Mitochondrial DNA-Activated cGAS-STING Signaling in Environmental Dry Eye.

Purpose: The cGAS-STING pathway has been shown to be an important mediator of inflammation. There is emerging evidence of the importance of this signaling cascade in a variety of inflammatory diseases settings. Here, we present evidence that the mitochondrial DNA (mtDNA) damage-mediated cGAS-STING pathway plays an important role in the induction of inflammation in environmental dry eye (DE). Methods: RT-qPCR and Western blot were used to assess the induction of the cGAS-STING pathway and inflammatory cytokines in environmental DE mouse model, primary human corneal epithelial cells (pHCECs), and patients with DE. RNA sequencing was used to determine mRNA expression patterns of high osmotic pressure (HOP)-stimulated pHCECs. mtDNA was detected with electron microscopy, flow cytometry, and immunofluorescent staining. mtDNA was isolated and transfected into pHCECs for evaluating the activation of the cGAS-STING pathway. Results: The expression levels of cGAS, STING, TBK1, IRF3, and IFNß were significantly increased in an environmental DE model and HOP-stimulated pHCECs. The STING inhibitor decreased the expression of inflammatory factors in DE. An upregulation of STING-mediated immune responses and IRF3 expression mediated by TBK1 were observed in the HOP group. HOP stimulation induced mitochondrial oxidative damage and the leakage of mtDNA into the cytoplasm. Then, mtDNA activated the cGAS-STING pathway and induced intracytoplasmic STING translocated to the Golgi apparatus. Finally, we also found activated cGAS-STING signaling in the human conjunctival blot cell of patients with DE. Conclusions: Our findings suggest that the cGAS-STING pathway is activated by recognizing cytoplasmic mtDNA leading to STING translocation, further exacerbating the development of inflammation in environmental DE.

Human studies of mitochondrial biology demonstrate an overall lack of binary sex differences: A multivariate meta-analysis.

Mitochondria are maternally inherited organelles that play critical tissue-specific roles, including hormone synthesis and energy production, that influence human development, health, and aging. However, whether mitochondria from women and men exhibit consistent biological differences remains unclear, representing a major gap in knowledge. This meta-analysis systematically examined four domains and six subdomains of mitochondrial biology (total 39 measures), including mitochondrial content, respiratory capacity, reactive oxygen species (ROS) production, morphometry, and mitochondrial DNA copy number. Standardized effect sizes (Hedge's g) of sex differences were computed for each measure using data in 2258 participants (51.5% women) from 50 studies. Only two measures demonstrated aggregate binary sex differences: higher mitochondrial content in women's WAT and isolated leukocyte subpopulations (g = 0.20, χ2 p = .01), and higher ROS production in men's skeletal muscle (g = 0.49, χ2 p < .0001). Sex differences showed weak to no correlation with age or BMI. Studies with small sample sizes tended to overestimate effect sizes (r = -.17, p < .001), and sex differences varied by tissue examined. Our findings point to a wide variability of findings in the literature concerning possible binary sex differences in mitochondrial biology. Studies specifically designed to capture sex- and gender-related differences in mitochondrial biology are needed, including detailed considerations of physical activity and sex hormones.

Mitochondrial Dysfunction in Diseases, Longevity, and Treatment Resistance: Tuning Mitochondria Function as a Therapeutic Strategy.

Mitochondria are very important intracellular organelles because they have various functions. They produce ATP, are involved in cell signaling and cell death, and are a major source of reactive oxygen species (ROS). Mitochondria have their own DNA (mtDNA) and mutation of mtDNA or change the mtDNA copy numbers leads to disease, cancer chemo/radioresistance and aging including longevity. In this review, we discuss the mtDNA mutation, mitochondrial disease, longevity, and importance of mitochondrial dysfunction in cancer first. In the later part, we particularly focus on the role in cancer resistance and the mitochondrial condition such as mtDNA copy number, mitochondrial membrane potential, ROS levels, and ATP production. We suggest a therapeutic strategy employing mitochondrial transplantation (mtTP) for treatment-resistant cancer.

CircTLK1 modulates sepsis-induced cardiomyocyte apoptosis via enhancing PARP1/HMGB1 axis-mediated mitochondrial DNA damage by sponging miR-17-5p.

