Mitochondrial Peroxidation Research Articles

RETRACTED: Intensified mitochondrial hydrogen peroxide release occurs in all brain regions, affects male as well as female Rett mice, and constitutes a life-long burden

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief and Authors. Professor Michael Müller approached the journal explaining that he had encountered an issue in the way the spectrofluorometric data analyses was performed. The normalization of the fluorescence curves to their respective starting points (as explained in Figure 1A) overestimated the changes in Mecp2-mutant mice, which usually started at lower levels. This overestimation applies to Figure 3 A-D as well as Table 2 and Table 3 and altered the outcomes of the study. Both the EiC and the authors agreed that a corrigendum would not be appropriate due to the change in conclusion and that the paper should therefore be retracted. The authors apologise for any confusion this paper may have resulted in.

On the mechanisms underlying attenuated redox responses to exercise in older individuals: A hypothesis

Responding appropriately to exercise is essential to maintenance of skeletal muscle mass and function at all ages and particularly during aging. Here, a hypothesis is presented that a key component of the inability of skeletal muscle to respond effectively to exercise in aging is a denervation-induced failure of muscle redox signalling. This novel hypothesis proposes that an initial increase in oxidation in muscle mitochondria leads to a paradoxical increase in the reductive state of specific cysteines of signalling proteins in the muscle cytosol that suppresses their ability to respond to normal oxidising redox signals during exercise. The following are presented for consideration:Transient loss of integrity of peripheral motor neurons occurs repeatedly throughout life and is normally rapidly repaired by reinnervation, but this repair process becomes less efficient with aging. Each transient loss of neuromuscular integrity leads to a rapid, large increase in mitochondrial peroxide production in the denervated muscle fibers and in neighbouring muscle fibers. This peroxide may initially act to stimulate axonal sprouting and regeneration, but also stimulates retrograde mitonuclear communication to increase expression of a range of cytoprotective proteins in an attempt to protect the fiber and neighbouring tissues against oxidative damage. The increased peroxide within mitochondria does not lead to an increased cytosolic peroxide, but the increases in adaptive cytoprotective proteins include some located to the muscle cytosol which modify the local cytosol redox environment to induce a more reductive state in key cysteines of specific signalling proteins. Key adaptations of skeletal muscle to exercise involve transient peroxiredoxin oxidation as effectors of redox signalling in the cytosol. This requires sensitive oxidation of key cysteine residues. In aging, the chronic change to a more reductive cytosolic environment prevents the transient oxidation of peroxiredoxin 2 and hence prevents essential adaptations to exercise, thus contributing to loss of muscle mass and function.Experimental approaches suitable for testing the hypothesis are also outlined.

Hyperbaric Oxygen Treatment Following Mid-Cervical Spinal Cord Injury Preserves Diaphragm Muscle Function.

Oxidative damage to the diaphragm as a result of cervical spinal cord injury (SCI) promotes muscle atrophy and weakness. Respiratory insufficiency is the leading cause of morbidity and mortality in cervical spinal cord injury (SCI) patients, emphasizing the need for strategies to maintain diaphragm function. Hyperbaric oxygen (HBO) increases the amount of oxygen dissolved into the blood, elevating the delivery of oxygen to skeletal muscle and reactive oxygen species (ROS) generation. It is proposed that enhanced ROS production due to HBO treatment stimulates adaptations to diaphragm oxidative capacity, resulting in overall reductions in oxidative stress and inflammation. Therefore, we tested the hypothesis that exposure to HBO therapy acutely following SCI would reduce oxidative damage to the diaphragm muscle, preserving muscle fiber size and contractility. Our results demonstrated that lateral contusion injury at C3/4 results in a significant reduction in diaphragm muscle-specific force production and fiber cross-sectional area, which was associated with augmented mitochondrial hydrogen peroxide emission and a reduced mitochondrial respiratory control ratio. In contrast, rats that underwent SCI followed by HBO exposure consisting of 1 h of 100% oxygen at 3 atmospheres absolute (ATA) delivered for 10 consecutive days demonstrated an improvement in diaphragm-specific force production, and an attenuation of fiber atrophy, mitochondrial dysfunction and ROS production. These beneficial adaptations in the diaphragm were related to HBO-induced increases in antioxidant capacity and a reduction in atrogene expression. These findings suggest that HBO therapy may be an effective adjunctive therapy to promote respiratory health following cervical SCI.

