Mitochondrial DNA Integrity Research Articles

Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long-lived animals.

Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (López‐Otín et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematode Caenorhabditis elegans also exhibits age‐dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role in C. elegans aging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age‐dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age‐dependent functional decline commonly observed in C. elegans. Computational modeling of mtDNA dynamics in C. elegans indicates that the lifespan of short‐lived animals such as C. elegans is likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long‐lived animals such as humans and rhesus monkey and possibly in rodents.

Activation of AMPK-SIRT3 signaling is chondroprotective by preserving mitochondrial DNA integrity and function

In osteoarthritis (OA), articular chondrocytes manifest mitochondrial damage, including mitochondrial DNA 4977-bp (mtDNA4977) deletion that impairs mitochondrial function. OA chondrocytes have decreased activity of AMPK, an energy biosensor that promotes mitochondrial biogenesis. Here, we tested if pharmacologic AMPK activation, via downstream activation of predominately mitochondrially localized sirtuin 3 (SIRT3), reverses existing decreases in mitochondrial DNA (mtDNA) integrity and function in human OA chondrocytes and limits mouse knee OA development. We assessed mtDNAintegrity and function including the common mtDNA4977 deletion and mtDNA content, mitochondrial reactive oxygen species (mtROS) generation, oxygen consumption and intracellular ATP levels. Phosphorylation of AMPKα, expression and activity of SIRT3, acetylation and expression of the mitochondrial antioxidant enzyme SOD2 and DNA repair enzyme 8-oxoguanine glycosylase (OGG1), and expression of subunits of mitochondrial respiratory complexes were examined. We assessed effect of pharmacologic activation of AMPK on age-related spontaneous mouse knee OA. The mtDNA4977 deletion was detected in both OA chondrocytes and menadione-treated normal chondrocytes, associated with increased mtROS, decreased SIRT3, and increased acetylation of SOD2 and OGG1. AMPKα1 deficient chondrocytes exhibited significantly reduced SIRT3 activity. AMPK pharmacologic activation attenuated existing mtDNA4977 deletion and improved mitochondrial functions in OA chondrocytes via SIRT3 by reducing acetylation and increasing expression of SOD2 and OGG1, and limited aging-associated mouse knee OA development and progression. AMPK activation, via SIRT3, limits oxidative stress and improves mtDNA integrity and function in OA chondrocytes. These effects likely contribute to chondroprotective effects of AMPK activity.

Mitochondrial HSP90 Accumulation Promotes Vascular Remodeling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival. We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis. Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats. We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat). Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.

Iron overload prevents oxidative damage to rat brain after chlorpromazine administration.

The hypothesis tested is that Fe administration leads to a response in rat brain modulating the effects of later oxidative challenges such as chlorpromazine (CPZ) administration. Either a single dose (acute Fe overload) or 6 doses every second day (sub-chronic Fe overload) of 500 or 50mg Fe-dextran/kg, respectively, were injected intraperitoneally (ip) to rats. A single dose of 10mg CPZ/kg was injected ip 8h after Fe treatment. DNA integrity was evaluated by quantitative PCR, lipid radical (LR·) generation rate by electron paramagnetic resonance (EPR), and catalase (CAT) activity by UV spectrophotometry in isolated brains. The maximum increase in total Fe brain was detected after 6 or 2h in the acute and sub-chronic Fe overload model, respectively. Mitochondrial and nuclear DNA integrity decreased after acute Fe overload at the time of maximal Fe content; the decrease in DNA integrity was lower after sub-chronic than after acute Fe overload. CPZ administration increased LR· generation rate in control rat brain after 1 and 2h; however, CPZ administration after acute or sub-chronic Fe overload did not affect LR· generation rate. CPZ treatment did not affect CAT activity after 1-4h neither in control rats nor in acute Fe-overloaded rats. However, CPZ administration to rats treated sub-chronically with Fe showed increased brain CAT activity after 2 or 4h, as compared to control values. Fe supplementation prevented brain damage in both acute and sub-chronic models of Fe overload by selectively activating antioxidant pathways.

