Cardiolipin is a key determinant for mtDNA stability and segregation during mitochondrial stress.

Mitochondria play a key role in adaptation during stressing situations. Cardiolipin, the main anionic phospholipid in mitochondrial membranes, is expected to be a determinant in this adaptive mechanism since it modulates the activity of most membrane proteins. Here, we used Saccharomyces cerevisiae subjected to conditions that affect mitochondrial metabolism as a model to determine the possible role of cardiolipin in stress adaptation. Interestingly, we found that thermal stress promotes a 30% increase in the cardiolipin content and modifies the physical state of mitochondrial membranes. These changes have effects on mtDNA stability, adapting cells to thermal stress. Conversely, this effect is cardiolipin-dependent since a cardiolipin synthase-null mutant strain is unable to adapt to thermal stress as observed by a 60% increase of cells lacking mtDNA (ρ0). Interestingly, we found that the loss of cardiolipin specifically affects the segregation of mtDNA to daughter cells, leading to a respiratory deficient phenotype after replication. We also provide evidence that mtDNA physically interacts with cardiolipin both in S. cerevisiae and in mammalian mitochondria. Overall, our results demonstrate that the mitochondrial lipid cardiolipin is a key determinant in the maintenance of mtDNA stability and segregation.

XRCC1 haploinsufficiency in mice has little effect on aging, but adversely modifies exposure-dependent susceptibility.

Oxidative DNA damage plays a role in disease development and the aging process. A prominent participant in orchestrating the repair of oxidative DNA damage, particularly single-strand breaks, is the scaffold protein XRCC1. A series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice were examined. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of ~26 months and a nearly identical maximal life expectancy of ~37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated-weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity-HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure.

Characterization of oxidative guanine damage and repair in mammalian telomeres.

8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1)-initiated DNA base excision repair (BER). Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH), by chromosome orientation-FISH (CO-FISH), and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/-)) mouse tissues and primary embryonic fibroblasts (MEFs) cultivated in hypoxia condition (3% oxygen), whereas telomere shortening was detected in Ogg1(-/-) mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen) or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/-) mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/-) mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/-) MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity in mammals.

Antineoplastic effects of sodium dichloroacetate and omeprazole, alone or in combination, on canine oral mucosal melanoma cells.

Oral mucosal melanoma (OMM) is a common neoplasm in canines, although it is rare in humans. Cancer cells present alterations in energetic metabolism, and the Warburg effect states that most cancer cells undergo aerobic glycolysis. This can be reversed by certain drugs, resulting in decreased cell viability and cell death. We sought to evaluate the effects of sodium dichloroacetate (DCA) and omeprazole (OMP) alone or in combination on canine OMM and human melanoma cells. CMGD5 and SK-MEL-28 cell lines were treated with DCA and OMP alone or in combination, and cell viability was assessed using the crystal violet assay. Cell death (apoptosis and necrosis) was assessed by Annexin V and propidium iodide (PI) staining assays using flow cytometry. In addition, the oxygen consumption rate (OCR) was evaluated using a SeaHorse XF assay. Treatment with DCA or OMP alone resulted in a significant, but not dose-dependent, reduction in cell viability in both cell lines; however, the combination of DCA and OMP resulted in a significant and dose-dependent decrease in viability in both cell lines. DCA and OMP, alone or in combination, did not alter OCR at the concentrations tested in either cell line. Since the combination of DCA and OMP potentialized the inhibition of viability and increased cell death in a synergistic manner in melanoma cells, this approach may represent a new repurposing strategy to treat cancer.

The Many Roles Mitochondria Play in Mammalian Aging.

