Interactions of Mitochondrial Transcription Factor a with DNA Damage: Mechanistic Insights and Functional Implications

Mitochondria have a plethora of functions in eukaryotic cells, including cell signaling, programmed cell death, protein cofactor synthesis, and various aspects of metabolism. The organelles carry their own genomic DNA, which encodes transfer and ribosomal RNAs and crucial protein subunits in the oxidative phosphorylation system. Mitochondria are vital for cellular and organismal functions, and alterations of mitochondrial DNA (mtDNA) have been linked to mitochondrial disorders and common human diseases. As such, how the cell maintains the integrity of the mitochondrial genome is an important area of study. Interactions of mitochondrial proteins with mtDNA damage are critically important for repairing, regulating, and signaling mtDNA damage. Mitochondrial transcription factor A (TFAM) is a key player in mtDNA transcription, packaging, and maintenance. Due to the extensive contact of TFAM with mtDNA, it is likely to encounter many types of mtDNA damage and secondary structures. This review summarizes recent research on the interaction of human TFAM with different forms of non-canonical DNA structures and discusses the implications on mtDNA repair and packaging.

The Mitochondrial Response to DNA Damage

Mitochondria are double membrane organelles in eukaryotic cells that provide energy by generating adenosine triphosphate (ATP) through oxidative phosphorylation. They are crucial to many aspects of cellular metabolism. Mitochondria contain their own DNA that encodes for essential proteins involved in the execution of normal mitochondrial functions. Compared with nuclear DNA, the mitochondrial DNA (mtDNA) is more prone to be affected by DNA damaging agents, and accumulated DNA damages may cause mitochondrial dysfunction and drive the pathogenesis of a variety of human diseases, including neurodegenerative disorders and cancer. Therefore, understanding better how mtDNA damages are repaired will facilitate developing therapeutic strategies. In this review, we focus on our current understanding of the mtDNA repair system. We also discuss other mitochondrial events promoted by excessive DNA damages and inefficient DNA repair, such as mitochondrial fusion, fission, and mitophagy, which serve as quality control events for clearing damaged mtDNA.

Amyloid-β Oligomers-induced Mitochondrial DNA Repair Impairment Contributes to Altered Human Neural Stem Cell Differentiation

Background: Amyloid-β42 oligomers (Aβ42O), the proximate effectors of neurotoxicity observed in Alzheimer’s disease (AD), can induce mitochondrial oxidative stress and impair mitochondrial function besides causing mitochondrial DNA (mtDNA) damage. Aβ42O also regulate the proliferative and differentiative properties of stem cells. Objective: We aimed to study whether Aβ42O-induced mtDNA damage is involved in the regulation of stem cell differentiation. Method: Human iPSCs-derived neural stem cell (NSC) was applied to investigate the effect of Aβ42O on reactive oxygen species (ROS) production and DNA damage using mitoSOX staining and long-range PCR lesion assay, respectively. mtDNA repair activity was measured by non-homologous end joining (NHEJ) in vitro assay using mitochondria isolates and the expression and localization of NHEJ components were determined by Western blot and immunofluorescence assay. The expressions of Tuj-1 and GFAP, detected by immunofluorescence and qPCR, respectively, were examined as an index of neurons and astrocytes production. Results: We show that in NSC Aβ42O treatment induces ROS production and mtDNA damage and impairs DNA end joining activity. NHEJ components, such as Ku70/80, DNA-PKcs, and XRCC4, are localized in mitochondria and silencing of XRCC4 significantly exacerbates the effect of Aβ42O on mtDNA integrity. On the contrary, pre-treatment with Phytic Acid (IP6), which specifically stimulates DNA-PK-dependent end-joining, inhibits Aβ42O-induced mtDNA damage and neuronal differentiation alteration. Conclusion: Aβ42O-induced mtDNA repair impairment may change cell fate thus shifting human NSC differentiation toward an astrocytic lineage. Repair stimulation counteracts Aβ42O neurotoxicity, suggesting mtDNA repair pathway as a potential target for the treatment of neurodegenerative disorders like AD.

