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International audience; Dozens of pathogenic mutations have been localized in the mitochondrial gene (MT-ATP6) that encodes the subunit a of ATP synthase. The subunit a together with a ring of identical subunits c moves protons across the mitochondrial inner membrane coupled to rotation of the subunit c-ring and ATP synthesis. One of these mutations, m.8851T > C, has been associated with bilateral striatal lesions of childhood (BSLC), a group of rare neurological disorders characterized by symmetric degeneration of the corpus striatum. It converts a highly conserved tryptophan residue into arginine at position 109 of subunit a (aW 109 R). We previously showed that an equivalent thereof in Saccharomyces cerevisiae (aW 126 R) severely impairs by an unknown mechanism the functioning of ATP synthase without any visible assembly/stability defect. Herein we show that ATP synthase function was recovered to varying degree by replacing the mutant arginine residue 126 with me-thionine, lysine or glycine or by replacing with methionine an arginine residue present at position 169 of subunit a (aR 169). In recently described atomic structures of yeast ATP synthase, aR 169 is at the center of a hydrophilic cleft along which protons are transported from the subunit c-ring to the mitochondrial matrix, in the proximity of the two residues known from a long time to be essential to the activity of F O (aR 176 and cE 59). We provide evidence that the aW 126 R change is responsible for electrostatic and steric hindrance that enables aR 169 to engage in a salt bridge with cE 59. As a result, aR 176 cannot interact properly with cE 5 and ATP synthase fails to effectively move protons across the mitochondrial membrane. In addition to insight into the pathogenic mechanism induced by the m.8851T > C mutation, the present study brings interesting information about the role of specific residues of subunit a in the energy-transducing activity of ATP synthase.

International audience; Flavonoids have antitumoral properties and may be attractive candidates as anticancer therapy. Isoliquiritigenin which is a constituent of licorice (Glycyrrhiza inflata), a plant commonly used in traditional Uyghur medicine in Xinjiang, China, was studied for antiproliferative and apoptotic activity in human cervical cancer cells, Ca Ski, SiHa, HeLa, and C-33A. Its molecular mechanism of action was specifically examined in Ca Ski cells. Isoliquiritigenin decreased cell viability, induced cell accumulation in G2/M and morphological and biochemical features of apoptosis in the four cancer cell lines. In Ca Ski cells, isoliquiritigenin led to a downregulation of HPV16 E6 expression associated with an increase of p53 and p21 levels, enhanced expression of Bax and decreased expression of Bcl-2 and Bid proform triggering dissipation of the mitochondrial membrane potential, released cytochrome c to the cytosol followed by activation of caspase cascade with cleavage of caspase-9, caspase-3, and PARP. Caspase-8 was also cleaved. Moreover treatment with a pan-caspase inhibitor prevented apoptosis. As Ca Ski cells are representative of carcinoma naturally occurring in the cervix, our results suggest a potential benefit of isoliquiritigenin for cervical cancer prevention and treatment.

International audience; AIMS: The translocator protein (TSPO) is located on the outer mitochondrial membrane where it is responsible for the uptake of cholesterol into mitochondria of steroidogenic organs. TSPO is also present in the heart where its role remains uncertain. We recently showed that TSPO ligands reduced infarct size and improved mitochondrial functions after ischaemia-reperfusion. This study, thus, sought to determine whether cholesterol could play a role in the cardioprotective effect of TSPO ligands. METHODS AND RESULTS: In a model of 30 min coronary occlusion/15 min reperfusion in Wistar rat, we showed that reperfusion induced lipid peroxidation as demonstrated by the increase in conjugated diene and thiobarbituric acid reactive substance formation and altered mitochondrial function (decrease in oxidative phosphorylation and increase in the sensitivity of mitochondrial permeability transition pore opening) in ex-vivo isolated mitochondria. This was associated with an increase in mitochondrial cholesterol uptake (89.5 ± 12.2 vs. 39.9 ± 3.51 nmol/mg protein in controls, P < 0.01) and a subsequent strong generation of auto-oxidized oxysterols, i.e. 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, and 5β,6β-epoxide (+173, +149, +165, +165, and +193% vs. controls, respectively; P < 0.01). Administration of the selective TSPO ligand 4'-chlorodiazepam inhibited oxidative stress, improved mitochondrial function, and abolished both mitochondrial cholesterol accumulation and oxysterol production. This was also observed with the new TSPO ligand TRO40303. CONCLUSION: These data suggest that 4'-chlorodiazepam inhibits oxidative stress and oxysterol formation by reducing the accumulation of cholesterol in the mitochondrial matrix at reperfusion and prevents mitochondrial injury. This new and original mechanism may contribute to the cardioprotective properties of TSPO ligands.

Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases and affects millions of people worldwide. Strong evidence supports the role of free radicals, oxidative stress, mitochondrial, and proteasomal dysfunctions underlying neuronal death in PD. Environmental factors, especially pesticides, represent one of the primary classes of neurotoxic agents associated with PD, and several epidemiological studies have identified the exposure of the herbicide paraquat (PQ) as a potential risk factor for the onset of PD. The objective of our study was to investigate the neuroprotective effects of the standardized extracts of Bacopa monniera (BM) against PQ-induced and 1-methyl-4-phenyl-pyridinium iodide (MPP(+))-induced toxicities and to elucidate the mechanisms underlying this protection. Our results show that a pretreatment with the BM extract from 50 μg/ml protected the dopaminergic SK-N-SH cell line against MPP(+)- and PQ-induced toxicities in various cell survival assays. We demonstrate that BM pretreatment prevented the depletion of glutathione (GSH) besides preserving the mitochondrial membrane potential and maintaining the mitochondrial complex I activity. BM pretreatment from 10.0 μg/ml also prevented the generation of intracellular reactive oxygen species and decreased the mitochondrial superoxide level. BM treatment activated the nuclear factor erythroid 2-related factor 2 pathway by modulating the expression of Keap1, thereby upregulating the endogenous GSH synthesis. The effect of BM on the phosphorylation of Akt further strengthens its role in the promotion of cell survival. By preserving the cellular redox homeostasis and mitochondrial activities and by promoting cell survival pathways, BM extract may have therapeutic uses in various age-related neurodegenerative diseases such as PD.

International audience; Inorganic arsenic is a major environmental contaminant known to exert immunosuppressive effects. In this study, we report toxicity of As2O3, a trivalent inorganic form, toward isolated human hematopoietic CD34+ progenitor cells. Our results demonstrate that low concentrations of As2O3 (0.1-5 microM) inhibit in vitro proliferation of CD34+ cells and their differentiation into various hematological cell lineages. These effects were associated with the induction of a necrotic process independent of caspases and likely related to mitochondrial damage. We conclude that As2O3 can impair in vitro human hematopoiesis by decreasing survival of CD34+ progenitor cells.

International audience; Bisphenol A (BPA) is an endocrine-disrupting chemical that leaches from polycarbonate plastics that consequently leads to low-dose human exposure. In addition to its known xenoendocrine action, BPA exerts a wide variety of metabolic effects, but no data are available on its actions on the functions of liver mitochondrial. To assess these effects, HepG2 cells were exposed to BPA (10(-4)-10(-12)M) and physiological parameters were measured by flow cytometry. We demonstrated a significant mitochondrial dysfunction including ROS production, ΔΨ(M) hyperpolarization, lipid accumulation, lipoperoxidation and the release of pro-inflammatory cytokines. In conclusion, we showed that low concentrations of BPA promote lipid accumulation in hepatic cells triggered by disturbances in mitochondrial function, alterations in lipid metabolism and by inflammation that can therefore contribute to steatosis.