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck’s length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.

Alterations in mitochondrial homeostasis in a potassium dichromate model of acute kidney injury and their mitigation by curcumin

Curcumin has protective effects in several acute kidney injury models, including that induced by potassium dichromate (K2Cr2O7). The protective effect of curcumin in this experimental model has been associated to the preservation of mitochondrial bioenergetics. This study is aimed at evaluating whether or not curcumin's protective effect in mitochondrial bioenergetics is related to the modulation of mitochondrial dynamics and biogenesis. Wistar rats were treated with a single subcutaneous dose of K2Cr2O7 (12.5 mg/kg) or received curcumin (400 mg/kg/day) by oral gavage 10 days before and one day after the K2Cr2O7 injection. K2Cr2O7 induced kidney dysfunction and increased mitochondrial hydrogen peroxide production, while decreasing the respiration directly attributable to oxidative phosphorylation and mitochondrial membrane potential. In mitochondria, K2Cr2O7 increased fission and reduced fusion. Structural analysis of mitochondria in the proximal tubular cells corroborated their fragmentation and loss of crests' integrity. Regarding mitochondrial biogenesis, K2Cr2O7 decreased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) levels. Conversely, curcumin treatment mitigated the aforementioned alterations and increased the expression of the mitochondrial transcription factor A (TFAM). Taken together, our results suggest that curcumin can protect against renal injury by modulating mitochondrial homeostasis, mitigating alterations in bioenergetics and dynamics, possibly by stimulating mitochondrial biogenesis.

Fgr kinase is required for proinflammatory macrophage activation during diet-induced obesity.

Proinflammatory macrophages are key in the development of obesity. In addition, reactive oxygen species (ROS), which activate the Fgr tyrosine kinase, also contribute to obesity. Here we show that ablation of Fgr impairs proinflammatory macrophage polarization while preventing high-fat diet (HFD)-induced obesity in mice. Systemic ablation of Fgr increases lipolysis and liver fatty acid oxidation, thereby avoiding steatosis. Knockout of Fgr in bone marrow (BM)-derived cells is sufficient to protect against insulin resistance and liver steatosis following HFD feeding, while the transfer of Fgr-expressing BM-derived cells reverts protection from HFD feeding in Fgr-deficient hosts. Scavenging of mitochondrial peroxides is sufficient to prevent Fgr activation in BM-derived cells and HFD-induced obesity. Moreover, Fgr expression is higher in proinflammatory macrophages and correlates with obesity traits in both mice and humans. Thus, our findings reveal the mitochondrial ROS-Fgr kinase as a key regulatory axis in proinflammatory adipose tissue macrophage activation, diet-induced obesity, insulin resistance and liver steatosis.

Assessment of ROS Production in the Mitochondria of Live Cells.

Production of reactive oxygen species (ROS) in the mitochondria plays multiple roles in physiology, and excessive production of ROS leads to the development of various pathologies. ROS in the mitochondria are generated by various enzymes, mainly in the electron transporvt chain, and it is important to identify not only the trigger but also the source of free radical production. It is important to measure mitochondrial ROS in live, intact cells, because activation of ROS production could be initiated by changes in extramitochondrial processes which could be overseen when using isolated mitochondria. Here we describe the approaches, which allow to measure production of ROS in the matrix of mitochondria in live cells. We also demonstrate how to measure kinetic changes in lipid peroxidation in mitochondria of live cells. These methods could be used for understanding the mechanisms of pathology in a variety of disease models and also for testing neuro- or cardioprotective chemicals.

Seaweeds' neuroprotective potential set in vitro on a human cellular stress model.