Mitochondria Transcription Factor A: A Putative Target for the Effect of Melatonin on U87MG Malignant Glioma Cell Line.

The disruption of mitochondrial activity has been associated with cancer development because it contributes to regulating apoptosis and is the main source of reactive oxygen species (ROS) production. Mitochondrial transcription factor A (TFAM) is a protein that maintains mitochondrial DNA (mtDNA) integrity, and alterations in its expression are associated with mitochondrial damage and cancer development. In addition, studies have shown that mitochondria are a known target of melatonin, the pineal gland hormone that plays an important anti-tumorigenic role. Thus, we hypothesized that melatonin decreases the expression of TFAM (RNA and protein) in the human glioblastoma cell line U87MG, which disrupts mtDNA expression and results in cell death due to increased ROS production and mitochondrial damage. Our results confirm the hypothesis, and also show that melatonin reduced the expression of other mitochondrial transcription factors mRNA (TFB1M and TFB2M) and interfered with mtDNA transcription. Moreover, melatonin delayed cell cycle progression and potentiated the reduction of cell survival due to treatment with the chemotherapeutic agent temozolomide. In conclusion, elucidating the effect of melatonin on TFAM expression should help to understand the signaling pathways involved in glioblastoma progression, and melatonin could be potentially applied in the treatment of this type of brain tumor.

Purification procedures meaningfully influence DNA quantification in milk

The isolation of highly purified DNA from milk has been one of the essential steps in an authentication process by PCR-based method. We considered the possibility that DNA quality could vary depending on the method of DNA purification used, and that these differences could substantively impact the subsequent PCR assay. In this study, we aimed to evaluate the effects of purification procedures on the DNA in milk. For this purpose, the DNA samples from milk were purified using organic solvent and two silica-based column methods, and the DNA quality was evaluated by amplifying long target sequences (>900bp) from mitochondrial and nuclear genes, as well as by monitoring total DNA integrity, yield, purity and ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). Results showed that the integrity of total DNA, mtDNA and nDNA was consistent, while the DNA yield, purity and mtDNA: nDNA ratio estimates were not uniform. The organic purification procedure produced the highest DNA yield (97 ng/mL), purity (1.58) and mtDNA: nDNA ratio (36). We concluded that purification method can profoundly influence DNA quantification in milk and organic purification can offer a potentially effective way of isolation high-quality DNA from milk.

Curcumin attenuates hepatic mitochondrial dysfunction through the maintenance of thiol pool, inhibition of mtDNA damage, and stimulation of the mitochondrial thioredoxin system in heat-stressed broilers.

The aim of this study was to investigate the effects of dietary curcumin supplementation on the performance, mitochondrial redox system, mitochondrial DNA (mtDNA) integrity, and antioxidant-related gene expression in the liver of broiler chickens after heat stress treatment. At day 21, a total of 400 Arbor Acres broiler chickens with similar body weight (BW) were divided into 5 groups with 8 replicates per group and then reared either at a normal temperature (22 ± 1 °C) or at a high ambient temperature (34 ± 1 °C for 8 h and 22 ± 1 °C for the remaining time) for 20 d. Broilers in the 5 groups were fed a basal diet at a normal temperature (NT group) and a basal diet with 0, 50, 100, and 200 mg/kg curcumin at a high ambient temperature (HT, CUR50, CUR100, and CUR200 groups), respectively. The serum and liver samples were analyzed for the parameters related to hepatic damage, mitochondrial function, and redox status. The results showed that the G:F was increased in the CUR50 and CUR100 groups, and the final BW was increased in CUR100 group in comparison with the HT group (P < 0.05). When compared with those in the HT group, both serum aspartate and alanine aminotransferase activities were decreased in the curcumin-supplemented groups (P < 0.05). Curcumin decreased the reactive oxygen species (ROS) production but increased the mitochondrial membrane potential in the hepatocytes of the broilers after heat stress (P < 0.05). The broilers in curcumin-supplemented groups had lower malondialdehyde and protein carbonyl concentrations as well as greater thiol concentrations (P < 0.05). The mitochondrial manganese superoxide dismutase (MnSOD) activity in the liver was increased (P < 0.05) in the CUR100 group compared with the HT group. Compared with the heat-stressed broilers, the broilers that were fed curcumin had greater (P < 0.05) mtDNA copy number and ATP concentrations than those in the HT group. Curcumin supplementation attenuated the depression of the thioredoxin 2 and peroxiredoxin-3 gene expressions (P < 0.05). The MnSOD gene expression was increased in the CUR100 and CUR200 groups, and the thioredoxin reductase 2 gene expression was increased in the CUR50 group in comparison with the HT group (P < 0.05). In conclusion, curcumin mitigated the mitochondrial dysfunction in heat-stressed broilers, as evidenced by the suppression of the ROS burst, the maintenance of the thiol pool and mtDNA content, and the enhanced mitochondrial antioxidant gene expression.