International audience; On the basis of our previous identification of aberrant phosphatidylinositol-3-kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235-induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235-induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase-dependent apoptosis. Lysosome-mediated apoptosis occurs in a ROS-dependent manner, as ROS scavengers significantly reduce BEZ235/CQ-induced loss of MMP, LMP and apoptosis. There is a mitochondrial-lysosomal cross-talk, since lysosomal enzyme inhibitors significantly decrease BEZ235- and CQ-induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome-mediated apoptosis via a mitochondrial-lysosomal cross-talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor-based combination therapies.

International audience; UNLABELLED: Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [3H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+ -dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. CONCLUSION: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes.

International audience; Mitochondria play central roles in cell life as a source of energy and in cell death by inducing apoptosis. Many important functions of mitochondria change in cancer, and these organelles can be a target of chemotherapy. The widely used anticancer drug doxorubicin (DOX) causes cell death, inhibition of cell cycle/proliferation and mitochondrial impairment. However, the mechanism of such impairment is not completely understood. In our study we used confocal and two-photon fluorescence imaging together with enzymatic and respirometric analysis to study short- and long-term effects of doxorubicin on mitochondria in various human carcinoma cells. We show that short-term (

International audience; Mitochondria are most important organelles in the survival of eukaryotic aerobic cells because they are the primary producers of ATP, regulators of ion homeostasis or redox state, and producers of free radicals. The key role of mitochondria in the generation of primordial ATP for the survival and proliferation of eukaryotic cells has been proven by extensive biochemical studies. In this context, it is crucial to understand the complexity of the mitochondrial compartment and its functionality and to develop experimental tools allowing the assessment of its nature and its function and metabolism. This review covers the role of the mitochondria in the cell, focusing on its structure, the mechanism of the mitochondrial respiratory chain, the maintenance of the transmembrane potential and the production of reactive oxygen species. The main probes used for mitochondrial compartment monitoring are described. In addition, various applications using mitochondrial-specific probes are detailed to illustrate the potential of flow and image cytometry in the study of the mitochondrial compartment. This review contains a panel of tools to explore mitochondria and to help researchers design experiments, determine the approach to be employed, and interpret their results.

International audience; BACKGROUND & AIMS: A high-fat diet affects liver metabolism, leading to steatosis, a complex disorder related to insulin resistance and mitochondrial alterations. Steatosis is still poorly understood since diverse effects have been reported, depending on the different experimental models used. METHODS: We hereby report the effects of an 8 week high-fat diet on liver energy metabolism in a rat model, investigated in both isolated mitochondria and hepatocytes. RESULTS: Liver mass was unchanged but lipid content and composition were markedly affected. State-3 mitochondrial oxidative phosphorylation was inhibited, contrasting with unaffected cytochrome content. Oxidative phosphorylation stoichiometry was unaffected, as were ATPase and adenine nucleotide translocator proteins and mRNAs. Mitochondrial acylcarnitine-related H(2)O(2) production was substantially higher and the mitochondrial quinone pool was smaller and more reduced. Cellular consequences of these mitochondrial alterations were investigated in perifused, freshly isolated hepatocytes. Ketogenesis and fatty acid-dependent respiration were lower, indicating a lower β-oxidation rate contrasting with higher RNA contents of CD36, FABP, CPT-1, and AcylCoA dehydrogenases. Concomitantly, the cellular redox state was more reduced in the mitochondrial matrix but more oxidized in the cytosol: these opposing changes are in agreement with a significantly higher in situ mitochondrial proton motive force. CONCLUSIONS: A high-fat diet results in both a decrease in mitochondrial quinone pool and a profound modification in mitochondrial lipid composition. These changes appear to play a key role in the resulting inhibition of fatty acid oxidation and of mitochondrial oxidative-phosphorylation associated with an increased mitochondrial ROS production. Mitochondrial quinone pool could have prospects as a crucial event, potentially leading to interesting therapeutic perspectives.

