Increased mtDNA Damage Research Articles

LRRK2 G2019S-induced mitochondrial DNA damage is LRRK2 kinase dependent and inhibition restores mtDNA integrity in Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with increased risk for developing Parkinson's disease (PD). Previously, we found that LRRK2 G2019S mutation carriers have increased mitochondrial DNA (mtDNA) damage and after zinc finger nuclease-mediated gene mutation correction, mtDNA damage was no longer detectable. While the mtDNA damage phenotype can be unambiguously attributed to the LRRK2 G2019S mutation, the underlying mechanism(s) is unknown. Here, we examine the role of LRRK2 kinase function in LRRK2 G2019S-mediated mtDNA damage, using both genetic and pharmacological approaches in cultured neurons and PD patient-derived cells. Expression of LRRK2 G2019S induced mtDNA damage in primary rat midbrain neurons, but not in cortical neuronal cultures. In contrast, the expression of LRRK2 wild type or LRRK2 D1994A mutant (kinase dead) had no effect on mtDNA damage in either midbrain or cortical neuronal cultures. In addition, human LRRK2 G2019S patient-derived lymphoblastoid cell lines (LCL) demonstrated increased mtDNA damage relative to age-matched controls. Importantly, treatment of LRRK2 G2019S expressing midbrain neurons or patient-derived LRRK2 G2019S LCLs with the LRRK2 kinase inhibitor GNE-7915, either prevented or restored mtDNA damage to control levels. These findings support the hypothesis that LRRK2 G2019S-induced mtDNA damage is LRRK2 kinase activity dependent, uncovering a novel pathological role for this kinase. Blocking or reversing mtDNA damage via LRRK2 kinase inhibition or other therapeutic approaches may be useful to slow PD-associated pathology.

Abstract 1782: Reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage in tumor hypoxia, poor radiotherapy response, and metastatic progression of rectal cancer

Abstract Background: Hypoxia is an important feature of the tumor microenvironment and correlates with radiotherapy resistance and metastatic progression. Additionally, as the malignant phenotype promotes aerobic glycolysis and contributes to the microenvironmental acidification, an underlying mechanism in radiation resistance and metastasis is likely linked to metabolic changes caused by mitochondrial reprogramming. We hypothesized that ROS and mtDNA damage might be indicators of poor therapy response and dissemination of rectal cancer. Methods: Three colorectal cancer (CRC) cell lines (HCT116, HT29, LoVo) were cultured under hypoxia (0.2% O2) or normoxia (21% O2) for 24 hours. In a prospective rectal cancer trial, serum was sampled from patients at the time of diagnosis. Levels of ROS were determined by the ability to oxidize dihydrochlorofluoroscein into a fluorescent derivative. mtDNA damage was quantified by the ability of modification on template DNA to inhibit restriction enzyme cleavage. For the study patients, histologic tumor response to neoadjuvant radiotherapy was evaluated according to standard ypTN staging and tumor regression grade (TRG) scoring. Groups were compared using Student’s t-test. Survival differences were assessed by the log-rank test. Results: Compared to normoxic conditions, hypoxia significantly reduced ROS levels in all CRC cell lines. Moreover, in serum samples, ROS levels were lower for T4 cases than for patients with T2-3 disease (p = 0.037), and accordingly, low circulating ROS was associated with adverse metastasis-free survival (MFS; p = 0.004) and overall survival (p < 0.001). Hypoxic HCT116 and HT29 cell lines, but not the LoVo cells, showed significantly higher mtDNA damage than under normoxia. Given the indicated dependence of ROS and mtDNA damage to hypoxia, a low ratio of ROS to mtDNA damage turned out to be a promising indicator of hypoxic CRC cells. This ratio was lower in serum samples from patients who obtained poor histologic tumor response to radiotherapy (ypT3-4 versus ypT0-2 and poor versus good TRG; p = 0.044 for both). Accordingly, a high ratio of circulating ROS to mtDNA damage was associated with longer MFS (p = 0.021). Conclusion: We showed that culturing of CRC cells under hypoxia caused decrease in ROS levels but increase in mtDNA damage. Interestingly, in serum samples from rectal cancer patients, low ROS levels were associated with T4 disease, representing a bulky and often hypoxic tumor, and thus adverse survival. Moreover, low ratio of ROS to mtDNA damage was correlated with poor histologic tumor response to neoadjuvant radiotherapy. These parameters may reflect an aggressive biological phenotype instigated by a hostile tumor microenvironment. The results are currently under validation in an independent patient cohort. Trial registration: NCT00278694 Citation Format: Paula A. Bousquet, Sebastian Meltzer, Linda Sønstevold, Ying Esbensen, Lars G. Lyckander, Svein Dueland, Kjersti Flatmark, Kathrine R. Redalen, Lars Eide, Anne H. Ree. Reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage in tumor hypoxia, poor radiotherapy response, and metastatic progression of rectal cancer [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 1782. doi:10.1158/1538-7445.AM2017-1782

