Trading mtDNA uncovers its role in metastasis

It has been controversial for many years of whether mtDNA mutations are involved in phenotypes related to cancer due to the difficulty in excluding possible involvement of nuclear DNA mutations in these phenotypes. We addressed this issue by complete trading of mtDNAs between tumor cells expressing different metastatic phenotypes. Resultant trans-mitochondrial cybrids share the same nuclear background, but possess mtDNA from tumor cells expressing different metastatic phenotypes, and thus can be used to uncover the role of mtDNA in these phenotypes. The results showed that mtDNA controls development of metastasis in tumor cells, while tumor development is controlled by nuclear genome.Key words: pathogenic mtDNA mutations, respiration defects, enhanced glycolysis, ROS overproduction, rho-zero cells, mtDNA transfer technology, metastasisHuman mtDNAs with pathogenic mutations inducing significant respiration defects have been shown to be closely associated with mitochondrial diseases.^1,2 Although mitochondrial respiratory function is controlled by both nuclear and mitochondrial genomes, the pathogenicity of these mtDNA mutations has been proven by co-transmission of the mutant mtDNAs and mitochondrial respiration defects to mtDNA-less (ρ⁰) human cells: the resultant trans-mitochondrial cybrids sharing the same nuclear backgrouond showed respiration defects, only when they accumulated the mutated mtDNA from the patients.^3–6 Moreover, we generated transmitochondrial mito-mice sharing the same nuclear background, but carrying various proportions of mtDNA with a pathogenic mutation, and provided model systems for studying exactly how mtDNAs with pathogenic mutations are transmitted and distributed in tissues resulting in the pathogenesis of mitochondrial diseases that show various clinical phenotypes.^7–9With respect to the involvement of mtDNA in tumor phenotypes, it has been proposed that most chemical carcinogens bind preferentially to mtDNA rather than to nuclear DNA in mammalian cells,^10–12 and thus, mtDNA should be the major cellular target of chemical carcinogens, and resultant creation of mutations in mtDNAs is responsible for expression of tumor phenotypes.¹²Although, there has been no direct evidence for creation of mtDNA mutations by chemical carcinogens, and for their contribution to tumor development in mammalian cells, recent studies showed that somatic mtDNA mutations accumulated in human colorectal tumors¹³ and in various tumor types¹⁴ rather than in normal cells of the same subjects, probably by the clonal expansion of the mutated mtDNAs along with the repeated division of tumor cells. Many subsequent studies supported preferential accumulation of mutated mtDNAs in tumor cells,^15–18 suggesting that mutated mtDNAs in tumor cells have acquired replication advantages to be homoplasmic. However, these studies did not address the fundamental question of whether the mutated mtDNAs are involved in tumor development.Our previous studies directly addressed this issue using transmitochondrial cybrids obtained by mtDNA trading between normal and tumor cells, and provided convincing evidence that mutations in nuclear DNA, but not in mtDNA were involved in tumor development in the mouse^19,20 and in human cultured cells.^21,22 The possibility that these observations may represent some specific tumor cases can be excluded since there has been no statistical evidence for association of tumor development and pathogenic mtDNA mutations in the patients with mitochondrial diseases expressing respiration defects caused by pathogenic mutations in mtDNA. The possibility that some polymorphic mtDNA mutations that do not induce respiration defects, but somehow contribute to tumor development also can be excluded, because there has been no statistical evidence for the presence of maternal inheritance of tumor development in spite of the strictly maternal inheritance of mammalian mtDNA.^23,24Nonetheless, it was still possible that mtDNA mutations are involved in other processes than oncogenic transformation of normal cells to develop tumors, such as in malignant progression of tumor cells to develop a metastatic potential. Recent studies demonstrated that mitochondrial respiration defects in TCA-cycle enzymes caused by nuclear DNA mutations controls tumor phenotypes as a consequence of induction of a pseudo-hypoxic pathway under normoxia.