Background Low concentrations of carbon monoxide (CO) protect hepatocytes against apoptosis and confers cytoprotection in several models of liver. Mitochondria are key organelles in cell death control via their membrane permeabilization and the release of pro-apoptotic factors. Results Herein, we show that CO prevents mitochondrial membrane permeabilization (MMP) in liver isolated mitochondria. Direct and indirect approaches were used to evaluate MMP inhibition by CO: mitochondrial swelling, mitochondrial depolarization and inner membrane permeabilization. Additionally, CO increases mitochondrial reactive oxygen species (ROS) generation, and their scavenging, by ß-carotene addition, decreases CO protection, which reveals the key role of ROS. Interestingly, cytochrome c oxidase transiently responds to low concentrations of CO by decreasing its activity in the first 5 min, later on there is an increase of cytochrome c oxidase activity, which were detected up to 30 min. Conclusion CO directly prevents mitochondrial membrane permeabilization, which might be implicated in the hepatic apoptosis inhibition by this gaseoustransmitter.

International audience; It is well established that cells exposed to the limiting oxygen microenvironment (hypoxia) of tumors acquire resistance to chemotherapy, through mechanisms not fully understood. We noted that a large number of cell lines showed protection from apoptotic stimuli, staurosporine, or etoposide, when exposed to long-term hypoxia (72 h). In addition, these cells had unusual enlarged mitochondria that were induced in a HIF-1-dependent manner. Enlarged mitochondria were functional as they conserved their transmembrane potential and ATP production. Here we reveal that mitochondria of hypoxia-induced chemotherapy-resistant cells undergo a HIF-1-dependent and mitofusin-1-mediated change in morphology from a tubular network to an enlarged phenotype. An imbalance in mitochondrial fusion/fission occurs since silencing of not only the mitochondrial fusion protein mitofusin 1 but also BNIP3 and BNIP3L, two mitochondrial HIF-targeted genes, reestablished a tubular morphology. Hypoxic cells were insensitive to staurosporine- and etoposide-induced cell death, but the silencing of mitofusin, BNIP3, and BNIP3L restored sensitivity. Our results demonstrate that some cancer cells have developed yet another way to evade apoptosis in hypoxia, by inducing mitochondrial fusion and targeting BNIP3 and BNIP3L to mitochondrial membranes, thereby giving these cells a selective growth advantage.

Background Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. Results Detailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs. Conclusions Both CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

International audience; Acrolein is one of the by-products of lipid peroxidation. Due to its high reactivity, it is not only a marker of lipid peroxidation but could also be an initiator of oxidative stress by adducting cellular nucleophilic groups. In brains of Alzheimer's disease (AD) patients, levels of acrolein are significantly higher in vulnerable brain region and, on primary hippocampal culture, it is more toxic than 4-hydroxyl-nonenal. The toxicity of the amyloid-beta peptide is mediated through the generation of hydrogen peroxide (H2O2). The actions of H2O2 include oxidative modifications of proteins, lipids, and DNA as observed in AD. Bacopa monniera (BM) has a long history of use in India as a memory-enhancing therapy. The objective of our study was to investigate the neuroprotective effects of the standardized extracts of BM against acrolein and H2O2 and to elucidate the mechanisms underlying this protection. Our results show that a pre-treatment with the BM extract protected the human neuroblastoma cell line SK-N-SH against H2O2 and acrolein. We demonstrated that BM pre-treatment significantly inhibited the generation of intracellular reactive oxygen species in addition to preserving the mitochondrial membrane potential. BM pre-treatment also prevented the modifications of the activity of several redox regulated proteins, i.e., NF-kappaB, Sirt1, ERK1/2, and p66Shc, so as to favor cell survival in response to oxidative stress. Thus, our findings demonstrate that BM can protect human neuroblastoma cells against H2O2 and acrolein through different mechanisms involved in the pathophysiology of AD and could have a therapeutic application in the prevention of AD.

