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Base excision repair of mitochondrial DNA damage in mammalian cells
Base Excision Repair of Mitochondrial DNA Damage in Mammalian Cells S. E L E D O U X AND G. L. WILSON
Department of Cell Biology and Neuroscience University of South Alabama Mobile, Alabama 36688 I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for BER in Mammalian Mitochondrial DNA . . . . . . . . . . . . . . . Repair of Oxidative Damage to mtDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . Repair of NO-Induced Damage in mtDNA . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Damage and Repair to mtDNA at the Level of Individual Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Mechanisms Involved in the Repair of mtDNA . . . . . . . . . . . . . . . . . . . . . VII. Cell-Specific Differences in mtDNA Repair . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusions and Future Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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This review of the work from our laboratory describes initial studies in which base excision repair in mtDNA was first seen. It considers the results of experiments in which the substrates for mtDNA repair were identified. The discussion then focuses on studies during which the sequence context for mtDNA damage and repair were explored. Next, it addresses factors that have been identified that influence mtDNA repair. Finally, it summarizes the results of studies that evaluated cell-specific differences in the repair of mtDNA and explored some of the biological consequences of these differences. © ~001 AcademicPress.
I. Inh'oduction O v e r t h e p a s t 10 years, i n t e r e s t i n m t D N A d a m a g e h a s r i s e n w i t h t h e discovery that defects in the mitochondrial genome are associated with several Abbreviations: BER, base excision repair; mtDNA, mitochondrial DNA; CNS, central nervous system; PCR, polymerase chain reaction; ROS, reactive oxygen species; RNS, reactive nitrogen species; SZ streptozotocin; MNU, methylnitrosourea; DMS, dimethyl sulfate; CHO, Chinese hamster ovary; LM-PCR, ligation-mediated polymerase chain reaction; XO, xanthine oxidase; NO, nitric oxide; XP, Xeroderma pigmentosum; CuZn SOD, copper-zinc superoxide dismutase; MnSOD, manganese superoxide dismutase. Progressin NucleicAcidResearch and MolecularBiology,Vol.68
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CopyrightO 2001byAcademicPress. Allrightsof reproductionin anyformreserved. 0079-6603/01$35.00
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human hereditary diseases such as Kearns-Sayre syndrome, Leber's hereditary optic neuropathy, Pearson's syndrome, and some cases of chronic progressive external ophthalmoplegia (1, 2). Additionally, accumulations of mutations and deletions in mtDNA with their associated defects in oxidative phosphorylation have been implicated in diabetes, ischemic heart disease, Parkinson's disease, demyelinating polyneuropathy, cancer, and aging (1-4). A key question to be answered is how alterations in the mitochondrial genome, which basically affect only electron transport, can cause different diseases. One mechanism that could lead to altered cellular function is related to differences in energy requirements in different cellular compartments. There is evidence that mitochondria are required to provide compartmentalized ATP to specific areas of the cell (5-8). For instance, the cell membrane requires ATP to energize specialized processes such as ion pumping, electrical transmission across the membrane as in Na+-K + exchange, and neurotransmitter release (9). Since different cells have unique energy requirements for these processes, it is likely that they would be functionally affected in different ways by mutations in mtDNA. Another way that mtDNA damage could differentially affect cells is through progressive cell death. Interest in the initiation and regulation of apoptosis has resulted in an exponential growth in research in this area over the last few years. Heterogeneous death signals precede a common effector phase during which cells pass a threshold of "no return," and are engaged in a degradation phase that results in the disassembly of the cellular scaffolding. There have been numerous hypotheses which postulate that specific mediators of pathways are responsible for apoptosis. One hypothesis is that the mitochondfion plays a key role in the regulation of apoptosis (10). Disruption of the mitochondrial membrane potential is associated with the induction of apoptosis following certain stimuli. The upstream factors that precede the disruption of the mitochondrial membrane potential have not been fully elucidated. Previously, the involvement of oxidative damage has been suggested. Becanse cells are continuously bombarded by reactive oxygen species (ROS), their survival depends upon a fine balance between radical production, damage repair, and antioxidant activity. The hypothesis that oxidative damage plays a role in apoptosis is supported by the observation that the addition of ROS or the removal of endogenous antioxidants results, in some cases, in apoptosis (11). Additionally, tumor necrosis factor-induced apoptosis is associated with increases in intracellular ROS levels (12). In attempting to provide a mechanism by which oxidative damage might induce apoptosis, a recent report presented data consistent with a pathway by which damage to mitochondrial DNA led to a bioenergetic crisis, disruption of mitochondrial membrane potential, and induction of apoptosis (13). Thus, an initial elevation in unrepaired oxidative damage to mtDNA could lead to defects in oxidative phosphorylation. This would cause additional ROS production which would heighten the stress and eventually push the cell into apoptosis.
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Therefore, mtDNA repair may play a pivotal role in normal cellular defense mechanisms. While complex enzymatic mechanisms for recognition and repair of nuclear DNA damage have been demonstrated, the study of mitochondrial DNA repair processes was impaired greatly by the difficultyin isolating sufficient quantities of mitochondrial DNA free of nuclear DNA contamination. It was originally thought that the mitochondrion did not repair damage to its DNA. Rather, when mtDNA was damaged, it was believed that it was degraded and new mtDNA was synthesized from undamaged templates. However, using sequencespecific repair analysis, it has been demonstrated that mitochondria possess efficient base excision repair (BER) capacity.
