Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing.

Human mitochondrial DNA (mtDNA) shows extensive within population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic,metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation,insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

Respiratory active mitochondrial supercomplexes.

The structural organization of the mitochondrial respiratory complexes as four big independently moving entities connected by the mobile carriers CoQ and cytochrome c has been challenged recently. Blue native gel electrophoresis reveals the presence of high-molecular-weight bands containing several respiratory complexes and suggesting an in vivo assembly status of these structures (respirasomes). However, no functional evidence of the activity of supercomplexes as true respirasomes has been provided yet. We have observed that (1) supercomplexes are not formed when one of their component complexes is absent; (2) there is a temporal gap between the formation of the individual complexes and that of the supercomplexes; (3) some putative respirasomes contain CoQ and cytochrome c; (4) isolated respirasomes can transfer electrons from NADH to O(2), that is, they respire. Therefore, we have demonstrated the existence of a functional respirasome and propose a structural organization model that accommodates these findings.

Differences in reactive oxygen species production explain the phenotypes associated with common mouse mitochondrial DNA variants.

Common mitochondrial DNA (mtDNA) haplotypes in humans and mice have been associated with various phenotypes, including learning performance and disease penetrance. Notably, no influence of mtDNA haplotype in cell respiration has been demonstrated. Here, using cell lines carrying four different common mouse mtDNA haplotypes in an identical nuclear background, we show that the similar level of respiration among the cell lines is only apparent and is a consequence of compensatory mechanisms triggered by different production of reactive oxygen species. We observe that the respiration capacity per molecule of mtDNA in cells with the NIH3T3 or NZB mtDNA is lower than in those with the C57BL/6J, CBA/J or BALB/cJ mtDNA. In addition, we have determined the genetic element underlying these differences. Our data provide insight into the molecular basis of the complex phenotypes associated with common mtDNA variants and anticipate a relevant contribution of mtDNA single nucleotide polymorphisms to phenotypic variability in humans.

Evolution meets disease: penetrance and functional epistasis of mitochondrial tRNA mutations.

About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNA(Ile) that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both.

Allotopic expression of mitochondrial-encoded genes in mammals: achieved goal, undemonstrated mechanism or impossible task?

Mitochondrial-DNA diseases have no effective treatments. Allotopic expression-synthesis of a wild-type version of the mutated protein in the nuclear-cytosolic compartment and its importation into mitochondria-has been proposed as a gene-therapy approach. Allotopic expression has been successfully demonstrated in yeast, but in mammalian mitochondria results are contradictory. The evidence available is based on partial phenotype rescue, not on the incorporation of a functional protein into mitochondria. Here, we show that reliance on partial rescue alone can lead to a false conclusion of successful allotopic expression. We recoded mitochondrial mt-Nd6 to the universal genetic code, and added the N-terminal mitochondrial-targeting sequence of cytochrome c oxidase VIII (C8) and the HA epitope (C8Nd6HA). The protein apparently co-localized with mitochondria, but a significant part of it seemed to be located outside mitochondria. Complex I activity and assembly was restored, suggesting successful allotopic expression. However, careful examination of transfected cells showed that the allotopically-expressed protein was not internalized in mitochondria and that the selected clones were in fact revertants for the mt-Nd6 mutation. These findings demonstrate the need for extreme caution in the interpretation of functional rescue experiments and for clear-cut controls to demonstrate true rescue of mitochondrial function by allotopic expression.

Restoration of electron transport without proton pumping in mammalian mitochondria.

We have restored the CoQ oxidative capacity of mouse mtDNA-less cells (rho degrees cells) by transforming them with the alternative oxidase Aox of Emericella nidulans. Cotransforming rho degrees cells with the NADH dehydrogenase of Saccharomyces cerevisiae, Ndi1 and Aox recovered the NADH DH/CoQ reductase and the CoQ oxidase activities. CoQ oxidation by AOX reduces the dependence of rho degrees cells on pyruvate and uridine. Coexpression of AOX and NDI1 further improves the recycling of NAD(+). Therefore, 2 single-protein enzymes restore the electron transport in mammalian mitochondria substituting >80 nuclear DNA-encoded and 11 mtDNA-encoded proteins. Because those enzymes do not pump protons, we were able to split electron transport and proton pumping (ATP synthesis) and inquire which of the metabolic deficiencies associated with the loss of oxidative phosphorylation should be attributed to each of the 2 processes.