B Mitochondrial function regulates arterial ageing in mice

BackgroundMitochondrial DNA (mtDNA) damage is present in ageing tissues, and may promote loss of tissue function. However whether mtDNA damage or mitochondrial dysfunction is either present in the ageing vasculature...

194 Mitochondrial function regulates arterial ageing in mice

BackgroundMitochondrial DNA (mtDNA) damage is present in ageing tissues, and may promote loss of tissue function. However whether mtDNA damage or mitochondrial dysfunction is either present in the ageing vasculature...

Fructose-Rich Diet Affects Mitochondrial DNA Damage and Repair in Rats.

Evidence indicates that many forms of fructose-induced metabolic disturbance 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 events involved in metabolic disease resulting from a fructose-rich diet. In the present study, we evaluated the degree of oxidative damage to liver mtDNA and its repair, in addition to the state of oxidative stress and antioxidant defense in the liver of rats fed a high-fructose diet. We used male rats feeding on a high-fructose or control diet for eight weeks. Our results showed an increase in mtDNA damage in the liver of rats fed a high-fructose diet and this damage, as evaluated by the expression of DNA polymerase γ, was not repaired; in addition, the mtDNA copy number was found to be significantly reduced. A reduction in the mtDNA copy number is indicative of impaired mitochondrial biogenesis, as is the finding of a reduction in the expression of genes involved in mitochondrial biogenesis. In conclusion, a fructose-rich diet leads to mitochondrial and mtDNA damage, which consequently may have a role in liver dysfunction and metabolic diseases.

O-GlcNAcylation of 8-Oxoguanine DNA Glycosylase (Ogg1) Impairs Oxidative Mitochondrial DNA Lesion Repair in Diabetic Hearts

mtDNA damage in cardiac myocytes resulting from increased oxidative stress is emerging as an important factor in the pathogenesis of diabetic cardiomyopathy. A prevalent lesion that occurs in mtDNA damage is the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which can cause mutations when not repaired properly by 8-oxoguanine DNA glycosylase (Ogg1). Although the mtDNA repair machinery has been described in cardiac myocytes, the regulation of this repair has been incompletely investigated. Here we report that the hearts of type 1 diabetic mice, despite having increased Ogg1 protein levels, had significantly lower Ogg1 activity than the hearts of control, non-type 1 diabetic mice. In diabetic hearts, we further observed increased levels of 8-OHdG and an increased amount of mtDNA damage. Interestingly, Ogg1 was found to be highly O-GlcNAcylated in diabetic mice compared with controls. In vitro experiments demonstrated that O-GlcNAcylation inhibits Ogg1 activity, which could explain the mtDNA lesion accumulation observed in vivo Reducing Ogg1 O-GlcNAcylation in vivo by introducing a dominant negative O-GlcNAc transferase mutant (F460A) restored Ogg1 enzymatic activity and, consequently, reduced 8-OHdG and mtDNA damage despite the adverse hyperglycemic milieu. Taken together, our results implicate hyperglycemia-induced O-GlcNAcylation of Ogg1 in increased mtDNA damage and, therefore, provide a new plausible biochemical mechanism for diabetic cardiomyopathy.

Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus.

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC.

Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy.