II. Evidence for BER in Mammalian Mitochondrial DNA In the initial studies that identified BER in mtDNA within mammalian cells
(14), mtDNA was distinguished in Southern transfers of total cellular DNA, using a probe that contained the entire 16.5-kb mouse mitochondrial genome. The formation and repair of N-methylpurines, which are alkali-labile, was measured in an insulinoma cell line (RINr 38) after exposure to the naturally occurring nitrosoamide streptozotocin (SZ). Alkali-labile sites were formed in mtDNA in a dose-dependent fashion. Eight hours after exposure to the toxin, 55% of the lesions were removed. The amount of repair increased to 70% after 24 h. In comparison, only 46% of NT-methylguanines were removed from the entire cellular genome at this time. These studies demonstrated that SZ causes appreciable mtDNA damage in a dose-dependent manner, and provided the first evidence that there is a repair mechanism in the mitochondrion for removal of these alkali-labile lesions. Following the demonstration of an apparent BER mechanism in mitochondria, it remained to be determined whether mitochondria could repair only lesions produced by simple alkylating toxins, or whether they have the capability for correcting damage caused by other agents. Accordingly, studies were undertaken to investigate the repair of DNA lesions in mtDNA from CHO Bll cells following exposure to different agents (15), namely methylnitrosourea (M N U), dimethyl sulfate (D M S), nitrogen mustard, ultraviolet (UV) irradiation, and c!tsplatin. CHO cells were used because a wealth of information is already available concerning repair of different types of lesions in nuclear DNA from these cells. The results revealed that repair of mtDNA damage depends upon the type of lesion produced by the damaging agent. There was efficient repair of methylation damage following exposure to MNU or DMS, with approximately 70% of the lesions being removed by 24 h. However, more complex alkylation damage, such as that resulting from exposure to nitrogen mustard, was not repaired. Additionally, no repair of pyrimidine dimers after exposure to UV light was detected. Cisplatin intrastrand crosslinks also were not repaired; however,
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interstrand crosslinks resulting from this toxin were repaired to a significant degree. More than 70% of these lesions were removed from mtDNA by 24 h. Therefore, these studies showed that, while the nuclei of CHO cells possess mechanisms to repair DNA damage by all the agents used, the mitochondria were able to repair only specific types of injury.
lU. Repair of Oxidative Damage to mlDNA Since mitochondria are constantly exposed to high levels of reactive oxygen species, it is likely that oxidative damage to mtDNA may be responsible for some of the maladies associated with aging. To determine whether mitochondria are able to repair this type of damage, a modification of the same Southern blot technique utilized to study repair of alkylation damage was employed (16). Alloxan was used as an oxygen-radical generator. Insulinoma cells were exposed to this toxin for 1 h and the DNA was isolated immediately, or after repair intervals of up to 8 h. Alkali treatment was used to identify abasic (AP) sites and sugar lesions, endonuclease III was used to identify a variety of lesions associated with thymine and cytosine, and FAPY glycosylase was employed to recognize formamidopyrimidines and 8-oxoguanines in the restricted DNA. The results showed that all the forms of damage studied were repaired completely by 4 h, indicating that mitochondria are able to efficiently repair injury to their DNA caused by ROS. This was the first report directly showing repair of oxidative damage in mtDNA. We subsequently expanded these studies to show that damage induced by the radiomimetic drug bleomycin was also repaired rapidly (17).
IV. Repair of NO-Induced Damage in mtDNA Most recently, we have directed our attention to the other reactive species to which mtDNA is frequently exposed, nitric oxide (NO). Initially, we showed that mtDNA from primary cultures of insulin-secreting/~-cells is a more vulnerable target than nuclear DNA for damage caused by NO produced endogenously and exogenously (18). Therefore, whether NO damage is repaired became a crucial question that needed to be answered. To address this question, experiments were initiated in which normal human fibroblasts were exposed to NO generated by PAPA NONOate (PAPA/NO) (19). Cells were subjected acutely to different concentrations of the NO generator, total cellular DNA was isolated, and a Southern blot procedure was performed to determine damage to mtDNA. Extensive damage to mtDNA was revealed which was blocked by the NO scavenger carboxy-PTIO. Thus, the damage to the mtDNA was likely the result of deamination reactions. In addition to the deamination of guanine to
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xanthine and adenine to hypoxanthine, both of which are alkali-labile, it was possible that cytosine was deaminated to uracil. However, treatment of mtDNA that had been exposed to the NO generator with uracil DNA glycosylase did not reveal any additional damage. To assess repair, cells were treated with NO and allowed to repair the damage before the DNA was isolated. Most of the damage was repaired by 4 h after exposure. Therefore, these were the first studies to show that NO selectively damages mtDNA, and that this damage is of sufficient consequence that the mitochondrion has had to evolve efficient mechanisms to rapidly repair it (18).
V. Evaluation of Damage and Repair to mlDNA at the Level of Individual Nucleotides In order to address mechanistic questions concerning mtDNA mutations, it was necessary to analyze damage and repair of mtDNA at the nucleotide level. Therefore, the technique of ligation-mediated PCR (LM-PCR), which had been used by Dr. Holmquist and colleagues to study nuclear repair (20), was adapted for the evaluation ofmtDNA (21). Initially, the frequencies of singlestrand breaks and oxidative base damage in mtDNA from insulinoma cells were measured. Addition of 5 mM alloxan to the cells increased the rate of oxidative base damage and the lesion frequency in mtDNA severalfold. Guanine positions showed the highest endogenous lesion frequencies and were the most responsive bases to alloxan-induced oxidative stress. Although specific bases were consistently hotspots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner. These data revealed nonrandom oxidative damage in several nucleotides which correlated with one of the break sites of the 5-kb "common deletion" seen with aging. Additionally, there was an apparent adaptive, non-strand-selective response for removal of such damage. Following this initial observation, the technique was used to look at other types of damage (19). When a 200-bp sequence of mtDNA from cells exposed to NO produced by the NO generator PAPA/NO was analyzed, guanine was the predominant base that was damaged. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites, indicating that the predominant lesion is the deamination of guanine to xanthine. Work with the ROS-generating system xanthine oxidase/hypoxanthine showed that many of the same guanines were vulnerable to attack. However, other base damage is also seen. As expected, the methylating agent MNU also selectively alkylated guanines. It is intriguing that the pattern of damaged guanines was not identical to that damaged by NO. The studies using LM-PCR have shed new light on the damage caused by NO in mtDNA. They revealed that guanine is the most frequently damaged base, although there were some damaged adenines
