Progress on the Physiological Function of Mitochondrial DNA and Its Specific Detection and Therapy.

Mitochondrial DNA (mtDNA) is the genetic information of mitochondrion, and its structure is circular double-stranded. Despite the diminutive size of the mitochondrial genome, mtDNA mutations are an important cause of mitochondrial diseases which are characterized by defects in oxidative phosphorylation (OXPHOS). Mitochondrial diseases are involved in multiple systems, particularly in the organs that are highly dependent on aerobic metabolism. The diagnosis of mitochondrial disease is more complicated since mtDNA mutations can cause various clinical symptoms. To realize more accurate diagnosis and treatment of mitochondrial diseases, the detection of mtDNA and the design of drugs acting on it are extremely important. Over the past few years, many probes and therapeutic drugs targeting mtDNA have been developed, making significant contributions to fundamental research including elucidation of the mechanisms of mitochondrial diseases at the genetic level. In this review, we summarize the structure, function, and detection approaches for mtDNA. The most current topics in this field, such as mechanistic exploration and treatment of mtDNA mutation-related disorders, are also reviewed. Specific attention is given to discussing the design and development of these probes and drugs for mtDNA. We hope that this review will provide readers with a comprehensive understanding of the importance of mtDNA, and promote the development of effective molecules for theragnosis of mtDNA mutation-related diseases.

Remdesivir; molecular and functional measures of mitochondrial safety.

Remdesivir is one of a few antiviral drugs approved for treating severe cases of coronavirus 2 (SARS-CoV-2) infection in hospitalized patients. The prodrug is a nucleoside analog that interferes with viral replication by inhibiting viral RNA-dependent RNA polymerase. The drug has also been shown to be a weak inhibitor of human mitochondrial RNA polymerase, leaving open the possibility of mitochondrial off-targets and toxicity. The investigation was designed to explore whether remdesivir causes mitochondrial toxicity, using both genomic and functional parameters in the assessment. Human-derived HepG2 liver cells were exposed for up to 48 h in culture to increasing concentrations of remdesivir. At sub-cytotoxic concentrations (<1 µM), the drug failed to alter either the number of copies or the expression of the mitochondrial genome. mtDNA copy number was unaffected as was the relative rates of expression of mtDNA-encoded and nuclear encoded subunits of complexes I and IV of the mitochondrial respiratory chain. Consistent with this is the observation that remdesivir was without effect on mitochondrial respiration, including basal respiration, proton leak, maximum uncoupled respiration, spare respiratory capacity or coupling efficiency. We conclude that although remdesivir has weak inhibitory activity towards mitochondrial RNA polymerase, mitochondria are not primary off-targets for the mechanism of cytotoxicity of the drug.

Cancer cell metabolism: Rewiring the mitochondrial hub.

To adapt to tumoral environment conditions or even to escape chemotherapy, cells rapidly reprogram their metabolism to handle adversities and survive. Given the rapid rise of studies uncovering novel insights and therapeutic opportunities based on the role of mitochondria in tumor metabolic programing and therapeutics, this review summarizes most significant developments in the field. Taking in mind the key role of mitochondria on carcinogenesis and tumor progression due to their involvement on tumor plasticity, metabolic remodeling, and signaling re-wiring, those organelles are also potential therapeutic targets. Among other topics, we address the recent data intersecting mitochondria as of prognostic value and staging in cancer, by mitochondrial DNA (mtDNA) determination, and current inhibitors developments targeting mtDNA, OXPHOS machinery and metabolic pathways. We contribute for a holistic view of the role of mitochondria metabolism and directed therapeutics to understand tumor metabolism, to circumvent therapy resistance, and to control tumor development.

The antiretroviral 2',3'-dideoxycytidine causes mitochondrial dysfunction in proliferating and differentiated HepaRG human cell cultures.