Neurodegenerative diseases, such as Parkinson's disease, represent a biggest challenge for medicine, imposing high social and economic impacts. As a result, it is of utmost importance to develop new therapeutic strategies. The present work evaluated the neuroprotective potential of seaweeds extracts on an in vitro dopamine (DA)-induced neurotoxicity cellular model. The neuroprotective effects on SH-SY5Y cells' viability were estimated by the MTT assay. Changes in mitochondrial membrane potential (MMP), caspase-3 activity, and hydrogen peroxide (H2O2) production were determined. DA (30-3000µM; 24h) treatment decreased SH-SY5Y cells' viability in concentration and time-dependent manner, increasing the H2O2 production, MMP depolarization, and caspase-3 activity. On the other hand, DA (1000µM; 24h) toxicity was reduced (10-15%) with Sargassum muticum and Codium tomentosum extracts (1000µg/mL; 24h). The highest neuroprotective activity was exhibited by a methanolic extract obtained from Saccorhiza polyschides, which completely blunted DA effects. Results show that the marine seaweed S. polyschides contain substances with high neuroprotective potential against the toxicity induced by DA, exhibiting anti-apoptotic effects associated with both mitochondrial protection and caspase-3 inhibition. S. polyschides reveals, therefore, to be an excellent source of bioactive molecules, for new drugs development aiming PD therapeutics.

MitoQ and CoQ10 supplementation mildly suppresses skeletal muscle mitochondrial hydrogen peroxide levels without impacting mitochondrial function in middle-aged men.

Excess production of reactive oxygen species (ROS) from the mitochondria can promote mitochondrial dysfunction and has been implicated in the development of a range of chronic diseases. As such there is interest in whether mitochondrial-targeted antioxidant supplementation can attenuate mitochondrial-associated oxidative stress. We investigated the effect of MitoQ and CoQ10 supplementation on oxidative stress and skeletal muscle mitochondrial ROS levels and function in healthy middle-aged men. Skeletal muscle and blood samples were collected from twenty men (50 ± 1 y) before and following six weeks of daily supplementation with MitoQ (20mg) or CoQ10 (200mg). High-resolution respirometry was used to determine mitochondrial respiration and H2O2 levels, markers of mitochondrial mass and antioxidant defences were measured in muscle samples and oxidative stress markers in urine and blood samples. Both MitoQ and CoQ10 supplementation suppressed mitochondrial net H2O2 levels during leak respiration, while MitoQ also elevated muscle catalase expression. However, neither supplement altered urine F2-isoprostanes nor plasma TBARS levels. Neither MitoQ nor CoQ10 supplementation had a significant impact on mitochondrial respiration or mitochondrial density markers (citrate synthase, mtDNA/nDNA, PPARGC1A, OXPHOS expression). Our results suggest that neither MitoQ and CoQ10 supplements impact mitochondrial function, but both can mildly suppress mitochondrial ROS levels in healthy middle-aged men, with some indication that MitoQ may be more effective than CoQ10.

Stimulating Embryo Polarization with Mitochondrial Peroxide.

Centrosomes break symmetry in the C.elegans one-cell embryo, triggering its anterior-posterior polarization and initiating segregation of somatic and germline cell lineages. In this issue of Developmental Cell, De Henau etal. show that mitochondria also contribute to symmetry breaking by producing hydrogen peroxide at the egg's future posterior pole.

Effects of atrial natriuretic peptide on mitochondria function in cortical collecting duct cells

