 phosphorylation of DRP1 was decreased in this cell line ( Fig. 5D and Supplementary Fig. S3). Phosphorylation of Ser616 of DRP1 was not altered (Fig. 5D and Supplementary  Fig. S3).
Superoxide production. Since mitochondrial respiratory chain dysfunction is often associated with increased ROS production 40,46 , we measured superoxide levels in patients' fibroblasts using the MitoSOX Red probe. Superoxide levels were elevated in all ETHE1 fibroblasts and the MOCS1 deficient cell line compared to control cells (Fig. 6A,B). Treatment with 40 and 200 nM JP4-039 decreased superoxide levels in MOCS1 and all of the ETHE1 deficient fibroblasts (Fig. 7A-E). In contrast, N-acetylcysteine, an antioxidant currently used as a therapeutic strategy for ETHE1 deficiency, had no effect on superoxide levels in ETHE1-4 (Fig. 7F).
Apoptosis. Cell death was evaluated by assay of annexin V (early apoptosis) and propidium iodide (PI) (late apoptosis and necrosis). Early stage apoptosis was increased in all patient cell lines (Fig. 8C,E, Supplementary  Figs S6 and S7), whereas late stage apoptosis and cell necrosis were increased only in ETHE1-1 and ETHE1-4 fibroblasts ( Fig. 8D and Supplementary Fig. S6).

Discussion
ETHE1 and MoCo deficiencies are devastating inborn errors in the metabolism of sulfur-containing amino acids. While respiratory chain dysfunction has been demonstrated and increased oxidative stress has been postulated, the precise mechanism of cellular pathophysiology remains unclear, and therapies remain largely ineffective. The aim of this study was to further examine cellular derangements in ETHE1 and MoCo deficient fibroblasts and evaluate their response to a novel mitochondrial antioxidant. After confirming the expected mutations in each cell line, we examined a series of parameters of mitochondrial bioenergetics and homeostasis. All of the patient fibroblasts demonstrated decreased mitochondrial respiration. This could be observed despite that previous studies showed reversion of H 2 S-induced inhibition of cytochrome c oxidase activity in ETHE1 deficient fibroblasts when exposed to air 14,49 . Furthermore, a recent article on ETHE1 deficient fibroblasts did not show impairment in mitochondrial respiration 50 . This discrepancy might be because they analyzed cells that had been adhering for more than a day whereas we seeded the cells in microplates pre-coated with poly-D-lysine and measured the respiration directly. On the other hand, this same study described a growth initiation phenotype of ETHE1 deficient  www.nature.com/scientificreports www.nature.com/scientificreports/ cells 50 , which is in line with our data on respiration defect of freshly adhered cells. The decreased respiration found here is further in line with data showing cytochrome c oxidase depletion in Ethe1 −/− mice, and impairment of mitochondrial energetic homeostasis caused by metabolites accumulating in ETHE1 deficiency and MoCD in rat tissues 15,27,28,51 . Additionally, MOCS1 and ETHE1 deficient cells showed reduced ATP production, confirming that bioenergetics is compromised in these cells.
Mitochondria are highly dynamic organelles that are constantly undergoing fusion and fission, controlling their shape and playing a fundamental role in the adaptation to disturbances in the cellular environment [52][53][54] . Changes in the cell bioenergetic state usually alter mitochondrial morphology and dynamics, leading to dysfunction 55 . We therefore investigated the content of the main proteins involved in mitochondrial dynamics, namely MFN1, MFN2, OPA1 (fusion) and DRP1 (fission). ETHE1-3 cells showed higher levels of all fusion proteins evaluated, while ETHE1-2 had increased levels of DRP1. Phosphorylation of DRP1 Ser637 and Ser616 was also determined as these post-translational modifications regulate DRP1-mediated fission. In this context, phosphorylation of Ser616 promotes Drp1-dependent mitochondrial fission, whereas Ser637 phosphorylation suppresses fission www.nature.com/scientificreports www.nature.com/scientificreports/ through the inhibition of the intramolecular interaction between GTPase and GED domains, thereby hampering the DRP1 GTPase activity and its recruitment to mitochondria 56,57 . While Ser637-phosphorylated DRP1 levels were markedly decreased in all cell lines, Ser616-phosphorylated DRP1 was mildly decreased in ETHE1-2, ETHE1-3 and ETHE1-4 deficient cells. Mitochondrial mass was further evaluated and an increase in this parameter was found in ETHE1-2 and ETHE1-4 cells. These changes suggest a variety of responses to maintain cellular energy homeostasis and reduce ROS levels [58][59][60][61] . MOCS1 deficient cells had a reduction in MFN1 and MFN2, an increase in DRP1, a decrease in Ser637-phosphorylated DRP1 and enhanced mitochondrial mass, indicating severe mitochondrial dysfunction and an increased number of smaller mitochondria.