^25–27 Thus, some mtDNA mutations also induce the pseudo-hypoxic pathway under normoxia by inducing mitochondrial respiration defects. However, there has been no direct evidence for involvement of mtDNA mutations in malignant progression or in the regulation of the pseudo-hypoxic pathway under normoxia, because of the difficulty in excluding possible contribution of nuclear DNA mutations in these processes.²⁸Recently, we addressed this issue using trans-mitochondrial cybrids²⁹ obtained by complete trading of mtDNAs between highly and poorly metastatic mouse lung carcinoma cells (Fig. 1). By this approach, we could provide convincing evidence for the control of malignant progression of tumor cells to develop metastatic potential by mtDNA:²⁹ all the trans-mitochondrial cybrids with mtDNA from highly metastatic tumor cells expressed high metastatic potential, while those with mtDNA from poorly metastatic tumor cells expressed low metastatic potential, irrespective of whether their nuclear genome was derived from highly or poorly metastatic tumor cells. The findings in our study²⁹ can be summarized as follows: (1) A missense G13997A mutation in the ND6 gene of mtDNA from highly metastatic lung tumor cells induces a complex I defect, and reversibly controls malignant progression of tumor cells to develop metastatic potential, but does not control oncogenic transformation of normal cells to develop tumors; (2) The complex I defect simultaneously induces enhanced glycolysis and ROS overproduction, but induction of metastasis is due to ROS overproduction; (3) ROS overproduction induces metastasis not by acceleration of genetic instability as usually proposed, but by reversible upregulation of nuclear-coded genes related to metastasis, such as Mcl-1; (4) ROS scavengers are therapeutically effective in suppressing mtDNA-mediated metastasis.Open in a separate windowFigure 1Scheme for the isolation of the trans-mitochondrial cybrids with completely exchanged mtDNA between parental cells expressing different metastatic phenotypes. Trading mtDNA shown in this scheme uncovered a role of mtDNA in metastasis. For trading mtDNA, parental P29 and A11 cells were treated with ditercalinium, an antitumor bis-intercalating agent, to isolate ρ⁰P29 and ρ⁰A11 cells (*), which have no mtDNA. Complete depletion of mtDNA was confirmed by PCR analysis. Enucleated cells of the mtDNA donors were prepared by their pretreatment with cytochalasin B and centrifugation. Resultant cytoplasts were fused with ρ⁰ cells by polyethylene glycol to obtain trans-mitochondrial cybrids. High metastatic potential is transferred to the P29mtA11 cybrids with the transfer of mtDNA from the A11 cells, and poor metastatic potential is transferred to the A11mtP29 cybrids with the transfer of mtDNA from the P29 cells. Involvement of cytoplasmic factors other than mtDNA from the A11 cells in expression of the high metastatic potential in the P29mtA11 cybrids can be ruled out by the observations that the A11mtP29 cybrids lost their high metastatic potential, even though they always contain cytoplasmic factors transcribed by the nuclear genes derived from the A11 cells.Thus, our study partly resolves the controversial issue on the relevance or irrelevance of mtDNA mutations in tumor development and/or tumor phenotypes by showing that mutations in mtDNA control development of metastasis in tumor cells.²⁹ Considering that complex I defects simultaneously induce enhanced glycolysis under normoxia (the Warburg effect) and ROS overproduction,²⁹ it remains possible that the Warburg effect alone can control metastasis independently from ROS overproduction. More recently, we examined this possibility by generating trans-mitochondrial cybrids with the deletion mutant mtDNA, which can be expected to induce overall respiration defects, and express enhanced glycolysis under normoxia, but not express ROS overproduction. The results showed that the Warburg effect alone did not control metastasis.     

Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (rho(0)) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties.     

Generation of trans-mitochondrial mito-mice by the introduction of a pathogenic G13997A mtDNA from highly metastatic lung carcinoma cells

To investigate the effects of respiration defects on the disease phenotypes, we generated trans-mitochondrial mice (mito-mice) by introducing a mutated G13997A mtDNA, which specifically induces respiratory complex I defects and metastatic potentials in mouse tumor cells. First, we obtained ES cells and chimeric mice containing the G13997A mtDNA, and then we generated mito-mice carrying the G13997A mtDNA via its female germ line transmission. The three-month-old mito-mice showed complex I defects and lactate overproduction, but showed no other phenotypes related to mitochondrial diseases or tumor formation, suggesting that aging or additional nuclear abnormalities are required for expression of other phenotypes.     

Enhanced glycolysis induced by mtDNA mutations does not regulate metastasis

We addressed the issue of whether enhanced glycolysis caused by mtDNA mutations independently induces metastasis in tumor cells using mtDNA transfer technology. The resultant trans-mitochondrial cybrids sharing the same nuclear background of poorly metastatic carcinoma P29 cells, P29mtA11 and P29mtDelta cybrids, possessed mtDNA with a G13997A mutation from highly metastatic carcinoma A11 cells and mtDNA with a 4696bp deletion mutation, respectively. The P29mtDelta cybrids expressed enhanced glycolysis, but did not express ROS overproduction and high metastatic potential, whereas P29mtA11 cybrids showed enhanced glycolysis, ROS overproduction, and high metastatic potential. Thus, enhanced glycolysis alone does not induce metastasis in the cybrids.     

"ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis"--a critical commentary

In a recent publication (K. Ishikawa et al., 2008, Science320, 661-664), the authors described how replacing the endogenous mitochondrial DNA (mtDNA) in a weakly metastatic mouse tumor cell line with mtDNA from a highly metastatic cell line enhanced tumor progression through enhanced production of reactive oxygen species (ROS). The authors attributed the transformation from a low-metastatic cell line to a high-metastatic phenotype to overproduction of ROS (hydrogen peroxide and superoxide) caused by a dysfunction in mitochondrial complex I protein encoded by mtDNA transferred from the highly metastatic tumor cell line. In this critical evaluation, using the paper by Ishikawa et al. as an example, we bring to the attention of researchers in the free radical field how the failure to appreciate the complexities of dye chemistry could potentially lead to pitfalls, misinterpretations, and erroneous conclusions concerning ROS involvement. Herein we make a case that the authors have failed to show evidence for formation of superoxide and hydrogen peroxide, presumed to be generated from complex I deficiency associated with mtDNA mutations in metastatic cells.     

Reversible regulation of metastasis by ROS-generating mtDNA mutations

It has been controversial whether mtDNA mutations are responsible for oncogenic transformation (normal cells to develop tumors), and for malignant progression (tumor cells to develop metastases). To clarify this issue, we created trans-mitochondrial cybrids with mtDNA exchanged between mouse tumor cells that express different metastatic phenotypes. The G13997A mutation in the ND6 gene of mtDNA from high metastatic tumor cells reversibly controlled development of metastases by overproduction of reactive oxygen species (ROS), but did not control development of tumors. The mtDNA-mediated reversible control of metastasis reveals a novel function of mtDNA, and suggests that ROS scavengers may be therapeutically effective in suppressing metastasis.     

Administration of an Antioxidant Prevents Lymphoma Development in Transmitochondrial Mice Overproducing Reactive Oxygen Species

Because of the difficulty to exclude possible involvement of nuclear DNA mutations, it has been a controversial issue whether pathogenic mutations in mitochondrial DNA (mtDNA) and the resultant respiration defects are involved in tumor development. To address this issue, our previous study generated transmitochondrial mice (mito-mice-ND6¹³⁹⁹⁷), which possess the nuclear and mtDNA backgrounds derived from C57BL/6J (B6) strain mice except that they carry B6 mtDNA with a G13997A mutation in the mt-Nd6 gene. Because aged mito-mice-ND6¹³⁹⁹⁷ simultaneously showed overproduction of reactive oxygen species (ROS) in bone marrow cells and high frequency of lymphoma development, current study examined the effects of administrating a ROS scavenger on the frequency of lymphoma development. We used N-acetylcysteine (NAC) as a ROS scavenger, and showed that NAC administration prevented lymphoma development. Moreover, its administration induced longevity in mito-mice-ND6¹³⁹⁹⁷. The gene expression profiles in bone marrow cells indicated the upregulation of the Fasl gene, which can be suppressed by NAC administration. Given that natural-killer (NK) cells mediate the apoptosis of various tumor cells via enhanced expression of genes encoding apoptotic ligands including Fasl gene, its overexpression would reflect the frequent lymphoma development in bone marrow cells. These observations suggest that continuous administration of an antioxidant would be an effective therapeutics to prevent lymphoma development enhanced by ROS overproduction.     

