Rotenone inhibits the mitochondrial permeability transition-induced cell death in U937 and KB cells

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca(2+)-sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca(2+)-induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death

We have investigated here the mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells (SH-SY5Y) treated with mitochondrial toxins, such as MPP(+) and rotenone. Mitochondrial respiratory chain inhibitors and uncouplers decreased mitochondrial membrane potential leading to DAPK dephosphorylation and activation. The class III phosphoinositide 3-kinase inhibitors attenuated DAPK dephosphorylation induced by mitochondrial toxins. Complex I inhibition by mitochondrial toxins (e.g. MPP(+)) resulted in mitochondrial swelling and lysosome reduction. Inhibition of class III phosphoinositide 3-kinase attenuated MPP(+)-induced lysosome reduction and cell death. The role of DAPK as a sensor of mitochondrial membrane potential in mitochondrial diseases was addressed.     

Coenzyme Q10 reduces the toxicity of rotenone in neuronal cultures by preserving the mitochondrial membrane potential

Defects in mitochondrial energy metabolism due to respiratory chain disorders lead to a decrease in mitochondrial membrane potential (DeltaPsim) and induce apoptosis. Since coenzyme Q10 (CoQ10) plays a dual role as an antioxidant and bioenergetic agent in the respiratory chain, it has attracted increasing attention concerning the prevention of apoptosis in mitochondrial diseases. In this study the potential of CoQ10 to antagonize the apoptosis-inducing effects of the respiratory chain inhibitor rotenone was explored by video-enhanced microscopy in SH-SY5Y neuroblastoma cells. The cationic fluorescent dye JC-1 which exhibits potential-dependent accumulation in mitochondria was used as an indicator to monitor changes in DeltaPsim. The relative changes in fluorescence intensity after incubation with rotenone for 15 minutes were calculated. Pre-treatment with CoQ10 (10 or 100 microM) for 48 h led to a significant reduction of rotenone-induced loss of DeltaPsim. These results suggest, that cytoprotection by CoQ10 may be mediated by raising cellular resistance against the initiating steps of apoptosis, namely the decrease of DeltaPsim. Whether these data may provide new directions for the development of neuroprotective strategies has to be investigated in future studies.     

In situ kinetic trapping reveals a fingerprint of reversible protein thiol oxidation in the mitochondrial matrix

Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation.     

鱼藤酮诱导线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平降低

观察鱼藤酮诱导的线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平的变化,探讨细胞氧化损伤的可能机制。通过荧光素发光法检测ATP生成、细胞内活性氧(ROS)水平的变化,流式细胞术检测线粒体膜电位,了解低剂量鱼藤酮对线粒体功能的影响;继而用H2O2诱导细胞氧化损伤,MTT法检测细胞活性,观察正常及线粒体缺陷细胞氧化应激时,胞内硫氧还蛋白(Trx)mRNA水平的变化。结果表明,鱼藤酮以剂量依赖方式抑制线粒体ATP的产生、降低线粒体膜电位,而细胞内ROS水平增高;当线粒体损伤细胞氧化应激时胞内Trx mRNA水平降低,提示鱼藤酮诱导线粒体轻度损伤细胞抗氧化能力降低与Trx转录受到抑制有关。     

Coenzyme Q cytoprotective mechanisms for mitochondrial complex I cytopathies involves NAD(P)H: quinone oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ1) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ1 cytoprotective mechanism required CoQ1 reduction by DT-diaphorase (NQO1). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ1 concentrations (5 microM). This suggests that the CoQ1H2 formed by NQO1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ1 concentrations (>10 microM), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ1 or menadione cytoprotection also involves the NQO1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ1H2 formed would then also act as a ROS scavenger.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.     

Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes

Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.     

Changes of mitochondrial respiratory function during odontogenic differentiation of rat dental papilla cells

Dental papilla cells (DPCs) belong to precursor cells differentiating to odontoblasts and play an important role in dentin formation and reproduction. This study aimed to explore the changes and and involvement of mitochondrial respiratory function during odontogenic differentiation. Primary DPCs were obtained from first molar dental papilla of neonatal rats and cultured in odontogenic medium for 7, 14, 21 days. DPCs, which expressed mesenchymal surface markers CD29, CD44 and CD90, had the capacity for self-renewal and multipotent differentiation. Odontoblastic induction increased mineralized matrix formation in a time-dependent manner, which was accompanied by elevated alkaline phosphatase (ALP), dentin sialophosphoprotein and dentin matrix protein 1 expression at mRNA and protein levels. Notably, odontogenic medium led to an increase in adenosine-5′-triphosphate content and mitochondrial membrane potential, whereas a decrease in intercellular reactive oxygen species production and NAD⁺/NADH ratio. Furthermore, odontogenic differentiation was significantly suppressed by treatment with rotenone, an inhibitor of mitochondrial respiratory chain. These results demonstrate that enhanced mitochondrial function is crucial for odontogenic differentiation of DPCs.     

Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.     

The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.     

Respiratory inhibitors and uncouplers prevent the aeration-induced increase in mitochondrial anion conductivity.

