Metabolic shift underlies recovery in reversible infantile respiratory chain deficiency.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues.

Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns of WGBS, and develop and assess the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). Our approach circumvents confounders by enriching for full-length molecules over nuclear DNA. Variant calling analysis against showed that 99.5% of homoplasmic mtDNA variants can be reliably identified providing there is adequate sequencing depth. We show that some of the mtDNA methylation signal detected by ONS is due to sequence-specific false positives introduced by the technique. The residual signal was observed across several human primary and cancer cell lines and multiple human tissues, but was always below the error threshold modelled using negative controls. We conclude that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies. Additionally, we developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.

Correction: Background sequence characteristics influence the occurrence and severity of disease-causing mtDNA mutations.

[This corrects the article DOI: 10.1371/journal.pgen.1007126.].

Background sequence characteristics influence the occurrence and severity of disease-causing mtDNA mutations.

Inherited mitochondrial DNA (mtDNA) mutations have emerged as a common cause of human disease, with mutations occurring multiple times in the world population. The clinical presentation of three pathogenic mtDNA mutations is strongly associated with a background mtDNA haplogroup, but it is not clear whether this is limited to a handful of examples or is a more general phenomenon. To address this, we determined the characteristics of 30,506 mtDNA sequences sampled globally. After performing several quality control steps, we ascribed an established pathogenicity score to the major alleles for each sequence. The mean pathogenicity score for known disease-causing mutations was significantly different between mtDNA macro-haplogroups. Several mutations were observed across all haplogroup backgrounds, whereas others were only observed on specific clades. In some instances this reflected a founder effect, but in others, the mutation recurred but only within the same phylogenetic cluster. Sequence diversity estimates showed that disease-causing mutations were more frequent on young sequences, and genomes with two or more disease-causing mutations were more common than expected by chance. These findings implicate the mtDNA background more generally in recurrent mutation events that have been purified through natural selection in older populations. This provides an explanation for the low frequency of mtDNA disease reported in specific ethnic groups.

Mutations in mitochondrial DNA causing tubulointerstitial kidney disease.

Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A>T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNAPhe, tRNALeu1 and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNAPhe (m.616T>C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNAPhe.

Heterozygous SSBP1 start loss mutation co-segregates with hearing loss and the m.1555A>G mtDNA variant in a large multigenerational family.

The m.1555A>G mtDNA variant causes maternally inherited deafness, but the reasons for the highly variable clinical penetrance are not known. Exome sequencing identified a heterozygous start loss mutation in SSBP1, encoding the single stranded binding protein 1 (SSBP1), segregating with hearing loss in a multi-generational family transmitting m.1555A>G, associated with mtDNA depletion and multiple deletions in skeletal muscle. The SSBP1 mutation reduced steady state SSBP1 levels leading to a perturbation of mtDNA metabolism, likely compounding the intra-mitochondrial translation defect due to m.1555A>G in a tissue-specific manner. This family demonstrates the importance of rare trans-acting genetic nuclear modifiers in the clinical expression of mtDNA disease.

Altered RNA metabolism due to a homozygous RBM7 mutation in a patient with spinal motor neuropathy.

The exosome complex is the most important RNA processing machinery within the cell. Mutations in its subunits EXOSC8 and EXOSC3 cause pontocerebellar hypoplasia, spinal muscular atrophy (SMA) and central nervous system demyelination. We present a patient with SMA-like phenotype carrying a homozygous mutation in RBM7-a subunit of the nuclear exosome targeting (NEXT) complex-which is known to bind and carry specific subtypes of coding and non-coding RNAs to the exosome. The NEXT complex with other protein complexes is responsible for the substrate specificity of the exosome. We performed RNA-sequencing (RNA-seq) analysis on primary fibroblasts of patients with mutations in EXOSC8 and RBM7 and gene knock-down experiments using zebrafish as a model system. RNA-seq analysis identified significantly altered expression of 62 transcripts shared by the two patient cell lines. Knock-down of rbm7, exosc8 and exosc3 in zebrafish showed a common pattern of defects in motor neurons and cerebellum. Our data indicate that impaired RNA metabolism may underlie the clinical phenotype by fine tuning gene expression which is essential for correct neuronal differentiation.

Recent mitochondrial DNA mutations increase the risk of developing common late-onset human diseases.