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found when alkaline conditions were employed. No damage was detected at any pyrimidines in the sequence evaluated. For comparison, studies with the ROS generator XO showed that we were able to detect oxidative lesions to both specific purines and pyrimidines. The pattern and frequency of base damage was similar to that observed previously using the ROS generator alloxan (21). Therefore, it appears likely that the inability to detect pyrimidine lesions following exposure to NO was because few, if any, lesions are formed at these bases. These findings support the notion that the damage that occurred to mtDNA is through the formation of N203, which causes deamination of guanine to xanthine and adenine to hypoxanthine. In both eases, this lesion preceded depurination to produce an AP site which was converted to a strand break with an appropriate end for ligation by alkali treatment. That AP sites predominantly are formed in DNA is in agreement with work by Tamir et al. (22) studying plasmid DNAs which were eleetroporated into CHO cells. When these cells were treated with NO, a significant number of AP sites were produced in the DNA. Additional work by these investigators, using both DNA exposed to NO in vivo and in vitro has revealed that xanthine followed by hypoxanthine are the predominant base alterations (23, 24). By comparing the pattern of damage produced by NO to that generated by the alkylating agent MNU or the ROS generator XO, it can be seen that, although all three of these agents damaged many of the same guanines, there were certain guanines that were only vulnerable to PAPA/NO. Therefore, it is possible to determine a signature damage pattern for reactive nitrogen species (RNS) that is different from that produced by ROS or methylating toxins. This finding may prove useful for future studies of mtDNA in which the identity of the damaging agent is unknown. Additionally, it will be important to compare damage produced in mtDNA by different agents with patterns of known mtDNA mutations. The fact that NO produced the same pattern of damage when exposed to a PCR-generated mtDNA sequence establishes that the pattern of damage produced is due to the chemical properties of NO interacting within the sequence context of the DNA, rather than being influenced by the association of the DNA with the mitoehondrial matrix proteins or other DNA-binding proteins. When considering lesions in mtDNA, it is important to mention that in cases where a single mutation has been shown to cause a disease, the degree of heteroplasmy is usually between 60 and 90% (mutated genomes to nonmutated). However, in major neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), this is not the ease. While increased mtDNA mutations are seen, none rises to a level sufficient by itself to cause disease. Thus, it is believed that it is a collection of mutations and heightened lesions in mtDNA that inactivates a critical number of mitoehondrial genomes in a specific way to cause disease. LM-PCR allows one
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to determine what mutations might be expected following exposure to specific types of DNA damaging agents.
VI. Mechanisms Involved in the Repair of mtDNA One approach to ascertain whether factors that affect repair in the nucleus also affect repair in the mitochondrion is to study repair in cells from individuals with known repair-deficient phenotypes such as xeroderma pigmentosum (XP). XP complementation group D (XP-D) is known to be defective in the repair of N-methylpurines in their nuclear DNA. To determine whether this defect also is seen in mtDNA from these cells, repair of MNU-induced N-methylpurines in XP-D cells was compared to that seen in normal WI-38 cells (25). At both 8 and 24 h after exposure to MNU, XP-D cells were found to normally repair N-methylpurine lesions in mtDNA, although the repair of the same lesions in the nuclear DNA from XP-D cells was significantly attenuated. These data indicated that the factors that retard repair of nuclear DNA in XP-D cells do not affect the repair of mtDNA in these cells. Extracts from cells of individuals with XP complementation group A (XP-A) have been reported to be defective in the repair of some types of oxidative damage. Therefore, studies were performed to determine whether there was a correlation between the inadequate repair of oxidatively damaged nuclear DNA in XP-A cells and the capacity to repair similar damage to their mtDNA (26). The ability of karyotypically normal human fibroblasts and XP-A fibroblasts to repair alloxan-generated oxidative damage to nuclear and mtDNA was assessed. These data indicate that both nuclear and mitochondrial repair of DNA damage are appreciably more efficient in normal human fibroblasts. These findings suggest a similarity between the process(es) used to repair oxidative damage to nuclear and mtDNA in that both are inefficient in XP-A cells.
VII. Cell-Specific Differences in mIDNA Repair Based on the studies described in the preceding sections, it is known that there are mechanisms for repair of endogenous damage in mtDNA. However, what is not as clear is how important mtDNA repair is to the cellular defenses of normal cells. The first indication of the importance of mtDNA repair to cellular defenses occurred when the simple question, "Are there cell-specific differences in repair of mtDNA?" was asked. Within the central nervous system (CNS) there are two predominant types of cells: neurons, which are the information processing cells of the CNS; and glial cells, which provide supportive functions
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for the neurons. Glial cells are composed mainly of three distinct populations of cells: astrocytes, oligodendrocytes, and microglia. While it was known that these cells play an important role in some diseases of the CNS, little was known about the repair mechanisms utilized by these cells in response to injury. Therefore, a well-characterized culture system (27) to generate pure primary cultures of astrocytes, oligodendrocytes, and microglia was used to evaluate mtDNA repair following both alkylation and oxidative damage. For the studies of simple alkylation damage, MNU was used as the alkylating agent, Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed no cell type-specific differences in initial mtDNA damage. However, mtDNA repair of N-methylpurines in oligodendrocytes and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, greater than 60% of N-methylpurines were removed from the mtDNA by 24 h. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes and microglia during the same time period. Since mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis, analyses using DNA laddering and ultrastructural examination were performed. DNA fragmentation and morphological changes consistent with apoptosis were apparent following MNU treatment of cultured oligodendrocyte progenitors and microglia, but not astroglia. These data were the first to demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis (28). Subsequently, oxidative mtDNA damage and repair also were assessed. Menadione, which undergoes redox cyclingwithin the mitochondria, was used to generate oxygen radicals. The results from these studies showed that exposure to equimolar concentrations of menadione resulted in more initial mtD NA damage in oligodendrocytes and microglia as compared to astrocytes. Repair experiments then were performed using both equimolar concentrations and concentrations that resulted in comparable strand breaks. Under both conditions, astrocytes repaired the damage efficiently with all of the lesions being removed by 6 h in mtDNA of astrocytes as compared to approximately 60% repair in oligodendrocytes or microglia. Our previous findings of a correlation between lack of mtDNA repair of N-methylpurines and induction of apoptosis led us to ask the question whether oligodendrocytes and microglia undergo apoptosis in response to an oxidative insult. ApoTag and annexin V staining, DNA laddering and electron microscopy were used to evaluate apoptosis. There were no indications of apoptosis in any of the experiments with astrocytes, while oligodendrocyte and microglial cultures were positive in all cases. We also questioned whether the increased sensitivity could be due to decreases in antioxidants within the oligodendrocytes and microglia; so, in collaboration with Dr. Doug Spitz at Washington University, we measured enzyme activities for glutathione peroxidase, catalase, CuZn, and MnSOD, along with total, reduced, and oxidized glutathione levels.
