Deletion of mtDNA disrupts mitochondrial function and structure, but not biogenesis

The role of nuclear DNA (nDNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mtDNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mtDNA-deficient rho0 mutants. Depletion of mtDNA in rho0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear beta-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by nDNA, remained unaffected in rho0 cells. Loss of mtDNA in rho0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mtDNA-deficient and parental cells. Thus, we conclude that cells lacking mtDNA still generate mitochondrial scaffolds, albeit with aberrant function.     

A stop-codon mutation in the human mtDNA cytochrome c oxidase I gene disrupts the functional structure of complex IV.

We have identified a novel stop-codon mutation in the mtDNA of a young woman with a multisystem mitochondrial disorder. Histochemical analysis of a muscle-biopsy sample showed virtually absent cytochrome c oxidase (COX) stain, and biochemical studies confirmed an isolated reduction of COX activity. Sequence analysis of the mitochondrial-encoded COX-subunit genes identified a heteroplasmic G-->A transition at nucleotide position 6930 in the gene for subunit I (COX I). The mutation changes a glycine codon to a stop codon, resulting in a predicted loss of the last 170 amino acids (33%) of the polypeptide. The mutation was present in the patient's muscle, myoblasts, and blood and was not detected in normal or disease controls. It was not detected in mtDNA from leukocytes of the patient's mother, sister, and four maternal aunts. We studied the genetic, biochemical, and morphological characteristics of transmitochondrial cybrid cell lines, obtained by fusing of platelets from the patient with human cells lacking endogenous mtDNA (rho0 cells). There was a direct relationship between the proportion of mutant mtDNA and the biochemical defect. We also observed that the threshold for the phenotypic expression of this mutation was lower than that reported in mutations involving tRNA genes. We suggest that the G6930A mutation causes a disruption in the assembly of the respiratory-chain complex IV.     

A pathogenic 15-base pair deletion in mitochondrial DNA-encoded cytochrome c oxidase subunit III results in the absence of functional cytochrome c oxidase

A 15-base pair, in-frame, deletion (9480del15) in the mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit III (COX III) gene was identified previously in a patient with recurrent episodes of myoglobinuria and an isolated COX deficiency. Transmitochondrial cell lines harboring 0, 97, and 100% of the 9480del15 deletion were created by fusing human cells lacking mtDNA (rho(0) cells) with platelet and lymphocyte fractions isolated from the patient. The COX III gene mutation resulted in a severe respiratory chain defect in all mutant cell lines. Cells homoplasmic for the mutation had no detectable COX activity or respiratory ATP synthesis, and required uridine and pyruvate supplementation for growth, a phenotype similar to rho(0) cells. The cells with 97% mutated mtDNA exhibited severe reductions in both COX activity (6% of wild-type levels) and rates of ATP synthesis (9% of wild-type). The COX III polypeptide in the mutant cells, although translated at rates similar to wild-type, had reduced stability. There was no evidence for assembly of COX I, COX II, or COX III subunits in a multisubunit complex in cells homoplasmic for the mutation, thus indicating that there was no stable assembly of COX I with COX II in the absence of wild-type COX III. In contrast, the COX I and COX II subunits were assembled in cells with 97% mutated mtDNA.     

Mice with only rat mtDNA are required as models of mitochondrial diseases

We examined the possibility of generation of mice expressing mitochondrial dysfunction by introduction of exogenous mtDNA from different species using mouse mtDNA-less (rho(0)) cells as mtDNA recipients. For determination of how genetically distant species of mtDNA could replicate in cells with only the mouse nuclear genome, we introduced mtDNA of the Syrian hamster (Mesocricetus auratus) into mouse rho(0) cells, and found that its replication was not sufficient to propagate to following generations, probably due to significant incompatibility between mouse-nuclear and Syrian hamster-mitochondrial genomes. On the other hand, rat mtDNA, which propagated stably and expressed mitochondrial dysfunction in mouse cells, also disappeared rapidly by exogenous introduction of mouse mtDNA, suggesting that mouse mtDNA in mouse cells must be excluded completely before introduction of rat mtDNA for generation of mice with rat mtDNA as mitochondrial disease models.     

