mtDNA Lesions Research Articles

Targeting human 8-oxoguanine glycosylase to mitochondria of oligodendrocytes protects against menadione-induced oxidative stress.

Within the central nervous system (CNS), there is a differential susceptibility among cell types to certain pathological conditions believed to involve oxidative stress. Oligodendrocytes are extremely sensitive to oxidative stress, which correlates with a decreased ability to repair damage in mitochondrial DNA (mtDNA), as we have shown previously. To determine whether there is a causal relationship, studies were carried out to correct the deficit in repair of the oxidative damage in mtDNA in cultured oligodendrocytes. A vector containing a mitochondrial transport sequence (MTS) upstream of the sequence for human 8-oxoguanine-DNA glycosylase (OGG) was transfected into the cells. The efficiency of transfection and the localization of recombinant protein were determined by fluorescence microscopy and by Western blot analysis. Subsequent mtDNA repair studies, employing 100 micro M menadione to produce reactive oxygen species, showed a significant enhancement in repair of oxidative lesions in mtDNA of MTS-OGG transfected oligodendrocytes compared with cells transfected with vector only. Experiments were also conducted to determine the effect of changing mtDNA repair capacity on menadione-induced apoptosis in oligodendrocytes. These experiments show that targeting the OGG repair enzyme to mitochondria reduces the release of cytochrome c from the intermitochondrial space and the activation of caspase 9 in oligodendrocytes after exposure to menadione. Therefore, targeting of DNA repair enzymes to mitochondria appears to be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.

Impaired adaptive resynthesis and prolonged depletion of hepatic mitochondrial DNA after repeated alcohol binges in mice

Background & Aims: A single dose of alcohol causes transient hepatic mitochondrial DNA (mtDNA) depletion in mice followed by increased mtDNA synthesis and an overshoot of mtDNA levels. We determined the effect of repeated alcohol binges on hepatic mtDNA in mice. Methods: Ethanol (5 g/kg) was administered by gastric intubation daily for 4 days, and mtDNA levels, synthesis, and integrity were assessed by slot blot hybridization, in organello [3H]deoxythymidine triphosphate incorporation, and long polymerase chain reaction analysis, respectively. Results: mtDNA levels were decreased for 48 hours after the last dose, with no overshoot phenomenon later on. Two and 24 hours after the fourth dose, long polymerase chain reaction experiments showed DNA lesions that blocked the progress of the polymerases and in organello mtDNA synthesis was decreased, although DNA polymerase γ activity was unchanged with synthetic templates. Mitochondria exhibited ultrastructural abnormalities, and respiration was impaired 2 and 24 hours after the fourth binge. Cytochrome P450 2E1, mitochondrial generation of peroxides, thiobarbituric acid reactants, and ethane exhalation were increased. Conclusions: After repeated doses of ethanol, the accumulation of unrepaired mtDNA lesions (possibly involving lipid peroxidation-induced adducts) blocks the progress of polymerase γ on mtDNA and prevents adaptive mtDNA resynthesis, causing prolonged hepatic mtDNA depletion.GASTROENTEROLOGY 2002;123:1278-1290

Mitochondrial repair of 8-oxoguanine and changes with aging

Reactive oxygen species (ROS) are formed in all living organisms as a by-product of normal metabolism (endogenous sources) and as a consequence of exposure to environmental compounds (exogenous sources). Endogenous ROS are largely formed during oxidative phosphorylation in the mitochondria and, therefore, mitochondrial DNA (mtDNA) is at particularly high risk of ROS-induced damage. Mitochondria are essential for cell viability, and oxidative damage to mtDNA has been implicated as a causative factor in a wide variety of degenerative diseases, and in cancer and aging. One of the most common oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which can introduce G/C to T/A transversions after DNA replication. Oxidative DNA base lesions, including 8-oxoG, are repaired primarily by the base excision repair (BER) pathway. While we know much about how this pathway functions in processing the nuclear DNA lesions, little is yet known about BER in mitochondria. We have used a number of different approaches to explore the mechanisms of DNA damage processing in the mtDNA. We have been able to demonstrate that mammalian mitochondria efficiently remove 8-oxoG from their genome, and that the efficiency of 8-oxoG incision increases with age in rats and mice. Yet 8-oxoG accumulates in mtDNA during aging. Changes in mitochondrial function with age have been observed in several organisms and accumulation of DNA lesions in mtDNA with age may be an underlying cause for numerous age-associated diseases including cancer.