AB001. Low mitochondrial DNA copy number is associated with low mitochondrial DNA integrity and advanced T-status in non-small cell lung cancer

AB001. Low mitochondrial DNA copy number is associated with low mitochondrial DNA integrity and advanced T-status in non-small cell lung cancer

Pharmacologic Protection of Mitochondrial DNA Integrity May Afford a New Strategy for Suppressing Lung Ischemia-Reperfusion Injury.

Lung ischemia-reperfusion (IR) injury contributes to post-transplant complications, including primary graft dysfunction. Decades of reports show that reactive oxygen species generated during lung IR contribute to pulmonary vascular endothelial barrier disruption and edema formation, but the specific target molecule(s) that "sense" injury-inducing oxidant stress to activate signaling pathways culminating in pathophysiologic changes have not been established. This review discusses evidence that mitochondrial DNA (mtDNA) may serve as a molecular sentinel wherein oxidative mtDNA damage functions as an upstream trigger for lung IR injury. First, the mitochondrial genome is considerably more sensitive than nuclear DNA to oxidant stress. Multiple studies suggest that oxidative mtDNA damage could be transduced to physiologic dysfunction by pathways that are either a direct consequence of mtDNA damage per se or involve formation of proinflammatory mtDNA damage-associated molecular patterns. Second, transgenic animals or cells overexpressing components of the base excision DNA repair pathway in mitochondria are resistant to oxidant stress-mediated pathophysiologic effects. Finally, published and preliminary studies show that pharmacologic enhancement of mtDNA repair or mtDNA damage-associated molecular pattern degradation suppresses reactive oxygen species-induced or IR injury in multiple organs, including preclinical models of lung procurement for transplant. Collectively, these findings point to the interesting prospect that pharmacologic enhancement of DNA repair during procurement or ex vivo lung perfusion may increase the availability of lungs for transplant and reduce the IR injury contributing to primary graft dysfunction.

Optimization of Storage Temperature for Retention of Undifferentiated Cell Character of Cultured Human Epidermal Cell Sheets

Cultured epidermal cell sheets (CES) containing undifferentiated cells are useful for treating skin burns and have potential for regenerative treatment of other types of epithelial injuries. The undifferentiated phenotype is therefore important for success in both applications. This study aimed to optimize a method for one-week storage of CES for their widespread distribution and use in regenerative medicine. The effect of storage temperatures 4 °C, 8 °C, 12 °C, 16 °C, and 24 °C on CES was evaluated. Analyses included assessment of viability, mitochondrial reactive oxygen species (ROS), membrane damage, mitochondrial DNA (mtDNA) integrity, morphology, phenotype and cytokine secretion into storage buffer. Lowest cell viability was seen at 4 °C. Compared to non-stored cells, ABCG2 expression increased between temperatures 8–16 °C. At 24 °C, reduced ABCG2 expression coincided with increased mitochondrial ROS, as well as increased differentiation, cell death and mtDNA damage. P63, C/EBPδ, CK10 and involucrin fluorescence combined with morphology observations supported retention of undifferentiated cell phenotype at 12 °C, transition to differentiation at 16 °C, and increased differentiation at 24 °C. Several cytokines relevant to healing were upregulated during storage. Importantly, cells stored at 12 °C showed similar viability and undifferentiated phenotype as the non-stored control suggesting that this temperature may be ideal for storage of CES.