Mitochondrial DNA (mtDNA) is highly polymorphic at the population level, and specific mtDNA variants affect mitochondrial function. With emerging evidence that mitochondrial mechanisms are central to common human diseases, it is plausible that mtDNA variants contribute to the "missing heritability" of several complex traits. Given the central role of mtDNA genes in oxidative phosphorylation, the same genetic variants would be expected to alter the risk of developing several different disorders, but this has not been shown to date. Here we studied 38,638 individuals with 11 major diseases, and 17,483 healthy controls. Imputing missing variants from 7,729 complete mitochondrial genomes, we captured 40.41% of European mtDNA variation. We show that mtDNA variants modifying the risk of developing one disease also modify the risk of developing other diseases, thus providing independent replication of a disease association in different case and control cohorts. High-risk alleles were more common than protective alleles, indicating that mtDNA is not at equilibrium in the human population, and that recent mutations interact with nuclear loci to modify the risk of developing multiple common diseases.

Mitochondrial dysfunction in liver failure requiring transplantation.

Liver failure is a heterogeneous condition which may be fatal and the primary cause is frequently unknown. We investigated mitochondrial oxidative phosphorylation in patients undergoing liver transplantation. We studied 45 patients who had liver transplantation due to a variety of clinical presentations. Blue native polyacrylamide gel electrophoresis with immunodetection of respiratory chain complexes I-V, biochemical activity of respiratory chain complexes II and IV and quantification of mitochondrial DNA (mtDNA) copy number were investigated in liver tissue collected from the explanted liver during transplantation. Abnormal mitochondrial function was frequently present in this cohort: ten of 40 patients (25 %) had a defect of one or more respiratory chain enzyme complexes on blue native gels, 20 patients (44 %) had low activity of complex II and/or IV and ten (22 %) had a reduced mtDNA copy number. Combined respiratory chain deficiency and reduced numbers of mitochondria were detected in all three patients with acute liver failure. Low complex IV activity in biliary atresia and complex II defects in cirrhosis were common findings. All six patients diagnosed with liver tumours showed variable alterations in mitochondrial function, probably due to the heterogeneity of the presenting tumour. In conclusion, mitochondrial dysfunction is common in severe liver failure in non-mitochondrial conditions. Therefore, in contrast to the common practice detection of respiratory chain abnormalities in liver should not restrict the inclusion of patients for liver transplantation. Furthermore, improving mitochondrial function may be targeted as part of a complex therapy approach in different forms of liver diseases.

Mitochondrial antibiograms in personalized medicine.

Some ribosomal antibiotics used in clinical practice to fight pathogenic bacteria can provoke serious adverse drug reactions in patients. Sensitivity to the antibiotics is a multifactorial trait but the genetic variation of sensitive individuals to off-target effects of the drugs might be one of the factors contributing to this condition. Thus, the protein synthesis apparatus of mitochondria is similar to that of bacteria because of its endosymbiotic origin and, therefore, mitochondrial ribosomes are frequently unintended off-targets of these antibiotics. Because of the limitations of epidemiologic studies of pharmacogenomics, we constructed 25 transmitochondrial cell lines using platelets from individuals belonging to high-frequency European mitochondrial DNA (mtDNA) haplogroups and grew them in the absence or presence of commonly used ribosomal antibiotics. Next, we analyzed the mitochondrial synthesis of proteins and the mitochondrial oxygen consumption to ascertain whether some side effects of ribosomal drugs are due to their interaction with particular mtDNA haplogroup-defining polymorphisms. The amount of mitochondrial translation products, the p.MT-CO1/succinate dehydrogenase subunit A ratio and the ratio of respiratory complex IV quantity to citrate synthase (CS)-specific activity were significantly lower, after the treatment with linezolid, in cybrids harboring the highly frequent m.3010A allele. These results suggest that mitochondrial antibiograms should be implemented for at least the most frequent mitochondrial ribosomal RNA (rRNA) polymorphisms and combinations of polymorphisms and the most frequently used ribosomal antibiotics. In this way, we would obtain individualized barcodes for antibiotic therapy, avoid the side effects of the antibiotics and enable appropriate personalized medicine.

Altered 2-thiouridylation impairs mitochondrial translation in reversible infantile respiratory chain deficiency.

Childhood-onset mitochondrial encephalomyopathies are severe, relentlessly progressive conditions. However, reversible infantile respiratory chain deficiency (RIRCD), due to a homoplasmic mt-tRNA(Glu) mutation, and reversible infantile hepatopathy, due to tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) deficiency, stand out by showing spontaneous recovery, and provide the key to treatments of potential broader relevance. Modification of mt-tRNA(Glu) is a possible functional link between these two conditions, since TRMU is responsible for 2-thiouridylation of mt-tRNA(Glu), mt-tRNA(Lys) and mt-tRNA(Gln). Here we show that down-regulation of TRMU in RIRCD impairs 2-thiouridylation and exacerbates the effect of the mt-tRNA(Glu) mutation by triggering a mitochondrial translation defect in vitro. Skeletal muscle of RIRCD patients in the symptomatic phase showed significantly reduced 2-thiouridylation. Supplementation with l-cysteine, which is required for optimal TRMU function, rescued respiratory chain enzyme activities in human cell lines of patients with RIRCD as well as deficient TRMU. Our results show that l-cysteine supplementation is a potential treatment for RIRCD and for TRMU deficiency, and is likely to have broader application for the growing group of intra-mitochondrial translation disorders.

Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging.

Macroautophagy decreases with age, and this change is considered a hallmark of the aging process. It remains unknown whether mitophagy, the essential selective autophagic degradation of mitochondria, also decreases with age. In our analysis of mitophagy in multiple organs in the mito-QC reporter mouse, mitophagy is either increased or unchanged in old versus young mice. Transcriptomic analysis shows marked upregulation of the type I interferon response in the retina of old mice, which correlates with increased levels of cytosolic mtDNA and activation of the cGAS/STING pathway. Crucially, these same alterations are replicated in primary human fibroblasts from elderly donors. In old mice, pharmacological induction of mitophagy with urolithin A attenuates cGAS/STING activation and ameliorates deterioration of neurological function. These findings point to mitophagy induction as a strategy to decrease age-associated inflammation and increase healthspan.

Oxidative phosphorylation differences between mitochondrial DNA haplogroups modify the risk of Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a maternally inherited optic atrophy caused by mitochondrial DNA point mutations. Previous epidemiological studies have shown that individuals from mitochondrial genetic backgrounds (haplogroups) J/Uk and H have a higher and a lower risk, respectively, of suffering this disorder. To analyze the bases of these associations at cellular and molecular levels, functional studies with cybrids provide high quality evidence. Cybrids from haplogroup J contain less mitochondrial deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and synthesize a smaller amount of mitochondrial DNA-encoded polypeptides than those from haplogroup H. Haplogroup J cybrids also display lower oxygen consumption, mitochondrial inner membrane potential and total adenosine-5'-triphosphate (ATP) levels. Moreover, mitochondrial DNA levels correlate with many parameters of the oxidative phosphorylation system. These results suggest that the mitochondrial DNA amount determines oxidative phosphorylation capacity and, along with other recently published observations, support the possibility that mitochondrial DNA levels may be responsible for the bias of the disorder toward males, for the incomplete penetrance of mutations causing Leber's hereditary optic neuropathy and for the association of the disease with particular mitochondrial DNA haplogroups.

'Progress' renders detrimental an ancient mitochondrial DNA genetic variant.

A human mitochondrial DNA (mtDNA) transition, m.1555A>G, in the 12S rRNA gene causes non-syndromic hearing loss. However, this pathological mutation is the wild-type allele in orangutan mtDNA. Here we rule out different genetic factors as the reason for its fixation in orangutans and show that aminoglycosides negatively affect the oxidative phosphorylation function by decreasing the synthesis of mtDNA-encoded proteins and the amount and activity of respiratory complex IV. These drugs also diminish the growth rate of orangutan cells. The m.1555G nucleotide is also the wild-type allele in other mammal species and they might be at risk of suffering a mitochondrial disorder if treated with aminoglycosides. Therefore, pharmacogenomic approaches should be used to confirm this possibility. These observations are important for human health. Due to the fact that old age and high frequency are criteria widely used in mitochondrial medicine to rule out a genetic change as being a pathological mutation, our results prevent against simplistic genetic approaches that do not consider the potential effect of environmental conditions. Hence, these results suggest that some ancient and highly frequent human population polymorphisms, such as those defining mtDNA haplogroups, in mitochondrial rRNA genes can be deleterious in association with new environmental conditions. Therefore, as the discovery of ribosomal antibiotics has allowed to fight infectious diseases and this breakthrough can be considered an important scientific advance or 'progress', our results suggest that 'progress' can also have a negative counterpart and render detrimental many of these mtDNA genotypes.

Stressed cybrids model demyelinated axons in multiple sclerosis.

Multiple sclerosis is likely caused by a complex interaction of multiple genes and environmental factors. The contribution of mitochondrial DNA genetic backgrounds has been frequently reported. To evaluate the effect of mitochondrial DNA haplogroups in the same genetic and environmental circumstances, we have built human transmitochondrial cell lines and simulated the effect of axon demyelination, one of the hallmarks of multiple sclerosis pathology, by altering the ionic gradients through the plasmalemma and increasing ATP consumption. In this model, mitochondrial biogenesis is observed. This process is larger in Uk cybrids, which mirrors their lower oxidative phosphorylation capacity in basal conditions. This model replicates a process occurring in both patients suffering from multiple sclerosis and several animal models of axon demyelination. Therefore, it can be used to analyze the contribution of various mitochondrial DNA genotypes to multiple sclerosis. In this sense, a longer or stronger energy stress, such as that associated with demyelinated axons in multiple sclerosis, could make Uk individuals more susceptible to this pathology. Finally, pharmacologic compounds targeted to mitochondrial biogenesis could be a potential therapy for multiple sclerosis.

