Enhanced ROS production and antioxidant defenses in cybrids harbouring mutations in mtDNA

It has been suggested that mutations in mitochondrial DNA (mtDNA) can produce an increase in reactive oxygen species (ROS) and that this can play a major role in the pathogenic mechanisms of mitochondrial encephalomyopathies. Many studies exist using electron transport chain (ETC) inhibitors, however there are only a few studies that examine ROS production associated with mutations in the mtDNA. To investigate this issue, we have studied ROS production, antioxidant defences and oxidative damage to lipids and proteins in transmitochondrial cybrids carrying different mtDNA mutations. Here, we report that two different mutant cell lines carrying mutations in their mitochondrial tRNA genes (A3243G in tRNA LeuUUR and A8344G in tRNA Lys) showed an increased ROS production with a parallel increase in the antioxidant enzyme activities, which may protect cells from oxidative damage in our experimental conditions (no overt oxidative damage to lipids and proteins has been observed). In contrast, cytochrome c oxidase (COX) mutant cybrids (carrying the stop-codon mutation G6930A in the COXI gene) showed neither an increase in ROS production nor elevation of antioxidant enzyme activities or oxidative damage. These results suggest that the specific location of mutations in mtDNA has a strong influence on the phenotype of the antioxidant response. Therefore, this issue should be carefully considered when antioxidant therapies are investigated in patients with mitochondrial disorders.     

The A3243G tRNALeu(UUR) mutation induces mitochondrial dysfunction and variable disease expression without dominant negative acting translational defects in complex IV subunits at UUR codons

Mutations in the mitochondrial tRNA(Leu(UUR)) gene are associated with a large variety of human diseases through a largely undisclosed mechanism. The A3243G tRNA(Leu(UUR)) mutation leads to reduction of mitochondrial DNA (mtDNA)-encoded proteins and oxidative phosphorylation activity even when the cells are competent in mitochondrial translation. These two aspects led to the suggestion that a dominant negative factor may underlie the diversity of disease expression. Here we test the hypothesis that A3243G tRNA(Leu(UUR)) generates such a dominant negative gain-of-function defect through misincorporation of amino acids at UUR codons of mtDNA-encoded proteins. Using an anti-complex IV immunocapture technique and mass spectrometry, we show that the mtDNA-encoded cytochrome c oxidase I (COX I) and COX II exist exclusively with the correct amino acid sequences in A3243G cells in a misassembled complex IV. A dominant negative component therefore cannot account for disease phenotype, leaving tissue-specific accumulation by mtDNA segregation as the most likely cause of variable mitochondrial disease expression.     

Repopulation of rho0 cells with mitochondria from a patient with a mitochondrial DNA point mutation in tRNA(Gly) results in respiratory chain dysfunction

Familial hypertrophic ventricular cardiomyopathy has been demonstrated to be associated with a number of mitochondrial DNA (mtDNA) mutations. A fibroblast cell line carrying a mutation in its mtDNA at position 9997 in the gene encoding tRNA glycine was obtained from a patient with hypertrophic cardiomyopathy. To demonstrate that the etiology of this disease was a result of the mtDNA mutation, cybrid clones were constructed by fusion of enucleated patient skin fibroblasts to rho0 osteosarcoma cells. Clones carrying high levels of mutant mtDNA showed predominantly cytochrome c oxidase and complex I deficiency, as well as an elevated lactate/pyruvate (L/P) ratio, a biochemical marker characteristic of respiratory chain deficiencies. Pulse-labeling experiments demonstrated a strong negative correlation between the levels of newly synthesized mtDNA-encoded polypeptides and glycine content. These data suggest that the T9997C mutation in mtDNA is causative of respiratory chain dysfunction when present at high levels of heteroplasmy.     