A novel defense system of mitochondria in mice and human subjects for preventing expression of mitochondrial dysfunction by pathogenic mutant mtDNAs

Recently, we generated mtDNA-based disease mice (mito mice) by introduction of respiration-deficient mitochondria possessing pathogenic mutant mtDNA with a 4696 bp deletion (ΔmtDNA4696) from somatic cells into mouse zygotes. Mito mice and cytochrome c oxidase (COX) electronmicrographs, that could identify the respiration enzyme activity at individual mitochondrial levels, enabled precise investigation of the pathogenesis of ΔmtDNA4696. All the observations represented unambiguous evidence for the presence of extensive and continuous exchange of genetic contents between mitochondria. Thus, the inter-mitochondrial interaction could correspond to a very unique and effective defense system of the highly oxidative organelles for preventing mice and human subjects from expressing mitochondrial dysfunction caused by mtDNA lesions, which have been continuously created by oxidative stresses during aging. Here, we would like to propose a new hypothesis on mitochondrial biogenesis, ‘the interaction theory of mammalian mitochondria’: mitochondria exchange genetic contents, and thus lose individuality and function as a single dynamic cellular unit.

Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria.

Extensive complementation between fused mitochondria is indicated by recombination of 'parental' mitochondrial (mt) DNA (ref. 1,2) of yeast and plant cells. It has been difficult, however, to demonstrate the occurrence of complementation between fused mitochondria in mammalian species through the presence of recombinant mtDNA molecules, because sequence of mtDNA throughout an individual tends to be uniform owing to its strictly maternal inheritance. We isolated two types of respiration-deficient cell lines, with pathogenic mutations in mitochondrial tRNAIle or tRNALeu(UUR) genes from patients with mitochondrial diseases. The coexistence of their mitochondria within hybrids restored their normal morphology and respiratory enzyme activity by 10-14 days after fusion, indicating the presence of an extensive and continuous exchange of genetic contents between the mitochondria. This complementation between fused mitochondria may represent a defence of highly oxidative organelles against mitochondrial dysfunction caused by the accumulation of mtDNA lesions with age.

Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes.

Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly repaired in MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA lesions at nucleotide resolution showed that damage patterns were similar between EC phenotypes, but there were disparities between XO and menadione in terms of the specific nucleotides damaged. These findings indicate that mtDNA in lung vascular ECs is damaged by XO- and menadione-derived free radicals and suggest that mtDNA damage and repair capacities differ between EC phenotypes.

A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA.

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30 degrees C and shows extensive vegetative petite induction by UV irradiation at 30 degrees C or when cultivated at a higher temperature (37 degrees C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37 degrees C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37 degrees C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.

Enhanced Mitochondrial DNA Repair and Cellular Survival after Oxidative Stress by Targeting the Human 8-Oxoguanine Glycosylase Repair Enzyme to Mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine DNA glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed that this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared with cells transfected with vector only, whereas nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of the tetrazolium compound to formazan, trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.

Poly(ADP-ribose) polymerase facilitates the repair of N-methylpurines in mitochondrial DNA.

This study was designed to test the hypothesis that poly(ADP-ribose) polymerase (PARP) plays a role in the repair of damage to mitochondrial DNA (mtDNA). A rat insulinoma cell line was transfected with a PARP antisense vector that was under the control of a dexamethasone promoter. Transfected cells were selected for stable integration of the antisense vector. Several cell lines containing the antisense vector were isolated. For these studies, one of these lines (clone 5) was chosen for further evaluation. When cells were treated with dexamethasone for 72 h, PARP activity was diminished by 60%. Western blot analysis revealed a concomitant reduction in PARP protein. When clone 5 cells were exposed to the simple methylating agent methylnitrosourea (MNU) without previous treatment with dexamethasone, repair of lesions in mtDNA was found to be similar to that seen in wild-type cells or in wild-type cells treated with dexamethasone. However, when clone 5 cells were pretreated with dexamethasone for 72 h, repair of MNU-induced damage was significantly inhibited. To ascertain whether the PARP activity that was inhibited by the antisense treatment was the same as that found in the nucleus, repair studies were performed on fibroblasts derived from PARP knockout mice and their normal wild-type controls. Attenuated repair was also seen in the cells in which the gene for PARP was inactivated. These are the first studies to demonstrate that PARP can facilitate the repair of simple alkylation damage to mtDNA.