PurposeIn the development of diabetic retinopathy, retinal mitochondria become dysfunctional, and mitochondrial DNA (mtDNA) is damaged. Because retinopathy is a progressive disease, and circulating glucose levels are high in diabetes, our aim was to investigate if peripheral blood mtDNA damage can serve as a potential biomarker of diabetic retinopathy.MethodsPeripheral blood mtDNA damage was investigated by extended-length PCR in rats and mice, diabetic for 10 to 12 months (streptozotocin-induced, type 1 model), and in 12- and 40-week-old Zucker diabetic fatty rats (ZDF, type 2). Mitochondrial copy number (in gDNA) and transcription (in cDNA) were quantified by qPCR. Similar parameters were measured in blood from diabetic patients with/without retinopathy.ResultsPeripheral blood from diabetic rodents had significantly increased mtDNA damage and decreased copy numbers and transcription. Lipoic acid administration in diabetic rats, or Sod2 overexpression or MMP-9 knockdown in mice, the therapies that prevent diabetic retinopathy, also ameliorated blood mtDNA damage and restored copy numbers and transcription. Although blood from 40-week-old ZDF rats had significant mtDNA damage, 12-week-old rats had normal mtDNA. Diabetic patients with retinopathy had increased blood mtDNA damage, and decreased transcription and copy numbers compared with diabetic patients without retinopathy and nondiabetic individuals.ConclusionsType 1 diabetic rodents with oxidative stress modulated by pharmacologic/genetic means, and type 2 animal model and patients with/without diabetic retinopathy, demonstrate a strong relation between peripheral blood mtDNA damage and diabetic retinopathy, and suggest the possibility of use of peripheral blood mtDNA as a noninvasive biomarker of diabetic retinopathy.

Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia.

Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia. Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA4977) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions. Compared with healthy controls, Mt/N and ΔmtDNA4977 frequency were elevated in thalassemia (P = 0.038 and P < 0.001, respectively). ΔmtDNA4977 was increased in the presence of either liver iron concentration > 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* < 20 ms) was present in 0%, 22%, and 46% of subjects with ΔmtDNA4977 frequency < 20, 20-40, and > 40/1 × 107 mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008). Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia.

Ogg1 -Dependent DNA Repair Regulates NLRP3 Inflammasome and Prevents Atherosclerosis

Activation of NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome-mediating interleukin (IL)-1β secretion has emerged as an important component of inflammatory processes in atherosclerosis. Mitochondrial DNA (mtDNA) damage is detrimental in atherosclerosis, and mitochondria are central regulators of the nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3 inflammasome. Human atherosclerotic plaques express increased mtDNA damage. The major DNA glycosylase, 8-oxoguanine glycosylase (OGG1), is responsible for removing the most abundant form of oxidative DNA damage. To test the role of OGG1 in the development of atherosclerosis in mouse. We observed that Ogg1 expression decreases over time in atherosclerotic lesion macrophages of low-density lipoprotein receptor (Ldlr) knockout mice fed a Western diet. Ogg1(-/-)Ldlr(-/-) mice fed a Western diet resulted in an increase in plaque size and lipid content. We found increased oxidized mtDNA, inflammasome activation, and apoptosis in atherosclerotic lesions and also higher serum IL-1β and IL-18 in Ogg1(-/-)Ldlr(-/-) mice than in Ldlr(-/-). Transplantation with Ogg1(-/-) bone marrow into Ldlr(-/-) mice led to larger atherosclerotic lesions and increased IL-1β production. However, transplantation of Ogg1(-/-)Nlrp3(-/-) bone marrow reversed the Ogg1(-/-) phenotype of increased plaque size. Ogg1(-/-) macrophages showed increased oxidized mtDNA and had greater amounts of cytosolic mtDNA and cytochrome c, increased apoptosis, and more IL-1β secretion. Finally, we found that proatherogenic miR-33 can directly inhibit human OGG1 expression and indirectly suppress both mouse and human OGG1 via AMP-activated protein kinase. OGG1 plays a protective role in atherogenesis by preventing excessive inflammasome activation. Our study provides insight into a new target for therapeutic intervention based on a link between oxidative mtDNA damage, OGG1, and atherosclerosis via NLRP3 inflammasome.