An intragenic suppressor in the cytochrome c oxidase I gene of mouse mitochondrial DNA

We report here the identification of a cell line containing single and double missense mutations in cytochrome c oxidase (COX) subunit I gene of mouse mitochondrial DNA. When present in homoplasmy, the single mutant displays a normal complex IV assembly but a significantly reduced COX activity, while the double mutant almost completely compensates the functional defect of the first mutation. We discuss the potential structural consequences of those mutations based on the modeled structure of mouse complex IV. Based on genetic, biochemical and molecular analyses of cultured mouse cells we infer that: (i) deleterious mutations can arise and become predominant; (ii) cultured cells can maintain several mtDNA haplotypes at stable frequencies; (iii) the respiratory chain has little spare COX capacity; and (iv) the size of a cavity in the vicinity of Val421 in CO I of animal COX may affect the function of the enzyme.     

A novel heteroplasmic tRNAleu(CUN) mtDNA point mutation in a sporadic patient with mitochondrial encephalomyopathy segregates rapidly in skeletal muscle and suggests an approach to therapy

A novel mtDNA point mutation was detected in the tRNAleu(CUN) gene (G to A at position 12315) in a sporadic patient with chronic progressive external ophthalmoplegia, ptosis, limb weakness, sensorineural hearing loss and a pigmentary retinopathy. The mutation disrupts base pairing in the T psi C stem at a site which has been conserved throughout evolution. Although the other mtDNA tRNAleu gene (UUR) is a hotspot for mutation, this is the first pathogenic mutation to be reported in the gene coding for tRNAleu(CUN). MtDNAs carrying the mutation constituted 94% of total mtDNAs in two separate muscle biopsies. Single fibre analysis showed that skeletal muscle fibres without detectable cytochrome c oxidase activity (COX-ve fibres) contained predominantly mutant mtDNAs (93-98%) while fibres with apparently normal COX activity had up to 90% mutant mtDNAs, demonstrating that the G12315A mutation is functionally recessive. Immunofluorescence studies with specific antibodies to mtDNA- or nuclear-encoded subunits of COX were consistent with a defect in mitochondrial protein translation. The mutation was not present in blood cells or cultured fibroblasts and surprisingly, it could not be detected in satellite cells cultured from the patient's muscle. This pattern, which may by typical of patients who have inherited new germline pathogenic mtDNA mutations, possibly reflects loss of the mutation by random genetic drift in mitotic tissues and proliferation of mitochondria containing the mutant mtDNA in post- mitotic cells. The absence of mtDNA carrying the mutation in satellite cells suggests that regeneration of skeletal muscle fibres from satellite cells could restore a wild-type mtDNA genotype and normal muscle function.      

The relationship between mitochondrial genotype and mitochondrial phenotype in lymphoblasts with a heteroplasmic mtDNA deletion

The relationship between mitochondrial genotype and mitochondrialphenotype was investigated in lymphoblasts derived from a patientwith the Pearson syndrome. In 70% of the mtDNA of this Pearsoncell line a deletion from within the COX II gene to within theND5 gene was present. The deletion led to a reduced expressionof the deleted genes, but the severely lowered synthesis ofe.g. subunlt II of cytochrome c oxidase was not reflected ina significant decrease in the cytochrome c oxidase activity.Moreover, there were no obvious differences between controlcells and Pearson cells regarding the capacity for oxidativephosphorylation. Analysis of the synthesis and assembly of bothnuclearly and mitochondrially encoded subunlts of cytochromec oxidase showed that normally mtDNA-encoded polypeptides areproduced in excess. This overproduction fully explained thediscrepancy between the severe defect in the expression of themitochondrial genome and the normal mitochondrial function inthe Pearson cells. These data demonstrate that the expressionof one or more mitochondrial genes can be reduced specificallyat intermediate percentages of deleted mtDNA. However, the dataalso suggest that whether or not a lower expression of mitochondrialgenes encoding subunits of enzymes involved In oxidative phosphorylationinfluences the normal function of these enzymes depends on therelative abundance of the mitochondrial subunits In tissuesor cells with deleted mtDNA.     

