Replicative Stress Coincides with Impaired Nuclear DNA Damage Response in COX4-1 Deficiency.

Cytochrome c oxidase (COX), a multimeric protein complex, is the final electron acceptor in the mitochondrial electron transfer chain. Primary COX deficiency, caused by mutations in either mitochondrial DNA or nuclear-encoded genes, is a heterogenous group of mitochondrial diseases with a wide range of presentations, ranging from fatal infantile to subtler. We previously reported a patient with primary COX deficiency due to a pathogenic variant in COX4I1 (encoding the common isoform of COX subunit 4, COX4-1), who presented with bone marrow failure, genomic instability, and short stature, mimicking Fanconi anemia (FA). In the present study, we demonstrated that accumulative DNA damage coincided primarily with proliferative cells in the patient's fibroblasts and in COX4i1 knockdown cells. Expression analysis implicated a reduction in DNA damage response pathways, which was verified by demonstrating impaired recovery from genotoxic insult and decreased DNA repair. The premature senescence of the COX4-1-deficient cells prevented us from undertaking additional studies; nevertheless, taken together, our results indicate replicative stress and impaired nuclear DNA damage response in COX4-1 deficiency. Interestingly, our in vitro findings recapitulated the patient's presentation and present status.

Two transgenic mouse models for ß-subunit components of succinate-CoA ligase yielding pleiotropic metabolic alterations.

Succinate-CoA ligase (SUCL) is a heterodimer enzyme composed of Suclg1 α-subunit and a substrate-specific Sucla2 or Suclg2 ß-subunit yielding ATP or GTP, respectively. In humans, the deficiency of this enzyme leads to encephalomyopathy with or without methylmalonyl aciduria, in addition to resulting in mitochondrial DNA depletion. We generated mice lacking either one Sucla2 or Suclg2 allele. Sucla2 heterozygote mice exhibited tissue- and age-dependent decreases in Sucla2 expression associated with decreases in ATP-forming activity, but rebound increases in cardiac Suclg2 expression and GTP-forming activity. Bioenergetic parameters including substrate-level phosphorylation (SLP) were not different between wild-type and Sucla2 heterozygote mice unless a submaximal pharmacological inhibition of SUCL was concomitantly present. mtDNA contents were moderately decreased, but blood carnitine esters were significantly elevated. Suclg2 heterozygote mice exhibited decreases in Suclg2 expression but no rebound increases in Sucla2 expression or changes in bioenergetic parameters. Surprisingly, deletion of one Suclg2 allele in Sucla2 heterozygote mice still led to a rebound but protracted increase in Suclg2 expression, yielding double heterozygote mice with no alterations in GTP-forming activity or SLP, but more pronounced changes in mtDNA content and blood carnitine esters, and an increase in succinate dehydrogenase activity. We conclude that a partial reduction in Sucla2 elicits rebound increases in Suclg2 expression, which is sufficiently dominant to overcome even a concomitant deletion of one Suclg2 allele, pleiotropically affecting metabolic pathways associated with SUCL. These results as well as the availability of the transgenic mouse colonies will be of value in understanding SUCL deficiency.

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion.

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the ß subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link. We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.

Combined OXPHOS complex I and IV defect, due to mutated complex I assembly factor C20ORF7.

Defects of the mitochondrial oxidative phosphorylation (OXPHOS) system are frequent causes of neurological disorders in children. Linkage analysis and DNA sequencing identified a new founder p.G250V substitution in the C20ORF7 complex I chaperone in five Ashkenazi Jewish patients from two families with a combined OXPHOS complex I and IV defect presenting with Leigh's syndrome in infancy. Complementation with the wild type gene restored complex I, but only partially complex IV activity. Although the pathogenic mechanism remains elusive, a C20ORF7 defect should be considered not only in isolated complex I deficiency, but also in combination with decreased complex IV. Given the significant 1:290 carrier rate for the p.G250V mutation among Ashkenazi Jews, this mutation should be screened in all Ashkenazi patients with Leigh's syndrome prior to muscle biopsy.

Variable effects of omaveloxolone (RTA408) on primary fibroblasts with mitochondrial defects.

Omaveloxolone (RTA408) is a second-generation oleanane triterpenoid Nrf2 inducer with antioxidant and anti-inflammatory properties and was reported to improve mitochondrial bioenergetics. It is currently being tested in medical trials for Friedrich ataxia, a genetic, multi-organ disease involving mitochondrial dysfunction. Thus, omaveloxolone could potentially be beneficial for additional disorders involving mitochondrial dysfunction. To this end, we investigated its effect on primary fibroblasts derived from patients with mitochondrial complex I deficiency, mitochondrial cytochrome oxidase deficiency, and two recessive forms of Parkinson's disease. Patients and control cells were incubated in the presence or absence of 50 nM omaveloxolone for 72 h prior to measurements. Generally, growth on galactose medium and ATP production were unaltered. Mitochondrial membrane potential was slightly but significantly decreased, while reactive oxygen species (ROS) production was variably decreased. Mitochondrial mass and mitochondrial DNA (mtDNA) contents were significantly increased in the patient's cells. These results were partially confirmed by the results of oxygen consumption studies which disclosed increased maximal oxygen consumption rates in most cells and increased energy status in all treated cells. Further investigation is required to explore the precise effect of omaveloxolone on mitochondrial function in disease.