外源性线粒体DNA在细胞内的重建（简报）

To create human mitochondrial DNA (mtDNA)-transferred cells as cell model of mitochondria defects-related diseases, platelet of normal young and old subjects were isolated as donor of mtDNA, then fused with mtDNA-depleted cells (rho0 cells) under induction of PEG1500. Auxtrophy test, cytochrome c oxidase activity assay and PCR amplification of mtDNA were done to confirm the transferring of mtDNA. Cell clones were visible in the medium 10 to 15 days after fusion, which grew well in medium lacking uridine, had positive COX activity and contained objective fragment of mtDNA by PCR, opposite to rho0 cells. Transferring of mtDNA to rho0 cells was identified, and mtDNA-transferred cell models were successfully created.     

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.     

Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype

We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-gamma to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-gamma harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in rho0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the rho0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-gamma can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.     

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.     

Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae.

Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant [( rho0]) of Saccharomyces cerevisiae. However, when cytoplasmic components were introduced by cytoduction from a wild-type [( rho+]) strain of S. cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones. The phenomenon was ascribed to the incompatibility with the introduced S. cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by [rho0] mutations in the cytoductant cells. Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and [rho+] nonkiller cells lacking the plasmids. High-frequency transmission of the plasmids was observed in crosses lacking mtDNA [( rho0] by [rho0] crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome. In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids. Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in [rho0] cells of S. cerevisiae.     

The mitochondrial nucleoid protein, Mgm101p, of Saccharomyces cerevisiae is involved in the maintenance of rho(+) and ori/rep-devoid petite genomes but is not required for hypersuppressive rho(-) mtDNA

The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional rho(+) genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a rho(+) strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have rho(-) genomes that are stably maintained. Interestingly, all surviving rho(-) mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS rho(-) mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of rho(+) and ori/rep-devoid rho(-) mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS rho(-) genome is stably maintained in a mgm101, rpo41 double mutant.     

Molecular characterization of mtDNA depleted and repleted NT2 cell lines

Transmitochondrial cytoplasmic hybrids (cybrids) enable functional assessment of mitochondrial DNA (mtDNA)-encoded proteins. Cybrid production often utilizes cell lines depleted of endogenous mtDNA (rho0 cells), and a number of suitable rho0 cell lines exist for this purpose. We now provide molecular data characterizing an NT2 human teratocarcinoma rho0 cell line, as well as NT2 cybrid derivatives. NT2 rho0 cells contained no detectable mtDNA on a sensitive PCR assay. Eight weeks after exogenous mtDNA transfer cybrids showed no evidence of endogenous mtDNA reversion, and heteroplasmic ratios of a single nucleotide substitution roughly reflected that of the blood samples used to repopulate their mtDNA.     

A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle.

We have identified a new mutation in mtDNA, involving tRNALeu(CUN) in a patient manifesting an isolated skeletal myopathy. This heteroplasmic A-->G transition at position 12320 affects the T psi C loop at a conserved site and was not found in 120 controls. Analysis of cultured fibroblasts, white blood cells/platelets, and skeletal muscle showed that only skeletal muscle contained the mutation and that only this tissue demonstrated a biochemical defect of respiratory-chain activity. In a series of four muscle-biopsy specimens taken over a 12-year period, there was a gradual increase, from 70% to 90%, in the overall level of mutation, as well as a marked clinical deterioration. Single-fiber PCR confirmed that the proportion of mutant mtDNA was highest in cytochrome c oxidase-negative fibers. This study, which reports a mutation involving tRNALeu(CUN), demonstrates clearly that mtDNA point mutations can accumulate over time and may be restricted in their tissue distribution. Furthermore, clinical deterioration seemed to follow the increase in the level of mutation, although, interestingly, the appearance of fibers deficient in respiratory-chain activity showed a lag period.     

