mROS Generation Research Articles

The interplay between mitochondrial reactive oxygen species formation and the coenzyme Q reduction level

Our aim was to elucidate the relationship between the rate of mitochondrial reactive oxygen species (mROS) formation and the reduction level of the mitochondrial coenzyme Q (mQ) pool under various levels of engagement of the mQ-reducing pathway (succinate dehydrogenase, complex II) and mQH2-oxidizing pathways (the cytochrome pathway and alternative oxidase pathway, (AOX)) in mitochondria isolated from the amoeba Acanthamoeba castellanii. The mQ pool was shifted to a more reduced state by inhibition of mQH2-oxidizing pathways (complex III and complex IV of the cytochrome pathway, and AOX) and the oxidative phosphorylation system. The mQ reduction level was lowered by decreasing the electron supply from succinate dehydrogenase and by stimulating the activity of the cytochrome or AOX pathways. The results indicate a direct dependence of mROS formation on the reduction level of the mQ pool for both mQH2-oxidizing pathways. A higher mQ reduction level leads to a higher mROS formation. For the cytochrome pathway, mROS generation depends nonlinearly upon the mQ reduction level, with a stronger dependency observed at values higher than the mQ reduction level of the phosphorylating state (~ 35%). AOX becomes more engaged at higher mQ pool reduction levels (above 40%), when mROS production via the cytochrome pathway increases. We propose that the mQ pool reduction level (endogenous mQ redox state) could be a useful endogenous reporter that allows indirect assessment of overall mROS production in mitochondria.

Alkaline pH Promotes NADPH Oxidase-Independent Neutrophil Extracellular Trap Formation: A Matter of Mitochondrial Reactive Oxygen Species Generation and Citrullination and Cleavage of Histone.

pH is highly variable in different tissues and affects many enzymatic reactions in neutrophils. In response to calcium ionophores such as A23187 and ionomycin, neutrophils undergo nicotinamide adenine dinucleotide phosphate oxidase (NOX)-independent neutrophil extracellular trap (NET) formation (NETosis). However, how pH influences calcium-dependent Nox-independent NET formation is not well understood. We hypothesized that increasing pH promotes Nox-independent NET formation by promoting calcium influx, mitochondrial reactive oxygen species (mROS) generation, histone citrullination, and histone cleavage. Here, we show that stimulating human neutrophils isolated from peripheral blood with calcium ionophore A23187 or ionomycin in the media with increasing extracellular pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.8) drastically increases intracellular pH within in 10–20 min. These intracellular pH values are much higher compared to unstimulated cells placed in the media with corresponding pH values. Raising pH slightly drastically increases intracellular calcium concentration in resting and stimulated neutrophils, respectively. Like calcium, mROS generation also increases with increasing pH. An mROS scavenger, MitoTempo, significantly suppresses calcium ionophore-mediated NET formation with a greater effect at higher pH, indicating that mROS production is at least partly responsible for pH-dependent suppression of Nox-independent NETosis. In addition, raising pH increases PAD4 activity as determined by the citrullination of histone (CitH3) and histone cleavage determined by Western blots. The pH-dependent histone cleavage is reproducibly very high during ionomycin-induced NETosis compared to A23187-induced NETosis. Little or no histone cleavage was noted in unstimulated cells, at any pH. Both CitH3 and cleavage of histones facilitate DNA decondensation. Therefore, alkaline pH promotes intracellular calcium influx, mROS generation, PAD4-mediated CitH3 formation, histone 4 cleavage and eventually NET formation. Calcium-mediated NET formation and CitH3 formation are often related to sterile inflammation. Hence, understanding these important mechanistic steps helps to explain how pH regulates NOX-independent NET formation, and modifying pH may help to regulate NET formation during sterile inflammation or potential damage caused by compounds such as ionomycin, secreted by Streptomyces, a group of Gram-positive bacteria well known for producing antibiotics.

ROS regulate differentiation of visceralizing Leishmania species into the virulent amastigote form.