Significance: Aging is a natural process that affects most living organisms, resulting in increased mortality. As the world population ages, the prevalence of age-associated diseases, and their associated health care costs, has increased sharply. A better understanding of the molecular mechanisms that lead to cellular dysfunction may provide important targets for interventions to prevent or treat these diseases. Recent Advances: Although the mitochondrial theory of aging had been proposed more than 40 years ago, recent new data have given stronger support for a central role for mitochondrial dysfunction in several pathways that are deregulated during normal aging and age-associated disease. Critical Issues: Several of the experimental evidence linking mitochondrial alterations to age-associated loss of function are correlative and mechanistic insights are still elusive. Here, we review how mitochondrial dysfunction may be involved in many of the known hallmarks of aging, and how these pathways interact in an intricate net of molecular relationships. Future Directions: As it has become clear that mitochondrial dysfunction plays causative roles in normal aging and age-associated diseases, it is necessary to better define the molecular interactions and the temporal and causal relationship between these changes and the relevant phenotypes seen during the aging process. Antioxid. Redox Signal. 36, 824-843.

Respiratory and TCA cycle activities affect S. cerevisiae lifespan, response to caloric restriction and mtDNA stability.

We studied the importance of respiratory fitness in S. cerevisiae lifespan, response to caloric restriction (CR) and mtDNA stability. Mutants harboring mtDNA instability and electron transport defects do not respond to CR, while tricarboxylic acid cycle mutants presented extended lifespans due to CR. Interestingly, mtDNA is unstable in cells lacking dihydrolipoyl dehydrogenase under CR conditions, and cells lacking aconitase under standard conditions (both enzymes are components of the TCA and mitochondrial nucleoid). Altogether, our data indicate that respiratory integrity is required for lifespan extension by CR and that mtDNA stability is regulated by nucleoid proteins in a glucose-sensitive manner.

Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane.

Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases. Most patients belong to complementation group B, and the CS group B (CSB) protein plays a role in genomic maintenance and transcriptome regulation. By immunocytochemistry, mitochondrial fractionation, and Western blotting, we demonstrate that CSB localizes to mitochondria in different types of cells, with increased mitochondrial distribution following menadione-induced oxidative stress. Moreover, our results suggest that CSB plays a significant role in mitochondrial base excision repair (BER) regulation. In particular, we find reduced 8-oxo-guanine, uracil, and 5-hydroxy-uracil BER incision activities in CSB-deficient cells compared to wild-type cells. This deficiency correlates with deficient association of the BER activities with the mitochondrial inner membrane, suggesting that CSB may participate in the anchoring of the DNA repair complex. Increased mutation frequency in mtDNA of CSB-deficient cells demonstrates functional significance of the presence of CSB in the mitochondria. The results in total suggest that CSB plays a direct role in mitochondrial BER by helping recruit, stabilize, and/or retain BER proteins in repair complexes associated with the inner mitochondrial membrane, perhaps providing a novel basis for understanding the complex phenotype of this debilitating disorder.

Mitochondrial base excision repair assays.

The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur. Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene-specific repair assays, chromatographic techniques as well as current optimizations for detecting 8-oxoG lesions in cells by immunofluorescence. Throughout the assay descriptions we will include methodological considerations that may help optimize the assays in terms of resolution and repeatability.

Manganese-Induced Neurotoxicity through Impairment of Cross-Talk Pathways in Human Neuroblastoma Cell Line SH-SY5Y Differentiated with Retinoic Acid.

Manganese (Mn) is an important element; yet acute and/or chronic exposure to this metal has been linked to neurotoxicity and neurodegenerative illnesses such as Parkinson's disease and others via an unknown mechanism. To better understand it, we exposed a human neuroblastoma cell model (SH-SY5Y) to two Mn chemical species, MnCl2 and Citrate of Mn(II) (0-2000 µM), followed by a cell viability assay, transcriptomics, and bioinformatics. Even though these cells have been chemically and genetically modified, which may limit the significance of our findings, we discovered that by using RA-differentiated cells instead of undifferentiated SH-SY5Y cell line, both chemical species induce a similar toxicity, potentially governed by disruption of protein metabolism, with some differences. The MnCl2 altered amino acid metabolism, which affects RNA metabolism and protein synthesis. Citrate of Mn(II), however, inhibited the E3 ubiquitin ligases-target protein degradation pathway, which can lead to the buildup of damaged/unfolded proteins, consistent with histone modification. Finally, we discovered that Mn(II)-induced cytotoxicity in RA-SH-SY5Y cells shared 84 percent of the pathways involved in neurodegenerative diseases.