The alterations in mitochondrial bioenergetics and dynamics were accompanied by increased superoxide production in all cell lines studied, the first direct demonstration of this finding, and consistent with previous data showing an abnormal proteome and decreased levels of reduced glutathione in ETHE1 deficiency cells, indicating disruption of redox homeostasis 30,50 . Moreover, accumulation of H 2 S and sulfite in induced ETHE1 and MoCo deficiency, respectively, elicits an oxidative stress response in rat brain 27,28,51,62 . Mitochondria are the main source of superoxide generation, predominantly in the redox centers of the respiratory chain complexes I and III from which electrons may leak during oxidative phosphorylation, leading to the incomplete reduction of oxygen. The physiological production of ROS by these sites plays an important role in cellular signaling, but the exacerbation of this process has been implicated in several diseases [63][64][65][66][67] since ROS may cause structural damage that exceeds cellular repair capacity, triggering cellular dysfunction 63 . Additionally, production of ROS induced by other cellular sources or accumulated metabolites may also result in secondary impairment of the respiratory chain components with further increase of superoxide production by mitochondria [68][69][70] .
Mitochondrial bioenergetic dysfunction and oxidative stress are associated with disruption of ER-mitochondria communication and ER stress 47,[71][72][73] . Crosstalk between mitochondria and ER is crucial for the regulation of mitochondrial dynamics and the signaling of calcium in cells, and is accomplished through the interaction of the VDAC1 in mitochondria and the IP3R in the ER, which are anchored by Grp75 [74][75][76] . In our ETHE1 and MOCS1 deficient cells, a reduced content of VDAC1 and IP3R was found, implying a disturbance in the ER-mitochondria crosstalk. Furthermore, DDIT3, an ER stress marker, was increased in all cell lines. The upregulation of DDIT3 is consistent with an increase in apoptosis induction observed in these cells, since this transcription factor is known to modulate this process 77 .
Since increased ROS production contributes to the damage of a series of biomolecules, including respiratory chain components [68][69][70] , it is reasonable to expect that the mitigation of ROS production would be of therapeutic value in ETHE1 and MoCo deficiencies. We have previously demonstrated that treatment with the novel mitochondria-targeted antioxidant and electron scavenger JP4-039, reduces ROS levels in cells with impairments in oxidative phosphorylation, while other clinically used antioxidants did not 40 . In the current study, we also verify that JP4-039 ameliorates ROS accumulation in ETHE1 and MOCS1 deficient fibroblasts and improves mitochondrial respiration. These findings are particularly interesting since previous data showed that JP4-039 crosses brain-blood barrier in mice 78 and ETHE1 and MOCS1 deficiencies are inborn errors mainly characterized by neurological dysfunction. Furthermore, N-acetylcysteine, an antioxidant currently used to treat ETHE1 deficient patients 10,13,79,80 , did not affect superoxide levels, demonstrating the importance of mitochondrial targeting for the biological efficacy of free radical scavengers.
Interestingly, the different ETHE1 deficient fibroblast cell lines showed some heterogenous alterations that could be at least partially explained by the different mutations. However, even different cell lines with the same mutation (ETHE1-1 and ETHE1-4) showed some variability in mitochondrial function, consistent with previous conclusions that there is no clear genotype-phenotype correlation in this disease [80][81][82] . The reasons for these variations and their correlation with clinical symptoms remain to be clarified, and may ultimately be useful for the identification of additional specific therapies. Furthermore, it is difficult to extrapolate the pathophysiological relevance of fibroblast findings to other tissues, though, in general, it is harder to demonstrate bioenergetic dysfunction in fibroblasts than in more mitochondria-rich tissues. Thus, it is likely that our fibroblast findings are relevant to tissues with higher energetic needs. www.nature.com/scientificreports www.nature.com/scientificreports/ Red. Data are presented as mean ± SD; number of replicates: 3-4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared to control (wild type) cells; # P < 0.05, ## P < 0.01, compared to patient cells (Tukey multiple range test).

Conclusions
In summary, we have demonstrated that fibroblasts from patients with ETHE1 deficiency and MoCD have impairments in mitochondrial bioenergetics and dynamics, as well as disruption of ER-mitochondria crosstalk. High superoxide levels identified in these cells presumably play an important role in the cellular pathophysiology, Figure 8. Disruption of endoplasmic reticulum-mitochondria crosstalk and apoptosis in ETHE1 and MOCS1 deficient fibroblasts. Inositol 1,4,5-trisphosphate receptor (IP3R), glucose-related protein 75 (Grp75), voltagedependent anion-selective channel 1 (VDAC1), glucose-related protein 78 (Grp78) and DNA damage inducible transcript 3 (DDIT3) protein content was evaluated in whole cell lysates prepared from ETHE1 (A) and MOCS1 (B) deficient fibroblasts. β-Actin or GAPDH was used as loading controls. Representative images are shown. Apoptosis and necrosis in ETHE1 (C,D) and MOCS1 (E,F) deficient fibroblasts were measured by flow cytometry. Data are presented as mean ± SD; number of replicates: 3-5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared to control (wild type) (t test for unpaired samples).