Leishmania virulence and disease development critically depends on the ability of Leishmania promastigotes to infect, differentiate into amastigote forms and replicate inside mammalian host macrophages. Understanding changes associated with amastigote differentiation in axenic culture conditions is key to identifying virulence factors. Here we compared efficiency of the conventional pH-temperature-dependent shift method to induce amastigote differentiation with the recently identified trigger for differentiation mediated by mitochondrial reactive oxygen species (ROS). Using two different visceral leishmaniasis species, L. infantum and. L. donovani, we show that ROS-generating methods such as iron deprivation or exposure to sub-lethal concentrations of H2O2 or menadione are significantly more effective in promoting promastigoteamastigote differentiation than the low pH-high temperature shift, leading to higher survival rates, morphological changes and gene expression patterns characteristic of the amastigote stage. Notably, both H2O2 and menadione-mediated differentiation did not require up-regulation of the mitochondrial electron transport chain (ETC)-associated protein p27, suggesting that treatment with oxidants bypasses the necessity to upregulate mitochondrial activity, a precondition for mROS generation. Our findings confirm that ROS-induced differentiation occurs in multiple Leishmania species, including the medically important visceralizing species, and provide mechanistic rationale for earlier reports demonstrating markedly increased virulence of L. infantum promastigotes pre-treated with oxidative reagents.

NLRX1 modulates differentially NLRP3 inflammasome activation and NF-κB signaling during Fusobacterium nucleatum infection

NOD-like receptors (NLRs) play a large role in regulation of host innate immunity, yet their role in periodontitis remains to be defined. NLRX1, a member of the NLR family that localizes to mitochondria, enhances mitochondrial ROS (mROS) generation. mROS can activate the NLRP3 inflammasome, yet the role of NLRX1 in NLRP3 inflammasome activation has not been examined. In this study, we revealed the mechanism by which NLRX1 positively regulates ATP-induced NLRP3 inflammasome activation through mROS in gingival epithelial cells (GECs). We found that depletion of NLRX1 by shRNA attenuated ATP-induced mROS generation and redistribution of the NLRP3 inflammasome adaptor protein, ASC. Furthermore, depletion of NLRX1 inhibited Fusobacterium nucleatum infection-activated caspase-1, suggesting that it also inhibits the NLRP3 inflammasome. Conversely, NLRX1 also acted as a negative regulator of NF-κB signaling and IL-8 expression. Thus, NLRX1 stimulates detection of the pathogen F. nucleatum via the inflammasome, while dampening cytokine production. We expect that commensals should not activate the inflammasome, and NLRX1 should decrease their ability to stimulate expression of pro-inflammatory cytokines such as IL-8. Therefore, NLRX1 may act as a potential switch with regards to anti-microbial responses in healthy or diseased states in the oral cavity.

Resveratrol rescues cadmium-induced mitochondrial injury by enhancing transcriptional regulation of PGC-1α and SOD2 via the Sirt3/FoxO3a pathway in TCMK-1 cells

Resveratrol has been reported to ameliorate Cd-induced nephrotoxicity. However, the beneficial effects of resveratrol on Cd-induced nephrotoxicity and the underlying mechanisms of this protection remain unclear. Here, we showed that mouse renal tubular epithelial (TCMK-1) cells exposed to Cd experienced significantly increased mitochondrial reactive oxygen species (mROS) production, as well as decreased mitochondrial biogenesis and function. Cd exposure dramatically decreased Sirt3 protein expression and activity and promoted the acetylation of forkhead box O3 (FoxO3a). Moreover, Cd exposure led to a decreased binding affinity of FoxO3a to the promoters of both peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α and superoxide dismutase 2 (SOD2), powerful and broad regulators of mitochondrial biogenesis and mROS metabolism. Meanwhile, resveratrol remarkably reduced mROS generation by promoting Sirt3 enrichment within the mitochondria and subsequent upregulation of FoxO3a-mediated mitochondria gene expression of PGC-1α and SOD2. Importantly, mechanistic study revealed that ERK1/2 activation was associated with increased apoptosis induced by Cd, resveratrol suppressed Cd-induced apoptosis in mice kidney. Taken together, our data suggest a novel mechanism of action for resveratrol-attenuated Cd-induced cellular damage, which, in part, was mediated through the activation of the Sirt3/FoxO3a signaling pathway.