Mitochondrial DNA Integrity Is Maintained by APE1 in Carcinogen-Induced Colorectal Cancer.

Changes in mitochondrial DNA (mtDNA) integrity have been reported in many cancers; however, the contribution of mtDNA integrity to tumorigenesis is not well understood. We used a transgenic mouse model that is haploinsufficient for the apurinic/apyrimidinic endonuclease 1 (Apex1+/-) gene, which encodes the base excision repair (BER) enzyme APE1, to determine its role in protecting mtDNA from the effects of azoxymethane (AOM), a carcinogen used to induce colorectal cancer. Repair kinetics of AOM-induced mtDNA damage was evaluated using qPCR after a single AOM dose and a significant induction in mtDNA lesions in colonic crypts from both wild-type (WT) and Apex1+/-animals were observed. However, Apex1+/- mice had slower repair kinetics in addition to decreased mtDNA abundance. Tumors were also induced using multiple AOM doses, and both WT and Apex1+/-animals exhibited significant loss in mtDNA abundance. Surprisingly, no major differences in mtDNA lesions were observed in tumors from WT and Apex1+/- animals, whereas a significant increase in nuclear DNA lesions was detected in tumors from Apex1+/- mice. Finally, tumors from Apex1+/- mice displayed an increased proliferative index and histologic abnormalities. Taken together, these results demonstrate that APE1 is important for preventing changes in mtDNA integrity during AOM-induced colorectal cancer.Implications: AOM, a colorectal cancer carcinogen, generates damage to the mitochondrial genome, and the BER enzyme APE1 is required to maintain its integrity. Mol Cancer Res; 15(7); 831-41. ©2017 AACR.

Abstract 1498: Melatonin inhibits mitochondrial transcription factor A expression in glioblastoma U87MG cell culture inducing an anti-tumorigenic effect

Abstract Melatonin, the major hormone of pineal gland, has been described to induce intrinsic apoptosis in tumor cell, while it is inhibited in non-tumor cell. In tumor cell, melatonin stimulates intracellular production of reactive oxygen species (ROS) by a mechanism not completely understood. Here we propose that melatonin inhibits the expression of the mitochondrial transcription factor A (TFAM) in culture of glioblastoma (GBM) cell lineage, leading to an increase of the production of ROS. TFAM is an important mitochondrial protein that maintains the mitochondrial DNA (mtDNA) integrity and consequently the oxidative phosphorylation. Human U87MG GBM cell lineage was treated with melatonin (1 mM) for 72 h to evaluate the expression of TFAM and the mtDNA copy number through Real Time PCR. Proliferation, apoptosis, cell cycle and ROS production were also evaluated by cytometry (Muse - Millipore Corporation) and the results were normalized to control (vehicle: ethanol 0.3 %). Melatonin reduced the expression of TFAM in 17.5 % compared to the control group, but had no effect on the mtDNA copy number. Moreover, the incubation of U87MG with melatonin induced a reduction in cell proliferation in 14.24 % ± 4.3 compared to control, with an arrest of cell cycle in G0/G1 phase, which increased from 67.4 ± 3.71 % to 72.78 ± 3.18 % in the group treated with melatonin compared to the control. Production of ROS presented an increase of 7.1 %, with no significant effect on apoptosis. These results suggest that TFAM maybe a target for the melatonin function for an anti-tumorigenic effect through an increase of ROS production, inducing a delay in the cell cycle and a reduction of cell proliferation. The melatonin effect onTFAM maybe a novel signaling pathway that could be explored for the control of GBM progression. Citation Format: Daiane G. Franco, Isabele F. Moretti, Suely K. Marie. Melatonin inhibits mitochondrial transcription factor A expression in glioblastoma U87MG cell culture inducing an anti-tumorigenic effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1498. doi:10.1158/1538-7445.AM2017-1498

High Sensitivity of SIRT3 Deficient Hearts to Ischemia-Reperfusion Is Associated with Mitochondrial Abnormalities.