In the ageing individual, the production of reactive oxygen species (ROS) accelerates with cell senescence. Depending on the heritability of the underlying processes that determine net ROS levels, this may influence ageing per se and its evolutionary direction and rate of change. In order to understand the inheritance and evolution of net ROS levels in free-ranging lizards, we used flow cytometry together with ROS-sensitive fluorogenic probes to measure ROS in lizard blood cells. We measured basal levels of (i) non-specific ROS (superoxide, singlet oxygen, H2O2 and peroxynitrite), (ii) superoxide specifically and (iii) superoxide after CCCP treatment, which elevated ROS production in the mitochondria. The cumulative level of non-specific ROS was higher in adults than juveniles and superoxide level showed high heritability and variability among families. We suggest that the evolution of ROS dynamics may be ROS species specific and perhaps depend on the relative degree of uni- or biparental inheritance of ROS main regulatory pathways.

International audience; OBJECTIVE: The first objective was to evaluate the influence of caloric intake on liver mitochondrial properties. The second objective was aimed at determining the impact of increasing fat intake on these properties. DESIGN: Lou/C rats, displaying an inborn low caloric intake and resistant to diet-induced obesity, were compared to Wistar rats fed either ad libitum or pair-fed. An additional group of Lou/C rats were allowed to increase their fat intake by adjusting their diet from a standard high carbohydrate low-fat diet to a high-fat carbohydrate-free diet. MEASUREMENTS: Hydrogen peroxide (H(2)O(2)) generation, oxygen consumption rate (J(O(2))), membrane potential (DeltaPsi), activity of respiratory chain complexes, cytochrome contents, oxidative phosphorylation efficiency (OPE) and uncoupling protein 2 (UCP2) expression were determined in liver mitochondria. RESULTS: H(2)O(2) production was higher in Lou/C than Wistar rats with glutamate/malate and/or succinate, octanoyl-carnitine, as substrates. These mitochondrial features cannot be mimicked by pair-feeding Wistar rats and remained unaltered by increasing fat intake. Enhanced H(2)O(2) production by mitochondria from Lou/C rats is due to an increased reverse electron flow through the respiratory-chain complex I and a higher medium-chain acyl-CoA dehydrogenase activity. While J(O(2)) was similar over a large range of DeltaPsi in both strains, Lou/C rats were able to sustain higher membrane potential and respiratory rate. In addition, mitochondria from Lou/C rats displayed a decrease in OPE that cannot be explained by increased expression of UCP2 but rather to a slip in proton pumping by cytochrome oxidase. CONCLUSIONS: Liver mitochondria from Lou/C rats display higher reactive oxygen species (ROS) generation but to deplete upstream electron-rich intermediates responsible for ROS generation, these animals increased intrinsic uncoupling of cytochrome oxidase. It is likely that liver mitochondrial properties allowed this strain of rat to display higher insulin sensitivity and resist diet-induced obesity.

International audience; Oxidative damage has been reported to be involved in the pathogenesis of diabetic neuropathy and neurodegenerative diseases. Recent evidence suggests that the antidiabetic drug metformin prevents oxidative stress-related cellular death in non-neuronal cell lines. In this report, we point to the direct neuroprotective effect of metformin, using the etoposide-induced cell death model. The exposure of intact primary neurons to this cytotoxic insult induced permeability transition pore (PTP) opening, the dissipation of mitochondrial membrane potential (DeltaPsim), cytochrome c release, and subsequent death. More importantly, metformin, together with the PTP classical inhibitor cyclosporin A (CsA), strongly mitigated the activation of this apoptotic cascade. Furthermore, the general antioxidant N-acetyl-L: -cysteine also prevented etoposide-promoted neuronal death. In addition, metformin was shown to delay CsA-sensitive PTP opening in permeabilized neurons, as triggered by a calcium overload, probably through its mild inhibitory effect on the respiratory chain complex I. We conclude that (1) etoposide-induced neuronal death is partly attributable to PTP opening and the disruption of DeltaPsim, in association with the emergence of oxidative stress, and (2) metformin inhibits this PTP opening-driven commitment to death. We thus propose that metformin, beyond its antihyperglycemic role, can also function as a new therapeutic tool for diabetes-associated neurodegenerative disorders.