Cisplatin-mediated impairment of mitochondrial DNA metabolism inversely correlates with glutathione levels.

Cisplatin accumulates in mitochondria, which are a major target for this drug in cancer cells. Thus alterations in mitochondrial function have been implicated in cancer cell resistance to chemotherapeutic agents. Moreover, cisplatin toxic side effects seem to be associated with mitochondrial injury in vivo and in vitro. In order to clarify the potential effect of cisplatin in mtDNA (mitochondrial DNA) maintenance and expression, we have analysed rat liver mtDNA and mtRNA (mitochondrial RNA) synthesis as well as their stability under the influence of in vivo treatment or in vitro exposure to cisplatin. We show that cisplatin causes a direct and significant impairment of mtDNA and mtRNA synthesis and decreases steady-state levels of mtRNAs in isolated mitochondria. Furthermore, in vivo treatment of the animals with cisplatin exerts a protective effect from the impairment of mtRNA metabolism caused by in vitro exposure to the drug, by means of increased mitochondrial GSH levels after in vivo cisplatin treatment.

Functional genetic analysis of the mammalian mitochondrial DNA encoded peptides: a mutagenesis approach.

Animal mitochondria are refractory to transformation. This fact has hampered the study of the oxidative phosphorylation system biogenesis by genetic manipulation of the mitochondrial DNA (mtDNA). In humans, a larger variety of mutants have been obtained from patients with mitochondrial diseases, but still we lack a great portion of the range of potential mutants and there is a major obstacle: Animal models cannot be derived from human mtDNA mutants. Until now the only source of mtDNA mutants in mouse was restricted to some drug-resistant-specific cell lines in which a given mtDNA mutation provided growth advantage in the presence of the inhibitor for a specific complex. To overcome these limitations, the authors have developed a protocol that allows the systematic generation of cells harboring mutations in their mtDNA affecting all types of mitochondrial genes. Chemical mutagenesis followed by mtDNA copy number reduction and the use of large-scale negative selection in duplicate cultures, are the key steps of the strategy used.

Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA.

Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

Revisiting the mouse mitochondrial DNA sequence.

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Consequently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.

Electrons feed into the mitochondrial electron transport chain (mETC) from NAD- or FAD-dependent enzymes. A shift from glucose to fatty acids increases electron flux through FAD, which can saturate the oxidation capacity of the dedicated coenzyme Q (CoQ) pool and result in the generation of reactive oxygen species. To prevent this, the mETC superstructure can be reconfigured through the degradation of respiratory complex I, liberating associated complex III to increase electron flux via FAD at the expense of NAD. Here, we demonstrate that this adaptation is driven by the ratio of reduced to oxidized CoQ. Saturation of CoQ oxidation capacity induces reverse electron transport from reduced CoQ to complex I, and the resulting local generation of superoxide oxidizes specific complex I proteins, triggering their degradation and the disintegration of the complex. Thus, CoQ redox status acts as a metabolic sensor that fine-tunes mETC configuration in order to match the prevailing substrate profile.

Mitochondria from ejaculated human spermatozoa do not synthesize proteins.

Sperm motility is dependent on mitochondrial ATP production that relies on the coordinated expression of the mitochondrial and nuclear genomes. It is generally accepted that mammalian ejaculated spermatozoa retain the ability to synthesize mtDNA-encoded proteins but not most of the nuclear ones. This implies an asynchronous regulation of the oxidative phosphorylation-related genes encoded by each genome. Trying to investigate this issue, we unexpectedly found that ejaculated human spermatozoa do not synthesize mtDNA-encoded proteins. Moreover, we estimated that the discrepancy between our observations and those published elsewhere was due to a chloramphenicol-sensitive protein synthesis attributed to mitochondria that instead corresponds to contaminating bacteria.

ROS-triggered phosphorylation of complex II by Fgr kinase regulates cellular adaptation to fuel use.