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus infection, and their use can cause mitochondrial toxicity, including mitochondrial DNA (mtDNA) depletion in several cases. The first-generation NRTIs, including 2',3'-dideoxycytidine (ddC), were originally and are still pursued as anticancer agents. NRTI-sensitive DNA polymerases localizing to mitochondria allow for the opportunity to poison proliferating cancer cell mtDNA replication as certain cancers rely heavily on mitochondrial functions. However, mtDNA replication is independent of the cell cycle creating a significant concern that toxicants such as ddC impair mtDNA maintenance in both proliferating and nonproliferating cells. To examine this possibility, we tested the utility of the HepaRG cell line to study ddC-induced toxicity in isogenic proliferating (undifferentiated) and nonproliferating (differentiated) cells. Following ddC exposures, we measured cell viability, mtDNA copy number, and mitochondrial bioenergetics utilizing trypan blue, Southern blotting, and extracellular flux analysis, respectively. After 13 days of 1 µM ddC exposure, proliferating and differentiated HepaRG harbored mtDNA levels of 0.9% and 17.9% compared with control cells, respectively. Cells exposed to 12 µM ddC contained even less mtDNA. By day 13, differentiated cell viability was maintained but declined for proliferating cells. Proliferating HepaRG bioenergetic parameters were severely impaired by day 8, with 1 and 12 µM ddC, whereas differentiated cells displayed defects of spare and maximal respiratory capacities (day 8) and proton-leak linked respiration (day 14) with 12 µM ddC. These results indicate HepaRG is a useful model to study proliferating and differentiated cell mitochondrial toxicant exposures.

Lutein upregulates the PGC-1α, NRF1, and TFAM expression by AMPK activation and downregulates ROS to maintain mtDNA integrity and mitochondrial biogenesis in hyperglycemic ARPE-19 cells and rat retina.

Hyperglycemia (HG) affects cellular organelle including mitochondrion in retina that diminishes mitochondrial biogenesis by downregulation of nuclear transcription factors peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α) and mitochondrial transcription factor A (TFAM). Mitochondrial dysfunction has been linked to diabetic retinopathy (DR). Carotenoids reported to modulate mitochondrial biogenesis in HG. Aim of the study was to explore the role of lutein, oxidized lutein (purified upon UV oxidation of lutein) and drug metformin, on mitochondrial biogenesis in HG-induced ARPE-19 cells and rat retina. Results showed higher uptake of lutein and oxidized lutein in ARPE-19 cells and rat retina of HG group than the control groups. Further, lutein and oxidized lutein augmented the AMPK phosphorylation and activation of mitochondrion signaling molecule TFAM (protein expression) and mRNA expression of PGC-1α, TFAM, and nuclear respiratory factor 1 (responsible for mitochondria biogenesis) along with lowered reactive oxygen species in HG compared with control and metformin groups. Higher mRNA expression of nicotinamide adenine dinucleotide dehydrogenase subunits mt-ND1, mt-ND4, mt-ND6, and cytochrome C that aid maintenance of mtDNA integrity was also evidenced. To conclude, lutein and oxidized lutein found to upsurge mitochondrial biogenesis in ARPE-19 cells and rat retina under HG, which may be due to upregulation of AMPK phosphorylation. Finally, lutein and oxidized lutein may provide a therapeutic basis to ameliorate HG-induced DR.

Tranexamic acid suppresses the release of mitochondrial DAMPs and reduces lung inflammation in a murine burn model.

BACKGROUND: Severe burn injuries are known to initiate a profound systemic inflammatory response (SIRS) that may lead to burn shock and other SIRS-related complications. Damage-associated molecular patterns (DAMPs) are important early signaling molecules that initiate SIRS after burn injury. Previous work in a rodent model has shown that application of a topical immune modulator (p38MAPK inhibitor) applied directly to the burn wound decreases cytokine expression, reduces pulmonary inflammation and edema. Our group has demonstrated that tranexamic acid (TXA)-in addition to its use as an antifibrinolytic-has cell protective in vitro effects. We hypothesized that administration of TXA after burn injury would attenuate DAMP release and reduce lung inflammation. METHODS: C57/BL6 male mice underwent a 40% Total Body Surface Area (TBSA) scald burn. Sham animals underwent the same procedure in room temperature water. One treatment group received the topical application of p38MAPK inhibitor after burn injury. The other treatment group received an intraperitoneal administration of TXA after burn injury. Animals were sacrificed at 5 hours. Plasma was collected by cardiac puncture. MtDNA levels in plasma were determined by quantitative Polymerase Chain Reaction (qPCR). Syndecan-1 levels in plasma were measured by ELISA. Lungs were harvested, fixed, and paraffin-embedded. Sections of lungs were stained for antigen to detect macrophages. RESULTS: Topical p38MAPK inhibitor and TXA significantly attenuated mtDNA release. Both TXA and the topical p38MAPK inhibitor reduced lung inflammation as represented by decreased macrophage infiltration. Syndecan-1 levels showed no difference between burn and treatment groups. CONCLUSION: Both p38 MAPK inhibitor and TXA demonstrated the ability to attenuate burn-induced DAMP release and lung inflammation. Beyond its role as an antifibrinolytic, TXA may have significant anti-inflammatory effects pertinent to burn resuscitation. Further study is required; however, TXA may be a useful adjunct in burn resuscitation.