Singlet Oxygen Mediates the UVA-induced Generation of the Photoaging-associated Mitochondrial Common Deletion

Mutations of mitochondrial (mt) DNA accumulate during normal aging. The most frequent mutation is a 4,977-base pair deletion also called the common deletion, which is increased in photoaged skin. Oxidative stress may play a major role in the generation of large scale mtDNA deletions, but direct proof for this has been elusive. We therefore assessed whether the common deletion can be generated in vitro through UV irradiation and whether reactive oxygen species are involved in this process. Normal human fibroblasts were repetitively exposed to sublethal doses of UVA radiation and assayed for the common deletion employing a semiquantitative polymerase chain reaction technique. There was a time/dose-dependent generation of the common deletion, attributable to the generation of singlet oxygen, since the common deletion was diminished when irradiating in the presence of singlet oxygen quenchers, but increased when enhancing singlet oxygen half-life by deuterium oxide. The induction of the common deletion by UVA irradiation was mimicked by treatment of unirradiated cells with singlet oxygen produced by the thermodecomposition of an endoperoxide. These studies provide evidence for the involvement of reactive oxygen species in the generation of aging-associated mtDNA lesions in human cells and indicate a previously unrecognized role of singlet oxygen in photoaging of human skin.

Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals.

The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.

Mitochondrial aging: open questions.

Interest in the role of mitochondria in aging has intensified in recent years. This focus on mitochondria originated in part from the free radical theory of aging, which argues that oxidative damage plays a key role in degenerative senescence. Among the numerous mechanisms known to generate oxidants, leakage of the superoxide anion and hydrogen peroxide from the mitochondrial electron transport chain are of particular interest, due to the correlation between species-specific metabolic rate ("rate of living") and life span. Phenomenological studies of mitochondrial function long ago noted a decline in mitochondrial function with age, and on-going research continues to add to this body of knowledge. The extranuclear somatic mutation theory of aging proposes that the accumulation of mutations in the mitochondrial genome may be responsible in part for the mitochondrial phenomenology of aging. Recent studies of mitochondrial DNA (mtDNA) deletions have shown that they increase with age in humans and other mammals. Currently, there exist numerous important and fundamental questions surrounding mitochondria and aging. Among these are (1) How important are mitochondrial oxidants in determining overall cellular oxidative stress? (2) What are the mechanisms of mitochondrial oxidant generation? (3) How are lesions and mutations in mtDNA formed? (4) How important are mtDNA lesions and mutations in causing mitochondrial dysfunction? (5) How are mitochondria regulated, and how does this regulation change during aging? (6) What are the dynamics of mitochondrial turnover? (7) What is the relationship between mitochondrial damage and lipofuscinogenesis? (8) What are the relationships among mitochondria, apopotosis, and aging? and (9) How can mitochondrial function (ATP generation and the establishment of a membrane potential) and dysfunction (oxidant generation) be modulated and degenerative senescence thereby treated?

PCR amplification using a single cell allows the detection of the mtDNA lesion associated with Leber's hereditary optic neuropathy

The development of the polymerase chain reaction (PCR), which routinely can amplify specific target sequences more than one billion-fold, has made it possible to produce readily detectable amounts of DNA from a few copies of very rare sequences. We have begun a study of mitochondrial myopathies with the purpose of developing a diagnostic test using PCR to amplify appropriate mitochondrial DNA (mtDNA) target sequences from small amounts of sample. We have developed a 15-min procedure for recovering mtDNA which can be amplified by PCR to detectable levels, from as little as 30 μl of blood or 5 μl of amniotic fluid. We have microscopically selected HL60 cells, and have found that 28 cycles of PCR allows the detection of mitochondrial targets from a single cell. Using micromanipulation techniques, we utilized this approach to analyze mtDNA from a single cell isolated from an 8-cell stage mouse blastocyst. Finally, a single cell cultured from a patient with Leber's hereditary optic neuropathy, a mitochondrial myopathy, provided sufficient mtDNA for detection of the single base substitution that leads to loss of a restriction endonuclease recognition site for SfaNI and generation of a site for MaeIII.

Muscle oxphos enzymes and mtDNA lesions in parkinson's disease patients

Muscle oxphos enzymes and mtDNA lesions in parkinson's disease patients