Deep sequencing of mitochondrial genomes reveals increased mutation load in Friedreich's ataxia

ObjectiveFriedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat expansion disorder caused by epigenetic silencing of the frataxin gene (FXN). Current research suggests that damage and variation of mitochondrial DNA (mtDNA) contribute to the molecular pathogenesis of FRDA. We sought to establish the extent of the mutation burden across the mitochondrial genome in FRDA cells and investigate the molecular mechanisms connecting FXN downregulation and the acquisition of mtDNA damage.MethodsDamage and mutation load in mtDNA of a panel of FRDA and control fibroblasts were determined using qPCR and next‐generation MiSeq sequencing, respectively. The capacity of FRDA and control cells to repair oxidative lesions in their mtDNA was measured using a quantitative DNA damage assay. Comprehensive RNA sequencing gene expression analyses were conducted to assess the status of DNA repair and metabolism genes in FRDA cells.ResultsAcute or prolonged downregulation of FXN expression resulted in a significant increase in mtDNA damage that translated to a significant elevation of mutation load in mtDNA. The predominant mutations identified throughout the mtDNA were C>T, G>A transitions (P = 0.007). Low FXN expression reduced capacity to repair oxidative damage in mtDNA. Downregulation of FXN expression strongly correlated (r = 0.73) with decreased levels of base excision repair (BER) DNA glycosylase NTHL1.InterpretationDownregulation of FXN expression in FRDA cells elevates mtDNA damage, increases mutation load of the mitochondrial genome, and diminishes DNA repair capacity. Progressive accumulation of mtDNA mutations in vulnerable FRDA patient cells reduces mitochondrial fitness ultimately leading to cell death.

Doxorubicin may damage cardiomyocytes more than cardiac progenitor cells

Long term survival of childhood cancers is now more than 70%. Unfortunately, adult survivors of childhood cancer are at risk for treatment‐related adverse health outcomes. Survivors with a median time from diagnosis of 25 years found 56% had cardiac dysfunction. Analysis of heart transplantation patients found doxorubicin as the underlying cause in 2–3% of all cases. Doxorubicin is a topoisomerase poison that accumulates in the nucleus and the mitochondria. Doxorubicin impacts heavily on the mitochondria as significant mtDNA depletion and mtDNA rearrangements have been reported. Unfortunately nearly all preclinical studies have only determined short effects. Very few studies have examined the long term effects of doxorubicin or used doses that were comparable to clinical treatment protocols. We have developed a murine model for late onset doxorubicin induced heart failure in which the onset was delayed and mimicked that seen clinically (~18 human years). Doxorubicin was injected (1–45 mg/kg, i.p.) at 3 weeks of age. We observed no change in cardiovascular function 2–4 months post‐injection, but a significant decrease in ejection shortening was observed after 6–8 months post‐injection. Low doses of doxorubicin (1–15 mg/kg) did not significantly alter cardiovascular function even out to 16 months post injection. Using flow cytometry we examined cell populations from the left ventricle, circulating non‐RBC cells, and bone marrow. Despite degradation of cardiovascular function we observed significant increases (p&lt;.05) in c‐kit+, SSEA1+, and Nkx2.5+ cells within the heart. In contrast, significant decreases of the c‐kit+, SSEA1+ cells were found in the circulating cells, while no changes were observed in the cell populations from the bone marrow compartment. We assessed mtDNA damage and observed significant increases in mtDNA damage in the ventricular cells and but not ventricular c‐kit+ cells. In cultured neonatal cardiomyocytes, doxorubicin increased mtDNA damage and promoted cell death below the threshold that significantly increase intramitochondrial superoxide levels. Measuring mitochondrial topoisomerase function we observed that H2O2 significantly increased DNA relaxation of supercoiled DNA suggesting that low levels of ROS alter mitochondrial topoisomerase function to promote DNA strand breakage. These findings suggest the heart attempted to regenerate by increased replacement of healthy cardiac progenitor cells but that degradation cardiomyocytes may have been the underlying cause of cardiovascular dysfunction. This suggests that the ability of the heart to direct self repair may be limited and that although the stem cell compartment attempted to compensate for cardiomyocyte loss, the degradation of cardiac function indicated that it was not successful.Support or Funding InformationSupported in part by NIH RO3HD065551

Effect of Aging and High Fat Diet on Mitochondrial and Cardiac Function in Mice with Altered Mitochondrial DNA Repair