C6ORF66 is an assembly factor of mitochondrial complex I.

Homozygosity mapping was performed in five patients from a consanguineous family who presented with infantile mitochondrial encephalomyopathy attributed to isolated NADH:ubiquinone oxidoreductase (complex I) deficiency. This resulted in the identification of a missense mutation in a conserved residue of the C6ORF66 gene, which encodes a 20.2 kDa mitochondrial protein. The mutation was also detected in a patient who presented with antenatal cardiomyopathy. In muscle of two patients, the levels of the C6ORF66 protein and of the fully assembled complex I were markedly reduced. Transfection of the patients' fibroblasts with wild-type C6ORF66 cDNA restored complex I activity. These data suggest that C6ORF66 is an assembly factor of complex I. Interestingly, the C6ORF66 gene product was previously shown to promote breast cancer cell invasiveness.

Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency.

Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the patient's cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were quantified by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a compensatory mechanism in COX4-1 deficiency.

The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria.

Mutations in mitochondrial small subunit ribosomal proteins MRPS16 or MRPS22 cause severe, fatal respiratory chain dysfunction due to impaired translation of mitochondrial mRNAs. The loss of either MRPS16 or MRPS22 was accompanied by the loss of most of another small subunit protein MRPS11. However, MRPS2 was reduced only about 2-fold in patient fibroblasts. This observation suggests that the small ribosomal subunit is only partially able to assemble in these patients. Two large subunit ribosomal proteins, MRPL13 and MRPL15, were present in substantial amounts suggesting that the large ribosomal subunit is still present despite a non-functional small subunit.

Mutation in the COX4I1 gene is associated with short stature, poor weight gain and increased chromosomal breaks, simulating Fanconi anemia.

We describe a novel autosomal recessive form of mitochondrial disease in a child with short stature, poor weight gain, and mild dysmorphic features with highly suspected Fanconi anemia due to a mutation in COX4I1 gene. Whole Exome Sequencing was performed then followed by Sanger confirmation, identified a K101N mutation in COX4I1, segregating with the disease. This nuclear gene encodes the common isoform of cytochrome c oxidase (COX) subunit 4 (COX 4-1), an integral regulatory part of COX (respiratory chain complex IV) the terminal electron acceptor of the mitochondrial respiratory chain. The patient's fibroblasts disclosed decreased COX activity, impaired ATP production, elevated ROS production, decreased expression of COX4I1 mRNA and undetectable (COX4) protein. COX activity and ATP production were restored by lentiviral transfection with the wild-type gene. Our results demonstrate the first human mutation in the COX4I1 gene linked to diseases and confirm its role in the pathogenesis. Thus COX4I1 mutations should be considered in any patient with features suggestive of this diagnosis.

Mitochondrial complex IV deficiency, caused by mutated COX6B1, is associated with encephalomyopathy, hydrocephalus and cardiomyopathy.

Isolated cytochrome c oxidase (COX) deficiency is a prevalent cause of mitochondrial disease and is mostly caused by nuclear-encoded mutations in assembly factors while rarely by mutations in structural subunits. We hereby report a case of isolated COX deficiency manifesting with encephalomyopathy, hydrocephalus and hypertropic cardiomyopathy due to a missense p.R20C mutation in the COX6B1 gene, which encodes an integral, nuclear-encoded COX subunit. This novel mutation was predicted to be severe in silico. In accord, enzymatic activity was undetectable in muscle and fibroblasts, was severely decreased in lymphocytes and the COX6B1 protein was barely detectable in patient's muscle mitochondria. Complementation with the wild-type cDNA by a lentiviral construct restored COX activity, and mitochondrial function was improved by 5-aminoimidazole-4-carboxamide ribonucleotide, resveratrol and ascorbate in the patient's fibroblasts. We suggest that genetic analysis of COX6B1should be included in the investigation of isolated COX deficiency, including patients with cardiac defects. Initial measurement of COX activity in lymphocytes may be useful as it might circumvent the need for invasive muscle biopsy. The evaluation of ascorbate supplementation to patients with mutated COX6B1 is warranted.

Human granulosa luteal cell oxidative phosphorylation function is not affected by age or ovarian response.