BackgroundAtrial natriuretic peptide (ANP) is a hormone released from cardiomyocytes in response to cardiac wall stretching. ANP acts on the kidney by promoting sodium and water excretion as well as providing a general vasodilatory effect. Direct application of ANP has been shown to reduce sodium reabsorption in the renal tubular cells. Recently, it has been discovered that natriuretic peptides (NPs) are involved in lipolysis, lipid oxidation, and mitochondrial respiration. Evidence of a correlation between the activity of the NP system and mitochondrial respiration and biogenesis has been explored to some extent; however, little is known about these pathways in the kidney. Here we hypothesized that ANP may be implicated in mitochondrial uncoupling in the cortical collecting ducts (CCD), affecting sodium transport in this segment.MethodsMouse cortical collecting duct (mpkCCD) cells were cultured in DMEM/F12 medium with supplements and grown on glass‐bottom dishes, 96‐well plates or permeable supports. Cells were treated with ANP (5 nM to 10 μM range) for 1 or 24 hours. Fluorescent dyes TMRM and Rhod123, CellRox Deep Red, AmplexRed, Rhod2 and Hoechst 33342 were used to label mitochondrial membrane potential (TMRM/Rhod123), production of mitochondrial ROS, hydrogen peroxide, calcium, and nuclei in live cells, respectively. A Leica TCS SP5 confocal microscope or a BMG Labtech microplate reader were employed to monitor fluorescence intensity changes in response to ANP. Mitochondrial stress test assays were performed using a Seahorse XF96 Extracellular Flux Analyzer. Sodium currents in the polarized mpkCCD monolayers were measured with an EVOM multimeter. One‐way ANOVA (OriginPro) was used for statistical comparisons.ResultsFluorescence microscopy with TMRM showed a mild (~30% at maximum effect) dose‐dependent decrease in mitochondrial membrane potential in mpkCCD cells treated with low dose ANP for 1, 2 and 24 h (1 nM to 500 nM), which was, interestingly, attenuated by higher ANP concentrations (&gt;5 μM). These data were confirmed in a 96‐well plate assay using Rhod 123 (1 h and 24 hours). In addition, we observed an acute increase in ROS (and specifically, hydrogen peroxide) production and mitochondrial Ca2+ level in response to ANP (at 1 h), which is in line with the mild uncoupling reported above. Preliminary seahorse analysis indicated that ANP decreases mitochondrial respiration in these cells in a dose‐dependent manner. We have also shown that direct uncoupling of mitochondria with FCCP results in a dose‐dependent decrease in sodium currents mediated by Epithelial Sodium Channel (ENaC) as measured in short circuit current experiments.ConclusionDecreased mitochondrial membrane potential is indicative of a disruption in the oxidative phosphorylation, which is the main mechanism of ATP synthesis in the cell and the source of energy for Na+/K+‐ATPase (NKA). Since NKA provides the gradient for Na+ reabsorption in CCD, the reported mitochondria‐mediated effect suggests a novel pathway through which ANP may regulate Na+ reabsorption in the collecting ducts, potentially affecting blood pressure.Support or Funding InformationAPS Summer Undergraduate Fellowship Program; NIH R00 DK105160, Dialysis Clinic Inc Reserve Funds.

Real-Time Imaging and Simultaneous Quantification of Mitochondrial H2O2 and ATP in Neurons with a Single Two-Photon Fluorescence-Lifetime-Based Probe.

Mitochondrial oxidative stress and energy metabolism are vital biological events and are involved in various physiological and pathological processes such as apoptosis and necrosis. However, it remains unclear how the dynamic patterns of mitochondrial hydrogen peroxide (H2O2) and adenosine-5'-triphosphate (ATP) change in these events and, more importantly, how they affect each other. Herein, we developed a single two-photon fluorescence-lifetime-based probe (TFP), which offered real-time imaging and the simultaneous determination of mitochondrial H2O2 and ATP changes in two well-separated fluorescence channels without spectral crosstalk. The fluorescence lifetime of TFP exhibited good responses and selectivity in the detection ranges of 0.4-10 μM H2O2 and 0.5-15 mM ATP, taking advantage of accuracy and the quantitative ability of fluorescence lifetime imaging. Using this useful probe, we studied the relationship between H2O2 and ATP in mitochondria and visualized the dynamic level changes of mitochondrial H2O2 and ATP induced by the superoxide anion (O2•-). It was discovered that O2•- stimulation in a short period of time (8 min) temporarily changes the levels of H2O2 and ATP in mitochondria, and neurons were capable of recovering to the initial state in a short time. However, increasing time of up to 50 min of O2•- stimulation led to permanent oxidative damage and an energy deficiency. Meanwhile, it was first found that the exogenous stimulation of O2•- and H2O2 had different impacts on the levels of mitochondrial H2O2 and ATP, in which O2•- demonstrated more severe and negative consequences. As a matter of fact, this work not only has provided a general molecular design methodology for multiple species imaging but also has revealed oxidative-stress-induced intracellular functions related to H2O2 and ATP in mitochondria based on this developed TFP probe.

Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations.

Mitochondria utilize the majority of oxygen (O2) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS) but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis, and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption (Jo2) from tissue mitochondrial preparations is an attractive investigative approach, but it introduces dynamic changes in media O2 concentration ([O2]) that can confound experimental results and interpretation. We utilized high-resolution fluorespirometry to evaluate Jo2 and hydrogen peroxide release (Jh2o2) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O2]s typically encountered during respirometry protocols (400-50 µM). Results demonstrate notable variations in Jh2o2 across tissues and sample preparations during nonphosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O2] but a linear decline in Jh2o2 of 5-15% per 50-µM decrease in chamber [O2] in all samples. Jo2 was generally stable in Mt and Hm across [O2]s above 50 µM but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of Jh2o2/O2 across chamber [O2]s and sample preparations. Development of chemical background fluorescence from the H2O2 probe (Amplex Red) was also O2 sensitive, emphasizing relevant calibration considerations. This study highlights the importance of monitoring and reporting the chamber [O2] at which Jo2 and Jh2o2 are recorded during fluorespirometry experiments and provides a basis for selecting sample preparations for studies addressing the role of mtROS in physiology and disease.

Epigenetic Metabolic Reprogramming of Right Ventricular Fibroblasts in Pulmonary Arterial Hypertension: A Pyruvate Dehydrogenase Kinase-Dependent Shift in Mitochondrial Metabolism Promotes Right Ventricular Fibrosis.

Right ventricular (RV) fibrosis in pulmonary arterial hypertension contributes to RV failure. While RV fibrosis reflects changes in the function of resident RV fibroblasts (RVfib), these cells are understudied. Examine the role of mitochondrial metabolism of RVfib in RV fibrosis in human and experimental pulmonary arterial hypertension. Male Sprague-Dawley rats received monocrotaline (MCT; 60 mg/kg) or saline. Drinking water containing no supplement or the PDK (pyruvate dehydrogenase kinase) inhibitor dichloroacetate was started 7 days post-MCT. At week 4, treadmill testing, echocardiography, and right heart catheterization were performed. The effects of PDK activation on mitochondrial dynamics and metabolism, RVfib proliferation, and collagen production were studied in RVfib in cell culture. Epigenetic mechanisms for persistence of the profibrotic RVfib phenotype in culture were evaluated. PDK expression was also studied in the RVfib of patients with decompensated RV failure (n=11) versus control (n=7). MCT rats developed pulmonary arterial hypertension, RV fibrosis, and RV failure. MCT-RVfib (but not left ventricular fibroblasts) displayed excess mitochondrial fission and had increased expression of PDK isoforms 1 and 3 that persisted for >5 passages in culture. PDK-mediated decreases in pyruvate dehydrogenase activity and oxygen consumption rate were reversed by dichloroacetate (in RVfib and in vivo) or siRNA targeting PDK 1 and 3 (in RVfib). These interventions restored mitochondrial superoxide and hydrogen peroxide production and inactivated HIF (hypoxia-inducible factor)-1α, which was pathologically activated in normoxic MCT-RVfib. Redox-mediated HIF-1α inactivation also decreased the expression of TGF-β1 (transforming growth factor-beta-1) and CTGF (connective tissue growth factor), reduced fibroblast proliferation, and decreased collagen production. HIF-1α activation in MCT-RVfib reflected increased DNMT (DNA methyltransferase) 1 expression, which was associated with a decrease in its regulatory microRNA, miR-148b-3p. In MCT rats, dichloroacetate, at therapeutic levels in the RV, reduced phospho-pyruvate dehydrogenase expression, RV fibrosis, and hypertrophy and improved RV function. In patients with pulmonary arterial hypertension and RV failure, RVfib had increased PDK1 expression. MCT-RVfib manifest a DNMT1-HIF-1α-PDK-mediated, chamber-specific, metabolic memory that promotes collagen production and RV fibrosis. This epigenetic mitochondrial-metabolic pathway is a potential antifibrotic therapeutic target.