In the developing heart it is important that mitochondrial biogenesis is precisely coordinated with cardiac growth, because impaired bioenergetic function could lead to heart failure. Oxidative mitochondrial (mt) DNA damage and repair play critical roles in mitochondrial biogenesis during postnatal cardiac development in rodents; therefore heart development could be adversely impacted by alterations in mtDNA repair. Our previous results showed that young mice deficient in the DNA repair glycosylase Ogg1, the first enzyme in the base excision repair pathway in mitochondria, displayed increased mtDNA damage, altered mitochondrial respiration and left ventricle hypertrophy. Surprisingly, despite these abnormalities, cardiac function in 3 week old Ogg1‐deficient animals remained unchanged. However, whether these cardiac compensatory mechanisms remain effective during aging or under stress conditions is unclear. Here we test the hypothesis that increased oxidative stress associated with aging and obesity decreases cardiac function and leads to development of cardiomyopathy in mice with altered mitochondrial DNA repair. Fifteen month old mice of three genotypes ‐ wild type (WT), Ogg1 knock‐out (KO) animals and KO mice transgenically overexpressing mitochondria‐targeted Ogg1 (KOTG) were fed a high fat diet for 14 weeks and analyzed for mtDNA damage and copy number, mitochondrial respiration, and for cardiac function. Despite approximately equal weight gain in all three groups of animals, mitochondrial and cardiac function characteristics were different for these genotypes. Compared to WT, KO mice displayed increased oxidative mtDNA damage and decreased mtDNA copy number in cardiac tissue. Conversely, mtDNA oxidation was decreased and mtDNA copy number restored in KOTG mice. Echocardiographic analysis demonstrated increased left ventricle mass and decreased cardiac contractility in KO mice. Compared to hearts from WT mice, KO animals displayed a significant increase in mitochondrial oxygen flux at state III that remained elevated after adding an uncoupling agent. Taking into account decreases in respiratory ratio and abnormally low ATP levels in KO mice, these results indicate that there is constitutive uncoupling in the cardiac tissue of Ogg1 deficient animals. These changes in cardiac function and mitochondrial bioenergetics observed in KO mice were prevented in KOTG mice, which were not different from WT animals. In summary, oxidative stress associated with aging and obesity lead to elevated oxidative mtDNA damage, decreased mtDNA copy number, mitochondrial dysfunction, and impaired cardiac contractility in Ogg1 deficient mice. Restoration of mtDNA repair in KOTG animals eliminated these effects on mitochondrial and cardiac function, thus demonstrating an important role of mtDNA integrity in maintenance of mitochondrial and cardiac health. Further, these results suggest that abnormalities in mitochondrial and cardiac function can be manipulated by altering mtDNA damage and repair.Support or Funding InformationSupported by NIH.

Sirt3 protects mitochondrial DNA damage and blocks the development of doxorubicin-induced cardiomyopathy in mice

Doxorubicin (Doxo) is a chemotherapeutic drug widely used to treat variety of cancers. One of the most serious side effects of Doxo is its dose-dependent and delayed toxicity to the heart. Doxo is known to induce cardiac mitochondrial damage. Recently, the mitochondrial sirtuin SIRT3 has been shown to protect mitochondria from oxidative stress. Here we show that overexpression of SIRT3 protects the heart from toxicity of Doxo by preventing the drug-induced mitochondrial DNA (mtDNA) damage. Doxo treatment caused depletion of Sirt3 levels both in primary cultures of cardiomyocytes and in mouse hearts, which led to massive acetylation of mitochondrial proteins. Doxo-induced toxicity to cardiomyocytes was associated with increased reactive oxygen species (ROS) production, mitochondrial fragmentation, and cell death. Overexpression of SIRT3 helped to attenuate Doxo-induced ROS levels and cardiomyocyte death. Sirt3 knockout (Sirt3.KO) mice could not endure the full dose of Doxo treatment, developed exacerbated cardiac hypertrophy, and died during the course of treatment, whereas Sirt3 transgenic (Sirt3.tg) mice were protected against Doxo-induced cardiotoxicity. Along with Sirt3, we also observed a concomitant decrease in levels of oxoguanine-DNA glycosylase-1 (OGG1), a major DNA glycosylase that hydrolyzes oxidized-guanine (8-oxo-dG) to guanine. Depletion of OGG1 levels was associated with increased mtDNA damage. Sirt3.KO mice and Doxo-treated mice showed increased 8-oxo-dG adducts in DNA and corresponding increase in mtDNA damage, whereas, 8-oxo-dG adducts and mtDNA damage were markedly reduced in Sirt3 overexpressing transgenic mice hearts. These results thus demonstrated that Sirt3 activation protects the heart from Doxo-induced cardiotoxicity by maintaining OGG1 levels and protecting mitochondria from DNA damage.

Endothelial Cell Bioenergetics and Mitochondrial DNA Damage Differ in Humans Having African or West Eurasian Maternal Ancestry.

We hypothesized that endothelial cells having distinct mitochondrial genetic backgrounds would show variation in mitochondrial function and oxidative stress markers concordant with known differential cardiovascular disease susceptibilities. To test this hypothesis, mitochondrial bioenergetics were determined in endothelial cells from healthy individuals with African versus European maternal ancestries. Bioenergetics and mitochondrial DNA (mtDNA) damage were assessed in single-donor human umbilical vein endothelial cells belonging to mtDNA haplogroups H and L, representing West Eurasian and African maternal ancestries, respectively. Human umbilical vein endothelial cells from haplogroup L used less oxygen for ATP production and had increased levels of mtDNA damage compared with those in haplogroup H. Differences in bioenergetic capacity were also observed in that human umbilical vein endothelial cells belonging to haplogroup L had decreased maximal bioenergetic capacities compared with haplogroup H. Analysis of peripheral blood mononuclear cells from age-matched healthy controls with West Eurasian or African maternal ancestries showed that haplogroups sharing an A to G mtDNA mutation at nucleotide pair 10398 had increased mtDNA damage compared with those lacking this mutation. Further study of angiographically proven patients with coronary artery disease and age-matched healthy controls revealed that mtDNA damage was associated with vascular function and remodeling and that age of disease onset was later in individuals from haplogroups lacking the A to G mutation at nucleotide pair 10398. Differences in mitochondrial bioenergetics and mtDNA damage associated with maternal ancestry may contribute to endothelial dysfunction and vascular disease.

Intracranial Glioblastomas: New Hope for an Effective Treatment

Intracranial Glioblastomas: New Hope for an Effective Treatment

Mitochondrial Topoisomerase Dysfunction Contributes to Myocardial Mitochondrial Dysfunction and Mitochondrial DNA (mtDNA) Degradation

Mitochondrial dysfunction has a significant role in the development of many different cardiomyopathies. Mitochondrial dysfunction and mtDNA mutations are also associated with other nonmyocardial diseases including cancer and neurodegenerative diseases such as Leigh Syndrome. One pathophysiologic response includes a decline in mtDNA integrity. Although it is widely accepted that mitochondrial‐derived oxidant stress is the sole cause of mtDNA damage, our work in models of diabetes and alcoholism has found involvement of the mitochondrial topoisomerases. To date, three unique mitochondrial topoisomerases have been identified in the myocardium. In models of type II diabetes or alcoholism we have observed a significant decreased in left ventricle cytochrome oxidase activity as an indicator of mitochondrial dysfunction, concomitant with significant increases in mtDNA damage. Using purified myocardial mitochondria, we observed significant increases in mitochondrial topoisomerase dependent DNA cleavage in response to diabetes or alcoholism. Mitochondrial topoisomerase is regulated at different levels and we found that DNA cleavage was significantly exacerbated by acute exposure to H2O2 as well as the topoisomerase inhibitor sobuzoxane to significantly increase DNA damage. Separate from a direct impact of oxidative stress on mtDNA, our studies find that alteration of mitochondrial topoisomerase reaction mechanism accelerates and propagates an increase in mtDNA damage. These findings indicate that the functional state of the mitochondrial topoisomerases have important consequences for the myocardium and participate in the pathophysiology leading to the development of cardiomyopathy.

Mitochondrial DNA: The Heart of the Matter

Cardiac conduction defects and cardiomyopathy are commonly reported in patients with inborn errors of metabolism harboring pathogenic mutations in either mitochondrial or nuclear DNA (mtDNA and nDNA, respectively). We hypothesized that the pathophysiology of inborn errors of metabolism varies due to synergistic heterozygosity between the two genomes. We examined a 13-generation Mennonite pedigree with autosomal recessive cardiomyopathy due a mutation in the adenine nucleotide translocator-1 (ANT1). Substantial variability in the progression of heart disease segregated with maternal lineage, and the severity of cardiomyopathy correlated with the mtDNA haplogroups (Strauss, et al 2013). To determine the causative nature of this correlation, we examined the influence of inherited mtDNA mutations on ANT1-cardiomyopathy in the mouse. We introduced homoplasmic mtDNA ND6 or COI missense mutations into the mouse female germ line, generating mice with complex I or IV deficiency, respectively, and analyzed Ant1-dependent cardiomyopathy on the different mtDNA backgrounds. On wt mtDNA background, the Ant1-/- mice developed a distinctive concentric dilated cardiomyopathy, characterized by substantial myocardial hypertrophy and ventricular dilation. Both COI and ND6 mtDNA mutations accelerated Ant1-/- age-dependent cardiomyopathy, as evidenced by ultrastructrural abnormalities, bioenergetic defects, mtROS production, sensitized mitochondrial permeability transition, increased mtDNA damage, and heart failure, which ultimately attenuated the lifespan of Ant1-deficient mice. Our results prove mtDNA dictates the penetrance of age-related cardiomyopathy and mammalian lifespan. Therefore, therapeutics that most effectively preserve mitochondrial DNA and bioenergetics will provide the most promise for healthy aging.

Asbestos-induced pulmonary fibrosis is augmented in 8-oxoguanine DNA glycosylase knockout mice.

Asbestos causes asbestosis and malignancies by mechanisms that are not fully established. Alveolar epithelial cell (AEC) injury and repair are crucial determinants of the fibrogenic potential of noxious agents such as asbestos. We previously showed that mitochondrial reactive oxygen species mediate asbestos-induced AEC intrinsic apoptosis and that mitochondrial human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme, prevents oxidant-induced AEC apoptosis. We reasoned that OGG1 deficiency augments asbestos-induced pulmonary fibrosis. Compared with intratracheal instillation of PBS (50 μl) or titanium dioxide (100 μg/50 μl), crocidolite or Libby amphibole asbestos (100 μg/50 μl) each augmented pulmonary fibrosis in wild-type C57BL/6J (WT) mice after 3 weeks as assessed by histology, fibrosis score, lung collagen via Sircol, and type 1 collagen expression; these effects persisted at 2 months. Compared with WT mice, Ogg1 homozygous knockout (Ogg1(-/-)) mice exhibit increased pulmonary fibrosis after crocidolite exposure and apoptosis in cells at the bronchoalveolar duct junctions as assessed via cleaved caspase-3 immunostaining. AEC involvement was verified by colocalization studies using surfactant protein C. Asbestos increased endoplasmic reticulum stress in the lungs of WT and Ogg1(-/-) mice. Compared with WT, alveolar type 2 cells isolated from Ogg1(-/-) mice have increased mtDNA damage, reduced mitochondrial aconitase expression, and increased P53 and cleaved caspase-9 expression, and these changes were enhanced 3 weeks after crocidolite exposure. These findings suggest an important role for AEC mtDNA integrity maintained by OGG1 in the pathogenesis of pulmonary fibrosis that may represent a novel therapeutic target.

Investigating AMD mechanisms using primary RPE cultures and donor tissue

AbstractStimuli that activate inflammasomes include mitochondrial (mt) reactive oxygen species, release of damaged mtDNA into the cytosol, or disruption of cellular ionic balance. These stimuli can arise from mt dysfunction, which may be a key pathogenic event in age‐related macular degeneration (AMD). This hypothesis is consistent with increased mtDNA damage reported in macular retinal pigment epithelium (RPE) of human donors with AMD. To investigate inflammasome activation, we developed primary cultures of RPE from donors with or without AMD. Cells express the RPE‐specific proteins bestrophin, CRALBP, and RPE65, are pigmented, express ZO‐1 at cell margins and Na‐K ATPase apically. These results show our primary cultures phenocopy the RPE in vivo. To examine inflammasome activation, cells were stimulated with LPS as a priming event, then treated with either the proteasome inhibitor MG132 to initiate formation of protein aggregates or rotenone to generate reactive oxygen species. We observed differences between cells from control and AMD donors in IL‐18 and IL‐1beta production and also caspase‐1 cleavage, the prototypic marker of inflammasome activation. These results suggest AMD affects inflammasome activation in the RPE. Commercial interest