INTRODUCTION: Septic cardiomyopathy is a common complication of sepsis with high morbidity and mortality, but lacks specific therapy. This study aimed to reveal the role of circTLK1 and its potential mechanisms in septic cardiomyopathy. MATERIALS AND METHODS: The in vitro and in vivo models of septic cardiomyopathy were established. Cell viability and apoptosis were detected by CCK8, TUNEL and flow cytometry, respectively. LDH, CK, SOD, MDA, ATP, 8-OHdG, NAD+/NADH ratio, ROS level, mitochondrial membrane potential and cytochrome C distribution were evaluated using commercial kits. qRT-PCR and western blotting were performed to detect RNA and protein levels. Mitochondrial DNA (mtDNA) copy number and transcription were assessed by quantitative PCR. Dual-luciferase assay, RNA immunoprecipitation and co-immunoprecipitation were performed to verify the interaction between circTLK1/PARP1 and miR-17-5p. RESULTS: CircTLK1, PARP1 and HMGB1 were up-regulated in the in vitro and in vivo models of septic cardiomyopathy. CircTLK1 inhibition restrained LPS-induced up-regulation of PARP1 and HMGB1. Moreover, circTLK1 knockdown repressed sepsis-induced mtDNA oxidative damage, mitochondrial dysfunction and consequent cardiomyocyte apoptosis by inhibiting PARP1/HMGB1 axis in vitro and in vivo. In addition, circTLK1 enhanced PARP1 expression via sponging miR-17-5p. Inhibition of miR-17-5p abolished the protective effects of circTLK1 silencing on oxidative mtDNA damage and cardiomyocyte apoptosis. CONCLUSION: CircTLK1 sponged miR-17-5p to aggravate mtDNA oxidative damage, mitochondrial dysfunction and cardiomyocyte apoptosis via activating PARP1/HMGB1 axis during sepsis, indicating that circTLK1 may be a putative therapeutic target for septic cardiomyopathy.

Evolution of eukaryotes as a story of survival and growth of mitochondrial DNA over two billion years.

Mitochondria's significance in human diseases and in functioning, health and death of eukaryotic cell has been acknowledged widely. Yet our perspective in cell biology and evolution remains nucleocentric. Mitochondrial DNA, by virtue of its omnipresence and species-level conservation, is used as a barcode in animal taxonomy. This article analyses various levels of containment structures that enclose mitochondrial DNA and advocates a fresh perspective wherein evolution of organic structures of the eukarya domain seem to support and facilitate survival and proliferation of mitochondrial DNA by splitting containers as they age and by directing them along two distinct pathways: destruction of containers with more mutant mitochondrial DNA and rejuvenation of containers with less mutant mitochondrial DNA.

Ovarian Aging: Role of Pituitary-Ovarian Axis Hormones and ncRNAs in Regulating Ovarian Mitochondrial Activity.

The number of mitochondria in the oocyte along with their functions (e.g., energy production, scavenger activity) decline with age progression. Such multifaceted functions support several processes during oocyte maturation, ranging from energy supply to synthesis of the steroid hormones. Hence, it is hardly surprising that their impairment has been reported in both physiological and premature ovarian aging, wherein they are crucial players in the apoptotic processes that arise in aged ovaries. In any form, ovarian aging implies the progressive damage of the mitochondrial structure and activities as regards to ovarian germ and somatic cells. The imbalance in the circulating hormones and peptides (e.g., gonadotropins, estrogens, AMH, activins, and inhibins), active along the pituitary-ovarian axis, represents the biochemical sign of ovarian aging. Despite the progress accomplished in determining the key role of the mitochondria in preserving ovarian follicular number and health, their modulation by the hormonal signalling pathways involved in ovarian aging has been poorly and randomly explored. Yet characterizing this mechanism is pivotal to molecularly define the implication of mitochondrial dysfunction in physiological and premature ovarian aging, respectively. However, it is fairly difficult considering that the pathways associated with ovarian aging might affect mitochondria directly or by altering the activity, stability and localization of proteins controlling mitochondrial dynamics and functions, either unbalancing other cellular mediators, released by the mitochondria, such as non-coding RNAs (ncRNAs). We will focus on the mitochondrial ncRNAs (i.e., mitomiRs and mtlncRNAs), that retranslocate from the mitochondria to the nucleus, as active players in aging and describe their role in the nuclear-mitochondrial crosstalk and its modulation by the pituitary-ovarian hormone dependent pathways. In this review, we will illustrate mitochondria as targets of the signaling pathways dependent on hormones and peptides active along the pituitary/ovarian axis and as transducers, with a particular focus on the molecules retrieved in the mitochondria, mainly ncRNAs. Given their regulatory function in cellular activities we propose them as potential diagnostic markers and/or therapeutic targets.