Increased mitochondrial ROS generation mediates the loss of the anti-contractile effects of perivascular adipose tissue in high-fat diet obese mice.

Obesity is associated with structural and functional changes in perivascular adipose tissue (PVAT), favouring release of reactive oxygen species (ROS), vasoconstrictor and proinflammatory factors. The cytokine TNF-α induces vascular dysfunction and is produced by PVAT. We tested the hypothesis that obesity-associated PVAT dysfunction was mediated by augmented mitochondrial ROS (mROS) generation due to increased TNF-α production in this tissue. C57Bl/6J and TNF-α receptor-deficient mice received control or high fat diet (HFD) for 18 weeks. We used pharmacological tools to determine the participation of mROS in PVAT dysfunction. Superoxide anion (O2.- ) and H2 O2 were assayed in PVAT and aortic rings were used to assess vascular function. Aortae from HFD-fed obese mice displayed increased contractions to phenylephrine and loss of PVAT anti-contractile effect. Inactivation of O2.- , dismutation of mitochondria-derived H2 O2 , uncoupling of oxidative phosphorylation and Rho kinase inhibition, decreased phenylephrine-induced contractions in aortae with PVAT from HFD-fed mice. O2.- and H2 O2 were increased in PVAT from HFD-fed mice. Mitochondrial respiration analysis revealed decreased O2 consumption rates in PVAT from HFD-fed mice. TNF-α inhibition reduced H2 O2 levels in PVAT from HFD-fed mice. PVAT dysfunction, i.e. increased contraction to phenylephrine in PVAT-intact aortae, was not observed in HFD-obese mice lacking TNF-α receptors. Generation of H2 O2 was prevented in PVAT from TNF-α receptor deficient obese mice. TNF-α-induced mitochondrial oxidative stress is a key and novel mechanism involved in obesity-associated PVAT dysfunction. These findings elucidate molecular mechanisms whereby oxidative stress in PVAT could affect vascular function. This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

Ionizing Radiation Induces Innate Immune Responses in Macrophages by Generation of Mitochondrial Reactive Oxygen Species.

During radiotherapy for tumors, the innate immune system also responds to ionizing radiation and induces immune modulation. However, little is known about the molecular mechanisms by which radiation modulates innate immune responses. In this study, we observed that radiation triggered the generation of mitochondrial reactive oxygen species (mROS), leading to innate immune responses in murine bone marrow-derived macrophages (BMDM). Radiation-induced mROS was essential for robust induction of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12p40 mRNA and protein in BMDM. Exposure to radiation also led to rapid activation of the mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways in BMDM. Notably, radiation-induced MAPK activation and NF-κB signaling were regulated by mROS in macrophages. Additionally, radiation-induced expression of TNF-α, IL-6 and IL-12p40 was dependent on JNK, p38 and NF-κB activation in BMDM. These data suggest a key role for radiation-induced pro-inflammatory responses and activation of the MAPK and NF-κB pathways through a triggering mechanism involving mROS generation.

Melatonin improves age-induced fertility decline and attenuates ovarian mitochondrial oxidative stress in mice.

Increasing evidence shows that melatonin protected against age-related mitochondrial oxidative damage. However, the protective effects of melatonin against ovarian aging has not been explored. Young Kunming females (aged 2–3 months) were fed with melatonin added to drinking water for 6 or 12 months (mo). We found that long-term (12 mo) melatonin treatment significantly reduced ovarian aging, as indicated by substantial increases in litter size, pool of follicles, and telomere length as well as oocyte quantity and quality. Melatonin treatment suppressed ovarian mitochondrial oxidative damage by decreasing mitochondrial reactive oxygen species (mROS) generation, inhibiting apoptosis, repressing collapse of mitochondrial membrane potential and preserving respiratory chain complex activities. Female mice fed with melatonin had enhanced mitochondrial antioxidant activities, thus reducing the risk of mitochondrial oxidative damage cause by free radicals. Notably, melatonin treatment enhanced SIRT3 activity but not the protein expression level, and increased the binding affinity of FoxO3a to the promoters of both superoxide dismutase 2 (SOD2) and catalase (CAT). In conclusion, melatonin exerted protection against aging-induced fertility decline and maintenance of mitochondrial redox balance.