Simultaneous imaging of mitochondrial viscosity and hydrogen peroxide in Alzheimer's disease by a single near-infrared fluorescent probe with a large Stokes shift.

It has been speculated that both the intracellular viscosity and H2O2 level in Alzheimer's disease (AD) brains are higher than that in healthy brains, but direct evidence from living beings is scarce. Herein, we report a NIR emissive fluorescent probe with a large Stokes shift for the associated detection of mitochondrial viscosity and H2O2 in live rat brains with AD for the first time.

Effects of copper and temperature on heart mitochondrial hydrogen peroxide production

High energy demand for continuous mechanical work and large number of mitochondria predispose the heart to excessive reactive oxygen species (ROS) production that may precipitate oxidative stress and heart failure. While mitochondria have been proposed as a unifying cellular target and driver of adverse effects induced by diverse stressful states, there is limited understanding of how heart mitochondrial ROS homeostasis is affected by combinations of stress factors. Thus, we probed the effect of copper (Cu) and thermal stress on ROS (as hydrogen peroxide, H2O2) emission and elucidated the effects of Cu on ROS production sites in rainbow trout heart mitochondria using the Amplex UltraRed-horseradish peroxidase detection system optimized for our model. Mitochondria oxidizing malate-glutamate or succinate were incubated at 4, 11 (control) and 23 °C and exposed to a range (1–100 μM) of Cu concentrations. We found that the rates and patterns of H2O2 emission depended on substrate type, Cu concentration and temperature. In mitochondria oxidizing malate-glutamate, Cu increased the rate of H2O2 emission with a spike at 1 μM while temperature had no effect. In contrast, both temperature and Cu increased the rate of H2O2 emission in mitochondria oxidizing succinate with a prominent spike at 25 μM Cu. The rates of H2O2 emission at the three temperatures during the spike imposed by 25 μM Cu were of the order 11 > 23 > 4 °C. Interestingly, 5 μM Cu supressed H2O2 emission in mitochondria oxidizing succinate or malate-glutamate suggesting a common mechanism of action independent of substrate type. In the absence of Cu, the site-specific capacities of H2O2 emission were: complex III outer ubiquinone binding site (site IIIQo) > complex II flavin site (site IIF) ≥ complex I flavin site (site IF) > complex I ubiquinone-binding site (site IQ). Rotenone marginally increased succinate-driven H2O2 emission suggesting either the absence of reverse electron transport (RET)-driven ROS production at site IQ or masking of the expected rotenone response (reduction) by H2O2 produced from other sites. Cu acted at multiple sites in the electron transport system resulting in different site-specific H2O2 emission responses depending on the concentration. Specifically, site IF H2O2 emission was suppressed by Cu concentration-dependently while H2O2 emission by site IIF was inhibited and stimulated by low and high concentrations of Cu, respectively. Additionally, emission from site IIIQo was stimulated by low and inhibited by high Cu concentrations. Overall, our study unveiled distinctive effects and sites of modulation of mitochondrial ROS production by Cu with implications for cardiac redox signaling networks and development of mitochondria-targeted Cu-based drugs.

Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson\u2019s disease

Mitochondrial degeneration is considered one of the major causes of Parkinson’s disease (PD). Improved mitochondrial functions are expected to be a promising therapeutic strategy for PD. In this study, we introduced a light-driven proton transporter, Delta-rhodopsin (dR), to Drosophila mitochondria, where the mitochondrial proton-motive force (Δp) and mitochondrial membrane potential are maintained in a light-dependent manner. The loss of the PD-associated mitochondrial gene CHCHD2 resulted in reduced ATP production, enhanced mitochondrial peroxide production and lower Ca2+-buffering activity in dopaminergic (DA) terminals in flies. These cellular defects were improved by the light-dependent activation of mitochondrion-targeted dR (mito-dR). Moreover, mito-dR reversed the pathology caused by the CHCHD2 deficiency to suppress α-synuclein aggregation, DA neuronal loss, and elevated lipid peroxidation in brain tissue, improving motor behaviors. This study suggests the enhancement of Δp by mito-dR as a therapeutic mechanism that ameliorates neurodegeneration by protecting mitochondrial functions.

Mitochondrial Derived Hydrogen Peroxide Results in an Increase in Aerobic Glycolysis in Pulmonary Endothelial Cells via the Activation of HIF-1α Through Disulfide Bond-mediated PHD2 Dimerization

Mitochondrial Derived Hydrogen Peroxide Results in an Increase in Aerobic Glycolysis in Pulmonary Endothelial Cells via the Activation of HIF-1α Through Disulfide Bond-mediated PHD2 Dimerization

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria.

Yeast (Saccharomyces cerevisiae) essential for respiration and viability 1 (Erv1; EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html), a member of the flavin adenine dinucleotide‐dependent Erv1/ALR disulphide bond generating enzyme family, works together with Mia40 to catalyse protein import and oxidative folding in the mitochondrial intermembrane space. Erv1/ALR functions either as an oxidase or cytochrome c reductase by passing electrons from a thiol substrate to molecular oxygen (O2) or cytochrome c, respectively. However, the substrate specificity for oxygen and cytochrome c is not fully understood. In this study, the oxidase and cytochrome c reductase kinetics of yeast Erv1 were investigated in detail, under aerobic and anaerobic conditions, using stopped‐flow absorption spectroscopy and oxygen consumption analysis. Using DTT as an electron donor, our results show that cytochrome c is ~ 7‐ to 15‐fold more efficient than O2 as electron acceptors for yeast Erv1, and that O2 is a competitive inhibitor of Erv1 cytochrome c reductase activity. In addition, Mia40, the physiological thiol substrate of Erv1, was used as an electron donor for Erv1 in a detailed enzyme kinetic study. Different enzyme kinetic k cat and K m values were obtained with Mia40 compared to DTT, suggesting that Mia40 modulates Erv1 enzyme kinetics. Taken together, this study shows that Erv1 is a moderately active enzyme with the ability to use both O2 and cytochrome c as the electron acceptors, indicating that Erv1 contributes to mitochondrial hydrogen peroxide production. Our results also suggest that Mia40‐Erv1 system may involve in regulation of the redox state of glutathione in the mitochondrial intermembrane space.Erv1EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html.

HyPer2 imaging reveals temporal and heterogeneous hydrogen peroxide changes in denervated and aged skeletal muscle fibers in vivo

To determine the role of denervation and motor unit turnover in the age-related increase in skeletal muscle oxidative stress, the hydrogen peroxide (H2O2) specific, genetically-encoded, fluorescent cyto-HyPer2 probe was expressed in mouse anterior tibialis (AT) muscle and compared with ex vivo measurements of mitochondrial oxidant generation. Crush of the peroneal nerve induced increased mitochondrial peroxide generation, measured in permeabilised AT fibers ex vivo and intra vital confocal microscopy of cyto-HyPer2 fluorescence showed increased cytosolic H2O2 in a sub-set (~24%) of individual fibers associated with onset of fiber atrophy. In comparison, mitochondrial peroxide generation was also increased in resting muscle from old (26 month) mice compared with adult (6–8 month) mice, but no age effect on fiber cytosolic H2O2in vivo was seen. Thus ageing is associated with an increased ability of muscle fibers to maintain cytosolic redox homeostasis in the presence of denervation-induced increase in mitochondrial peroxide generation.