Pericardial Mitochondrial DNA Levels Are Associated With Atrial Fibrillation After Cardiac Surgery.

BACKGROUND: Postoperative atrial fibrillation (POAF) is the most common complication after cardiac surgery, and is associated with increased morbidity and mortality. Inflammation has been implicated as an etiology of POAF. Mitochondrial DNA (mtDNA) has been shown to initiate inflammation. This study analyzed inflammatory mechanisms of POAF by evaluating mtDNA, neutrophils, and cytokines/chemokines in the pericardial fluid and blood after cardiac surgery. METHODS: Blood and pericardial fluid from patients who underwent coronary artery bypass or heart valve surgery, or both, were collected intraoperatively and at 4, 12, 24, and 48 hours postoperatively. Real-time polymerase chain reaction was used to quantify mtDNA in the pericardial fluid and blood. A Luminex (Luminex Corp, Austin, TX) assay was used to study cytokine and chemokine levels. Flow cytometry was used to analyze neutrophil infiltration and activation in the pericardial fluid. RESULTS: Samples from 100 patients were available for analysis. Postoperatively, mtDNA and multiple cytokine levels were higher in the pericardial fluid versus blood. Patients who had POAF had significantly higher levels of mtDNA in the pericardial fluid compared with patients who did not (P < .001, area under the curve 0.74). There was no difference in the mtDNA concentration in the blood between the POAF group and non-POAF group (P = .897). Neutrophil concentration increased in the pericardial fluid over time from a baseline of 0.8% to 56% at 48 hours (P < .01). CONCLUSIONS: The pericardial space has a high concentration of inflammatory mediators postoperatively. Mitochondrial DNA in the pericardial fluid was strongly associated with the development of POAF. This finding provides insight into a possible mechanism of inflammation that may contribute to POAF, and may offer novel therapeutic targets.

Mitochondrial behavior during nuclear and mitochondrial DNA repair in Trypanosoma cruzi epimastigotes.

Different genotoxic agents can lead to DNA single- and double-strand breaks, base modification and oxidation. As most living organisms, Trypanosoma cruzi is subjected to oxidative stress during its life cycle; thus, DNA repair is essential for parasite survival and establishment of infection. The mitochondrion plays important roles beyond the production of ATP. For example, it is a source of signaling molecules, such as the superoxide anion and H2O2. Since T. cruzi has only one mitochondrion, the integrity of this organelle is pivotal for parasite viability. H2O2 and methyl methanesulfonate cause DNA lesions in T. cruzi that are repaired by different DNA repair pathways. Herein, we evaluate mitochondrial involvement during the repair of nuclear and mitochondrial DNA in T. cruzi epimastigotes incubated with these two genotoxic agents under conditions that induce repairable DNA damage. Overall, in both treatments, an increase in oxygen consumption rates and in mitochondrial H2O2 release was observed, as well as maintenance of ATP levels compared to control. Interestingly, these changes coincided with DNA repair kinetics, suggesting the importance of the mitochondrion for this energy-consuming process.

Isosteviol sodium protects the cardiomyocyte response associated with the SIRT1/PGC-1α pathway.

Cardiomyocyte dysfunction is attributed to excess oxidative damage, but the molecular pathways involved in this process have not been completely elucidated. Evidence indicates that isosteviol sodium (STVNa) has cardioprotective effects. We therefore aimed to identify the effect of STVNa on cardiomyocytes, as well as the potential mechanisms involved in this process. We established two myocardial hypertrophy models by treating H9c2 cells with high glucose (HG) and isoprenaline (ISO). Our results showed that STVNa reduced H9c2 mitochondrial damage by attenuating oxidative damage and altering the morphology of mitochondria. The results also indicated that STVNa had a positive effect on HG- and ISO-induced damages via mitochondrial biogenesis. The protective effects of STVNa on cardiomyocytes were associated with the regulation of the SIRT1/PGC-1α signalling pathway. Importantly, the effects of STVNa involved different methods of regulation in the two models, which was confirmed by experiments using an inhibitor and activator of SIRT1. Together, the results provide the basis for using STVNa as a therapy for the prevention of cardiomyocyte dysfunctions.