Dimethyl fumarate ameliorates dextran sulfate sodium-induced murine experimental colitis by activating Nrf2 and suppressing NLRP3 inflammasome activation

In the present study, we examined the effects of dimethyl fumarate (DMF) on dextran sulfate sodium (DSS)-induced murine colitis, an animal model which mimics human IBD. Oral administration of DMF dose-dependently attenuated body weight loss, colon length shortening and colonic pathological damage including decreased myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activities in DSS-treated mice. Increased glutathione (GSH) induced by DMF demonstrated its potential antioxidant capacity. In addition, Nrf2 and its downstream genes were markedly activated by DMF. Furthermore, protein and mRNA levels of pro-inflammatory cytokines, including IL-1β, TNF-α and IL-6 were markedly suppressed by DMF. At the same time, decreased activation of caspase-1 was detected in DMF-treated mice, indicating that the NLRP3 inflammasome activation was suppressed. The in vitro study verified a negative regulation of DMF and its intestinal metabolite on NLRP3 inflammasome. Moreover, the inhibitory effect was found to be mostly dependent on Nrf2 which decreased mitochondrial ROS (mROS) generation and mitochondrial DNA (mtDNA) release. Taken together, our results demonstrated the ability of DMF to inhibit NLRP3 inflammasome activation and its potential use in the treatment of NLRP3-associated diseases.

NLRX1 modulates the balance between NLRP3 inflammasome activation and NF-kB signaling during Fusobacterium nucleatum infection.

Abstract NOD-like receptors (NLRs) play an important role in regulation of host innate immunity, yet their role in periodontitis remains to be defined. NLRX1, a member of the NLR family that localizes to mitochondria, modulates mitochondrial ROS (mROS) generation. mROS has been shown to activate the NLRP3 inflammasome, yet the role of NLRX1 in NLRP3 inflammasome activation has not been examined. In this study, we revealed the mechanism by which NLRX1 positively regulates ATP-elicited NLRP3 inflammasome activation through mROS in gingival epithelial cells (GECs). Fluorescence microscopy showed that depletion of NLRX1 by shRNA attenuates ATP-induced mROS generation and redistribution of the NLRP3 inflammasome adaptor protein, ASC. Furthermore, depletion of NLRX1 inhibits Fusobacterium nucleatum infection-activated caspase-1, suggesting that it also inhibits the NLRP3 inflammasome. Therefore, we propose that NLRX1 may promote F. nucleatum-caused dysregulated pro-inflammatory responses in periodontitis. On the other hand, the mechanism by which F. nucleatum infection-induces IL-8 expression is still unclear. We showed that NLRX1 also acts as a negative regulator in NF-kB signaling to modulate IL-8 expression. Thus, NLRX1 stimulates detection of the pathogen F. nucleatum via the inflammasome, while dampening cytokine production. We expect that commensals should not activate the inflammasome, but NLRX1 should still decrease their ability to stimulate inflammation. We conclude that NLRX1 may act as a potential switch in regards to the virulence of F. nucleatum in healthy or diseased oral cavity.

Tyrosine Phosphorylation of Mitochondrial Ca2+ Uniporter Dictates Mitochondrial Ca2+ Overload and Cardiomyocyte Death

IntroductionMitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) is critical for the cardiomyocyte survival and death. Basal tyrosine phosphorylation (P‐Y) of MCU was reported from mass spectroscopy of human tissues. However, it is still unclear whether the post‐translational modifications of the MCU (e.g. phosphorylation) modulates its channel properties as well as mitochondrial and cellular functions in cardiomyocytes.HypothesisP‐Y of MCU activates mitochondrial Ca2+ uptake and induces mitochondrial Ca2+ overload and cardiomyocyte death.MethodsNon‐phospho‐mimetic MCUs (MCU‐YFs; changed from Y to F) were generated by PCR‐based site direct mutagenesis. Mitochondrial Ca2+ concentration was measured by matrix‐targeted Ca2+ biosensors using confocal microscopy.Resultsa1‐adrenoceptor (a1‐AR) signaling activated mitochondrial matrix‐localized proline‐rich tyrosine kinase 2 (Pyk2) and enhanced the interaction between Pyk2 and MCU, which subsequently increased P‐Y of MCU and mitochondrial Ca2+ uptake in rat cardiomyocytes. We confirmed that two tyrosine sites showed an increase of phosphorylation levels in response to a1‐AR stimulation in situ (Fig. A). In addition, the overexpression of these MCU‐YFs failed to increase mitochondrial Ca2+ uptake in response to cytosolic Ca2+ elevation, whereas wild‐type MCU overexpression dramatically accelerated mitochondrial Ca2+ uptake compared to control cells (Fig. B). Moreover, persistent a1‐AR stimulation increased mitochondrial reactive oxygen species (mROS) generation, cytochrome C release, and cardiomyocyte death. These effects were abolished by the overexpression of a dominant‐negative mutant of MCU in cardiomyocytes.ConclusionMCU contains Pyk2‐specific phosphorylation site(s) and P‐Y of MCU activates its channel function. Persistent P‐Y of MCU induces mitochondrial Ca2+ overload, mROS generation and apoptosis in cardiomyocytes.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

Gene Expression Profiling Reveals Key Mitochondrial Gene Changes in Stored Platelets

IntroductionAs of June 2015 the FDA has approved an alternative procedure under 21 CFR 640.120 that allows for storage of apheresis platelets at refrigerator temperature (1-6 C; 4°C) without agitation for up to 3 days for use in the resuscitation of actively bleeding patients. Understanding underlying mechanisms responsible for enhanced hemostatic function at 4°C will be critical for such improvements in platelet transfusion. We hypothesized that 4°C platelets display better mitochondrial respiratory function for up to 7 days compared to standard 5-day RT platelets and that mitochondrial gene expression differences between RT and 4°C -stored platelets will correlate with mitochondrial function.MethodsPlatelets were collected from healthy donors by apheresis according to an IRB-approved protocol. Apheresis platelets (AP) were rested for 1 h before allocation into platelet minibags (Blood Cell Storage, Seattle, WA) and stored for 4 storage durations (Baseline (BL), Day 3, 5, and 7). Mitochondrial respiration, maximal oxygen utilization, and individual mitochondrial complex-dependent respiration were assessed with high-resolution respirometry (O2k, Oroboros). Mitochondrial ROS generation in response to storage condition or stimulation (to assess oxidative burst capacity as a measure of function) was visualized with fluorescent imaging and assayed with flow cytometry using a superoxide stain (Life Technologies). Total RNA was extracted both immediately following apheresis (BL) and on Day 5 from RT and 4°C-stored platelets using Trizol (Molecular Research Center, Cincinnati, OH) after centrifuging the platelets at 900 x g for 10 min. Platelet RNA was quantified using the NanoDrop 2000. RNA quality was examined using gel electrophoresis with the Reliant Gel System (Cambrex, Rockland, ME). Platelet mitochondrial gene expression analysis was evaluated using the 96-well RT2 Profiler PCR Array (Qiagen, Valencia, CA) which profiled 84 mitochondria-focused targets and 12 control genes per sample. Gene expression data analysis was based on the ΔΔCt method with normalization of the raw data to housekeeping genes located on each 96-well plate.ResultsMitochondrial respiration was lower in platelets stored at 4°C compared to RT on Days 3, 5, and 7 (Day 5= -57%±0.3; P < 0.05), demonstrating that refrigeration slows metabolism. Additionally, maximal mitochondrial oxygen utilization (electron transport system capacity) was better preserved in platelets stored at 4°C (Figure 1). Fluorescent imaging and flow cytometry demonstrated that mROS generation was higher in RT-stored platelets compared to 4°C, reflecting mitochondrial damage. Mitochondrial burst during de novo mROS generation due to stimulation was also preserved at 4°C. Mitochondrial gene expression studies revealed distinct differences in expression profiles for 4°C versus RT-stored platelets after 5 days of storage when normalized to BL measures. Storage at 4°C resulted in significantly greater preservation of 15 gene products at Day 5 (P<0.05). In contrast, Day 5 RT samples resulted in a marked decrease or loss of gene products when compared to BL levels of gene expression (P<0.05).DiscussionPlatelet mitochondrial respiratory function (mitochondrial respiration and maximal oxygen utilization) decreased in RT-stored platelets over 7 days, but the impairment was attenuated by 4°C storage. We previously noted that intracellular ROS flux was higher at room temperature, and here the gene expression analysis in combination with oximetry data showed that mitochondrial damage is likely responsible. Furthermore, gene expression profiling of mitochondrial-related genes revealed that distinct differences exist in key mitochondrial genes between the storage conditions. This work illustrates that 4°C storage of platelets preserved and enhanced critical mitochondrial genes compared to RT; this finding combined with improved mitochondrial respiratory measures and reduced ROS demonstrates a significant improvement in current efforts to mitigate platelet dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rotenone-induced Impairment of Mitochondrial Electron Transport Chain Confers a Selective Priming Signal for NLRP3 Inflammasome Activation