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For these studies, cells were prepared exactly as for the repair studies. The results demonstrated that under our culture conditions, there were no significant differences in catalase, glutathione peroxidase, or CuZnSOD between any of the cell types. Concerning glutathione, astrocytes had significantly lower levels than oligodendrocytes or microglia. The same was true for MnSOD (29). Thus, while antioxidant and repair capacity are both involved in protecting cells from oxidant insults, it appears that cells with efficient repair capacity may be spared, even in the presence of very low antioxidant levels, and that cells with less efficient repair are susceptible, even in the presence of higher levels of antioxidants. This observation of cell-specific differences in repair of oxidative damage among glial cells leads to the obvious question "Is there repair of oxidative mtDNA damage in neurons?" To address this question, primary cultures of cerebellar granule cells were exposed to comparable concentrations of menadione. More initial damage was observed in the neuronal cultures as compared to the glial cells. Because of the increased sensitivity, a 50-#M concentration was used for subsequent repair experiments. The results of these experiments demonstrated that the repair kinetics were slower in neurons as compared to glial cells, but by 48 h the lesions had been removed. As in the glial cultures with decreased repair capacity, apoptosis was induced in cerebellar granule cells by menadione exposure using quantitative electron microscopic evaluation, annexin V positive staining, or Apotag positive staining as the marker for apoptosis. Since menadione redox cycles with complex I of the electron transport chain to produce superoxide, one could hypothesize that mitochondria may play a substantia~ role through activation of caspases. Of the caspases, caspase 9 has been associated with mitochondrial changes. The release of cytochrome c from the mitochondrial intermembrane space to the cytosol is necessary for the activation of this caspase. Western blots were performed using mitochondrial and cytosolic proteins and a monoclonal antibody to cytochrome c. No cytochrome c protein was detected in the cytosolic fraction prior to menadione treatment, indicating that the cell fractionation procedure had not disrupted the outer mitochondrial membrane. However, 2 h after exposure to 100/zM menadione, there was a decrease in the intensity of the mitochondrial cytochrome c band with a concomitant increase in the cytosolic band of oligodendrocytes and microglia. As expected, astrocytes, which show no evidence of cell death in response to menadione exposure, showed no increase in the cytosolic cytochrome c band. Therefore, release of cytochrome c from the mitochondria into the cytosol correlates with induction of apoptosis in CNS glial cells (29). To determine if this release of cytochrome c resulted in the activation of caspase 9, a colorimetric activity assay based on cleavage of a caspase 9-specific substrate was performed. For a positive control, staurosporine, which has been shown to cause cytochrome c release in cerebellar granule neurons, was employed. After 3 h, caspase 9 activity in oligodendrocytes, microglia, and Jurkat
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cells exposed to menadione was elevated compared to control cells. No detectable increase in astrocyte caspase 9 activity was found. Subsequent experiments were performed using xanthine oxidase and hypoxanthine to generate the ROS. Again, activation of caspase 9 was observed (29). Since it has been suggested that caspase 8 may play a role in the mitochondrial pathway of apoptosis, its activity following menadione exposure was tested in each of the cell types. Activation of caspase 8 has been well documented in the Fas-pathway of apoptosis; thus, Jurkat cells treated with an anti-Fas antibody were used for a positive control. No caspase 8 activity was detected in any of the cell types following exposure to menadione. Following treatment with the antiFas antibody, however, oligodendrocytes and microglia demonstrated caspase 8 activity. Conversely, astrocytes remained unresponsive to induction ofapoptosis, showing no caspase 8 activity. From these experiments, it appears that mtDNA repair plays a pivotal role in cellular defense mechanisms. This statement is based on the evidence that there are cell-specific differences in the repair of mtDNA damage. The importance of these differences is exemplified in the observation that decreases in mtDNA repair capacity correlates with increased cell killing. Cell death occurs through the induction of apoptosis involving a mitochondrial pathway that includes the release of cytochrome c and the activation of caspase 9.
VIII. Conclusionsand Future Questions It is becoming increasingly clear that lesions in mtDNA play an important role in a number of human diseases. Owing to the crucial role that mtDNA integrity plays in cellular processes, a new area for investigation into the pathogenesis of a number of age-related diseases has been identified. The premise upon which these investigations are based is that an acceptable lesion equilibrium must be maintained in mtDNA for normal mitochondrial function. This lesion equilibrium is controlled by a balance in the rate at which mtDNA is damaged and the rate at which these lesions are removed. If this critical balance is disrupted either by an increase in the rate of mtDNA damage or a decrease in the rate of repair, fewer functioning mitochondrial genomes will be available for transcription and cellular bioenergetics will decrease. This will cause cellular functions to diminish due to energy depletion. Additionally, there will be an increase in oxidative stress in the cell due to defects in electron transport caused by the heightened damage to mtDNA. If this deleterious process is allowed to continue, the cell will ultimately die via apoptotic or necrotic mechanisms. There are several ways in which intervention could alter this catastrophic cascade of events. However, before such intervention can be initiated, a more thorough understanding of the mechanisms involved in the alteration of the lesion
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equilibrium in mtDNA needs to be obtained. Therefore, many questions remain to be answered. The problems remaining to be addressed include a more precise definition of the components involved in mtDNA repair, a better comprehension ofhow they are regulated, a more thorough understanding of how they can malfunction to precipitate disease states, and the development of gene therapy protocols to reverse repair defects and prevent or delay the onset of disease.