Different in situ hybridization patterns of mitochondrial DNA in cytochrome c oxidase-deficient extraocular muscle fibres in the elderly

Summary Previous studies have revealed an increase of cytochrome c oxidase-deficient fibres/cells in the skeletal and heart muscle of humans during ageing. The enzyme defect is due to a lack of both mitochondrial and nuclear coded enzyme subunits. In the present investigation in situ hybridization of mitochondrial DNA (mtDNA) has been performed on extraocular muscles of humans over 70 years of age to show whether mutated mtDNA with the so called common deletion of 4,977 basepairs at position 8,482–13,460 of mtDNA accumulates in the cytochrome c oxidase-deficient fibres. The cytochrome c oxidase-deficient fibres revealed different hybridization patterns: a normal hybridization signal with three different mtDNA probes, a reduced or lacking signal with all three probes indicating depletion of mtDNA and a selective hybridization defect with the probe recognizing the common deletion region of mtDNA as evidence of mtDNA deletion. The results suggest that during ageing defects of cytochrome c oxidase are associated with different molecular alterations of mtDNA. Deletion and depletion of mtDNA are not the only nor probably the leading mechanisms responsible for the loss of respiratory chain capacity during ageing. The normal hybridization signal in most of the cytochrome c oxidase-deficient fibres and the loss of mitochondrial and nuclear protein subunits indicate the involvement of other, especially nuclear factors.     

Altered mitochondrial energy metabolism may play a role in vascular aging

Epidemiological studies demonstrated that even in the absence of other risk factors (e.g., diabetes, hypertension, hypercholesterolemia), vascular aging significantly increases cardiovascular morbidity. Previous studies revealed that vascular aging is characterized by an age-dependent decline in endothelial function due to a decreased bioavailability of NO and increased production of reactive oxygen species. Yet, the mechanisms underlying the process of vascular aging are still poorly understood. Many authors consider that aging is a mitochondrial disease. Indeed, there is evidence that aging is associated with an increase in mtDNA damage and a decline in expression/activity of mitochondrial enzymes in various organs. On the basis of recent observations we predict that similar changes in mitochondrial gene expression profile are present in the aged cardiovascular system as well. It is significant, that components of the electron transport chain (including cytochrome c oxidase) seem to be similarly down-regulated with age in many species. Because pharmacological inhibition of mitochondrial energy metabolism significantly impairs endothelium-dependent vascular relaxation and may increase the production of reactive oxygen species, we propose that alterations of mitochondrial energetic phenotype may contribute to endothelial dysfunction in aging.     

Men with oligoasthenoteratozoospermia harbour higher numbers of multiple mitochondrial DNA deletions in their spermatozoa, but individual deletions are not indicative of overall aetiology

It is believed that one cause of sperm dysfunction might arise through multiple mitochondrial DNA deletions (Delta mtDNA) resulting in the formation of an incomplete electron transport chain. This study investigates the incidence of multiple Delta mtDNA in human spermatozoa prepared on Percoll gradients. Firstly, we investigated for the presence of two frequently analysed Delta mtDNA, the 4977 and 7.4 kb deletions, using conventional polymerase chain reaction (PCR). These two deletions are characteristically flanked by direct repeats. We further analysed the incidence of one other deletion, the 15 bp deletion in the cytochrome c oxidase subunit III (COX III) of complex IV to determine whether other deletions flanked by direct repeats could be equally predictive. The incidence of these three deletions was not clearly associated with the diagnostic categorization of male infertility. However, the use of long PCR showed that samples harbouring high numbers of Delta mtDNA were associated with the diagnostic categorization of male infertility. We propose that these deletions could arise through a free radical-driven event occurring at the spermatogonial cell stage resulting in the replication of Delta mtDNA molecules at the expense of wild-type molecules. These anomalies in ejaculated sperm mtDNA could account for reproductive failure in some men.     