Polymorphic mutations in mouse mitochondrial DNA regulate a tumor phenotype

To examine whether polymorphic mtDNA mutations that do not induce significant respiration defects regulate phenotypes of tumor cells, we used mouse transmitochondrial tumor cells (cybrids) with nuclear DNA from C57BL/6 (B6) strain and mtDNA from allogenic C3H strain. The results showed that polymorphic mutations of C3H mtDNA in the cybrids induced hypoxia sensitivity, resulting in a delay of tumor formation on their subcutaneous inoculation into B6 mice. Therefore, the effects of polymorphic mutations in normal mtDNA have to be carefully considered, particularly when we apply the gene therapy to the embryos to replace their pathogenic mtDNA by normal mtDNA.     

Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.     

Determination of normal ranges of mitochondrial respiratory activities by mtDNA transfer from 54 Human subjects to mtDNA-less HeLa cells for identification of the pathogenicities of mutated mtDNAs

To determine the pathogenicities of mutated mtDNAs in patients with respiration defects, the possible involvement of nuclear DNA mutations has to be excluded, since respiratory function is controlled by both nuclear DNA and mtDNA. This was achieved by showing that the mutated mtDNAs and respiration defects were co-transferred from patients to mtDNA-less human cells, and the resultant cybrid clones carrying mutated mtDNAs expressed respiration defects. To decide whether the cybrid clones expressed respiration defects, in this study the lowest limits of normal respiratory function were evaluated by transfer of mtDNAs from 54 normal subjects to mtDNA-less HeLa cells. The resultant cybrid clones showed that 71% respiratory function was the lowest limit of mtDNAs from normal subjects. On the other hand, cybrid clones carrying pathogenic mtDNAs from patients with mitochondrial diseases showed 0-64% respiratory function, suggesting that less than 71% respiratory function in cybrid clones should be a reliable indicator of whether the mutated mtDNAs of the patients were pathogenic.     

Bioenergetics of mitochondrial diseases associated with mtDNA mutations

This mini-review summarizes our present view of the biochemical alterations associated with mitochondrial DNA (mtDNA) point mutations. Mitochondrial cytopathies caused by mutations of mtDNA are well-known genetic and clinical entities, but the biochemical pathogenic mechanisms are often obscure. Leber's hereditary optic neuropathy (LHON) is due to three main mutations in genes for complex I subunits. Even if the catalytic activity of complex I is maintained except in cells carrying the 3460/ND1 mutation, in all cases there is a change in sensitivity to complex I inhibitors and an impairment of mitochondrial respiration, eliciting the possibility of generation of reactive oxygen species (ROS) by the complex. Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP), is due to a mutation in the ATPase-6 gene. In NARP patients ATP synthesis is strongly depressed to an extent proportional to the mutation load; nevertheless, ATP hydrolysis and ATP-driven proton translocation are not affected. It is suggested that the NARP mutation affects the ability of the enzyme to couple proton transport to ATP synthesis. A point mutation in subunit III of cytochrome c oxidase is accompanied by a syndrome resembling MELAS: however, no major biochemical defect is found, if we except an enhanced production of ROS. The mechanism of such enhancement is at present unknown. In this review, we draw attention to a few examples in which the overproduction of ROS might represent a common step in the induction of clinical phenotypes and/or in the progression of several human pathologies associated with mtDNA point mutations.     