Exogenous administration of mitochondrial DNA promotes ischemia reperfusion injury via TLR9-p38 MAPK pathway.

Previous studies have shown a role of mitochondrial DNA (mtDNA) in innate immunity. However, the specific role of mtDNA in acute myocardial infarction remains elusive. This study was designed to examine the damaging effect of mtDNA on cardiomyocytes. H9c2s cells were incubated with purified mtDNA or nuclear DNA with or without pretreatment by chloroquine, an inhibitor of Toll-like receptor 9(TLR9). The cell viability was tested by MTT. To demonstrate the toxicity of mtDNA, mtDNA fragments were injected into rats 10 min before ischemia for 30 min and reperfusion for 24 h. Infarct size was measured by TTC staining. Apoptosis of myocardium was detected by TUNEL staining and caspase-3 activity. The levels of TLR9, p-p38 MAPK, and p38 MAPK were detected by western blotting. The results showed that exogenous mtDNA reduced the viability of H9c2s cells and induced TLR9 expression, caspase 3 activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation. However, these effects were inhibited by chloroquine. In contrast, nuclear DNA did not have these effects. Intravenous injection of mtDNA into rats aggravated ischemia-reperfusion injury and increased infarction area through TLR9-p38 MAPK activation. We concluded that mtDNA released into the circulation by AMI may has detrimental effect on myocardium through aggravating ischemia-reperfusion injury via TLR9-p38 MAPK pathway.

Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

MTERF4 regulates the mitochondrial dysfunction induced by MPP(+) in SH-SY5Y cells.

Mitochondrial transcription termination factor 4, MTERF4, a member of the MTERF family, has been implicated in the regulation of mitochondrial translation by targeting NSUN4 to the large mitochondrial ribosome. Here, we found a novel role for MTERF4 in regulating mitochondrial dysfunction induced by MPP(+). We observed that knockdown of MTERF4 in SH-SY5Y cells resulted in increased mitochondrial DNA transcription levels and decreased mitochondrial DNA translation levels. In addition, after treatment with 2 mM MPP(+) for 24 h, the expression levels of MTERF4 were decreased compared to wide-type SH-SY5Y cells. Moreover, after exposure to 2 mM MPP(+) for 24 h, knockdown of MTERF4 in SH-SY5Y cells worsened the mitochondrial dysfunction induced by MPP(+), including increased reactive oxygen species, accumulated cleaved PARP-1, decreased mitochondrial membrane potential and depressed mitochondrial complexes. Furthermore, overexpression of MTERF4 in SH-SY5Y cells partially alleviated the mitochondrial dysfunction induced by MPP(+). Based on these findings, we suggest that the main function of MTERF4 is regulating mtDNA expression, and it is the crucial factor in the mechanism of mitochondrial dysfunction in SH-SY5Y cells induced by MPP(+). MTERF4 probably is the triggering of the pathogenesis of Parkinson's disease induced by environmental toxin.

Caenorhabditis elegans as a Model System for Studying Drug Induced Mitochondrial Toxicity.

Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial. We therefore set out to develop the use of Caenorhabditis elegans to study drug induced mitochondrial toxicity. Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry. Additionally we show that mitochondrial toxicity of antiretroviral drugs cannot be exclusively attributed to interference with the mitochondrial DNA polymerase.

Bioenergetic adaptation in response to autophagy regulators during rotenone exposure.

Parkinson's disease is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing Parkinson's disease. We previously reported that in differentiated SH-SY5Y cells, rotenone-induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10-100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 h, caused cell death at 24 h with an LD50 of 10 nM. Overall, autophagic flux was decreased by 10 nM rotenone at both 2 and 24 h, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 h, suggesting that a mitochondrial-specific lysosomal degradation pathway may be activated. Up-regulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine exacerbated cell death. Interestingly, while 3-methyladenine exacerbated the rotenone-dependent effects on bioenergetics, rapamycin did not prevent rotenone-induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor.