Unmasking the causes of multifactorial disorders: OXPHOS differences between mitochondrial haplogroups.

Many epidemiologic studies have associated human mitochondrial haplogroups to rare mitochondrial diseases like Leber's hereditary optic neuropathy or to more common age-linked disorders such as Parkinson's disease. However, cellular, biochemical and molecular-genetic evidence that is able to explain these associations is very scarce. The etiology of multifactorial diseases is very difficult to sort out because such diseases are due to a combination of genetic and environmental factors that individually only contribute in small part to the development of the illness. Thus, the haplogroup-defining mutations might behave as susceptibility factors, but they could have only a small effect on oxidative phosphorylation (OXPHOS) function. Moreover, these effects would be highly dependent on the 'context' in which the genetic variant is acting. To homogenize this 'context' for mitochondrial DNA (mtDNA) mutations, a cellular approach is available that involves the use of what is known as 'cybrids'. By using this model, we demonstrate that mtDNA and mtRNA levels, mitochondrial protein synthesis, cytochrome oxidase activity and amount, normalized oxygen consumption, mitochondrial inner membrane potential and growth capacity are different in cybrids from the haplogroup H when compared with those of the haplogroup Uk. Thus, these inherited basal differences in OXPHOS capacity can help to explain why some individuals more quickly reach the bioenergetic threshold below which tissue symptoms appear and progress toward multifactorial disorders. Hence, some population genetic variants in mtDNA contribute to the genetic component of complex disorders. The existence of mtDNA-based OXPHOS differences opens possibilities for the existence of a new field, mitochondrial pharmacogenomics. New sequence accession nos: HM103354-HM103363.

p38 MAPK priming boosts VSMC proliferation and arteriogenesis by promoting PGC1α-dependent mitochondrial dynamics.

Vascular smooth muscle cell (VSMC) proliferation is essential for arteriogenesis to restore blood flow after artery occlusion, but the mechanisms underlying this response remain unclear. Based on our previous findings showing increased VSMC proliferation in the neonatal aorta of mice lacking the protease MT4-MMP, we aimed at discovering new players in this process. We demonstrate that MT4-MMP absence boosted VSMC proliferation in vitro in response to PDGF-BB in a cell-autonomous manner through enhanced p38 MAPK activity. Increased phospho-p38 in basal MT4-MMP-null VSMCs augmented the rate of mitochondrial degradation by promoting mitochondrial morphological changes through the co-activator PGC1α as demonstrated in PGC1α-/- VSMCs. We tested the in vivo implications of this pathway in a novel conditional mouse line for selective MT4-MMP deletion in VSMCs and in mice pre-treated with the p38 MAPK activator anisomycin. Priming of p38 MAPK activity in vivo by the absence of the protease MT4-MMP or by anisomycin treatment led to enhanced arteriogenesis and improved flow recovery after femoral artery occlusion. These findings may open new therapeutic opportunities for peripheral vascular diseases.

Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro.

Most humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1-2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

An atlas of mitochondrial DNA genotype-phenotype associations in the UK Biobank.

Mitochondrial DNA (mtDNA) variation in common diseases has been underexplored, partly due to a lack of genotype calling and quality-control procedures. Developing an at-scale workflow for mtDNA variant analyses, we show correlations between nuclear and mitochondrial genomic structures within subpopulations of Great Britain and establish a UK Biobank reference atlas of mtDNA-phenotype associations. A total of 260 mtDNA-phenotype associations were new (P < 1 × 10-5), including rs2853822 /m.8655 C>T (MT-ATP6) with type 2 diabetes, rs878966690 /m.13117 A>G (MT-ND5) with multiple sclerosis, 6 mtDNA associations with adult height, 24 mtDNA associations with 2 liver biomarkers and 16 mtDNA associations with parameters of renal function. Rare-variant gene-based tests implicated complex I genes modulating mean corpuscular volume and mean corpuscular hemoglobin. Seven traits had both rare and common mtDNA associations, where rare variants tended to have larger effects than common variants. Our work illustrates the value of studying mtDNA variants in common complex diseases and lays foundations for future large-scale mtDNA association studies.