MtDNA sequence features associated with 'selfish genomes' predict tissue-specific segregation and reversion.

Mitochondrial DNA (mtDNA) encodes cellular machinery vital for cell and organism survival. Mutations, genetic manipulation, and gene therapies may produce cells where different types of mtDNA coexist in admixed populations. In these admixtures, one mtDNA type is often observed to proliferate over another, with different types dominating in different tissues. This 'segregation bias' is a long-standing biological mystery that may pose challenges to modern mtDNA disease therapies, leading to substantial recent attention in biological and medical circles. Here, we show how an mtDNA sequence's balance between replication and transcription, corresponding to molecular 'selfishness', in conjunction with cellular selection, can potentially modulate segregation bias. We combine a new replication-transcription-selection (RTS) model with a meta-analysis of existing data to show that this simple theory predicts complex tissue-specific patterns of segregation in mouse experiments, and reversion in human stem cells. We propose the stability of G-quadruplexes in the mtDNA control region, influencing the balance between transcription and replication primer formation, as a potential molecular mechanism governing this balance. Linking mtDNA sequence features, through this molecular mechanism, to cellular population dynamics, we use sequence data to obtain and verify the sequence-specific predictions from this hypothesis on segregation behaviour in mouse and human mtDNA.

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.

Insertions of mitochondrial DNA into the nucleus-effects and role in cell evolution.

We review the insertion of mitochondrial DNA (mtDNA) fragments into nuclear DNA (NUMTS) as a general and ongoing process that has occurred many times during genome evolution. Fragments of mtDNA are generated during the lifetime of organisms in both somatic and germinal cells, by the production of reactive oxygen species in the mitochondria. The fragments are inserted into the nucleus during the double-strand breaks repair via the non-homologous end-joining machinery, followed by genomic instability, giving rise to the high variability observed in NUMT patterns among species, populations, or genotypes. Some de novo produced mtDNA insertions show harmful effects, being involved in human diseases, carcinogenesis, and ageing. NUMT generation is a non-stop process overpassing the Mendelian transmission. This parasitic property ensures their survival even against their harmful effects. The accumulation of mtDNA fragments mainly at pericentromeric and subtelomeric regions is important to understand the transmission and integration of NUMTs into the genomes. The possible effect of female meiotic drive for mtDNA insertions at centromeres remains to be studied. In spite of the harmful feature of NUMTs, they are important in cell evolution, representing a major source of genomic variation.

Mitochondrial DNA from hepatocytes induces upregulation of interleukin-33 expression of macrophages in nonalcoholic steatohepatitis.

OBJECTIVE: In the present study, we propose that lipotoxicity induces the release of mitochondrial DNA (mtDNA) from hepatocytes, which in turn upregulates IL-33 expression in macrophages. METHODS: The mtDNA levels of plasma were determined in methionine- and mholine-deficient diet (MCD)-fed mice and NASH patients. Cultured hepatocytes were pre-incubated with Mito-TEMPO or rapamycin and were then stimulated with palmitic acid. The mtDNA levels in the cytosol were measured. The mtDNA from hepatocytes of mice was added to bone marrow-derived macrophages (BMDMs) in the presence of IRS (TLR9 antagonist). The expression of IL-33 in BMDMs was measured. RESULTS: Levels of mtDNA were higher in NASH patients and MCD-fed mice. Treatment of hepatocytes with palmitic acid in vitro induced mtDNA release into cytosol, which was attenuated by mito-TEMPO or rapamycin, and aggravated by inhibition of autophagy. Treatment of BMDMs with mtDNA enhanced IL-33 expression, which was attenuated by knockdown of TLR9. Treatment of BMDMs with mtDNA enhanced lipopolysaccharide (LPS)-induced production of IL-1ß and TNF-α, which was attenuated by pretreatment with soluble ST2. CONCLUSION: mtDNA released from injured hepatocytes under lipid overload induced the upregulation of IL-33 expression in macrophages via TLR9, and enhanced LPS-induced inflammatory cytokine production.

Comparative evaluation of two informative markers in the phylogeny of Babesia and Theileria parasites.