Aim: Sirtuins are NAD+-dependent deacetylases that regulate cell metabolism through protein acetylation/deacetylation, and SIRT3 is the major deacetylase among mitochondrial isoforms. Here, we elucidated the possible role of acetylation of cyclophilin D, a key regulator of the mitochondrial permeability transition pore (mPTP), in mitochondria-mediated cardiac dysfunction induced by ischemia-reperfusion (IR) in wild type (WT) and SIRT3 knockout (SIRT3-/-) mice.Materials and Methods: Isolated and Langendorff-mode perfused hearts of WT and SIRT3-/- mice were subjected to 25-min global ischemia followed by 60-min of reperfusion in the presence or absence of the mPTP inhibitor, sanglifehrin A (SfA).Results: Analysis of mitochondrial sirtuins demonstrated that SIRT3 deficiency upregulated SIRT4 with no effect on SIRT5 expression. Hearts of SIRT3-/- mice exhibited significantly less recovery of cardiac function at the end of IR compared to WT mice. Intact (non-perfused) SIRT3-/- hearts exhibited an increased rate of Ca2+-induced swelling in mitochondria as an indicator of mPTP opening. However, there was no difference in mPTP opening and cyclophilin D acetylation between WT and SIRT3-/- hearts subjected to IR injury. Ca2+-stimulated H2O2 production was significantly higher in SIRT3-/- mitochondria that was prevented by SfA. Superoxide dismutase activity was lower in SIRT3-/- heart mitochondria subjected to IR which correlated with an increase in protein carbonylation. However, mitochondrial DNA integrity was not affected in SIRT3-/- hearts after IR.Conclusion: SIRT3 deficiency exacerbates cardiac dysfunction during post-ischemic recovery, and increases mPTP opening and ROS generation without oxidative damage to mitochondrial proteins and DNA.

Klotho, an antiaging molecule, attenuates oxidant-induced alveolar epithelial cell mtDNA damage and apoptosis.

Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from "exaggerated" lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an antiaging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression, whereas Klotho overexpression is protective while Klotho silencing augments AEC mtDNA damage. Compared with wild-type, Klotho heterozygous hypomorphic allele (kl/+) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in wild-type but not mitochondrial catalase overexpressing (MCAT) mice 3 wk following exposure to asbestos and that EUK-134, a MnSOD/catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent on AKT activation arising from upstream fibroblast growth factor receptor 1 activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.

Effect of cholesterol loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa

The beneficial effects of cholesterol loaded cyclodextrin (CLC) addition were evaluated in cryopreserved bull semen. Forty ejaculates were collected from Hariana bulls (n = 4), pooled and divided into 4 aliquots. All the aliquots were initially diluted in to egg yolk tris citrate and supplemented with CLC @ 0.5 mg (Group-II), 1.0 mg (G-III) and 2.0 mg (G-IV) CLC/120 × 106 spermatozoa or without CLC (G-I) that served as control. Extended semen was cryopreserved at −196 °C for 24 h. Seminal attributes like motility, viability, cryocapacitation like changes, tyrosine phosphorylation, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA integrity were evaluated after equilibration and thawing. Results showed a significant increase in the motility, viability and acrosome intact spermatozoa in Group II as compared to other three groups. Further, the proportion of spermatozoa showing capacitation and acrosome reaction was also decreased (P < 0.05) significantly in Group II as compared to Group I, III, and IV. Immunoblot demonstrated a 32 kDa (p32) protein showing differential variation in the band intensity in all the four groups being lower in Group II. Further, the immunolocalization study revealed positive immune reactivity for tyrosine phosphorylated proteins at middle piece and neck (high fluorescence), post-acrosomal region (medium fluorescence), and principal piece (low fluorescence) of spermatozoa. Addition of CLC significantly increased (P < 0.05) the percentage of spermatozoa showing high transmembrane mitochondrial potential, and also, CLC @ 0.5 mg/120 × 106 in semen extender significantly decreased (P < 0.05) spermatozoa showing fragmented DNA after thawing as compared to control. Results of the present study indicate beneficial effects of CLC supplementation on cryodamage of spermatozoa by reducing the cryocapacitation and apoptosis like changes.