Mitochondrial dysfunction is considered crucial for NLRP3 inflammasome activation partly through its release of mitochondrial toxic products, such as mitochondrial reactive oxygen species (mROS)(2) and mitochondrial DNA (mtDNA). Although previous studies have shown that classical NLRP3-activating stimulations lead to mROS generation and mtDNA release, it remains poorly understood whether and how mitochondrial damage-derived factors may contribute to NLRP3 inflammasome activation. Here, we demonstrate that impairment of the mitochondrial electron transport chain by rotenone primes NLRP3 inflammasome activation only upon costimulation with ATP and not with nigericin or alum. Rotenone-induced priming of NLRP3 in the presence of ATP triggered the formation of specklike NLRP3 or ASC aggregates and the association of NLRP3 with ASC, resulting in NLRP3-dependent caspase-1 activation. Mechanistically, rotenone confers a priming signal for NLRP3 inflammasome activation only in the context of aberrant high-grade, but not low-grade, mROS production and mitochondrial hyperpolarization. By contrast, rotenone/ATP-mediated mtDNA release and mitochondrial depolarization are likely to be merely an indication of mitochondrial damage rather than triggering factors for NLRP3 inflammasome activation. Our results provide a molecular insight into the selective contribution made by mitochondrial dysfunction to the NLRP3 inflammasome pathway.

Activation of the Nlrp3 inflammasome by mitochondrial reactive oxygen species: A novel mechanism of albumin-induced tubulointerstitial inflammation

Albuminuria is not only an important marker of chronic kidney disease but also a crucial contributor to tubulointerstitial inflammation (TIF). In this study, we determined whether activation of the Nlrp3 inflammasome is involved in albuminuria induced-TIF and the underlying mechanisms of inflammasome activation by mitochondrial reactive oxygen species (mROS). We established an albumin-overload induced rat nephropathy model characterised by albuminuria, renal infiltration of inflammatory cells, tubular dilation and atrophy. The renal expression levels of the Nlrp3 inflammasome, IL-1β and IL-18 were significantly increased in this animal model. In vitro, albumin time- and dose-dependently increased the expression levels of the Nlrp3 inflammasome, IL-1β and IL18. Moreover, the silencing of the Nlrp3 gene or the use of the caspase-1 inhibitor Z-VAD-fmk significantly attenuated the albumin-induced increase in IL-1β and IL-18 expression in HK2 cells. In addition, mROS generation was elevated by albumin stimulation, whereas the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited Nlrp3 expression and the release of IL-1β and IL-18. In kidney biopsy specimens obtained from patients with IgA nephropathy, Nlrp3 expression was localised to the proximal tubular epithelial cells, and this result is closely correlated with the extent of proteinuria and TIF. In summary, this study demonstrates that albuminuria may serve as an endogenous danger-associated molecular pattern (DAMP) that stimulates TIF via the mROS-mediated activation of the cytoplasmic Nlrp3 inflammasome.