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. In the study, we assessed the relative resolution capabilities of the DNA sequences of the nuclear genes 40S ribosomal protein S5 (RPS5) and mitochondrial DNA Cytochrome c oxidase subunit III (cox3) gene in the phylogeny of Babesia and Theileria species isolates. We demonstrated that by using the cox3 gene can recover a better supported species tree for some Theileria species than when using the nuclear RPS5 gene alone, it tends to intra-specific diversity and considerable inter-specific difference. Additionally, the combined DNA sequences of the nuclear RPS5 and cox3 gene improved the inference of evolutionary relationships among Babesia and Theileria species. The mitochondrial cox3 gene outperforms nuclear RPS5 gene and yields better resolution on the intra-specific diversity of Babesia and Theileria species. However, the combined RPS5 nuclear DNA and cox3 DNA tree had more advantage in the phylogeny of Babesia and Theileria species than that of single gene alone.

Mitochondrial Dynamics and Its Involvement in Disease.

The dynamic properties of mitochondria-including their fusion, fission, and degradation-are critical for their optimal function in energy generation. The interplay of fusion and fission confers widespread benefits on mitochondria, including efficient transport, increased homogenization of the mitochondrial population, and efficient oxidative phosphorylation. These benefits arise through control of morphology, content exchange, equitable inheritance of mitochondria, maintenance of high-quality mitochondrial DNA, and segregation of damaged mitochondria for degradation. The key components of the machinery mediating mitochondrial fusion and fission belong to the dynamin family of GTPases that utilize GTP hydrolysis to drive mechanical work on biological membranes. Defects in this machinery cause a range of diseases that especially affect the nervous system. In addition, several common diseases, including neurodegenerative diseases and cancer, strongly affect mitochondrial dynamics.

An Evaluation of the DNA-Protective Effects of Extracts from Menyanthes trifoliata L. Plants Derived from In Vitro Culture 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-1ß, 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.

Reciprocal interactions between mtDNA and lifespan control in budding yeast.

Loss of mitochondrial DNA (mtDNA) results in loss of mitochondrial respiratory activity, checkpoint-regulated inhibition of cell cycle progression, defects in growth, and nuclear genome instability. However, after several generations, yeast cells can adapt to the loss of mtDNA. During this adaptation, rho0 cells, which have no mtDNA, exhibit increased growth rates and nuclear genome stabilization. Here, we report that an immediate response to loss of mtDNA is a decrease in replicative lifespan (RLS). Moreover, we find that adapted rho0 cells bypass the mtDNA inheritance checkpoint, exhibit increased mitochondrial function, and undergo an increase in RLS as they adapt to the loss of mtDNA. Transcriptome analysis reveals that metabolic reprogramming to compensate for defects in mitochondrial function is an early event during adaptation and that up-regulation of stress response genes occurs later in the adaptation process. We also find that specific subtelomeric genes are silenced during adaptation to loss of mtDNA. Moreover, we find that deletion of SIR3, a subtelomeric gene silencing protein, inhibits silencing of subtelomeric genes associated with adaptation to loss of mtDNA, as well as adaptation-associated increases in mitochondrial function and RLS extension.

Mitochondrial DNA: Distribution, Mutations, and Elimination.

Mitochondrion harbors its own DNA (mtDNA), which encodes many critical proteins for the assembly and activity of mitochondrial respiratory complexes. mtDNA is packed by many proteins to form a nucleoid that uniformly distributes within the mitochondrial matrix, which is essential for mitochondrial functions. Defects or mutations of mtDNA result in a range of diseases. Damaged mtDNA could be eliminated by mitophagy, and all paternal mtDNA are degraded by endonuclease G or mitophagy during fertilization. In this review, we describe the role and mechanism of mtDNA distribution and elimination. In particular, we focus on the regulation of paternal mtDNA elimination in the process of fertilization.

Nanoparticles and danger signals: Oral delivery vehicles as potential disruptors of intestinal barrier homeostasis.

Gut immune system homeostasis involves diverse structural interactions among resident microbiota, the protective mucus layer, and a variety of cells (intestinal epithelial, lymphoid, and myeloid). Due to the substantial surface area in direct contact with an "external" environment and the diversity of xenobiotic, abiotic, and self-interactions coordinating to maintain gut homeostasis, there is enhanced potential for the generation of endogenous danger signals when this balance is lost. Here, we focus on the potential generation and reception of damage in the gut resulting from exposure to nanoparticles (NPs), common food and drug additives. Specifically, we describe recent evidence in the literature showing that certain NPs are potential generators of damage-associated molecular patterns, as well as potential immune-stimulating molecular patterns themselves.