Mutations in AAC2, equivalent to human adPEO-associated ANT1 mutations, lead to defective oxidative phosphorylation in Saccharomyces cerevisiae and affect mitochondrial DNA stability

Autosomal dominant and recessive forms of progressive external ophthalmoplegia (adPEO and arPEO) are mitochondrial disorders characterized by the presence of multiple deletions of mitochondrial DNA in affected tissues. Four adPEO-associated missense mutations have been identified in the ANT1 gene. In order to investigate their functional consequences on cellular physiology, we introduced three of them at equivalent positions in AAC2, the yeast orthologue of human ANT1. We demonstrate here that expression of the equivalent mutations in aac2-defective haploid strains of Saccharomyces cerevisiae results in (a) a marked growth defect on non-fermentable carbon sources, and (b) a concurrent reduction of the amount of mitochondrial cytochromes, cytochrome c oxidase activity and cellular respiration. The efficiency of ATP and ADP transport was variably affected by the different AAC2 mutations. However, irrespective of the absolute level of activity, the AAC2 pathogenic mutants showed a significant defect in ADP versus ATP transport compared with wild-type AAC2. In order to study whether a dominant phenotype, as in humans, could be observed, the aac2 mutant alleles were also inserted in combination with the endogenous wild-type AAC2 gene. The heteroallelic strains behaved as recessive for oxidative growth and petite-negative phenotype. In contrast, reduction in cytochrome content and increased mtDNA instability appeared to behave as dominant traits in heteroallelic strains. Our results indicate that S. cerevisiae is a suitable in vivo model to study the pathogenicity of the human ANT1 mutations and the pathophysiology leading to impairment of oxidative phosphorylation and damage of mtDNA integrity, as found in adPEO.     

In situ lineage tracking of human prostatic epithelial stem cell fate reveals a common clonal origin for basal and luminal cells

Stem cells accumulate mitochondrial DNA (mtDNA) mutations resulting in an observable respiratory chain defect in their progeny, allowing the mapping of stem cell fate. There is considerable uncertainty in prostate epithelial biology where both basal and luminal stem cells have been described, and in this study the clonal relationships within the human prostate epithelial cell layers were explored by tracing stem cell fate. Fresh-frozen and formalin-fixed histologically-benign prostate samples from 35 patients were studied using sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) enzyme histochemistry and COX subunit I immunofluorescence to identify areas of respiratory chain deficiency; mtDNA mutations were identified by whole mitochondrial genome sequencing of laser-captured areas. We demonstrated that cells with respiratory chain defects due to somatic mtDNA point mutations were present in prostate epithelia and clonally expand in acini. Lineage tracing revealed distinct patterning of stem cell fate with mtDNA mutations spreading throughout the whole acinus or, more commonly, present as mosaic acinar defects. This suggests that individual acini are typically generated from multiple stem cells, and the presence of whole COX-deficient acini suggests that a single stem cell can also generate an entire branching acinar subunit of the gland. Significantly, a common clonal origin for basal, luminal and neuroendocrine cells is demonstrated, helping to resolve a key area of debate in human prostate stem cell biology.     

Disorders Associated with Depletion of Mitochondrial DNA

Quantitative defects of mtDNA have been recently described in patients with fatal mitochondrial disease of early infancy or mitochondrial myopathy of childhood. There was variable tissue expression and depletion of up to 98% of mtDNA in affected tissues. Pedigree analysis was compatible with mendelian inheritance, suggesting faulty communication between nuclear and mitochondrial genomes, but the primary molecular lesion is unknown. In muscle, morphological studies allowed to correlate mtDNA depletion, absence of mtDNA-encoded peptides, mitochondrial proliferation, and loss of cytochrome c oxidase (COX) activity in individual fibers.     

Characterization of mouse nuclear and mitochondrial mutants with increased resistance to cytochromeb inhibitors

A series of mouse lines with increased resistance to respiratory inhibitors which block electron transport through the protonmotive cytochrome bof complex III have been isolated in this laboratory. We describe here the isolation of a mutant with increased resistance to HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) whose phenotype is due to a nuclear mutation. At the cellular level, there is a severe reduction in respiration with the residual oxygen consumption being resistant to inhibitors of both ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase. At the mitochondrial level, there was a severe derangement in NADH oxidase activity. Electron transport through the succinate oxidase span of the respiratory chain and its coupling to oxidative phosphorylation are also reduced in this nuclear mutant but not to the same extent. It is concluded that the primary defect in the mutant lies within a nuclear gene encoding a component of complex I (NADH-ubiquinol oxidoreductase). In addition, further biochemical characterization of the mitochondrially inherited inhibitor-resistant mutants has demonstrated that they also show significant reductions in the efficiency of energy transduction and in the rate of cytochrome belectron transport.     