NEK5 interacts with LonP1 and its kinase activity is essential for the regulation of mitochondrial functions and mtDNA maintenance.

Little is known about Nima-related kinase (NEKs), a widely conserved family of kinases that have key roles in cell-cycle progression. Nevertheless, it is now clear that multiple NEK family members act in networks, not only to regulate specific events of mitosis, but also to regulate metabolic events independently of the cell cycle. NEK5 was shown to act in centrosome disjunction, caspase-3 regulation, myogenesis, and mitochondrial respiration. Here, we demonstrate that NEK5 interacts with LonP1, an AAA+ mitochondrial protease implicated in protein quality control and mtDNA remodeling, within the mitochondria and it might be involved in the LonP1-TFAM signaling module. Moreover, we demonstrate that NEK5 kinase activity is required for maintaining mitochondrial mass and functionality and mtDNA integrity after oxidative damage. Taken together, these results show a new role of NEK5 in the regulation of mitochondrial homeostasis and mtDNA maintenance, possibly due to its interaction with key mitochondrial proteins, such as LonP1.

Accumulation of (5'S)-8,5'-cyclo-2'-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice.

Cockayne syndrome (CS) is a human genetic disorder characterized by sensitivity to UV radiation, neurodegeneration, premature aging among other phenotypes. CS complementation group B (CS-B) gene (csb) encodes the CSB protein (CSB) that is involved in base excision repair of a number of oxidatively induced lesions in genomic DNA in vivo. We hypothesized that CSB may also play a role in cellular repair of the DNA helix-distorting tandem lesion (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA). Among many DNA lesions, S-cdA is unique in that it represents a concomitant damage to both the sugar and base moieties of the same nucleoside. Because of the presence of the C8-C5' covalent bond, S-cdA is repaired by nucleotide excision repair unlike most of other oxidatively induced lesions in DNA, which are subject to base excision repair. To test our hypothesis, we isolated genomic DNA from brain, kidney and liver of wild type and csb knockout (csb(-/-)) mice. Animals were not exposed to any exogenous oxidative stress before the experiment. DNA samples were analysed by liquid chromatography/mass spectrometry with isotope-dilution. Statistically greater background levels of S-cdA were observed in all three organs of csb(-/-) mice than in those of wild type mice. These results suggest the in vivo accumulation of S-cdA in genomic DNA due to lack of its repair in csb(-/-) mice. Thus, this study provides, for the first time, the evidence that CSB plays a role in the repair of the DNA helix-distorting tandem lesion S-cdA. Accumulation of unrepaired S-cdA in vivo may contribute to the pathology associated with CS.

Novel DNA mismatch-repair activity involving YB-1 in human mitochondria.

Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch-binding and recognition steps.

Base excision repair of oxidative DNA damage and association with cancer and aging.

Aging has been associated with damage accumulation in the genome and with increased cancer incidence. Reactive oxygen species (ROS) are produced from endogenous sources, most notably the oxidative metabolism in the mitochondria, and from exogenous sources, such as ionizing radiation. ROS attack DNA readily, generating a variety of DNA lesions, such as oxidized bases and strand breaks. If not properly removed, DNA damage can be potentially devastating to normal cell physiology, leading to mutagenesis and/or cell death, especially in the case of cytotoxic lesions that block the progression of DNA/RNA polymerases. Damage-induced mutagenesis has been linked to various malignancies. The major mechanism that cells use to repair oxidative damage lesions, such as 8-hydroxyguanine, formamidopyrimidines, and 5-hydroxyuracil, is base excision repair (BER). The BER pathway in the nucleus is well elucidated. More recently, BER was shown to also exist in the mitochondria. Here, we review the association of BER of oxidative DNA damage with aging, cancer and other diseases.