Reverse genetic analysis of yeast YPR099C/MRPL51 reveals a critical role of both overlapping ORFs in respiratory growth and MRPL51 in mitochondrial DNA maintenance

ABSTRACT The Saccharomyces cerevisiae genome contains 6572 ORFs, of which 680 ORFs are classified as dubious ORFs. A dubious ORF is a small, noncoding, nonconserved ORF that overlaps with another ORF of the complementary strand. Our study characterizes a dubious/nondubious ORF pair, YPR099C/MRPL51, and shows the transcript and protein level expression of YPR099C. Its subcellular localization was observed in the mitochondria. The overlapping ORF, MRPL51, encodes a mitochondrial ribosomal protein of large subunit. Deletion of any ORF from YPR099C/MRPL51 pair induces common phenotypes, i.e. loss of mtDNA, lack of mitochondrial fusion and lack of respiratory growth, due to the double deletion (ypr099cΔ/Δmrpl51Δ/Δ) caused by sequence overlap. Hence, we created the single deletions of each ORF of the YPR099C/MRPL51 pair by an alternative approach to distinguish their phenotypes and identify the specific functions. Both the ORFs were found essential for the functional mitochondria and respiratory growth, but MRPL51 showed its specific requirement in mtDNA stability. The mechanism of mtDNA maintenance by Mrpl51 is probably Mhr1 dependent that physically interacts with Mrpl51 and also regulates mtDNA repair. Overall, our study provides strong evidence for the protein level expression of a dubious ORF YPR099C and the bifunctional role of Mrpl51 in mtDNA maintenance.

A novel mtDNA repair fusion protein attenuates maladaptive remodeling and preserves cardiac function in heart failure

Oxidative stress results in mtDNA damage and contributes to myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We investigated a novel, mitochondria-targeted fusion protein (Exscien1-III) containing endonuclease III in myocardial ischemia-reperfusion injury and transverse aortic constriction (TAC)-induced heart failure. Male C57/BL6J mice (10–12 wk) were subjected to 45 min of myocardial ischemia and either 24 h or 4 wk of reperfusion. Exscien1-III (4 mg/kg ip) or vehicle was administered at the time of reperfusion. Male C57/BL6J mice were subjected to TAC, and Exscien1-III (4 mg/kg i.p) or vehicle was administered daily starting at 3 wk post-TAC and continued for 12 wk. Echocardiography was performed to assess left ventricular (LV) structure and function. Exscien1-III reduced myocardial infarct size ( P < 0.01) at 24 h of reperfusion and preserved LV ejection fraction at 4 wk postmyocardial ischemia. Exscien1-III attenuated TAC-induced LV dilation and dysfunction at 6–12 wk post-TAC ( P < 0.05). Exscien1-III reduced ( P < 0.05) cardiac hypertrophy and maladaptive remodeling after TAC. Assessment of cardiac mitochondria showed that Exscien1-III localized to mitochondria and increased mitochondrial antioxidant and reduced apoptotic markers. In conclusion, our results indicate that administration of Exscien1-III provides significant protection against myocardial ischemia and preserves myocardial structure and LV performance in the setting of heart failure. NEW & NOTEWORTHY Oxidative stress-induced mitochondrial DNA damage is a prominent feature in the pathogenesis of cardiovascular diseases. In the present study, we demonstrate the efficacy of a novel, mitochondria-targeted fusion protein that traffics endonuclease III specifically for mitochondrial DNA repair in two well-characterized murine models of cardiac injury and failure.

MMS19 localizes to mitochondria and protects the mitochondrial genome from oxidative damage

MMS19 localizes to the cytoplasmic and nuclear compartments involved in transcription and nucleotide excision repair (NER). However, whether MMS19 localizes to mitochondria, where it plays a role in maintaining mitochondrial genome stability, remains unknown. In this study, we provide the first evidence that MMS19 is localized in the inner membrane of mitochondria and participates in mtDNA oxidative damage repair. MMS19 knockdown led to mitochondrial dysfunctions including decreased mtDNA copy number, diminished mtDNA repair capacity, and elevated levels of mtDNA common deletion after oxidative stress. Immunoprecipitation – mass spectrometry analysis identified that MMS19 interacts with ANT2, a protein associated with mitochondrial ATP metabolism. ANT2 knockdown also resulted in a decreased mtDNA repair capacity after oxidative damage. Our findings suggest that MMS19 plays an essential role in maintaining mitochondrial genome stability.