Dual Phases of Respiration Chain Defect-Augmented mROS-Mediated mCa2+Stress during Oxidative Insult in Normal andρ0RBA1 Astrocytes

Mitochondrial respiratory chain (RC) deficits, resulting in augmented mitochondrial ROS (mROS) generation, underlie pathogenesis of astrocytes. However, mtDNA-depleted cells (ρ 0) lacking RC have been reported to be either sensitive or resistant to apoptosis. In this study, we sought to determine the effects of RC-enhanced mitochondrial stress following oxidative insult. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy, the ability to resist oxidative stress and levels of mROS formation and mitochondrial calcium (mCa2+) were compared between two different astrocyte cell lines, control and ρ 0 astrocytes, over time upon oxidative stress. Our results showed that the cytoplasmic membrane becomes permeated with YO-PRO-1 dye at 150 and 130 minutes in RBA-1 and ρ 0 astrocytes, respectively. In contrast to RBA-1, 30 minutes after 20 mM H2O2 exposure, ρ 0 astrocytes formed marked plasma membrane blebs, lost the ability to retain Mito-R, and showed condensation of nuclei. Importantly, H2O2-induced ROS and accompanied mCa2+ elevation in control showed higher levels than ρ 0 at early time point but vice versa at late time point. Our findings underscore dual phase of RC-defective cells harboring less mitochondrial stress due to low RC activity during short-term oxidative stress but augmented mROS-mediated mCa2+ stress during severe oxidative insult.

Matairesinol inhibits angiogenesis via suppression of mitochondrial reactive oxygen species

Mitochondrial reactive oxygen species (mROS) are involved in cancer initiation and progression and function as signaling molecules in many aspects of hypoxia and growth factor-mediated signaling. Here we report that matairesinol, a natural small molecule identified from the cell-based screening of 200 natural plants, suppresses mROS generation resulting in anti-angiogenic activity. A non-toxic concentration of matairesinol inhibited the proliferation of human umbilical vein endothelial cells. The compound also suppressed in vitro angiogenesis of tube formation and chemoinvasion, as well as in vivo angiogenesis of the chorioallantoic membrane at non-toxic doses. Furthermore, matairesinol decreased hypoxia-inducible factor-1α in hypoxic HeLa cells. These results demonstrate that matairesinol could function as a novel angiogenesis inhibitor by suppressing mROS signaling.

TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria, and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase (Phox) machinery1. However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to macrophage bactericidal activity, although the mechanisms linking innate immune signaling to mitochondria for mROS generation remain unclear2-4. Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of the TLR signaling adapter tumor necrosis factor receptor-associated factor 6 (TRAF6) to mitochondria where it engages evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), a protein implicated in mitochondrial respiratory chain assembly5. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT and TRAF6 depleted macrophages exhibit decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results therefore reveal a novel pathway linking innate immune signaling to mitochondria, implicate mROS as important components of antibacterial responses, and further establish mitochondria as hubs for innate immune signaling.

Balancing Mitochondrial Redox Signaling: A Key Point in Metabolic Regulation

Mitochondrial reactive oxygen species (mROS) have emerged as signaling molecules in physiology primarily as a result of studies of uncoupling mechanisms in mitochondrial respiration. The discovery that this mechanism negatively regulates mROS generation in many cell types has drawn the attention of the scientific community to the pathological consequences of excess mROS production. From reports of the energetic fluxes in cells grown under normal conditions, the hypothesis that mROS are an integrated physiological signal of the metabolic status of the cell has emerged. Here, we consider recent studies that support this point of view in two key nutrient sensors of the body, beta cells and the hypothalamus, which are the main coordinators of endocrine and nervous controls of energy metabolism and adipose tissue, which is of paramount importance in controlling body weight and, therefore, the development of obesity and type 2 diabetes. In this context, finely balanced mROS production may be at the core of proper metabolic maintenance, and unbalanced mROS production, which is largely documented, might be an important trigger of metabolic disorders.