Mitochondrial DNA, base excision repair and neurodegeneration.

Neurodegeneration is a growing public health concern because of the rapid increase in median and maximum life expectancy in the developed world. Mitochondrial dysfunction seems to play a critical role in neurodegeneration, likely owing to the high energy demand of the central nervous system and its sole reliance on oxidative metabolism for energy production. Loss of mitochondrial function has been clearly demonstrated in several neuropathologies, most notably those associated with age, like Alzheimer's, Parkinson's and Huntington's diseases. Among the common features observed in such conditions is the accumulation of oxidative DNA damage, in particular in the mitochondrial DNA, suggesting that mitochondrial DNA instability may play a causative role in the development of these diseases. In this review we examine the evidence for the accumulation of oxidative DNA damage in mitochondria, and its relationship with loss of mitochondrial function and cell death in neural tissues. Oxidative DNA damage is repaired mainly by the base excision repair pathway. Thus, we review the molecular events and enzymes involved in base excision repair in mitochondria, and explore the possible role of alterations in mitochondrial base excision repair activities in premature aging and age-associated neurodegenerative diseases.

Human embryonic stem cells have enhanced repair of multiple forms of DNA damage.

Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic maintenance pathways are enhanced in human embryonic stem cells, relative to differentiated human cells.

Defective DNA base excision repair in brain from individuals with Alzheimer's disease and amnestic mild cognitive impairment.

Oxidative stress is thought to play a role in the pathogenesis of Alzheimer's disease (AD) and increased oxidative DNA damage has been observed in brain tissue from AD patients. Base excision repair (BER) is the primary DNA repair pathway for small base modifications such as alkylation, deamination and oxidation. In this study, we have investigated alterations in the BER capacity in brains of AD patients. We employed a set of functional assays to measure BER activities in brain tissue from short post-mortem interval autopsies of 10 sporadic AD patients and 10 age-matched controls. BER activities were also measured in brain samples from 9 amnestic mild cognitive impairment (MCI) subjects. We found significant BER deficiencies in brains of AD patients due to limited DNA base damage processing by DNA glycosylases and reduced DNA synthesis capacity by DNA polymerase beta. The BER impairment was not restricted to damaged brain regions and was also detected in the brains of amnestic MCI patients, where it correlated with the abundance of neurofibrillary tangles. These findings suggest that BER dysfunction is a general feature of AD brains which could occur at the earliest stages of the disease. The results support the hypothesis that defective BER may play an important role in the progression of AD.

p53-Dependent and p53-Independent Responses of Cells Challenged by Photosensitization.

The p53 protein exerts fundamental roles in cell responses to a variety of stress stimuli. It has clear roles in controlling cell cycle, triggering apoptosis, activating autophagy and modulating DNA damage response. Little is known about the role of p53 in autophagy-associated cell death, which can be induced by photoactivation of photosensitizers within cells. The photosensitizer 1,9-dimethyl methylene blue (DMMB) within nanomolar concentration regimes has specific intracellular targets (mitochondria and lysosomes), photoinducing a typical scenario of cell death with autophagy. Importantly, in consequence of its subcellular localization, photoactive DMMB induces selective damage to mitochondrial DNA, saving nuclear DNA. By challenging cells having different p53 protein levels, we investigated whether p53 modulates DMMB/light-induced phototoxicity and cell cycle dynamics. Cells lacking p53 activity were slightly more resistant to photoactivated DMMB, which was correlated with a smaller sub-G1 population, indicative of a lower level of apoptosis. DMMB photosensitization seems to induce mostly autophagy-associated cell death and S-phase cell cycle arrest with replication stress. Remarkably, these responses were independent on the p53 status, indicating that p53 is not involved in either process. Despite describing some p53-related responses in cells challenged by photosensitization, our results also provide novel information on the consequences of DMMB phototoxicity.

Enzymology of mitochondrial DNA repair.