Abstract 85: A Novel Mitochondrial Targeted Fusion Protein Containing Endonuclease III Protects the Heart Against Myocardial Infarction and Pressure Overload Heart Failure

Background: Oxidative stress is a primary cause of mitochondrial DNA (mtDNA) damage and plays a role in myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We tested the efficacy of a novel, mitochondrial targeted fusion protein that traffics Endonuclease III (Exscien1-III) in murine models of myocardial ischemia/reperfusion (MI/R) injury and transverse aortic constriction (TAC) heart failure (HF). We previously demonstrated that Exscien1-III administered at R reduced myocardial infarct size and preserved left ventricular ejection fraction (LVEF) following MI/R. We hypothesized that delayed administration of Exscien1-III would promote mtDNA repair and protect the myocardium against MI/R and TAC heart failure. Methods: Male C57/BL6J (10-12 wks) were subjected to 45 min of MI and 24 hrs of R. Exscien1-III (4 mg/kg, i.p., n=13) or vehicle (VEH, n=13) was administered 30 min after R. Male C57/BL6J were subjected to TAC (27 g needle) and Exscien1-III (4 mg/kg/d, i.p., n=10) or VEH (n=6) were administered starting at 3 wks post TAC. Echocardiography was performed at baseline and following TAC to assess LVEF. Results: Exscien1-III reduced myocardial INF/AAR by 24% (p < 0.05 vs. VEH). Exscien1-III preserved LVEF (49.1 ± 4.0% vs. 32.9 ± 3.2%, p < 0.01) and reduced LV dilation (LVEDD/LVESD; 3.8/2.8 vs. 4.4/3.4, p < 0.05) at 8 wks compared to vehicle. Conclusion: These results demonstrate that delayed administration of Exscien1-III significantly attenuates myocardial cell death and preserves LV function in acute MI and HF. Studies are currently underway to define the molecular mechanisms involved in Exscien1-III induced cardioprotection.

Abstract 15768: Mitochondria-Targeted DNA Repair Glycosylase Ogg1 Suppresses Early Stage of Atherogenesis in Ogg1 Deficient Mice

Introduction: Reactive oxygen species (ROS) play a key role in the development of atherosclerosis. Mitochondria are a main source of endogenous ROS in the cell. Mitochondrial DNA (mtDNA) is sensitive to oxidation and our previous results from cultured cell and intact animal models suggest that increasing mtDNA repair prevents both oxidative mtDNA damage and associated cytotoxicity and cellular dysfunction. Involvement of oxidative mtDNA damage in disorders characterized by chronic oxidative stress has been less thoroughly studied. Hypothesis: In the present study we tested the hypothesis that transgenic modulation of Ogg1, a DNA glycosylase mediating the first step in the base excision repair of oxidative mtDNA damage, coordinately regulates atherogenesis in mice fed a high fat diet. Methods: Wild type (WT) mice, Ogg1 knock-out (KO) mice and KO mice transgenically overexpressing mitochondria-targeted Ogg1 (KO-Tg) were fed pro-atherogenic Western type diet for 14 weeks and analyzed for mtDNA damage and signs of atherogenesis. Results: KO mice fed a high fat diet had increased oxidative mtDNA damage in cardiac tissue, whereas KO-Tg animals did not differ from WT mice (WT: 0.18 ± 0.04; KO: 0.35 ± 0.04; KO-Tg: 0.15 ± 0.01 lesions per 10 4 bp; n=3, P<0.05; quantitative Southern blot analysis). We did not observe significant atherosclerotic plaque formation in the aortic valve of animals from any group; however appearance of fatty streaks, indicative of early plaque development, was more evident in KO mice (WT: 179 ± 20; KO: 384 ± 59 pixels, n=4, P<0.05; immunohistochemistry for Fc receptor - general marker of inflammatory cells). This effect was completely blocked in KO-Tg mice. We found increased number of apoptotic cells in the aortic valve of KO, but not KO-Tg mice (WT: 2.00 ± 0.50; KO: 4.25 ± 0.48; KO-Tg: 2.14 ± 0.85 apoptotic cells, n=4, P<0.05; TUNEL assay). Conclusion: Our data demonstrate that Ogg1 deficiency in mice fed a high fat diet leads to increased oxidative mtDNA damage, appearance of fatty streaks and cell apoptosis. In contrast, enhancement of mtDNA repair with mitochondria-targeted Ogg1 reduces fatty streaks formation and apoptosis induced by a high fat diet. These results suggest mtDNA damage and repair could be important targets for atheroprotection.