1. When mitochondria are stirred in air the rate of anion conductivity increases, this effect being enhanced by the addition of respiratory substrate. 2. This effect is reversible if the mitochondria are stored for a period of time under N2. 3. The aeration-induced increase in mitochondrial anion conductivity can also be prevented by the addition of respiratory inhibitors rotenone and antimycin A, as well as by 30 microM-cyanide. 4. A decrease in this aeration-induced anion conductivity can also be observed upon the addition of the uncouplers carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (2 microM) and 2,4-dinitrophenol (100 microM). 5. Simultaneous measurements of mitochondrial anion conductivity and membrane potential show a relationship between the level of membrane potential and anion conductivity. 6. It is suggested that the level of membrane potential is either directly or indirectly responsible for the level of mitochondrial anion conductivity.     

Titrating the effects of mitochondrial complex I impairment in the cell physiology.

The mitochondrial oxidative phosphorylation system consists of five multimeric enzymes (complexes I-V). NADH dehydrogenase or complex I (CI) is affected in most of the mitochondrial diseases and in some neurodegenerative disorders. We have studied the physiological consequences of a partial CI inhibition at the cellular level. We used a genetic model (40% CI-inhibited human-ape xenomitochondrial cybrids) and a drug-induced model (0-100% CI-inhibited cells using different concentrations of rotenone). We observed a quantitative correlation between the level of CI impairment and cell respiration, cell growth, free radical production, lipid peroxidation, mitochondrial membrane potential, and apoptosis. We showed that cell death was quantitatively associated with free radical production rather than with a decrease in respiratory chain function. The results obtained with human xenomitochondrial cybrid cells were compatible with those observed in rotenone-induced 40% CI-inhibited cells. At high concentrations (5-6-fold higher than the concentration necessary for 100% CI inhibition), rotenone showed a second toxic effect at the level of microtubule assembly, which also led to apoptosis. The correlation found among all the parameters studied helped clarify the physiological consequences of partial CI inhibitions at the cellular level.     

Megakaryocytic Differentiation of K562 Cells Induced by PMA Reduced the Activity of Respiratory Chain Complex IV

Mitochondria are involved in the regulation of cell differentiation processes, but its function changes and molecular mechanisms are not yet clear. In this study, we found that mitochondrial functions changed obviously when K562 cells were induced to megakaryocytic differentiation by phorbol 12-myristate 13-acetate (PMA). During the cell differentiation, the reactive oxygen species (ROS) level was increased, mitochondrial membrane potential declined and respiratory chain complex IV activity was decreased. Treatment with specific inhibitor of mitochondrial respiratory chain complex IV led to a significant inhibition in mitochondrial membrane potential and reduction of PMA-induced cell differentiation. However, treatment with cyclosporine A, a stabilization reagent of mitochondrial membrane potential, did not improve the down-regulation of mitochondrial respiratory chain complex IV induced by PMA. Furthermore, we found that the level of the complex IV core subunit COX3 and mitochondrial transport-related proteins Tim9 and Tim10 were decreased during the differentiation of K562 cells induced by PMA, suggesting an important role of these factors in mitochondrial functional changes. Our results suggest that changes in mitochondrial functions are involved in the PMA-induced K562 cell differentiation process, and the maintenance of the steady-state of mitochondrial functions plays a critical role in the regulation of cell differentiation.     

DiS-C3(3) monitoring of in vivo mitochondrial membrane potential in C. elegans

The mitochondrial respiratory chain plays a crucial role in cellular and organismal health. In addition to being the major source of energy for most cells, mitochondrial respiratory chain function regulates or modulates redox and metabolite homeostasis, apoptosis and the generation of reactive oxygen species. In order to measure the relative in vivo mitochondrial membrane potential of different strains of the nematode, Caenorhabditis elegans, we have developed a fluorescence assay using the cationic, lipophilic carbocyanine dye, diS-C(3)(3). We demonstrate that two complex I-deficient mutants have significantly lower mitochondrial membrane potentials in vivo than wild type animals. Our fluorescence assay will enable us to better dissect and understand the complex phenotypic consequences of mitochondrial dysfunction.     

Resveratrol preserves mitochondrial function in a human post-mitotic cell model

Dysfunctions caused by genetic defects in the mitochondrial DNA (mtDNA) of humans are called mitochondrial diseases; however, mtDNA mutations are also associated with aging and age-related diseases. Here, we present an original cellular model that allows gathering information on molecules that might contrast or prevent mitochondrial dysfunctions and their related diseases. This model allowed us to show that resveratrol (RSV), a phytochemical present in food, exerts protective effects at low concentrations on resting human fibroblasts carrying dysfunctional respiratory chain Complex I. Cells were maintained both in resting condition, to mimic the high energy demanding post-mitotic tissues (serum absence and gramicidin presence), and under glucose deficiency to push the synthesis of ATP via oxidative phosphorylation. Pre-incubation with RSV prolonged the viability of the fibroblasts exposed to rotenone, a well-known specific inhibitor of the respiratory chain Complex I, and decreased mitochondrial fragmentation. It significantly prevented the oxidative phosphorylation impairment indirectly caused by the rotenone-mediated Complex I inhibition, allowing for an almost complete preservation of the cellular ATP level. Indeed, RSV limited the rotenone-induced reactive oxygen species increase, allowing for the maintenance of a functional mitochondrial membrane potential. These findings indicate the potential usage of resveratrol to prevent or possibly treat many disorders, in which the bioenergetic defects and oxidative stress are the primary (mitochondrial encephalomyopathy), or the secondary (age-related diseases) causes of the pathology; and to also assist cell senescence during aging.