High mutational burden in the mtDNA control region from aged muscles: a single-fiber study

The ageing process is associated with the accumulation of somatic mutations of mitochondrial DNA (mtDNA). The aged human skeletal muscle tissue presents a mosaic of fibers when stained histochemically for cytochrome c oxidase (COX) activity with a proportion of COX negative fibers. Given the potential relevance of any alteration in the mtDNA control region for replication, we analysed the correlation between the presence of mutations and their degree of heteroplasmy and the COX phenotype in individual muscle fibers of aged healthy donors.A region of the mtDNA D-loop was cloned from single fiber-derived DNA and multiple clones were analysed. This strategy showed that a high level of mutational burden is present in all fibers and that several types of mtDNA rearrangements are detectable: recurrent (A189G, T408A and T414G) and rare point mutations, length variations affecting the homopolymeric tract and the (CA)(n) repeat and macrodeletions. The aggregate mutational load in the D-loop region correlated with the single fiber COX phenotype, suggesting that the cumulative burden of multiple, individually rare, mtDNA alterations might functionally impair the mitochondrial genetic machinery.     

Renal and skin involvement in a patient with complete Kearns-Sayre syndrome.

We report on a 13-year-old girl with complete Kearns-Sayre syndrome (KSS) and unusual manifestations of anhidrosis and de Toni-Fanconi-Debré syndrome which preceded by several years the onset of KSS triad. Histochemical examination of skeletal muscle showed focal deficiency of cytochrome c oxidase (CCO). Southern blot analysis of mitochondrial DNA (mtDNA) demonstrated a deletion of 5.4 kb in 60% of the total mtDNAs isolated from the muscle and kidney. On electron microscopy, epithelial cells of the proximal and distal renal tubules and the sweat glands showed an increased number of giant mitochondria with complicated and concentric cristae. This appears to be the first report of complete KSS associated with renal and skin involvement. Data obtained in this patient provide important information on the clinical heterogeneity and tissue specificity of CCO deficiency.     

Phylogenetic analysis of the mitochondrial genome indicates significant differences between patients with Alzheimer disease and controls in a French-Canadian founder population.

The activity of cytochrome oxidase (CO), the terminal enzyme of the mitochondrial electron transport chain, has been reported to be lower in the brains of Alzheimer disease (AD) patients. This suggests that a modification of mitochondrial DNA (mtDNA) may be responsible for this decrease of CO activity. Many mtDNA variants were found by different studies at a higher frequency in AD patients, suggesting that mtDNA variants could confer a genetic susceptibility to AD. In this study, we sequenced the entire mitochondrial genome region that encompasses the three CO genes and the 22 mitochondrial tRNA in 69 AD patients and 83 age-matched controls. We detected a total of 95 mtDNA variants. The allele frequencies of the majority of these variants were similar in patients and controls. However, a haplotype composed of three different modifications (positions: 5633, 7476, and 15812) was present in three of the 69 late-onset AD patients (4.3%) and also in 1 of 16 early-onset AD patients (6.2%) but not in control individuals. Given that one of these variants (15812) has already been shown to be associated with another neurodegenerative disease and that all three modifications are relatively conserved and their frequencies in the general population is only 0.1%, our data suggest that the presence of this haplotype may represent a risk factor for AD. We also found a significant association (P < 0.05) of two other variants at positions 709 (rRNA 12S) and 15928 (tRNA(Thr)). These two mtDNA variants are three times more frequent in control individuals compared with AD patients, suggesting that they may be protective against AD.