OBJECTIVE: To safely prepare a functional autologous mitochondrial concentrate (MC) from follicular fluid (FF) cells, and to determine the effect of age and ovarian response on the oxidative phosphorylation (OXPHOS). DESIGN: The nontoxicity of the MC was confirmed in human and mouse oocytes. The OXPHOS function was assessed by measuring the activity of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX), and adenosine triphosphate (ATP) production in comparison with citrate synthase. The integrity of the mitochondrial DNA (mtDNA) was demonstrated by polymerase chain reaction (PCR). SETTING: Tertiary hospital. PATIENT(S): A total of 40 patients undergoing IVF of heterogeneous ages and ovarian response. ANIMAL(S): Superovulated 8- to 12-week-old female B(6)C(3)F(1) mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): A system for the preparation of functional nontoxic MC was established. The correlation between the mitochondrial mass and function to age and ovarian response was calculated. The integrity of mtDNA was demonstrated. RESULT(S): After injection into mouse oocytes, the MC did not interfere with parthenogenetic development. The MC OXPHOS function was intact. Total activity of SDH and COX was in correlation with the retrieved oocytes number, and in reverse correlation with age. However, after correction to the mitochondrial mass, COX and SDH activities were constant, unaffected by age or ovarian response. The mtDNA was intact in all samples, regardless of age and ovarian response. CONCLUSION(S): The function of the respiratory chain in mitochondria of FF cells is constant, unaffected by age or ovarian response.

Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy.

The oxidative phosphorylation (OXPHOS) system is under control of both the mitochondrial and the nuclear genomes; 13 subunits are synthesized by the mitochondrial translation machinery. We report a patient with Cornelia de Lange-like dysmorphic features, brain abnormalities and hypertrophic cardiomyopathy, and studied the genetic defect responsible for the combined OXPHOS complex I, III and IV deficiency observed in fibroblasts. The combination of deficiencies suggested a primary defect associated with the synthesis of mitochondrially encoded OXPHOS subunits. Analysis of mitochondrial protein synthesis revealed a marked impairment in mitochondrial translation. Homozygosity mapping and sequence analysis of candidate genes revealed a homozygous mutation in MRPS22, a gene encoding a mitochondrial ribosomal small subunit protein. The mutation predicts a Leu215Pro substitution at an evolutionary conserved site. Mutations in genes implicated in Cornelia de Lange syndrome or copy number variations were not found. Transfection of patient fibroblasts, in which MRPS22 was undetectable, with the wild-type MRPS22 cDNA restored the amount and activity of OXPHOS complex IV, as well as the 12S rRNA transcript level to normal values. These findings demonstrate the pathogenicity of the MRPS22 mutation and stress the significance of mutations in nuclear genes, including genes that have no counterparts in lower species like bacteria and yeast, for mitochondrial translation defects.

Antibiotic effects on mitochondrial translation and in patients with mitochondrial translational defects.

The infantile presentation of mitochondrial respiratory chain defects frequently simulates acute bacterial infection and sepsis. Consequently, broad spectrum antibiotic therapy is often initiated before definitive diagnosis is reached and without taking into consideration the potential harm of antibiotics affecting mitochondrial translation. Here, we demonstrate that some commonly used translation-targeted antibiotics adversely affect the growth of fibroblasts from patients with defective mitochondrial translation systems. In addition, we show that these antibiotics inhibit mitochondrial translation in vitro. Our results suggest that patients with mitochondrial translation defects may be more vulnerable to toxic-side-effects following the administration of certain translation-targeted antibiotics.

Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion.

The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the beta subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome.

Evaluation of enzymatic assays and compounds affecting ATP production in mitochondrial respiratory chain complex I deficiency.

Isolated complex I deficiency is the most common oxidative phosphorylation defect and is associated with substantial morbidity and mortality. The diagnosis is made by enzymatic analysis and for most patients the molecular pathology remains undefined. Various cofactors and vitamins are frequently administered, but their efficacy have been difficult to assess. We employed determination of ATP production in fibroblast cell lines from patients with complex I deficiency to evaluate the usefulness of therapeutic agents. The effect of each additive varied among the different patients with certain agents favorably affecting ATP production rate in some of the patients and adversely affecting it in others. The reduced nicotinamide adenine dinucleotide (NADH)-ferricyanide reductase assay in muscle mitochondria correlated better than the NADH-coenzyme Q and NADH-cytochrome c assays with ATP production rate in fibroblasts. Our results underscore the necessity of evaluation of different agents for each patient separately. The NADH-ferricyanide reductase assay play a helpful role in directing mutation analysis and identifying patients which are more likely to have their cells amenable for ATP production assessment.

Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency.

Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.

Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation.

The mitochondrial respiratory chain comprises 85 subunits, 13 of which are mitochondrial encoded. The synthesis of these 13 proteins requires many nuclear-encoded proteins that participate in mitochondrial DNA replication, transcript production, and a distinctive mitochondrial translation apparatus. We report a patient with agenesis of corpus callosum, dysmorphism, and fatal neonatal lactic acidosis with markedly decreased complex I and IV activity in muscle and liver and a generalized mitochondrial translation defect identified in pulse-label experiments. The defect was associated with marked reduction of the 12S rRNA transcript level likely attributed to a nonsense mutation in the MRPS16 gene. A new group of mitochondrial respiratory chain disorders is proposed, resulting from mutations in nuclear encoded components of the mitochondrial translation apparatus.