Electron flux in the mitochondrial electron transport chain is determined by the superassembly of mitochondrial respiratory complexes. Different superassemblies are dedicated to receive electrons derived from NADH or FADH2, allowing cells to adapt to the particular NADH/FADH2 ratio generated from available fuel sources. When several fuels are available, cells adapt to the fuel best suited to their type or functional status (e.g., quiescent versus proliferative). We show that an appropriate proportion of superassemblies can be achieved by increasing CII activity through phosphorylation of the complex II catalytic subunit FpSDH. This phosphorylation is mediated by the tyrosine-kinase Fgr, which is activated by hydrogen peroxide. Ablation of Fgr or mutation of the FpSDH target tyrosine abolishes the capacity of mitochondria to adjust metabolism upon nutrient restriction, hypoxia/reoxygenation, and T cell activation, demonstrating the physiological relevance of this adaptive response.

Length variation in the mouse mitochondrial tRNA(Arg) DHU loop size promotes oxidative phosphorylation functional differences.

The efficiency of the cellular oxidative phosphorylation system was recently shown to be modulated by common mitochondrial tRNA(A) (rg) haplotypes. The molecular mechanism by which some mt-Tr haplotypes induce these functional differences remains undetermined. Common polymorphisms in mouse mt-Tr genes affect the size of the dihydrouridine loop in the mature tRNA, producing loops of between five and seven nucleotides, the largest being a rare variant among mammals. Here, we analyzed a new mt-Tr variant identified in C3H mice, and found that it is mitochondrial tRNA loop size, but not the specific sequence, that is responsible for the observed differences in cellular respiration. We further found that the sensitivity of mitochondrial protein synthesis to specific inhibitors is dependent on the mt-Tr gene haplotype, and confirmed that the differences in oxidative phosphorylation performance are masked by a reactive oxygen species-induced compensatory increase in mitochondrial biogenesis.

Supercomplex assembly determines electron flux in the mitochondrial electron transport chain.

The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.

A genome-wide shRNA screen for new OxPhos related genes.

The mitochondrial oxidative phosphorylation (OxPhos) system produces most of the ATP required by the cell. The structural proteins of the OxPhos holoenzymes are well known, but important aspects of their biogenesis and regulation remain to be uncovered and a significant fraction of mitochondrial proteins have yet to be identified. We have used a high throughput, genome-wide RNA interference (RNAi) approach to identify new OxPhos-related genes. We transduced a mouse fibroblast cell line with a lentiviral-based shRNA-library, and screened the cell population for growth impairment in galactose-based medium, which requires an intact OxPhos system. Candidate genes were ranked according to their co-expression with known genes encoding OxPhos mitochondria-located proteins. For the top ranking candidates the cellular process in which they are involved was evaluated. Our results show that the use of genome-wide RNAi together with screening for deficient growth in galactose medium is a suitable approach to identifying OxPhos-related and cellular energy metabolism-related genes. Interestingly also ubiquitin-proteasome related genes were selected.

Tissue-specific differences in mitochondrial activity and biogenesis.

Each cell type develops and maintains a specific oxidative phosphorylation (OXPHOS) capacity to satisfy its metabolic and energetic demands. This implies that there are differences between tissues in mitochondrial number, function, protein composition and morphology. The OXPHOS system biogenesis requires the coordinated expression of both mitochondrial and nuclear genomes. Mitochondrial DNA (mtDNA) expression can be regulated at different levels (replication, transcription, translation and post-translational levels) to contribute to the final observed OXPHOS activities. By analyzing five mammalian tissues, we evaluated the differences in the cellular amount of mtDNA and its correlation with the final observed mitochondrial activity.

Isolation of mitochondria for biogenetical studies: An update.

The use of good quality preparations of isolated mitochondria is necessary when studying the mitochondrial biogenetical activities. This article explains a fast and simple method for the purification of mammalian mitochondria from different tissues and cultured cells, that is suitable for the analysis of many aspects of the organelle's biogenesis. The mitochondria isolated following the protocol described here, are highly active and capable of DNA, RNA and protein synthesis. Mitochondrial tRNA aminoacylation, mtDNA-protein interactions and specific import of added proteins into the organelles, can also be studied using this kind of preparations.

Five entry points of the mitochondrially encoded subunits in mammalian complex I assembly.

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.