Reduced expression of Connexin26 and its DNA promoter hypermethylation in the inner ear of mimetic aging rats induced by d-galactose.

Connexin26 (Cx26), one of the major protein subunits forming gap junctions (GJs), is important in maintaining homeostasis in the inner ear and normal hearing. Cx26 mutation is one of the most common causes for inherited nonsyndromic deafness, but the relationship between Cx26 and presbycusis is unknown. Our study aimed at exploring the expression and the aberrant methylation of the promoter region of Cx26 gene in the cochlea of inner ear mimetic aging rats. We applied a mimetic aging of inner ear rat model with mtDNA common deletion by d-gal injection for 8weeks. Real-time RT-PCR and Western blot of rat inner ear tissue indicated that the Cx26 expression decreased in the d-gal group. Further bisulfite sequencing analysis revealed that the methylation status of the promoter region of Cx26 gene in the d-gal group was higher than that in control group. These results indicated that the decrease of Cx26 expression might contribute to the development of presbycusis and the hypermethylation of promoter region of GJB2 might be associated with the Cx26 downregulation.

α7 nicotinic acetylcholine receptor signaling inhibits inflammasome activation by preventing mitochondrial DNA release.

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide-induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release.

Disease progression in patients with single, large-scale mitochondrial DNA deletions.

Single, large-scale deletions of mitochondrial DNA are a common cause of mitochondrial disease and cause a broad phenotypic spectrum ranging from mild myopathy to devastating multi-system syndromes such as Kearns-Sayre syndrome. Studies to date have been inconsistent on the value of putative predictors of clinical phenotype and disease progression such as mutation load and the size or location of the deletion. Using a cohort of 87 patients with single, large-scale mitochondrial DNA deletions we demonstrate that a variety of outcome measures such as COX-deficient fibre density, age-at-onset of symptoms and progression of disease burden, as measured by the Newcastle Mitochondrial Disease Adult Scale, are significantly (P < 0.05) correlated with the size of the deletion, the deletion heteroplasmy level in skeletal muscle, and the location of the deletion within the genome. We validate these findings with re-analysis of 256 cases from published data and clarify the previously conflicting information of the value of these predictors, identifying that multiple regression analysis is necessary to understand the effect of these interrelated predictors. Furthermore, we have used mixed modelling techniques to model the progression of disease according to these predictors, allowing a better understanding of the progression over time of this strikingly variable disease. In this way we have developed a new paradigm in clinical mitochondrial disease assessment and management that sidesteps the perennial difficulty of ascribing a discrete clinical phenotype to a broad multi-dimensional and progressive spectrum of disease, establishing a framework to allow better understanding of disease progression.

Characterization of chemically modified oligonucleotides targeting a pathogenic mutation in human mitochondrial DNA.

Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Most of the deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mtDNA coexist in the same cell. Therefore, a shift in the proportion between mutant and wild type molecules could restore mitochondrial functions. The anti-replicative strategy aims to induce such a shift in heteroplasmy by mitochondrial targeting specifically designed molecules in order to inhibit replication of mutant mtDNA. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and impact heteroplasmy level, however, the effect was mainly transient, probably due to a rapid degradation of RNA molecules. In the present study, we introduced various chemically modified oligonucleotides in anti-replicative RNAs. We show that the most important increase of anti-replicative molecules' lifetime can be achieved by using synthetic RNA-DNA chimerical molecules or by ribose 2'-O-methylation in nuclease-sensitive sites. The presence of inverted thymidine at 3' terminus and modifications of 2'-OH ribose group did not prevent the mitochondrial uptake of the recombinant molecules. All the modified oligonucleotides were able to anneal specifically with the mutant mtDNA fragment, but not with the wild-type one. Nevertheless, the modified oligonucleotides did not cause a significant effect on the heteroplasmy level in transfected transmitochondrial cybrid cells bearing a pathogenic mtDNA deletion, proving to be less efficient than non-modified RNA molecules.

Mitochondria as a pharmacological target: magnum overview.

Mitochondria, responsible for energy metabolism within the cell, act as signaling organelles. Mitochondrial dysfunction may lead to cell death and oxidative stress and may disturb calcium metabolism. Additionally, mitochondria play a pivotal role in cardioprotective phenomena and a variety of neurodegenerative disorders ranging from Parkinson's to Alzheimer's disease. Mitochondrial DNA mutations may lead to impaired respiration. Hence, targeting the mitochondria with drugs offers great potential for new therapeutic approaches. The purpose of this overview is to present the recent state of knowledge concerning the interactions of various substances with mitochondria.