Expression of Rattus norvegicus mtDNA in Mus musculus cells results in multiple respiratory chain defects

The production of in vitro and in vivo models of mitochondrial DNA (mtDNA) defects is currently limited by a lack of characterized mouse cell mtDNA mutants that may be expected to model human mitochondrial diseases. Here we describe the creation of transmitochondrial mouse (Mus musculus) cells repopulated with mtDNA from different murid species (xenomitochondrial cybrids). The closely related Mus spretus mtDNA is readily maintained when introduced into M. musculus mtDNA-less (rho(0)) cells, and the resulting cybrids have normal oxidative phosphorylation (OXPHOS). When the more distantly related Rattus norvegicus mtDNA is transferred to the mouse nuclear background the mtDNA is replicated, transcribed, and translated efficiently. However, function of several OXPHOS complexes that depend on the coordinated assembly of nuclear and mtDNA-encoded proteins is impaired. Complex I activity in the Rattus xenocybrid was 46% of the control mean; complex III was 37%, and complex IV was 78%. These defects combined to restrict maximal respiration to 12-31% of the control and M. spretus xenocybrids, as measured polarographically using isolated cybrid mitochondria. These defects are distinct to those previously reported for human/primate xenocybrids. It should be possible to produce other mouse xenocybrid constructs with less severe OXPHOS phenotypes, to model human mtDNA diseases.     

Mitochondrial DNA Mutations in Mutator Mice Confer Respiration Defects and B-Cell Lymphoma Development

Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ⁰) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.     

肝癌组织中线粒体DNA D-Loop区碱基变异与ROS水平

为了探讨ROS水平与突变的关系,对原发性肝癌线粒体DNA区的突变情况进行研究,同时对原发性肝癌患者组织细胞内ROS进行测定。选择20例原发性肝癌组织及其邻近的癌旁组织,用PCR方法将线粒体DNA D-Loop扩增后测序。组织内ROS的水平采用流式细胞技术测定。结果表明在20对原发性肝癌组织中存在8对mtDNA突变,突变率为40%,共发现突变位点53个,包括2个插入,11个缺失,40个点突变,其中T-C,C-T的转换占75%,4个属于微卫星结构。癌组织突变一般伴有癌旁组织突变,癌组织突变位点高于癌旁组织。发现一例标本的癌组织和癌旁组织均有大片段丢失。原发性肝癌组织内ROS水平明显高于癌旁对照( P<0.01),同时我们发现在区发生突变的患者的组织中ROS水平明显高于未发生突变的肝癌组织标本（P<0.01）,发生突变的癌旁组织内ROS水平明显高于未发生突变的癌旁组织（P<0.01）。结论 (1)线粒体DNA D-Loop区是一个高度多态性和突变性的区域,在原发性肝癌中突变率较高。(2)肝癌患者组织细胞内ROS异常,提示肝癌的线粒体DNA发生的点突变及肝癌的发生可能与ROS升高有关。     

Regulation of energy metabolism in human cells in aging and diabetes: FoF(1), mtDNA, UCP, and ROS

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF(1)), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H(+) (Delta muH(+)). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Delta muH(+) rotates an axis in FoF(1) that is regulated by inhibitors and ATP-sensitive K(+)-channels. We cultured human rho(o) cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Delta muH(+), UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O(2). Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Delta muH(+) and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic rho(o) cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Delta muH(+). Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Delta muH(+) was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and rho(o) cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells.     

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.     

Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA (rho0 cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in rho0 cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and rho0 cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and rho0 cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.     

Mitochondrial DNA variants influence mitochondrial bioenergetics in Drosophila melanogaster

The influence of mitochondrial DNA (mtDNA) mutations on human disease has been extensively studied, but the impact of mutations within the adaptive range is debated. We studied males from lines of Drosophila melanogaster that have a highly standardized nuclear genome but different mtDNA, at two ages. We measured mitochondrial respiration on permeabilized muscle fibers, hydrogen peroxide production of isolated mitochondria and mtDNA copy number of whole individuals. The results show that a small set of naturally occurring mtDNA mutations can have a significant influence on mitochondrial bioenergetics that may change as the organism ages.