Mitochondrial DNA Ligase Is Dispensable for the Viability of Cultured Cells but Essential for mtDNA Maintenance

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ⁰ phenotype.     

Cytoplasmic transfer of platelet mtDNA from elderly patients with Parkinson's disease to mtDNA-less HeLa cells restores complete mitochondrial respiratory function

For determination of whether platelet mtDNA in patients with Parkinson's disease (PD) possesses some lesions to reduce respiratory enzyme activities, platelet mtDNA was transferred into mtDNA-less (rho0) HeLa cells from aged PD patients and age-matched normal subjects, since their activities were controlled by both mitochondrial and nuclear genomes. The resultant mtDNA-repopulated cybrid clones containing the HeLa nuclear genome as a common background were used for comparison of respiratory enzyme activities. Remarkable variations of the enzyme activities were observed in the cybrid clones, irrespective of whether their mtDNA was transferred from normal subjects or PD patients, and some of them showed 20% reduction of average activities. Thus, the mtDNA mutations responsible for inducing 20% reduction should be polymorphic rather than pathogenic. On the other hand, pathogenic control cybrid clones possessing mtDNA mutations from patients with mitochondrial disorders showed significant and specific decline of respiratory enzyme complex I activity beyond the normal range of the variations. These observations warrant reassessment of the conventional concept that complex I activity in platelets of PD patients is defective due to mtDNA mutations.     

A "petite obligate" mutant of Saccharomyces cerevisiae: functional mtDNA is lethal in cells lacking the delta subunit of mitochondrial F1-ATPase

Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.     

Oltipraz-induced phase 2 enzyme response conserved in cells lacking mitochondrial DNA

Oltipraz, a member of a class of 1,2-dithiolethiones, is a potent phase 2 enzyme inducing agent used as a cancer chemopreventive. In this study, we investigated regulation of the phase 2 enzyme response and protection against endogenous oxidative stress in lymphoblastic leukemic parental CEM cells and cells lacking mitochondrial DNA (mtDNA) (rho0) by oltipraz. Glutathione (GSH) levels (total and mitochondrial) and glutathione S-transferase (GST) activity were significantly increased after pretreatment with oltipraz in both parental (rho+) and rho0 cells, and both cell lines were resistant to mitochondrial oxidation, loss of mitochondrial membrane potential, and cell death in response to the GSH depleting agent diethylmaleate. These results show that the phase 2 enzyme response, by enhancing GSH-dependent systems involved in xenobiotic metabolism, blocks endogenous oxidative stress and cell death, and that this response is intact in cells lacking mtDNA.     

Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia

CPEO (chronic progressive external ophthalmoplegia) is a common mitochondrial disease phenotype in adults which is due to mtDNA (mitochondrial DNA) point mutations in a subset of patients. Attributing pathogenicity to novel tRNA mtDNA mutations still poses a challenge, particularly when several mtDNA sequence variants are present. In the present study we report a CPEO patient for whom sequencing of the mitochondrial genome revealed three novel tRNA mtDNA mutations: G5835A, del4315A, T1658C in tRNATyr, tRNAIle and tRNAVal genes. In skeletal muscle, the tRNAVal and tRNAIle mutations were homoplasmic, whereas the tRNATyr mutation was heteroplasmic. To address the pathogenic relevance, we performed two types of functional tests: (i) single skeletal muscle fibre analysis comparing G5835A mutation loads and biochemical phenotypes of corresponding fibres, and (ii) Northern-blot analyses of mitochondrial tRNATyr, tRNAIle and tRNAVal. We demonstrated that both the G5835A tRNATyr and del4315A tRNAIle mutation have serious functional consequences. Single-fibre analyses displayed a high threshold of the tRNATyr mutation load for biochemical phenotypic expression at the single-cell level, indicating a rather mild pathogenic effect. In contrast, skeletal muscle tissue showed a severe decrease in respiratory-chain activities, a reduced overall COX (cytochrome c oxidase) staining intensity and abundant COX-negative fibres. Northern-blot analyses showed a dramatic reduction of tRNATyr and tRNAIle levels in muscle, with impaired charging of tRNAIle, whereas tRNAVal levels were only slightly decreased, with amino-acylation unaffected. Our findings suggest that the heteroplasmic tRNATyr and homoplasmic tRNAIle mutation act together, resulting in a concerted effect on the biochemical and histological phenotype. Thus homoplasmic mutations may influence the functional consequences of pathogenic heteroplasmic mtDNA mutations.     