Mitochondrial transcription factor A (TFAM) rs1937 and AP endonuclease 1 (APE1) rs1130409 alleles are associated with reduced cognitive performance

Mitochondrial dysfunction and DNA damage is intimately connected to ageing and neurodegeneration, including Alzheimer’s disease (AD). A particular culprit in this context is oxidative stress, which is a result of increased reactive oxygen species (ROS) due to hyperactive or dysfunctional mitochondria and/or reduced DNA repair capacity. Base excision repair (BER) is the major pathway for repairing oxidative damage events in chromosomal and mitochondrial DNA. Defects in BER have been detected in ageing and neurodegeneration. Mitochondrial transcription factor A (TFAM) plays an important role in the maintenance of mitochondrial DNA integrity. The present study investigated single nucleotide polymorphisms (SNPs) in the genes encoding the BER components MutYH, OGG1, APE1, PolB and PolG and the gene encoding mitochondrial TFAM in a cohort of 161 AD patients, 96 non-AD patient controls (PC) and 192 healthy controls (HC). Notably, the minor allele carriers of APE1 rs1130409 and the common allele carriers of TFAM rs1937 were associated with reduced mini-mental state examination score in AD patients, PC and HC, with no distinction of SNP frequencies in either of these sub-groups. Collectively, the results suggest an association between DNA maintenance and decline in cognitive function. These studies enlighten the normal brain aging process and point to potential new biomarkers for cognitive function and impairment.

Effects of adenosine receptor antagonists in MPTP mouse model of Parkinson's disease: mitochondrial DNA integrity.

IntroductionIn Parkinson’s disease (PD), compelling data indicate a functional link between adenosine/dopamine receptors and the progression of the neurodegenerative process. The present study was carried out to evaluate the effect of the non-selective adenosine receptor (ADR) antagonist caffeine, as well as the selective antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an ADRsA1 antagonist, and ((E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione) (KW-6002), an ADRsA2A antagonist, on the prevention of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism in mice.Material and methodsMice were allocated to five groups: group I – control group; group II: MPTP group, received four injections of MPTP (20 mg/kg, i.p.) at 2 h intervals; groups III, IV, V: received MPTP and i.p. caffeine (20 mg/kg/day) or DPCPX (5 mg/kg/day) or KW-6002 (10 mg/kg/day) starting one week before MPTP injection and continuing for 2 weeks.ResultsTherapy with caffeine or KW-6002 not only led to the reversibility of movement dysfunction and increased the concentrations of dopamine and ATP levels (p < 0.05), but also, ameliorates the dopaminergic neuron loss and restored the mtDNA and nDNA integrity (p < 0.05). Furthermore, in passive avoidance test, caffeine and DPCPX significantly (p < 0.05) reversed the MPTP-induced memory deficits, whereas the specific ADRsA2A antagonist did not.ConclusionsThe current results provide evidence that blockade of both ADRsA1 and ADRsA2A has therapeutic implications in alleviating MPTP-induced motor and cognitive dysfunction and might be a promising candidate for treatment of PD.

The Role of DNA Repair in Maintaining Mitochondrial DNA Stability.

