Involvement of autophagy and mitochondrial dynamics in determining the fate and effects of irreparable mitochondrial DNA damage

Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes—many of which have homologs involved in human mitochondrial diseases—had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.     

Involvement of autophagy and mitochondrial dynamics in determining the fate and effects of irreparable mitochondrial DNA damage

Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes—many of which have homologs involved in human mitochondrial diseases—had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.     

Oxidants and not alkylating agents induce rapid mtDNA loss and mitochondrial dysfunction

Mitochondrial DNA (mtDNA) is essential for proper mitochondrial function and encodes 22 tRNAs, 2 rRNAs and 13 polypeptides that make up subunits of complex I, III, IV, in the electron transport chain and complex V, the ATP synthase. Although mitochondrial dysfunction has been implicated in processes such as premature aging, neurodegeneration, and cancer, it has not been shown whether persistent mtDNA damage causes a loss of oxidative phosphorylation. We addressed this question by treating mouse embryonic fibroblasts with either hydrogen peroxide (H(2)O(2)) or the alkylating agent methyl methanesulfonate (MMS) and measuring several endpoints, including mtDNA damage and repair rates using QPCR, levels of mitochondrial- and nuclear-encoded proteins using antibody analysis, and a pharmacologic profile of mitochondria using the Seahorse Extracellular Flux Analyzer. We show that a 60min treatment with H(2)O(2) causes persistent mtDNA lesions, mtDNA loss, decreased levels of a nuclear-encoded mitochondrial subunit, a loss of ATP-linked oxidative phosphorylation and a loss of total reserve capacity. Conversely, a 60min treatment with 2mM MMS causes persistent mtDNA lesions but no mtDNA loss, no decrease in levels of a nuclear-encoded mitochondrial subunit, and no mitochondrial dysfunction. These results suggest that persistent mtDNA damage is not sufficient to cause mitochondrial dysfunction.     

Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins

The mitochondrial genome is a significant target of exogenous and endogenous genotoxic agents; however, the determinants that govern this susceptibility and the pathways available to resist mitochondrial DNA (mtDNA) damage are not well characterized. Here we report that oxidative mtDNA damage is elevated in strains lacking Ntg1p, providing the first direct functional evidence that this mitochondrion-localized, base excision repair enzyme functions to protect mtDNA. However, ntg1 null strains did not exhibit a mitochondrial respiration-deficient (petite) phenotype, suggesting that mtDNA damage is negotiated by the cooperative actions of multiple damage resistance pathways. Null mutations in ABF2 or PIF1, two genes implicated in mtDNA maintenance and recombination, exhibit a synthetic-petite phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strains. This phenotype was partially rescued by malonic acid, indicating that reactive oxygen species generated by the electron transport chain contribute to mitochondrial dysfunction in abf2 Delta strains. In contrast, when two other genes involved in mtDNA recombination, CCE1 and NUC1, were inactivated a strong synthetic-petite phenotype was not observed, suggesting that the effects mediated by Abf2p and Pif1p are due to novel activities of these proteins other than recombination. These results document the existence of recombination-independent mechanisms in addition to base excision repair to cope with oxidative mtDNA damage in Saccharomyces cerevisiae. Such systems are likely relevant to those operating in human cells where mtDNA recombination is less prevalent, validating yeast as a model system in which to study these important issues.     

Mitochondrial DNA damage is a hallmark of chemically induced and the R6/2 transgenic model of Huntington's disease

Many forms of neurodegeneration are associated with oxidative stress and mitochondrial dysfunction. Mitochondria are prominent targets of oxidative damage, however, it is not clear whether mitochondrial DNA (mtDNA) damage and/or its lack of repair are primary events in the delayed onset observed in Huntington's disease (HD). We hypothesize that an age-dependent increase in mtDNA damage contributes to mitochondrial dysfunction in HD. Two HD mouse models were studied, the 3-nitropropionic acid (3-NPA) chemically induced model and the HD transgenic mice of the R6/2 strain containing 115-150 CAG repeats in the huntingtin gene. The mitochondrial toxin 3-NPA inhibits complex II of the electron transport system and causes neurodegeneration that resembles HD in the striatum of human and experimental animals. We measured nuclear and mtDNA damage by quantitative PCR (QPCR) in striatum of 5- and 24-month-old untreated and 3-NPA treated C57BL/6 mice. Aging caused an increase in damage in both nuclear and mitochondrial genomes. 3-NPA induced 4-6 more damage in mtDNA than nuclear DNA in 5-month-old mice, and this damage was repaired by 48h in the mtDNA. In 24-month-old mice 3NPA caused equal amounts of nuclear and mitochondrial damage and this damage persistent in both genomes for 48h. QPCR analysis showed a progressive increase in the levels of mtDNA damage in the striatum and cerebral cortex of 7-12-week-old R6/2 mice. Striatum exhibited eight-fold more damage to the mtDNA compared with a nuclear gene. These data suggest that mtDNA damage is an early biomarker for HD-associated neurodegeneration and supports the hypothesis that mtDNA lesions may contribute to the pathogenesis observed in HD.     