A novel mutation in the mitochondrial 16S rRNA gene in a patient with MELAS syndrome, diabetes mellitus, hyperthyroidism and cardiomyopathy

Using RNase protection analysis, we found a novel C to G mutation at nucleotide position 3093 of mitochondrial DNA (mtDNA) in a previously reported 35-year-old woman exhibiting clinical features of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome together with diabetes mellitus, hyperthyroidism and cardiomyopathy. The patient also had an A3243G mutation in the tRNA(Leu(UUR)) gene and a 260-base pair duplication in the D-loop of mtDNA. The fibroblasts of the patient were cultured and used for the construction of cybrids using cytoplasmic transfer of the patient's mtDNA to the mtDNA-less rho(0) cells. RNA isolated from the cybrids was subjected to RNase protection analysis, and a C3093G transversion at the 16S rRNA gene and a MELAS-associated A3243G mutation of mtDNA were detected. The novel C3093G mutation together with the A3243G transition were found in muscle biopsies, hair follicles and blood cells of this patient and also in her skin fibroblasts and cybrids. The proportion of the C3093G mutant mtDNA in muscle biopsies of the patient was 51%. In contrast, the mutation was not detected in three sons of the proband. To characterize the impact of the mtDNA mutation-associated defects on mitochondrial function, we determined the respiratory enzyme activities of the primary culture of fibroblasts established from the proband, her mother and her three sons. The proportions of mtDNA with the C3093G transversion and the A3243G transition in the fibroblasts of the proband were 45 and 58%, respectively. However, the fibroblasts of the proband's mother and children harbored lower levels of mtDNA with the A3243G mutation but did not contain the C3093G mutation. The complex I activity in the proband's fibroblasts was decreased to 47% of the control but those of the fibroblasts of the mother and three sons of the proband were not significantly changed. These findings suggest that the C3093G transversion together with the A3243G transition of mtDNA impaired the respiratory function of mitochondria and caused the atypical MELAS syndrome associated with diabetes mellitus, hyperthyroidism and cardiomyopathy in this patient.     

CXCL12 induces lung cancer cell migration by polarized mtDNA redistribution

Instability of mitochondrial DNA (mtDNA) has been associated with the initiation and development of cancer, but the specific role of mtDNA in the invasiveness and migration of cancer cells remains unclear. In this study, we investigated whether the chemokine CXCL12 causes intact mitochondria to redistribute in cancer cells and, in this way, to increase cell invasiveness and migration. A549 lung cancer cells with intact mtDNA (mtDNA+) and ρ⁰A549 cells depleted of mtDNA (mtDNA?) by long-term ethidium bromide incubation were examined for their responses to CXCL12 in a transwell migration assay and for mitochondrial distribution by fluorescence microscopy. Intact A549 cells showed significantly increased migration and increased polar distribution of mitochondria (asymmetry) in response to CXCL12. However, ρ⁰A549 cells showed no changes in mitochondrial distribution in response to CXCL12, and only a few ρ⁰A549 cells migrated across the transwell membrane after CXCL12 treatment. These results demonstrate that, in A549 lung cancer cells, intact mitochondrial DNA is necessary for mitochondrial redistribution and a chemotactic response to CXCL12.