ACKNOWLEDGMENTS This work was supported in part by the United States Public Health Service Grants ESO5865, ESO0313, and ESO3456 from the National Institute of Environmental Health Sciences, and AG12442 from the Institute of Aging,
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Department of Cell Biology and Neuroscience University of South Alabama Mobile, Alabama 36688 I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for BER in Mammalian Mitochondrial DNA . . . . . . . . . . . . . . . Repair of Oxidative Damage to mtDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . Repair of NO-Induced Damage in mtDNA . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Damage and Repair to mtDNA at the Level of Individual Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Mechanisms Involved in the Repair of mtDNA . . . . . . . . . . . . . . . . . . . . . VII. Cell-Specific Differences in mtDNA Repair . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusions and Future Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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This review of the work from our laboratory describes initial studies in which base excision repair in mtDNA was first seen. It considers the results of experiments in which the substrates for mtDNA repair were identified. The discussion then focuses on studies during which the sequence context for mtDNA damage and repair were explored. Next, it addresses factors that have been identified that influence mtDNA repair. Finally, it summarizes the results of studies that evaluated cell-specific differences in the repair of mtDNA and explored some of the biological consequences of these differences. © ~001 AcademicPress.
I. Inh'oduction O v e r t h e p a s t 10 years, i n t e r e s t i n m t D N A d a m a g e h a s r i s e n w i t h t h e discovery that defects in the mitochondrial genome are associated with several Abbreviations: BER, base excision repair; mtDNA, mitochondrial DNA; CNS, central nervous system; PCR, polymerase chain reaction; ROS, reactive oxygen species; RNS, reactive nitrogen species; SZ streptozotocin; MNU, methylnitrosourea; DMS, dimethyl sulfate; CHO, Chinese hamster ovary; LM-PCR, ligation-mediated polymerase chain reaction; XO, xanthine oxidase; NO, nitric oxide; XP, Xeroderma pigmentosum; CuZn SOD, copper-zinc superoxide dismutase; MnSOD, manganese superoxide dismutase. Progressin NucleicAcidResearch and MolecularBiology,Vol.68
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human hereditary diseases such as Kearns-Sayre syndrome, Leber's hereditary optic neuropathy, Pearson's syndrome, and some cases of chronic progressive external ophthalmoplegia (1, 2). Additionally, accumulations of mutations and deletions in mtDNA with their associated defects in oxidative phosphorylation have been implicated in diabetes, ischemic heart disease, Parkinson's disease, demyelinating polyneuropathy, cancer, and aging (1-4). A key question to be answered is how alterations in the mitochondrial genome, which basically affect only electron transport, can cause different diseases. One mechanism that could lead to altered cellular function is related to differences in energy requirements in different cellular compartments. There is evidence that mitochondria are required to provide compartmentalized ATP to specific areas of the cell (5-8). For instance, the cell membrane requires ATP to energize specialized processes such as ion pumping, electrical transmission across the membrane as in Na+-K + exchange, and neurotransmitter release (9). Since different cells have unique energy requirements for these processes, it is likely that they would be functionally affected in different ways by mutations in mtDNA. Another way that mtDNA damage could differentially affect cells is through progressive cell death. Interest in the initiation and regulation of apoptosis has resulted in an exponential growth in research in this area over the last few years. Heterogeneous death signals precede a common effector phase during which cells pass a threshold of "no return," and are engaged in a degradation phase that results in the disassembly of the cellular scaffolding. There have been numerous hypotheses which postulate that specific mediators of pathways are responsible for apoptosis. One hypothesis is that the mitochondfion plays a key role in the regulation of apoptosis (10). Disruption of the mitochondrial membrane potential is associated with the induction of apoptosis following certain stimuli. The upstream factors that precede the disruption of the mitochondrial membrane potential have not been fully elucidated. Previously, the involvement of oxidative damage has been suggested. Becanse cells are continuously bombarded by reactive oxygen species (ROS), their survival depends upon a fine balance between radical production, damage repair, and antioxidant activity. The hypothesis that oxidative damage plays a role in apoptosis is supported by the observation that the addition of ROS or the removal of endogenous antioxidants results, in some cases, in apoptosis (11). Additionally, tumor necrosis factor-induced apoptosis is associated with increases in intracellular ROS levels (12). In attempting to provide a mechanism by which oxidative damage might induce apoptosis, a recent report presented data consistent with a pathway by which damage to mitochondrial DNA led to a bioenergetic crisis, disruption of mitochondrial membrane potential, and induction of apoptosis (13). Thus, an initial elevation in unrepaired oxidative damage to mtDNA could lead to defects in oxidative phosphorylation. This would cause additional ROS production which would heighten the stress and eventually push the cell into apoptosis.
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Therefore, mtDNA repair may play a pivotal role in normal cellular defense mechanisms. While complex enzymatic mechanisms for recognition and repair of nuclear DNA damage have been demonstrated, the study of mitochondrial DNA repair processes was impaired greatly by the difficultyin isolating sufficient quantities of mitochondrial DNA free of nuclear DNA contamination. It was originally thought that the mitochondrion did not repair damage to its DNA. Rather, when mtDNA was damaged, it was believed that it was degraded and new mtDNA was synthesized from undamaged templates. However, using sequencespecific repair analysis, it has been demonstrated that mitochondria possess efficient base excision repair (BER) capacity.