Mitochondrial DNA damage Is associated with reduced mitochondrial bioenergetics in Huntington's disease

Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.     

Effect of IGF-1 on the balance between autophagy of dysfunctional mitochondria and apoptosis

Mutations in mitochondrial DNA (mtDNA) cause excessive production of mitochondrial reactive oxygen species (ROS) and shorten animal life span. We examined the mechanisms responsible for removal of mitochondria with deleterious mtDNA mutations by autophagy. Incubation of primary cells and cell lines in the absence of serum promotes autophagy of mitochondria with deleterious mtDNA mutations but spares their normal counterparts. The effect of serum withdrawal on the autophagy of dysfunctional mitochondria is prevented by the addition of IGF-1. As a result of the elimination of mitochondria with deleterious mutations, excessive ROS production, characteristic of dysfunctional mitochondria, is greatly reduced. Mitochondrial autophagy shares a common mechanism with mitochondrial-induced cell apoptosis, including mitochondrial transition pore formation and increased ROS production.     

Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA.

Genomic DNA is constantly being damaged and repaired and our genomes exist at lesion equilibrium for damage created by endogenous mutagens. Mitochondrial DNA (mtDNA) has the highest lesion equilibrium frequency recorded; presumably due to damage by H2O2 and free radicals generated during oxidative phosphorylation processes. We measured the frequencies of single strand breaks and oxidative base damage in mtDNA by ligation-mediated PCR and a quantitative Southern blot technique coupled with digestion by the enzymes endonuclease III and formamidopyrimidine DNA glycosylase. Addition of 5 mM alloxan to cultured rat cells increased the rate of oxidative base damage and, by several fold, the lesion frequency in mtDNA. After removal of this DNA damaging agent from culture, the single strand breaks and oxidative base damage frequency decreased to levels slightly below normal at 4 h and returned to normal levels at 8 h, the overshoot at 4 h being attributed to an adaptive up-regulation of mitochondrial excision repair activity. Guanine positions showed the highest endogenous lesion frequencies and were the most responsive positions to alloxan-induced oxidative stress. Although specific bases were consistently hot spots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner. The data reveal non-random oxidative damage to several nucleotides in mtDNA and an apparent adaptive, non-strand selective response for removal of such damage. These are the first studies to characterize oxidative damage and its subsequent removal at the nucleotide level in mtDNA.     

Mitochondrial DNA Damage via Augmented Oxidative Stress Regulates Endoplasmic Reticulum Stress and Autophagy: Crosstalk,Links and Signaling

Saturated free fatty acids (FFAs) have been implicated in the increase of oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, autophagy, and insulin resistance (IR) observed in skeletal muscle. Previously, we have shown that palmitate-induced mitochondrial DNA (mtDNA) damage triggers mitochondrial dysfunction, mitochondrial reactive oxygen species (mtROS) production, apoptosis and IR in L6 myotubes. The present study showed that mitochondrial overexpression of human 8-oxoguanine DNA glycosylase/AP lyase (hOGG1) decreased palmitate-induced carbonylation of proteins in mitochondria. Additionally, we found that protection of mtDNA from palmitate-induced damage significantly diminished markers of both ER stress and autophagy in L6 myotubes. Moreover, we observed that the addition of ROS scavenger, N-acetylcystein (NAC), to palmitate diminished both ER stress and autophagy markers mimicking the effect of mitochondrial overexpression of hOGG1. This is the first study to show that mtDNA damage is upstream of palmitate-induced ER stress and autophagy in skeletal muscle cells.     

Persistent damage induces mitochondrial DNA degradation

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6 h after induction of mutant uracil-N-glycosylase and by 12 h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144 h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and “foaming” of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.     