The mitochondrial genome encodes proteins essential for the oxidative phosphorylation and, consequently, for proper mitochondrial function. Its localization and, possibly, structural organization contribute to higher DNA damage accumulation, when compared to the nuclear genome. In addition, the mitochondrial genome mutates at rates several times higher than the nuclear, although the causal relationship between these events are not clearly established. Maintaining mitochondrial DNA stability is critical for cellular function and organismal fitness, and several pathways contribute to that, including damage tolerance and bypass, degradation of damaged genomes and DNA repair. Despite initial evidence suggesting that mitochondria lack DNA repair activities, most DNA repair pathways have been at least partially characterized in mitochondria from several model organisms, including humans. In this chapter, we review what is currently known about how the main DNA repair pathways operate in mitochondria and contribute to mitochondrial DNA stability, with focus on the enzymology of mitochondrial DNA repair.

Investigation of base excision repair gene variants in late-onset Alzheimer's disease.

Base excision repair (BER) defects and concomitant oxidative DNA damage accumulation play a role in the etiology and progression of late-onset Alzheimer's disease (LOAD). However, it is not known whether genetic variant(s) of specific BER genes contribute to reduced BER activity in LOAD patients and whether they are associated with risk, development and/or progression of LOAD. Therefore, we performed targeted next generation sequencing for three BER genes, uracil glycosylase (UNG), endonuclease VIII-like DNA glycosylase 1 (NEIL1) and polymerase ß (POLß) including promoter, exonic and intronic regions in peripheral blood samples and postmortem brain tissues (temporal cortex, TC and cerebellum, CE) from LOAD patients, high-pathology control and cognitively normal age-matched controls. In addition, the known LOAD risk factor, APOE was included in this study to test whether any BER gene variants associate with APOE variants, particularly APOE ε4. We show that UNG carry five significant variants (rs1610925, rs2268406, rs80001089, rs1018782 and rs1018783) in blood samples of Turkish LOAD patients compared to age-matched controls and one of them (UNG rs80001089) is also significant in TC from Brazilian LOAD patients (p<0.05). The significant variants present only in CE and TC from LOAD are UNG rs2569987 and POLß rs1012381950, respectively. There is also significant epistatic relationship (p = 0.0410) between UNG rs80001089 and NEIL1 rs7182283 in TC from LOAD subjects. Our results suggest that significant BER gene variants may be associated with the risk of LOAD in non-APOE ε4 carriers. On the other hand, there are no significant UNG, NEIL1 and POLß variants that could affect their protein level and function, suggesting that there may be other factors such as post-transcriptional or-translational modifications responsible for the reduced activities and protein levels of these genes in LOAD pathogenesis. Further studies with increased sample size are needed to confirm the relationship between BER variants and LOAD risk.

Lower mitochondrial DNA content but not increased mutagenesis associates with decreased base excision repair activity in brains of AD subjects.

Accumulation of oxidative mitochondrial DNA (mtDNA) damage and impaired base excision repair (BER) in brains have been associated with Alzheimer's disease (AD). However, it is still not clear how these affect mtDNA stability, as reported levels of mtDNA mutations in AD are conflicting. Thus, we investigated whether alterations in BER correlate with mtDNA instability in AD using postmortem brain samples from cognitively normal AD subjects and individuals who show neuropathological features of AD, but remained cognitively normal (high-pathology control). To date, no data on DNA repair and mtDNA stability are available for these individuals. BER activities, mtDNA mutations, and mtDNA copy number were measured in the nuclear and mitochondrial extracts. Significantly lower uracil DNA glycosylase activity was detected in nuclear and mitochondrial extracts from AD subjects, while apurinic/apyrimidinic endonuclease activity was similar in all groups. Although mtDNA mutation frequency was similar in all groups, mtDNA copy number was significantly decreased in the temporal cortex of AD brains but not of high-pathology control subjects. Our results show that lower mitochondrial uracil DNA glycosylase activity does not result in increased mutagenesis, but rather in depletion of mtDNA in early-affected brain regions during AD development.