II. Evidence for BER in Mammalian Mitochondrial DNA In the initial studies that identified BER in mtDNA within mammalian cells
(14), mtDNA was distinguished in Southern transfers of total cellular DNA, using a probe that contained the entire 16.5-kb mouse mitochondrial genome. The formation and repair of N-methylpurines, which are alkali-labile, was measured in an insulinoma cell line (RINr 38) after exposure to the naturally occurring nitrosoamide streptozotocin (SZ). Alkali-labile sites were formed in mtDNA in a dose-dependent fashion. Eight hours after exposure to the toxin, 55% of the lesions were removed. The amount of repair increased to 70% after 24 h. In comparison, only 46% of NT-methylguanines were removed from the entire cellular genome at this time. These studies demonstrated that SZ causes appreciable mtDNA damage in a dose-dependent manner, and provided the first evidence that there is a repair mechanism in the mitochondrion for removal of these alkali-labile lesions. Following the demonstration of an apparent BER mechanism in mitochondria, it remained to be determined whether mitochondria could repair only lesions produced by simple alkylating toxins, or whether they have the capability for correcting damage caused by other agents. Accordingly, studies were undertaken to investigate the repair of DNA lesions in mtDNA from CHO Bll cells following exposure to different agents (15), namely methylnitrosourea (M N U), dimethyl sulfate (D M S), nitrogen mustard, ultraviolet (UV) irradiation, and c!tsplatin. CHO cells were used because a wealth of information is already available concerning repair of different types of lesions in nuclear DNA from these cells. The results revealed that repair of mtDNA damage depends upon the type of lesion produced by the damaging agent. There was efficient repair of methylation damage following exposure to MNU or DMS, with approximately 70% of the lesions being removed by 24 h. However, more complex alkylation damage, such as that resulting from exposure to nitrogen mustard, was not repaired. Additionally, no repair of pyrimidine dimers after exposure to UV light was detected. Cisplatin intrastrand crosslinks also were not repaired; however,
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interstrand crosslinks resulting from this toxin were repaired to a significant degree. More than 70% of these lesions were removed from mtDNA by 24 h. Therefore, these studies showed that, while the nuclei of CHO cells possess mechanisms to repair DNA damage by all the agents used, the mitochondria were able to repair only specific types of injury.
lU. Repair of Oxidative Damage to mlDNA Since mitochondria are constantly exposed to high levels of reactive oxygen species, it is likely that oxidative damage to mtDNA may be responsible for some of the maladies associated with aging. To determine whether mitochondria are able to repair this type of damage, a modification of the same Southern blot technique utilized to study repair of alkylation damage was employed (16). Alloxan was used as an oxygen-radical generator. Insulinoma cells were exposed to this toxin for 1 h and the DNA was isolated immediately, or after repair intervals of up to 8 h. Alkali treatment was used to identify abasic (AP) sites and sugar lesions, endonuclease III was used to identify a variety of lesions associated with thymine and cytosine, and FAPY glycosylase was employed to recognize formamidopyrimidines and 8-oxoguanines in the restricted DNA. The results showed that all the forms of damage studied were repaired completely by 4 h, indicating that mitochondria are able to efficiently repair injury to their DNA caused by ROS. This was the first report directly showing repair of oxidative damage in mtDNA. We subsequently expanded these studies to show that damage induced by the radiomimetic drug bleomycin was also repaired rapidly (17).
IV. Repair of NO-Induced Damage in mtDNA Most recently, we have directed our attention to the other reactive species to which mtDNA is frequently exposed, nitric oxide (NO). Initially, we showed that mtDNA from primary cultures of insulin-secreting/~-cells is a more vulnerable target than nuclear DNA for damage caused by NO produced endogenously and exogenously (18). Therefore, whether NO damage is repaired became a crucial question that needed to be answered. To address this question, experiments were initiated in which normal human fibroblasts were exposed to NO generated by PAPA NONOate (PAPA/NO) (19). Cells were subjected acutely to different concentrations of the NO generator, total cellular DNA was isolated, and a Southern blot procedure was performed to determine damage to mtDNA. Extensive damage to mtDNA was revealed which was blocked by the NO scavenger carboxy-PTIO. Thus, the damage to the mtDNA was likely the result of deamination reactions. In addition to the deamination of guanine to
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xanthine and adenine to hypoxanthine, both of which are alkali-labile, it was possible that cytosine was deaminated to uracil. However, treatment of mtDNA that had been exposed to the NO generator with uracil DNA glycosylase did not reveal any additional damage. To assess repair, cells were treated with NO and allowed to repair the damage before the DNA was isolated. Most of the damage was repaired by 4 h after exposure. Therefore, these were the first studies to show that NO selectively damages mtDNA, and that this damage is of sufficient consequence that the mitochondrion has had to evolve efficient mechanisms to rapidly repair it (18).
V. Evaluation of Damage and Repair to mlDNA at the Level of Individual Nucleotides In order to address mechanistic questions concerning mtDNA mutations, it was necessary to analyze damage and repair of mtDNA at the nucleotide level. Therefore, the technique of ligation-mediated PCR (LM-PCR), which had been used by Dr. Holmquist and colleagues to study nuclear repair (20), was adapted for the evaluation ofmtDNA (21). Initially, the frequencies of singlestrand breaks and oxidative base damage in mtDNA from insulinoma cells were measured. Addition of 5 mM alloxan to the cells increased the rate of oxidative base damage and the lesion frequency in mtDNA severalfold. Guanine positions showed the highest endogenous lesion frequencies and were the most responsive bases to alloxan-induced oxidative stress. Although specific bases were consistently hotspots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner. These data revealed nonrandom oxidative damage in several nucleotides which correlated with one of the break sites of the 5-kb "common deletion" seen with aging. Additionally, there was an apparent adaptive, non-strand-selective response for removal of such damage. Following this initial observation, the technique was used to look at other types of damage (19). When a 200-bp sequence of mtDNA from cells exposed to NO produced by the NO generator PAPA/NO was analyzed, guanine was the predominant base that was damaged. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites, indicating that the predominant lesion is the deamination of guanine to xanthine. Work with the ROS-generating system xanthine oxidase/hypoxanthine showed that many of the same guanines were vulnerable to attack. However, other base damage is also seen. As expected, the methylating agent MNU also selectively alkylated guanines. It is intriguing that the pattern of damaged guanines was not identical to that damaged by NO. The studies using LM-PCR have shed new light on the damage caused by NO in mtDNA. They revealed that guanine is the most frequently damaged base, although there were some damaged adenines