International audience; The cis-diamminedichloroplatinum(II), known as cis-DDP or cisplatin is a widely used drug in cancer chemotherapy. Although a recent study has shown the anti-Leishmania activity of some cis-DDP derivatives, the cytotoxic properties were measured only on promastigotes, the insect vector form of the parasite. In this study the effect of cis-DDP on promastigotes and amastigotes, the vertebrate stage of the parasite is reported. The IC50, determined by flow cytometry, after 72 h of drug incubation was four times higher, 7.73+/-1.03 microM in the case of promastigotes compared to axenic amastigotes, 1.88+/-0.10 microM. In intracellular amastigotes the IC50, determined by counting the parasite index was 1.85+/-0.22 microM. By using flow cytometry, two patterns of cell cycle changes was observed: cis-DDP treated promastigotes and amastigotes accumulated in S phase and G2 phase, respectively. The cis-DDP response was also found to involve an "apoptosis-like" death of both promastigotes and amastigotes. However, DNA fragmentation was only detected in promastigote forms. In contrast mitochondrial transmembrane potential loss was observed for both stages of the parasite. Upon incubation of parasites with the drug an increase on GSH and GSSG levels and reactive oxygen species could be detected in the case of promastigote. Moreover, a slight increase of GSH level was detected on amastigote form. Taken together, these observations indicate that amastigotes are more sensitive to cis-DDP when compared to promastigotes. However, the signaling pathways leading to cell death could be different.

International audience; We have studied the effect of nitric oxide (NO) and potassium cyanide (KCN) on oxidative phosphorylation efficiency. Concentrations of NO or KCN that decrease resting oxygen consumption by 10-20% increased oxidative phosphorylation efficiency in mitochondria oxidizing succinate or palmitoyl-L-carnitine, but not in mitochondria oxidizing malate plus glutamate. When compared to malate plus glutamate, succinate or palmitoyl-L-carnitine reduced the redox state of cytochrome oxidase. The relationship between membrane potential and oxygen consumption rates was measured at different degrees of ATP synthesis. The use of malate plus glutamate instead of succinate (that changes the H(+)/2e(-) stoichiometry of the respiratory chain) affected the relationship, whereas a change in membrane permeability did not affect it. NO or KCN also affected the relationship, suggesting that they change the H(+)/2e(-) stoichiometry of the respiratory chain. We propose that NO may be a natural short-term regulator of mitochondrial physiology that increases oxidative phosphorylation efficiency in a redox-sensitive manner by decreasing the slipping in the proton pumps.

Summary The mitochondrial inner membrane harbors complexes of the respiratory chain and translocase complexes for preproteins. The membrane potential generated by the respiratory chain is essential for ATP production by the mitochondrial ATP synthase and as a driving force for protein import [1–7]. It is generally believed that the preprotein translocases just use the membrane potential without getting into physical contact with respiratory-chain complexes [3, 6, 7]. Here, we show that the presequence translocase interacts with the respiratory chain. Tim21, a specific subunit of the sorting-active presequence translocase [8, 9], recruits proton-pumping respiratory-chain complexes and stimulates preprotein insertion. Thus, the presequence translocase cooperates with the respiratory chain and promotes membrane-potential-dependent protein sorting into the inner mitochondrial membrane. These findings suggest a new coupling mechanism in an energy-transducing membrane.

International audience; We investigated the effects of the general anesthetic Etomidate on oxidative phosphorylation in isolated rat liver mitochondria. The study of each electron transfer site shows that there is an inhibition: mainly at complex I but also, to a lesser extent, at complex III. Moreover, with succinate as substrate, the increase in non-phosphorylating respiration is accompanied by a decrease in DeltaPsi. However, this effect is not due to classical uncoupling of oxidative phosphorylation, since ADP addition at high Etomidate concentrations restores the transmembrane difference of electrical potential. Also, in the same range of Etomidate concentration, the ATP/O ratio is not significantly affected. In conclusion, the main effect of Etomidate is to decrease the oxidative phosphorylation rate without changing yield. The H(+) leak which appears under non-phosphorylating conditions becomes negligible in physiological conditions.

International audience; The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.