Radon-induced reduced apoptosis in human bronchial epithelial cells with knockdown of mitochondria DNA.

Radon and radon progeny inhalation exposure are recognized to induce lung cancer. To explore the role of mitochondria in radon-induced carcinogenesis in humans, an in vitro partially depleted mitochondrial DNA (mtDNA) cell line (ρ-) was generated by treatment of human bronchial epithelial (HBE) cells (ρ+) with ethidium bromide (EB). The characterization of ρ- cells indicated the presence of dysfunctional mitochondria and might thus serve a reliable model to investigate the role of mitochondria. In a gas inhalation chamber, ρ- and ρ+ cells were exposed to radon gas produced by a radium source. Results showed that apoptosis was significantly increased both in ρ- and ρ+ cells irradiated by radon. Moreover, apoptosis in ρ- cells showed a lower level than in ρ+ cells. Radon was further found to depress mitochondrial membrane potential (MMP) of HBE cells with knockdown mtDNA. Production of reactive oxygen species (ROS) was markedly elevated both in ρ- and ρ+ cells exposed to radon. The distribution of phases of cell cycle was different in ρ- compared to ρ+ cells. Radon irradiation induced a rise in G2/M and decrease in S phase in ρ+ cells. In ρ- cells, G1, G2/M, and S populations remained similar to cells exposed to radon. In conclusion, radon-induced changes in ROS generation, MMP and cell cycle are all attributed to reduction of apoptosis, which may trigger and promote cell transformation, leading to carcinogenesis. Our study indicates that the use of the ρ- knockdown mtDNA HBE cells may serve as a reliable model to study the role played by mitochondria in carcinogenic diseases.

Neutrophil cerebrovascular transmigration triggers rapid neurotoxicity through release of proteases associated with decondensed DNA.

Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1-stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone-DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.

Impaired mitochondrial biogenesis in hippocampi of rats with chronic seizures.

Mitochondrial dysfunction has been suggested to be a contributing factor of epilepsy, but the underlying mechanisms are not completely explored. Mitochondrial biogenesis is involved in regulation of mitochondrial content, morphology, and function. In the current study, we show mitochondrial biogenesis severely impaired in hippocampi of rats with chronic seizures induced by pilocarpine, as evidenced by decreased mitochondrial DNA (mtDNA) content and decreased mtDNA-encoded protein level. Furthermore, we show mtDNA transcription and replication reduced in rats with chronic seizures. These defects were independent of downregulation of mitochondrial biogenesis-related factors, such as peroxisome proliferator-activated receptor gamma coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcription factor A (Tfam), but depended on reduced Tfam-DNA binding activity. The present study suggests novel mechanisms for mitochondrial dysfunction during chronic seizures.

Antimycin A-induced mitochondrial apoptotic cascade is mitigated by phenolic constituents of Phyllanthus amarus aqueous extract in Hep3B cells.

Antimycin A (AMA) treatment of cells blocks mitochondrial electron transport chain, and leads to elevated ROS generation, thereby causing damage to mtDNA, proteins and lipids, along with mitochondrial membrane depolarization, release of pro-apoptotic proteins into the cytoplasm, and induction of apoptosis. Prevention of such oxidative cellular damage by the aqueous extract of Phyllanthus amarus has been investigated in this study. The extract demonstrated significant potential in mitigating H(2)O(2)-induced membrane damage along with considerable recession in AMA-governed mitochondrial protein and lipid degradation in Hep3B cells. 8-OHdG analysis of mtDNA damage revealed substantial protective potential of the extract against mtDNA damage. SQ-PCR of selected mtDNA sequences confirmed the potential of the extract to alleviate levels of mtDNA damage. FACS analysis with JC-1 fluorescent dye established significant escalation of mitochondrial membrane potential by the extract in AMA-treated cells. Extract treatment resulted in a distinct decline in the degrees of AMA-induced release of cytochrome c and AIF into the cytoplasm along with consequent pacification of apoptosis. All protective efficiencies of the extract reported in this study were found to hold strong and significant (P<0.05) positive correlation to its total phenolic contents, thereby proving that polyphenolic constituents of P. amarus aqueous extract mitigate oxidative stress-induced cellular degeneration and aging.