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found when alkaline conditions were employed. No damage was detected at any pyrimidines in the sequence evaluated. For comparison, studies with the ROS generator XO showed that we were able to detect oxidative lesions to both specific purines and pyrimidines. The pattern and frequency of base damage was similar to that observed previously using the ROS generator alloxan (21). Therefore, it appears likely that the inability to detect pyrimidine lesions following exposure to NO was because few, if any, lesions are formed at these bases. These findings support the notion that the damage that occurred to mtDNA is through the formation of N203, which causes deamination of guanine to xanthine and adenine to hypoxanthine. In both eases, this lesion preceded depurination to produce an AP site which was converted to a strand break with an appropriate end for ligation by alkali treatment. That AP sites predominantly are formed in DNA is in agreement with work by Tamir et al. (22) studying plasmid DNAs which were eleetroporated into CHO cells. When these cells were treated with NO, a significant number of AP sites were produced in the DNA. Additional work by these investigators, using both DNA exposed to NO in vivo and in vitro has revealed that xanthine followed by hypoxanthine are the predominant base alterations (23, 24). By comparing the pattern of damage produced by NO to that generated by the alkylating agent MNU or the ROS generator XO, it can be seen that, although all three of these agents damaged many of the same guanines, there were certain guanines that were only vulnerable to PAPA/NO. Therefore, it is possible to determine a signature damage pattern for reactive nitrogen species (RNS) that is different from that produced by ROS or methylating toxins. This finding may prove useful for future studies of mtDNA in which the identity of the damaging agent is unknown. Additionally, it will be important to compare damage produced in mtDNA by different agents with patterns of known mtDNA mutations. The fact that NO produced the same pattern of damage when exposed to a PCR-generated mtDNA sequence establishes that the pattern of damage produced is due to the chemical properties of NO interacting within the sequence context of the DNA, rather than being influenced by the association of the DNA with the mitoehondrial matrix proteins or other DNA-binding proteins. When considering lesions in mtDNA, it is important to mention that in cases where a single mutation has been shown to cause a disease, the degree of heteroplasmy is usually between 60 and 90% (mutated genomes to nonmutated). However, in major neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), this is not the ease. While increased mtDNA mutations are seen, none rises to a level sufficient by itself to cause disease. Thus, it is believed that it is a collection of mutations and heightened lesions in mtDNA that inactivates a critical number of mitoehondrial genomes in a specific way to cause disease. LM-PCR allows one
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to determine what mutations might be expected following exposure to specific types of DNA damaging agents.
VI. Mechanisms Involved in the Repair of mtDNA One approach to ascertain whether factors that affect repair in the nucleus also affect repair in the mitochondrion is to study repair in cells from individuals with known repair-deficient phenotypes such as xeroderma pigmentosum (XP). XP complementation group D (XP-D) is known to be defective in the repair of N-methylpurines in their nuclear DNA. To determine whether this defect also is seen in mtDNA from these cells, repair of MNU-induced N-methylpurines in XP-D cells was compared to that seen in normal WI-38 cells (25). At both 8 and 24 h after exposure to MNU, XP-D cells were found to normally repair N-methylpurine lesions in mtDNA, although the repair of the same lesions in the nuclear DNA from XP-D cells was significantly attenuated. These data indicated that the factors that retard repair of nuclear DNA in XP-D cells do not affect the repair of mtDNA in these cells. Extracts from cells of individuals with XP complementation group A (XP-A) have been reported to be defective in the repair of some types of oxidative damage. Therefore, studies were performed to determine whether there was a correlation between the inadequate repair of oxidatively damaged nuclear DNA in XP-A cells and the capacity to repair similar damage to their mtDNA (26). The ability of karyotypically normal human fibroblasts and XP-A fibroblasts to repair alloxan-generated oxidative damage to nuclear and mtDNA was assessed. These data indicate that both nuclear and mitochondrial repair of DNA damage are appreciably more efficient in normal human fibroblasts. These findings suggest a similarity between the process(es) used to repair oxidative damage to nuclear and mtDNA in that both are inefficient in XP-A cells.
VII. Cell-Specific Differences in mIDNA Repair Based on the studies described in the preceding sections, it is known that there are mechanisms for repair of endogenous damage in mtDNA. However, what is not as clear is how important mtDNA repair is to the cellular defenses of normal cells. The first indication of the importance of mtDNA repair to cellular defenses occurred when the simple question, "Are there cell-specific differences in repair of mtDNA?" was asked. Within the central nervous system (CNS) there are two predominant types of cells: neurons, which are the information processing cells of the CNS; and glial cells, which provide supportive functions
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for the neurons. Glial cells are composed mainly of three distinct populations of cells: astrocytes, oligodendrocytes, and microglia. While it was known that these cells play an important role in some diseases of the CNS, little was known about the repair mechanisms utilized by these cells in response to injury. Therefore, a well-characterized culture system (27) to generate pure primary cultures of astrocytes, oligodendrocytes, and microglia was used to evaluate mtDNA repair following both alkylation and oxidative damage. For the studies of simple alkylation damage, MNU was used as the alkylating agent, Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed no cell type-specific differences in initial mtDNA damage. However, mtDNA repair of N-methylpurines in oligodendrocytes and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, greater than 60% of N-methylpurines were removed from the mtDNA by 24 h. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes and microglia during the same time period. Since mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis, analyses using DNA laddering and ultrastructural examination were performed. DNA fragmentation and morphological changes consistent with apoptosis were apparent following MNU treatment of cultured oligodendrocyte progenitors and microglia, but not astroglia. These data were the first to demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis (28). Subsequently, oxidative mtDNA damage and repair also were assessed. Menadione, which undergoes redox cyclingwithin the mitochondria, was used to generate oxygen radicals. The results from these studies showed that exposure to equimolar concentrations of menadione resulted in more initial mtD NA damage in oligodendrocytes and microglia as compared to astrocytes. Repair experiments then were performed using both equimolar concentrations and concentrations that resulted in comparable strand breaks. Under both conditions, astrocytes repaired the damage efficiently with all of the lesions being removed by 6 h in mtDNA of astrocytes as compared to approximately 60% repair in oligodendrocytes or microglia. Our previous findings of a correlation between lack of mtDNA repair of N-methylpurines and induction of apoptosis led us to ask the question whether oligodendrocytes and microglia undergo apoptosis in response to an oxidative insult. ApoTag and annexin V staining, DNA laddering and electron microscopy were used to evaluate apoptosis. There were no indications of apoptosis in any of the experiments with astrocytes, while oligodendrocyte and microglial cultures were positive in all cases. We also questioned whether the increased sensitivity could be due to decreases in antioxidants within the oligodendrocytes and microglia; so, in collaboration with Dr. Doug Spitz at Washington University, we measured enzyme activities for glutathione peroxidase, catalase, CuZn, and MnSOD, along with total, reduced, and oxidized glutathione levels.