Mitochondrial DNA repair of oxidative damage in mammalian cells

Nuclear and mitochondrial DNA are constantly being exposed to damaging agents, from endogenous and exogenous sources. In particular, reactive oxygen species (ROS) are formed at high levels as by-products of the normal metabolism. Upon oxidative attack of DNA many DNA lesions are formed and oxidized bases are generated with high frequency. Mitochondrial DNA has been shown to accumulate high levels of 8-hydroxy-2'-deoxyguanosine, the product of hydroxylation of guanine at carbon 8, which is a mutagenic lesion. Most of these small base modifications are repaired by the base excision repair (BER) pathway. Despite the initial concept that mitochondria lack DNA repair, experimental evidences now show that mitochondria are very proficient in BER of oxidative DNA damage, and proteins necessary for this pathway have been isolated from mammalian mitochondria. Here, we examine the BER pathway with an emphasis on mtDNA repair. The molecular mechanisms involved in the formation and removal of oxidative damage from mitochondria are discussed. The pivotal role of the OGG1 glycosylase in removal of oxidized guanines from mtDNA will also be examined. Lastly, changes in mtDNA repair during the aging process and possible biological implications are discussed.     

Effects of X-irradiation on mitochondrial DNA damage and its supercoiling formation change

There have been a small number of reports of radiation-induced mtDNA damage, and mtDNA supercoiling formation change induced by ionizing radiation has not been investigated before. This study evaluated mtDNA damage and supercoiling formation change after X-irradiation. The human breast cancer cell line, MCF-7 cells were used for analysis. Modified supercoiling-sensitive real-time PCR approach was used to evaluate mitochondrial DNA supercoiling formation change and copy number; long-PCR method was applied for the quantification of mtDNA damage. MtDNA damage and formation change induced by high-dose irradiation was persistent in 24 h after irradiation and was not significant after low-dose irradiation. MtDNA copy number was slightly increased after high-dose irradiation and a transit increase was observed after low-dose irradiation. This is the first study to evaluate radiation-induced mitochondrial DNA supercoiling formation change using real-time PCR. Combined with data of ROS generation and dynamics of mitochondrial mass, our findings suggested that mtDNA is sensitive to radiation hazards, indicating mitochondrial biogenesis play an important role in radiation-induced cellular response.     

Autophagy,exosomes and drusen formation in age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of loss of vision in developed countries. AMD is characterized by a progressive degeneration of the macula of the retina, usually bilateral, leading to a severe decrease in central vision. An early sign of AMD is the appearance of drusen, which are extracellular deposits that accumulate on Bruch’s membrane below the retinal pigment epithelium (RPE). Drusen are a risk factor for developing AMD. Some of the protein components of drusen are known, yet we know little about the processes that lead to formation of drusen. We have previously reported increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we used in vitro modeling of increased mtDNA damage. Under conditions of increased mtDNA damage, autophagy markers and exosome markers were upregulated. In addition, we found autophagy markers and exosome markers in the region of Bruch’s membrane in the retinas of old mice. Furthermore, we found that drusen in AMD donor eyes contain markers for autophagy and for exosomes. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients.     

Genetic insights into OXPHOS defect and its role in cancer

Warburg proposed that cancer originates from irreversible injury to mitochondrial oxidative phosphorylation (mtOXPHOS), which leads to an increase rate of aerobic glycolysis in most cancers. However, despite several decades of research related to Warburg effect, very little is known about the underlying genetic cause(s) of mtOXPHOS impairment in cancers. Proteins that participate in mtOXPHOS are encoded by both mitochondrial DNA (mtDNA) as well as nuclear DNA. This review describes mutations in mtDNA and reduced mtDNA copy number, which contribute to OXPHOS defects in cancer cells. Maternally inherited mtDNA renders susceptibility to cancer, and mutation in the nuclear encoded genes causes defects in mtOXPHOS system. Mitochondria damage checkpoint (mitocheckpoint) induces epigenomic changes in the nucleus, which can reverse injury to OXPHOS. However, irreversible injury to OXPHOS can lead to persistent mitochondrial dysfunction inducing genetic instability in the nuclear genome. Together, we propose that "mitocheckpoint" led epigenomic and genomic changes must play a key role in reversible and irreversible injury to OXPHOS described by Warburg. These epigenetic and genetic changes underlie the Warburg phenotype, which contributes to the development of cancer.     

Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle's high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5′ flap structures generated during DNA synthesis. Furthermore, removal of 5′ flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity.