mtDNA Lesions Research Articles

Mitochondrial Probe for Glutathione Depletion Reveals NME3 Essentiality for Mitochondrial Redox Response.

Maintenance of the mitochondrial thiol redox state is essential for cell survival. However, we lack a comprehensive understanding of the redox response to mitochondrial glutathione depletion. We developed a mitochondria-penetrating peptide, mtCDNB, to specifically deplete mitochondrial glutathione. A genome-wide CRISPR/Cas9 screen in tandem with mtCDNB treatment was employed to uncover regulators of the redox response to mitochondrial glutathione depletion. We identified nucleoside diphosphate kinase 3 (NME3) as a regulator of mitochondrial dynamics. We show that NME3 is recruited to the mitochondrial outer membrane when under redox stress. In the absence of NME3, there is impaired mitophagy, which leads to the accumulation of dysfunctional mitochondria. NME3 knockouts depleted of mitochondrial glutathione have increased mitochondrial ROS production, accumulate mtDNA lesions, and present a senescence-associated secretory phenotype. Our findings suggest a novel role for NME3 in selecting mitochondria for degradation through mitophagy under conditions of mitochondrial redox stress.

A blood-based marker of mitochondrial DNA damage in Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and neuroprotective or disease-modifying interventions remain elusive. High-throughput markers aimed at stratifying patients on the basis of shared etiology are required to ensure the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific accumulation of mitochondrial DNA (mtDNA) damage in PD neuronal culture and animal models, as well as in human PD postmortem brain tissue. To investigate mtDNA damage as a potential blood-based marker for PD, we describe herein a PCR-based assay (Mito DNADX) that allows for the accurate real-time quantification of mtDNA damage in a scalable platform. We found that mtDNA damage was increased in peripheral blood mononuclear cells derived from patients with idiopathic PD and those harboring the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation in comparison with age-matched controls. In addition, mtDNA damage was elevated in non-disease-manifesting LRRK2 mutation carriers, demonstrating that mtDNA damage can occur irrespective of a PD diagnosis. We further established that Lrrk2 G2019S knock-in mice displayed increased mtDNA damage, whereas Lrrk2 knockout mice showed fewer mtDNA lesions in the ventral midbrain, compared with wild-type control mice. Furthermore, a small-molecule kinase inhibitor of LRRK2 mitigated mtDNA damage in a rotenone PD rat midbrain neuron model and in idiopathic PD patient-derived lymphoblastoid cell lines. Quantifying mtDNA damage using the Mito DNADX assay may have utility as a candidate marker of PD and for measuring the pharmacodynamic response to LRRK2 kinase inhibitors.

The Acute Effects of Exercise on Saliva-Derived Regional Mitochondrial DNA from Trained Runners

ABSTRACT Introduction/Purpose Research has identified two regions within mitochondrial deoxyribonucleic acid (mtDNA), which seem to be more (major arc; mtMAJ) and less (minor arc; mtMIN) prone to oxidation and mtDNA lesion formation. Oxidation of mtDNA base pairs can result in reduced copy number and has been linked to chronic diseases. Interestingly, exercise can also induce acute copy number reductions; however, in a healthy population, there seems to be a restorative effect (i.e., gaining mtDNA copy numbers) that occurs within a couple of hours to days after exercise. Few have investigated these regional mtDNA changes in trained runners (>90th fitness percentile). Typical mtDNA procedures are invasive and inconvenient, requiring muscle biopsies, and/or blood draws; therefore, a simpler, less invasive method of specimen collection is appealing. The purpose of this study was to examine how salivary-derived molecular (mtMAJ and mtMIN), hormonal (cortisol), and oxidative (8-hydroxy-2′-deoxyguanosine (8-OHdG)) markers change after acute exercise. Methods Trained runners (n = 7; 52 ± 12 miles·wk−1 run in the past year; 28.8 ± 3.3 yr old; height, 1.76 ± 0.07 m; weight, 67.9 ± 11.3 kg; maximum oxygen consumption, 62.4 ± 5.7 mL·kg−1·min−1) completed a 60-min treadmill run at 70% peak oxygen consumption. Saliva samples were collected before exercise, 30 min after exercise (30Post), and 6 h after exercise (6hPost) and analyzed for changes in regional mtDNA via real-time quantitative polymerase chain reaction and stress (cortisol and 8-OHdG) markers using competitive enzyme-linked immunosorbent assays. Results mtMIN copy number decreased 30Post (−44.5%, P = 0.04) and 6hPost (−45.3% P = 0.03), but mtMAJ copy numbers were unchanged after exercise (P = 0.44). Neither of the stress-related saliva biomarkers changed after exercise (cortisol, P = 0.13; 8-OHdG, P = 0.77). Conclusions Saliva-derived DNA findings point to mtMIN, supposedly the more stable of the two regions, as a greater contributor to copy number reductions within our cohort of trained runners. These results contribute to the understanding of aerobic exercise’s influence over saliva-derived, regional mtDNA dynamics.

Mitochondrial DNA Repair in Neurodegenerative Diseases and Ageing.

Mitochondria are the only organelles, along with the nucleus, that have their own DNA. Mitochondrial DNA (mtDNA) is a double-stranded circular molecule of ~16.5 kbp that can exist in multiple copies within the organelle. Both strands are translated and encode for 22 tRNAs, 2 rRNAs, and 13 proteins. mtDNA molecules are anchored to the inner mitochondrial membrane and, in association with proteins, form a structure called nucleoid, which exerts a structural and protective function. Indeed, mitochondria have evolved mechanisms necessary to protect their DNA from chemical and physical lesions such as DNA repair pathways similar to those present in the nucleus. However, there are mitochondria-specific mechanisms such as rapid mtDNA turnover, fission, fusion, and mitophagy. Nevertheless, mtDNA mutations may be abundant in somatic tissue due mainly to the proximity of the mtDNA to the oxidative phosphorylation (OXPHOS) system and, consequently, to the reactive oxygen species (ROS) formed during ATP production. In this review, we summarise the most common types of mtDNA lesions and mitochondria repair mechanisms. The second part of the review focuses on the physiological role of mtDNA damage in ageing and the effect of mtDNA mutations in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Considering the central role of mitochondria in maintaining cellular homeostasis, the analysis of mitochondrial function is a central point for developing personalised medicine.

Abstract 2343: Andrographolide treatment results in decreased mtDNA damage and loss of the mitochondrial membrane potential while increasing ROS production in prostate cancer

Abstract Introduction: Prostate Cancer (PCa) is the second most diagnosed malignancy and the fifth leading cause of cancer deaths in men worldwide. Andrographolide, a labdane diterpenoid that is the main bioactive component of the medicinal plant Andrographis paniculata, has shown a wide range of therapeutic potential against many factors including cancer. Previous studies in our laboratory showed that Andrographolide induces expression of DNA repair genes associated with double-strand break repair, cell growth suppression, cell cycle arrest, and apoptosis in PCa in vitro. Andrographolide also showed an altered ATP production ratio between mitochondrial-ATP and glycolysis-ATP production in PC3 and 22RV1 cell lines treated with Andrographolide. However, the mechanism of action of Andrographolide in prostate cancer is not completely known. The objective of this study is to determine the role of Andrographolide in mtDNA damage, mitochondrial ROS production, and mitochondrial membrane potential in prostate cancer cell lines. Methods: Prostate cancer cell lines (PC3 and 22Rv1) were treated with Andrographolide (25μM) for 24 and 48 hours. DNA isolated from treated and control cells was used to detect mtDNA damage using a PCR-based method. To evaluate ROS production, cells were incubated with MitoSOX and fluorescence detected using fluorescence microscopy. Membrane potential status was also determined with fluorescence microscopy, using the dye JC-1. Results: Detection of mtDNA damage in PC3 cells treated with Andrographolide for 48 hours and in 22Rv1 cells treated with Andrographolide for 24 and 48 hours, show decreased mtDNA lesions in treated cells. Evaluation of mitochondrial membrane potential shows membrane depolarization in 22Rv1 cells treated with Andrographolide. In addition, mitochondrial ROS production shows a 1.4-fold increase in 22Rv1 cells treated with Andrographolide for 24 hours. Conclusion: This study shows that Andrographolide disrupts mitochondrial function in PC3 and 22Rv1 PCa cells, which in turn may trigger a metabolic phenotype leading to cell death PCa. This research could provide new insights into the role of Andrographolide in PCa altering bioenergetics and mitochondrial function, making Andrographolide a promising therapeutic candidate as a metabolic modulator and a chemo preventive agent in prostate cancer. Citation Format: Yarelis M. Roque-Reyes, Jesus Sosa-Rivera, María M. Sánchez-Vázquez, Sylvette Ayala-Peña, Carlos A. Torres-Ramos, Magaly Martínez-Ferrer. Andrographolide treatment results in decreased mtDNA damage and loss of the mitochondrial membrane potential while increasing ROS production in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2343.

Laminar Shear Stress Enhances Cytosolic PINK1 Expression: Increased Mitophagic Sensitivity Toward Mitochondrial Dysfunction

PURPOSEPhosphatase and tensin homolog (PTEN)‐induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process and known to act as a cytoprotective protein in mammalian cells. Mutation or deficiency of PINK1 has been closely related to several disease conditions. The purpose of this study was to determine PINK1 expression levels and subcellular localization under exercise‐mimic laminar shear stress (LSS) condition in human aortic endothelial cells (HAECs) or in exercising mice, and its implication on endothelial homeostasis and cardiovascular disease (CVD) prevention.METHODS & RESULTSFirst, we measured full‐length PINK1 (FL‐PINK1) mRNA and protein expression levels in HAECs under unidirectional laminar shear stress (LSS) at 15 dyne/cm2 for 48h. LSS significantly elevated both FL‐PINK1 mRNA and protein expressions in HAECs compared to static control. Mitochondrial fractionation assays showed a decrease in FL‐PINK1 accumulation in the mitochondria with a compensatory increase in the cytosolic FL‐PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Also, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential (JC‐1) showed mitochondrial elongation and increased mitochondrial membrane potential, respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. Based on this observation, we further hypothesized that the elevation of the cytosolic pool of FL‐PINK1 would increase the sensitivity to mitophagic signals in pathological conditions. Preconditioned HAECs with LSS showed lower mtDNA lesions under angiotensin II stimulation (100 nM, 6h). Moreover, LSS‐preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e. CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS‐enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise‐preconditioned mice were more protective to angiotensin II‐induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1‐dependent manner.CONCLUSIONLSS increases a cytosolic pool of FL‐PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in endothelial cells. Our data suggest that exercise may support mitochondrial homeostasis in vascular endothelial cells by enhancing PINK1‐dependent cell protection mechanisms.

Flow-induced endothelial mitochondrial remodeling mitigates mitochondrial reactive oxygen species production and promotes mitochondrial DNA integrity in a p53-dependent manner

Tumor suppressor p53 plays a pivotal role in orchestrating mitochondrial remodeling by regulating their content, fusion/fission processes, and intracellular signaling molecules that are associated with mitophagy and apoptosis pathways. In order to determine a molecular mechanism underlying flow-mediated mitochondrial remodeling in endothelial cells, we examined, herein, the role of p53 on mitochondrial adaptations to physiological flow and its relevance to vascular function using endothelial cell-specific p53 deficient mice. We observed no changes in aerobic capacity, basal blood pressure, or endothelial mitochondrial phenotypes in the endothelial p53 mull animals. However, after 7 weeks of voluntary wheel running exercise, blood pressure reduction and endothelial mitochondrial remodeling (biogenesis, elongation, and mtDNA replication) were substantially blunted in endothelial p53 null animals compared to the wild-type, subjected to angiotensin II-induced hypertension. In addition, endothelial mtDNA lesions were significantly reduced following voluntary running exercise in wild-type mice, but not in the endothelial p53 null mice. Moreover, in vitro studies demonstrated that unidirectional laminar flow exposure significantly increased key putative regulators for mitochondrial remodeling and reduced mitochondrial reactive oxygen species generation and mtDNA damage in a p53-dependent manner. Mechanistically, unidirectional laminar flow instigated translocalization of p53 into the mitochondrial matrix where it binds to mitochondrial transcription factor A, TFAM, resulting in improving mtDNA integrity. Taken together, our findings suggest that p53 plays an integral role in mitochondrial remodeling under physiological flow condition and the flow-induced p53-TFAM axis may be a novel molecular intersection for enhancing mitochondrial homeostasis in endothelial cells.

Beyond base excision repair: an evolving picture of mitochondrial DNA repair.

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function – deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria.Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.

Grape Seed Proanthocyanidin Extract Moderated Retinal Pigment Epithelium Cellular Senescence Through NAMPT/SIRT1/NLRP3 Pathway.

BackgroundRetinal pigment epithelium (RPE) cellular senescence is an important process in degenerative retinal disorders. Grape seed proanthocyanidin extract (GSPE) alleviates senescence-related degenerative disorders; however, the potential effects of GSPE intake on RPE cellular senescence through regulating NAMPT/SIRT1/NLRP3 pathway remain unclear.MethodsThe effects of GSPE on NAMPT expression and NAD+ contents were detected with Western blot and assay kit in both in-vivo and in-vitro AMD models. Senescence-related biomarkers, including p16, p21 expressions and β-gal staining, were conducted in different groups. The protective effects of GSPE treatment on the mitochondrial homeostasis and barrier function of RPE cells were detected using mtDNA lesions analyses, JC-1 staining, ZO1 staining and trans-epithelial cell resistance (TEER) detection. The expression of senescence-associated secretory phenotype (SASP) in different groups would be conducted with qPCR. To demonstrate the potential effects of NAMPT/SIRT1/NLRP3 pathway after GSPE treatment, the protein levels of relevant key regulators after applications of NAMPT inhibitor, Fk866, and SIRT1 inhibitor, EX-527.ResultsGSPE significantly improves the NAMPT expression and NAD+ content in aging mice, and thus alleviates the RPE cellular senescence. In advanced in-vitro studies, GSPE significantly up-regulated NAMPT content and thus relieved H2O2 induced NAD+ depression through analyzing the NAD+ contents in different groups. In advanced analyses, it was reported that GSPE could alleviate mitochondrial permeability, mtDNA damage, ZO1 expression and SASP levels in aging RPE cells. Thus, GSPE treatment significantly decreased senescence-related protein p16 and p21, as well as SASP levels in in-vitro aging model, and it was demonstrated that GSPE could illustrate a significant anti-aging effect. The Western blot data in GSPE treatment of aging RPE cells demonstrated that GSPE could significantly improve NAMPT and SIRT1 levels, and thus depressed NLRP3 expression.ConclusionThis study indicated that GSPE alleviated RPE cellular senescence through NAMPT/SIRT1/NLRP3 pathway. This study highlighted the potential effects of GSPE on degenerative retinopathy through the crosstalk of NAD+ metabolism, SIRT1 function and NLRP3 activation.

Mitochondrial Function Are Disturbed in the Presence of the Anticancer Drug, 3-Bromopyruvate.

3-bromopuryvate (3-BP) is a compound with unique antitumor activity. It has a selective action against tumor cells that exhibit the Warburg effect. It has been proven that the action of 3-BP is pleiotropic: it acts on proteins, glycolytic enzymes, reduces the amount of ATP, induces the formation of ROS (reactive oxygen species), and induces nuclear DNA damage. Mitochondria are important organelles for the proper functioning of the cell. The production of cellular energy (ATP), the proper functioning of the respiratory chain, or participation in the production of amino acids are one of the many functions of mitochondria. Here, for the first time, we show on the yeast model that 3-BP acts in the eukaryotic cell also by influence on mitochondria and that agents inhibiting mitochondrial function can potentially be used in cancer therapy with 3-BP. We show that cells with functional mitochondria are more resistant to 3-BP than rho0 cells. Using an MTT assay (a colorimetric assay for assessing cell metabolic activity), we demonstrated that 3-BP decreased mitochondrial activity in yeast in a dose-dependent manner. 3-BP induces mitochondrial-dependent ROS generation which results in ∆sod2, ∆por1, or ∆gpx1 mutant sensitivity to 3-BP. Probably due to ROS mtDNA lesions rise during 3-BP treatment. Our findings may have a significant impact on the therapy with 3-BP.

Role of Mitochondrial DNA in Inflammatory Airway Diseases.

The mitochondrial genome is a small, circular, and highly conserved piece of DNA which encodes only 13 protein subunits yet is vital for electron transport in the mitochondrion and, therefore, vital for the existence of multicellular life on Earth. Despite this importance, mitochondrial DNA (mtDNA) is located in one of the least-protected areas of the cell, exposing it to high concentrations of intracellular reactive oxygen species (ROS) and threat from exogenous substances and pathogens. Until recently, the quality control mechanisms which ensured the stability of the nuclear genome were thought to be minimal or nonexistent in the mitochondria, and the thousands of redundant copies of mtDNA in each cell were believed to be the primary mechanism of protecting these genes. However, a vast network of mechanisms has been discovered that repair mtDNA lesions, replace and recycle mitochondrial chromosomes, and conduct alternate RNA processing for previously undescribed mitochondrial proteins. New mtDNA/RNA-dependent signaling pathways reveal a mostly undiscovered biochemical landscape in which the mitochondria interface with their host cells/organisms. As the myriad ways in which the function of the mitochondrial genome can affect human health have become increasingly apparent, the use of mitogenomic biomarkers (such as copy number and heteroplasmy) as toxicological endpoints has become more widely accepted. In this article, we examine several pathologies of human airway epithelium, including particle exposures, inflammatory diseases, and hyperoxia, and discuss the role of mitochondrial genotoxicity in the pathogenesis and/or exacerbation of these conditions. © 2021 American Physiological Society. Compr Physiol 11:1485-1499, 2021.

Huntingtin Maintains Mitochondrial Genome Integrity and Function

SUMMARYThe function of huntingtin (HTT) is enigmatic in that the native protein provides neuroprotection while mutant HTT (mHTT) carrying an expanded stretch of glutamines triggers neurotoxicity in Huntington’s disease (HD). We recently reported that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase and DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP). This complex resolves DNA lesions during transcription to maintain genome integrity, while mHTT impairs the activity of this complex, resulting in DNA lesion accumulation, activating the DNA damage-response in HD. Here we report that HTT forms a DNA repair complex within the mitochondria with mitochondrial RNA polymerase, transcription factors, and PNKP. This complex resolves mitochondrial DNA (mtDNA) lesions to preserve mtDNA integrity and function. Pathogenic mHTT reduces the complex activity, resulting in persistence of mtDNA lesions, impairing mitochondrial function in HD. Restoring the activity of the mtDNA repair complex in a Drosophila model of HD dramatically improves mtDNA integrity, and motor defects.

Mitochondrial Oxidative Stress Induces Rapid Intermembrane Space/Matrix Translocation of Apurinic/Apyrimidinic Endonuclease 1 Protein through TIM23 Complex

Mitochondria are essential cellular organelles that import the majority of proteins to sustain their function in cellular metabolism and homeostasis. Due to their role in oxidative phosphorylation, mitochondria are constantly affected by oxidative stress. Stability of mitochondrial DNA (mtDNA) is essential for mitochondrial physiology and cellular well-being and for this reason mtDNA lesions have to be rapidly recognized and repaired. Base excision repair (BER) is the main pathway responsible for repairing non-helix distorting base lesions both into the nucleus and in mitochondria. Apurinic/Apyrimidinic Endonuclease 1 (APE1) is a key component of BER pathway and the only protein that can recognize and process an abasic (AP) site. Comprehensions of the mechanisms regulating APE1 intracellular trafficking are still fragmentary. In this study we focused our attention on the mitochondrial form of APE1 protein and how oxidative stress induces its translocation to maintain mtDNA integrity. Our data proved that: (i) the rise of mitochondrial ROS determines a very rapid translocation of APE1 from the intermembrane space (IMS) into the matrix; and (ii) TIM23/PAM machinery complex is responsible for the matrix translocation of APE1. Moreover, our data support the hypothesis that the IMS, where the majority of APE1 resides, could represent a sort of storage site for the protein.

Mitochondrial DNA lesions and copy number are strain dependent in endurance-trained mice.

In this pilot work, we selected two inbred strains that respond well to endurance training (ET) (FVB/NJ, and SJL/J strains), and two strains that respond poorly (BALB/cByJ and NZW/LacJ), to determine the effect of a standardized ET treadmill program on mitochondrial and nuclear DNA (nucDNA) integrity, and mitochondrial DNA (mtDNA) copy number. DNA was isolated from plantaris muscles (n = 37) and a gene‐specific quantitative PCR‐based assay was used to measure DNA lesions and mtDNA copy number. Mean mtDNA lesions were not different within strains in the sedentary or exercise‐trained states. However, mtDNA lesions were significantly higher in trained low‐responding NZW/LacJ mice (0.24 ± 0.06 mtDNA lesions/10 Kb) compared to high‐responding strains (mtDNA lesions/10 Kb: FVB/NJ = 0.11 ± 0.01, p = .049; SJL/J = 0.04 ± 0.02; p = .003). ET did not alter mean mtDNA copy numbers for any strain, although both sedentary and trained FVB/NJ mice had significantly higher mtDNA copies (99,890 ± 4,884 mtDNA copies) compared to low‐responding strains (mtDNA copies: BALB/cByJ = 69,744 ± 4,675; NZW/LacJ = 65,687 ± 5,180; p < .001). ET did not change nucDNA lesions for any strain, however, SJL/J had the lowest mean nucDNA lesions (3.5 ± 0.14 nucDNA lesions/6.5 Kb) compared to all other strains (nucDNA lesions/6.5 Kb: FVB/NJ = 4.4 ± 0.11; BALB/cByJ = 4.7 ± 0.09; NZW/LacJ = 4.4 ± 0.11; p < .0001). Our results demonstrate strain differences in plantaris muscle mtDNA lesions in ET mice and, independent of condition, differences in mean mtDNA copy and nucDNA lesions between strains.

Mitochondrial apurinic/apyrimidinic endonuclease 1 enhances mtDNA repair contributing to cell proliferation and mitochondrial integrity in early stages of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the leading cause of primary liver cancers. Surveillance of individuals at specific risk of developing HCC, early diagnostic markers, and new therapeutic approaches are essential to obtain a reduction in disease-related mortality. Apurinic/apyrimidinic endonuclease 1 (APE1) expression levels and its cytoplasmic localization have been reported to correlate with a lower degree of differentiation and shorter survival rate. The aim of this study is to fully investigate, for the first time, the role of the mitochondrial form of APE1 in HCC.MethodsAs a study model, we analyzed samples from a cohort of patients diagnosed with HCC who underwent surgical resection. Mitochondrial APE1 content, expression levels of the mitochondrial import protein Mia40, and mtDNA damage of tumor tissue and distal non-tumor liver of each patient were analyzed. In parallel, we generated a stable HeLa clone for inducible silencing of endogenous APE1 and re-expression of the recombinant shRNA resistant mitochondrially targeted APE1 form (MTS-APE1). We evaluated mtDNA damage, cell growth, and mitochondrial respiration.ResultsAPE1’s cytoplasmic positivity in Grades 1 and 2 HCC patients showed a significantly higher expression of mitochondrial APE1, which accounted for lower levels of mtDNA damage observed in the tumor tissue with respect to the distal area. In the contrast, the cytoplasmic positivity in Grade 3 was not associated with APE1’s mitochondrial accumulation even when accounting for the higher number of mtDNA lesions measured. Loss of APE1 expression negatively affected mitochondrial respiration, cell viability, and proliferation as well as levels of mtDNA damage. Remarkably, the phenotype was efficiently rescued in MTS-APE1 clone, where APE1 is present only within the mitochondrial matrix.ConclusionsOur study confirms the prominent role of the mitochondrial form of APE1 in the early stages of HCC development and the relevance of the non-nuclear fraction of APE1 in the disease progression. We have also confirmed overexpression of Mia40 and the role of the MIA pathway in the APE1 import process. Based on our data, inhibition of the APE1 transport by blocking the MIA pathway could represent a new therapeutic approach for reducing mitochondrial metabolism by preventing the efficient repair of mtDNA.

Mitochondrial Mechanisms of LRRK2 G2019S Penetrance.

Several mutations in leucine-rich repeat kinase-2 (LRRK2) have been associated with Parkinson's disease (PD). The most common substitution, G2019S, interferes with LRRK2 kinase activity, which is regulated by autophosphorylation. Yet, the penetrance of this gain-of-function mutation is incomplete, and thus far, few factors have been correlated with disease status in carriers. This includes (i) LRRK2 autophosphorylation in urinary exosomes, (ii) serum levels of the antioxidant urate, and (iii) abundance of mitochondrial DNA (mtDNA) transcription-associated 7S DNA. In light of a mechanistic link between LRRK2 kinase activity and mtDNA lesion formation, we previously investigated mtDNA integrity in fibroblasts from manifesting (LRRK2+/PD+) and non-manifesting carriers (LRRK2+/PD−) of the G2019S mutation as well as from aged-matched controls. In our published study, mtDNA major arc deletions correlated with PD status, with manifesting carriers presenting the highest levels. In keeping with these findings, we now further explored mitochondrial features in fibroblasts derived from LRRK2+/PD+ (n = 10), LRRK2+/PD− (n = 21), and control (n = 10) individuals. In agreement with an accumulation of mtDNA major arc deletions, we also detected reduced NADH dehydrogenase activity in the LRRK2+/PD+ group. Moreover, in affected G2019S carriers, we observed elevated mitochondrial mass and mtDNA copy numbers as well as increased expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates antioxidant signaling. Taken together, these results implicate mtDNA dyshomeostasis—possibly as a consequence of impaired mitophagy—in the penetrance of LRRK2-associated PD. Our findings are a step forward in the pursuit of unveiling markers that will allow monitoring of disease progression of LRRK2 mutation carriers.

INFLUENCE OF GENETIC BACKGROUND ON HEART MITOCHONDRIAL DNA LESIONS AND COPY NUMBER IN INBRED MICE

PURPOSE: The heart is a critical tissue responsible for facilitating a multitude of endurance training adaptations in aerobic capacity. Mechanisms of heart mitochondrial DNA (mtDNA) repair remain incompletely understood, and genetic susceptibility to exercise-induced mitochondrial-derived oxidative damage may be present. mtDNA damage presents as an indirect measure of mitochondrial-derived oxidative stress, while mtDNA copy number is a correlate of mitochondrial biogenesis. As two critical aspects for enhancing trainability in aerobic capacity, the purpose of this study was to characterize heart mtDNA lesions and copy number in a genetically diverse panel of male inbred mouse strains. METHODS: A genetically diverse panel of 34 inbred mouse strains were selected, and hearts of male mice (n = 184; 2 - 6 mice per strain) were removed and weighed. DNA was and a gene-specific quantitative PCR-based assay was used to measuremtDNA lesions and copy number. RESULTS: Among the strains, we found significant interstrain varitation in mtDNA lesions (range = -0.15 - 4.0 mtDNA lesions/10Kb) and copy number (range = 3682 - 111895 mtDNA copies). We observed that the wild-derived PWD/PhJ strain had the highest overall heart mtDNA lesions (PWD/PhJ = 4.31 ± 0.07 mtDNA lesions/10Kb vs all other strains = 0.25 ± 0.42 mtDNA lesions/10Kb; p<0.0001), while having the lowest overall mean mtDNA copy number (PWD/PhJ = 6,538 ± 898 mtDNA copies vs all other strains = 54,943 ± 27,085 mtDNA copies; p<0.0001). We did not find an association between mtDNA lesions or copy number with exercise capacity or heart weight. CONCLUSIONS: Our results demonstrate that there are inherent differences in heart mtDNA damage and copy number. Interestingly, the wild-derived PWD/PhJ strain had higher overall mtDNA lesions, complimented by lower mtDNA copies, possibly suggesting the hearts of this strain undergomore significant mitochondrial-derived oxidative stress (e.g. higher fission vs fusion; mitophagy) compared to others. Thus, ongoing work aims to 1) characterize markers of mitochondrial-derived oxidative stress (i.e., fission/fusion; mitophagy), 2) sequence the complete mitochondrial genome and determine levels of heteroplasmy and indels that may associate with mtDNA damage and copy number.

Mitochondrial reactive oxygen species cause major oxidative mitochondrial DNA damages and repair pathways

Mitochondria-derived reactive oxygen species (mROS) are produced at a variety of sites and affect the function of bio-molecules. The anti-oxidant system from both mitochondria and cytosol tightly coordinate to maintain the redox balance of cells and reduce damage from mROS. Mitochondrial DNA (mtDNA) are highly susceptible to mROS, and are easily oxidized to accumulate DNA modifications. Frequent oxidative damages in mtDNA have been associated with neurological degeneration, inflammasomes, tumorigenesis, and malignant progression. Among mitochondrial DNA repair pathways, the base excision repair pathway has been extensively characterized to remove some of oxidative damages in mtDNA as efficiently as the nuclear base excision repair. The implications of other pathways remain unclear. This review focuses on: (i) Sources of mROS and the antioxidant system to balance redox status; (ii) major mtDNA lesions or damages from mROS-mediated oxidation and the reported repair pathways or repairing factors; (iii) cellular response of oxidized mtDNA and methods to identify oxidatively generated DNA modifications in pathological conditions. DNA damages caused by mROS have been increasingly implicated in diseases and aging, and thus we critically discuss methods of the oxidative modifications evaluation and the complexity of non-canonical DNA repair pathways in mitochondria.

An Evaluation of the DNA-Protective Effects of Extracts fromMenyanthes trifoliataL. Plants Derived fromIn VitroCulture Associated with Redox Balance and Other Biological Activities

Menyanthes trifoliata L. is a valuable medical plant found in Europe, North America, and Asia, which grows on peat bogs and swamps. It has long been used in folk medicine as a remedy for various ailments. This is the first report to demonstrate the protective antioxidant and anti-inflammatory properties of aqueous methanolic extracts derived from the aerial parts (MtAPV) and roots (MtRV) of in vitro grown plants on human umbilical vein endothelial cells (HUVECs). It describes the influence of the tested extracts on the expression of antioxidant (HO-1, NQO1, NRF2, kEAP1, and GCLC) and inflammation-related genes (IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ) in cells stimulated with H2O2 or LPS, respectively. In addition, M. trifoliata extracts were found to moderately affect the growth of certain bacterial and fungal pathogens, with the strongest antibacterial effect found against Pseudomonas aeruginosa and Enterococcus faecalis. M. trifoliata extracts demonstrated protective effects against mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage caused by ROS, decreasing the numbers of mtDNA lesions in the ND1 and ND2 genes and nDNA damage in the TP53 and HPRT1 genes and reducing cleavage in PARP1- and γ-H2A.X-positive cells. The root extract of in vitro M. trifoliata (MtRV) appears to have better anti-inflammatory, antioxidant, antimicrobial, and protective properties than the extract from the aerial part (MtAPV). These differences in biological properties may result from the higher content of selected phenolic compounds and betulinic acid in the MtRV than in the MtAPV extract.

Abstract 4219: Mitochondrial DNA damage: A promising tool to screen cervical cancer risk

Abstract Background: Cervical cancer is the 4th most common female cancer, accounting for 500,000 new cases and 200,000 deaths across the world. 99% of cervical cancer is due to infection by human papillomavirus (HPV). The factors leading to HPV integration and induced carcinogenesis are not entirely clear, but high DNA damage levels have been proposed to play a role in HPV integration. Chronic HPV infection increases DNA damage and reduces DNA repair, leading to even higher DNA damage levels. Recently, we showed that the levels of nuclear DNA (nDNA) damage are a potential biomarker predictive of cancer risk. Mitochondrial DNA (mtDNA) damage has been reported to be more persistent than nDNA damage. We have previously demonstrated a good correlation between mtDNA and nDNA damage. Herein, we performed a pilot study to assess whether the levels of mtDNA damage can be used as a potential tool to screen cancer risk. Aims: (1) To measure determine whether the amount of mtDNA damage differs between cervical samples with distinct pathologies. (2) To determine whether the number of mtDNA lesions correlates with the grade of cervical dysplasia. Methods: Ethics Committee approval and written informed consent was obtained from all participants. Samples from 25 patients were collected from the endocervix during a pelvic exam in the Dysplasia Clinic. Samples for DNA damage studies were collected by cytobrush and DNA extracted as previously reported. Demographic and risk factor data were collected through detailed questionnaires. Clinicopathologic data was abstracted from the medical chart. The amount of mtDNA damage was quantified by a long-run real-time PCR-based DNA-damage quantification (LORDQ) assay. Data were analyzed by Student's t-tests. Logistic regression analysis was done modeling the probability of having present pathology risk. Results: Histopathological reports indicated that 9 cervical samples were normal (C0) and 16 had cervical dysplasia. Dysplasia samples were as follows: 9 low-grade squamous intraepithelial lesions (LSILs or C1) and 7 high-grade squamous intraepithelial lesions (HSILs or C2/3). The amount of mtDNA lesions per 10,000 bases were increased two-fold in LSILs or C1 cases and three-fold in HSILs or C2/3 cases when compared to C0. Regression analysis showed no significant correlation between age, race, smoking and drinking status compare with pathology risk. Conclusion: Our data show that mtDNA damage is the lowest in patients without cervical dysplasia and increases progressively with higher grades of dysplasia and correspondent cancer risk. This pilot study shows the feasibility of the approach and stresses the potential of using mtDNA damage to predict cervical cancer risk. Measuring mtDNA damage detection by LORDQ is less time consuming and more cost-effective than the measurement of nDNA damage. Larger population studies are urgently needed to fully assess the potential of this approach for cervical cancer risk. Citation Format: Balaji Sadhasivam, Camille C. Gunderson, Elizabeth H. Hahn, Sarah E. Johnston, Yan D. Zhao, Vengatesh Ganapathy, Lurdes Queimado. Mitochondrial DNA damage: A promising tool to screen cervical cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4219.