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For these studies, cells were prepared exactly as for the repair studies. The results demonstrated that under our culture conditions, there were no significant differences in catalase, glutathione peroxidase, or CuZnSOD between any of the cell types. Concerning glutathione, astrocytes had significantly lower levels than oligodendrocytes or microglia. The same was true for MnSOD (29). Thus, while antioxidant and repair capacity are both involved in protecting cells from oxidant insults, it appears that cells with efficient repair capacity may be spared, even in the presence of very low antioxidant levels, and that cells with less efficient repair are susceptible, even in the presence of higher levels of antioxidants. This observation of cell-specific differences in repair of oxidative damage among glial cells leads to the obvious question "Is there repair of oxidative mtDNA damage in neurons?" To address this question, primary cultures of cerebellar granule cells were exposed to comparable concentrations of menadione. More initial damage was observed in the neuronal cultures as compared to the glial cells. Because of the increased sensitivity, a 50-#M concentration was used for subsequent repair experiments. The results of these experiments demonstrated that the repair kinetics were slower in neurons as compared to glial cells, but by 48 h the lesions had been removed. As in the glial cultures with decreased repair capacity, apoptosis was induced in cerebellar granule cells by menadione exposure using quantitative electron microscopic evaluation, annexin V positive staining, or Apotag positive staining as the marker for apoptosis. Since menadione redox cycles with complex I of the electron transport chain to produce superoxide, one could hypothesize that mitochondria may play a substantia~ role through activation of caspases. Of the caspases, caspase 9 has been associated with mitochondrial changes. The release of cytochrome c from the mitochondrial intermembrane space to the cytosol is necessary for the activation of this caspase. Western blots were performed using mitochondrial and cytosolic proteins and a monoclonal antibody to cytochrome c. No cytochrome c protein was detected in the cytosolic fraction prior to menadione treatment, indicating that the cell fractionation procedure had not disrupted the outer mitochondrial membrane. However, 2 h after exposure to 100/zM menadione, there was a decrease in the intensity of the mitochondrial cytochrome c band with a concomitant increase in the cytosolic band of oligodendrocytes and microglia. As expected, astrocytes, which show no evidence of cell death in response to menadione exposure, showed no increase in the cytosolic cytochrome c band. Therefore, release of cytochrome c from the mitochondria into the cytosol correlates with induction of apoptosis in CNS glial cells (29). To determine if this release of cytochrome c resulted in the activation of caspase 9, a colorimetric activity assay based on cleavage of a caspase 9-specific substrate was performed. For a positive control, staurosporine, which has been shown to cause cytochrome c release in cerebellar granule neurons, was employed. After 3 h, caspase 9 activity in oligodendrocytes, microglia, and Jurkat
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cells exposed to menadione was elevated compared to control cells. No detectable increase in astrocyte caspase 9 activity was found. Subsequent experiments were performed using xanthine oxidase and hypoxanthine to generate the ROS. Again, activation of caspase 9 was observed (29). Since it has been suggested that caspase 8 may play a role in the mitochondrial pathway of apoptosis, its activity following menadione exposure was tested in each of the cell types. Activation of caspase 8 has been well documented in the Fas-pathway of apoptosis; thus, Jurkat cells treated with an anti-Fas antibody were used for a positive control. No caspase 8 activity was detected in any of the cell types following exposure to menadione. Following treatment with the antiFas antibody, however, oligodendrocytes and microglia demonstrated caspase 8 activity. Conversely, astrocytes remained unresponsive to induction ofapoptosis, showing no caspase 8 activity. From these experiments, it appears that mtDNA repair plays a pivotal role in cellular defense mechanisms. This statement is based on the evidence that there are cell-specific differences in the repair of mtDNA damage. The importance of these differences is exemplified in the observation that decreases in mtDNA repair capacity correlates with increased cell killing. Cell death occurs through the induction of apoptosis involving a mitochondrial pathway that includes the release of cytochrome c and the activation of caspase 9.
VIII. Conclusionsand Future Questions It is becoming increasingly clear that lesions in mtDNA play an important role in a number of human diseases. Owing to the crucial role that mtDNA integrity plays in cellular processes, a new area for investigation into the pathogenesis of a number of age-related diseases has been identified. The premise upon which these investigations are based is that an acceptable lesion equilibrium must be maintained in mtDNA for normal mitochondrial function. This lesion equilibrium is controlled by a balance in the rate at which mtDNA is damaged and the rate at which these lesions are removed. If this critical balance is disrupted either by an increase in the rate of mtDNA damage or a decrease in the rate of repair, fewer functioning mitochondrial genomes will be available for transcription and cellular bioenergetics will decrease. This will cause cellular functions to diminish due to energy depletion. Additionally, there will be an increase in oxidative stress in the cell due to defects in electron transport caused by the heightened damage to mtDNA. If this deleterious process is allowed to continue, the cell will ultimately die via apoptotic or necrotic mechanisms. There are several ways in which intervention could alter this catastrophic cascade of events. However, before such intervention can be initiated, a more thorough understanding of the mechanisms involved in the alteration of the lesion
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equilibrium in mtDNA needs to be obtained. Therefore, many questions remain to be answered. The problems remaining to be addressed include a more precise definition of the components involved in mtDNA repair, a better comprehension ofhow they are regulated, a more thorough understanding of how they can malfunction to precipitate disease states, and the development of gene therapy protocols to reverse repair defects and prevent or delay the onset of disease.
ACKNOWLEDGMENTS This work was supported in part by the United States Public Health Service Grants ESO5865, ESO0313, and ESO3456 from the National Institute of Environmental Health Sciences, and AG12442 from the Institute of Aging,
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