Mitochondria are vital double-membrane organelles that act as a "powerhouse" inside the cell and have essential roles to maintain cellular functions, e.g., ATP production, iron-sulfur synthesis metabolism, and steroid synthesis. An important difference with other organelles is that they contain their own mitochondrial DNA (mtDNA). Such powerful organelles are also sensitive to both endogenous and exogenous factors that can cause lesions to their structural components and their mtDNA, resulting in gene mutations and eventually leading to diseases. In this review, we will mainly focus on mammalian mitochondrial DNA repair pathways that safeguard mitochondrial DNA integrity and several important factors involved in the repair process, especially on an essential pathway, base excision repair. We eagerly anticipate to explore more methods to treat related diseases by constantly groping for these complexes and precise repair mechanisms.

Effects of airborne toxicants on pulmonary function and mitochondrial DNA damage in rodent lungs.

Inhalation of airborne toxicants such as cigarette smoke and ozone is a shared health risk among the world's populations. The use of toxic herbicides like paraquat (PQ) is restricted by many countries, yet in the developing world PQ has demonstrable ill effects. The present study examined changes in pulmonary function, mitochondrial DNA (mtDNA) integrity and markers of DNA repair induced by acute or repeated exposure of PQ to rats. Similar to cigarette smoke and ozone, PQ promotes oxidative stress, and the impact of PQ on mtDNA was compared with that obtained with these agents. Tracheal instillation (i.t.) of PQ (0.01-0.075 mg/kg) dose dependently increased Penh (dyspnoea) by 48 h while body weight and temperature declined. Lung wet weight and the wet/dry weight ratio rose; for the latter, by as much as 52%. At low doses (0.02 and 0.03 mg/kg), PQ increased Penh by about 7.5-fold at 72 h. It quickly waned to near baseline levels. The lung wet/dry weight ratio remained elevated 7 days after administration coincident with marked inflammatory cell infiltrate. Repeated administration of PQ (1 per week for 8 weeks) resulted in a similar rise in Penh on the first instillation, but the magnitude of this response was markedly attenuated upon subsequent exposures. Pulmonary [lactate] and catalase activity, [8-oxodG] and histone fragmentation (cell death) were significantly increased. Repeated PQ instillation downregulated the expression of the mitochondrial-encoded genes, mtATP8, mtNd2 and mtcyB and nuclear ones for the DNA glycosylases, Ogg1, Neil1, Neil2 and Neil3. Ogg1 protein content decreased after acute and repeated PQ administration. mtDNA damage or changes in mtDNA copy number were evident in lungs of PQ-, cigarette smoke- and ozone-exposed animals. Taken together, these data indicate that loss of pulmonary function and inflammation are coupled to the loss of mtDNA integrity and DNA repair capability following exposure to airborne toxicants.

Restoration of Mitochondrial Integrity, Telomere Length, and Sensitivity to Oxidation by In Vitro Culture of Fuchs' Endothelial Corneal Dystrophy Cells.

Fuchs' endothelial corneal dystrophy (FECD), a degenerative disease of the corneal endothelium that leads to vision loss, is a leading cause of corneal transplantation. The cause of this disease is still unknown, but the implication of oxidative stress is strongly suggested. In this study, we analyzed the impact of FECD on mitochondrial DNA (mtDNA) integrity and telomere length, both of which are affected by the oxidative status of the cell. We compared the levels of total mtDNA, mtDNA common deletion (4977 bp), and relative telomere length in the corneal endothelial cells of fresh Descemet's membrane-endothelium explants and cultured cells from healthy and late stage FECD subjects. Oxidant-antioxidant gene expression and sensitivity to ultraviolet A (UVA)- and H2O2-induced cell death were assessed in cultured cells. Our results revealed increased mtDNA levels and telomere shortening in FECD explants. We also found that cell culture restores a normal phenotype in terms of mtDNA levels, telomere length, oxidant-antioxidant gene expression balance, and sensitivity to oxidative stress-induced cell death in the FECD cells compared with the healthy cells. Taken together, these results bring new evidence of the implication of oxidative stress in FECD. They also show that FECD does not evenly affect the integrity of corneal endothelial cells and that cell culture can rehabilitate the molecular phenotypes related to oxidative stress by selecting the more functional FECD cells.