MtDNA T8993G-Augmented Mitochondrial Stresses Upon mCa2+ Overload and its Protection by Melatonin in a NARP Cybrid

Mitochondrial DNA (mtDNA) T8993G mutation inhibits specifically mitochondrial F1F0-ATPase (complex V) for severe ATP deficiency and is clinically associated with neurological muscle weakness, ataxia, and retinitis pigmentosa so called NARP mutation. At present, detail T8993G-associated mitochondrial mechanisms as well as its therapeutic strategies are limited. Using time-lapse laser scanning dual fluorescence imaging microscopy, this study investigated T8993G-altered apoptotic mitochondrial pathology particular upon mCa2+ stress and protection by melatonin, previously reported to protect mCa2+ stress-mediated apoptosis (Hsu et al., 2009 JPR in press). In comparison to its parental osteosarcoma 143B and mtDNA less (ρ0) cells, T8993G induced significant hyperpolarization of mitochondrial membrane potential (ΔΨm) and potentiated greatly ionomycin-induced mCa2+ stress. T8993G-augmented mCa2+ stress subsequently elicited rigorously generation of mitochondrial oxygen species (mROS), depletion of cardiolipin (CL) and activation of the mitochondrial permeability transition (MPT). In contract, ρ0 cells, with much depolarized ΔΨm, suffered less mCa2+ stress, mROS formation, CL depletion and the MPT opening. Interestingly, melatonin reduced significantly peak amplitude of the ionomycin-induced mCa2+ transient and antagonized efficiently mCa2+-augmented mROS generation for a reduced depletion of CL and activation of the MPT. In addition, melatonin prevented “oxidation free mCa2+”-mediated MPT suggesting its direct targeting on the MPT. Melatonin-enhanced tail amplitude of mCa2+ transient possibly due to the reduced MPT-dependent depolarization of ΔΨm, however, did not enhance mCa2+ stress-mediated pathology in NARP cybrids possibly as melatonin-elevated mCa2+ improved mitochondrial respiratory. Thus, the administration of melatonin may provide potential improvement for the treatment of mtDNA T8993G-associated NARP syndromes and diseases.

Dual Role of Mitochondrial Reactive Oxygen Species in Hypoxia Signaling: Activation of Nuclear Factor-κB via c-SRC– and Oxidant-Dependent Cell Death

Hypoxia is a prominent feature of solid tumor development and is known to stimulate mitochondrial ROS (mROS), which, in turn, can activate hypoxia-inducible transcription factor-1alpha and nuclear factor-kappaB (NF-kappaB). Because NF-kappaB plays a central role in carcinogenesis, we examined the mechanism of mROS-mediated NF-kappaB activation and the fate of cancer cells during hypoxia after mitochondrial reduced glutathione (mGSH) depletion. Hypoxia generated mROS in hepatoma (HepG2, H35), neuroblastoma (SH-SY5Y), and colon carcinoma (DLD-1) cells, leading to hypoxia-inducible transcription factor-1alpha-dependent gene expression and c-Src activation that was prevented in cells expressing a redox-insensitive c-Src mutant (C487A). c-Src stimulation activated NF-kappaB without IkappaB-alpha degradation due to IkappaB-alpha tyrosine phosphorylation that was inhibited by rotenone/TTFA or c-Src antagonism. The c-Src-NF-kappaB signaling contributed to the survival of cells during hypoxia as c-Src inhibition or p65 down-regulation by small interfering RNA-sensitized HepG2 cells to hypoxia-induced cell death. Moreover, selective mGSH depletion resulted in an accelerated and enhanced mROS generation by hypoxia that killed SH-SY5Y and DLD-1 cells without disabling the c-Src-NF-kappaB pathway. Thus, although mROS promote cell survival by NF-kappaB activation via c-Src, mROS overgeneration may be exploited to sensitize cancer cells to hypoxia.