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Mitochondrial function is coupled to metabolic and survival pathways through both direct signaling cascades and dynamic changes in mitochondrial morphology. For example, a hyperfused mitochondrial reticulum is activated upon cellular stress and is protective against cell death. As part of a genome-wide small inhibitory ribonucleic acid screen, we identified the central redox regulator, Keap1, as a novel regulator of mitochondrial morphology. Here, we aimed to determine the mechanism through which redox signaling and Keap1 mediate changes in mitochondrial morphology.We found that the Nrf2 transcription factor is required for mitochondrial hyperfusion induced by knockdown of Keap1. Nrf2, which is negatively regulated by Keap1, mediates the cell's response to stress by controlling the expression of several hundred genes, including proteasome expression. We next showed that increased proteasome activity, a result of increased Nrf2 activity, is responsible for the degradation of the mitochondrial fission protein Drp1, which occurs in an ubiquitin-independent manner.Our study described a novel pathway by which Nrf2 activation, known to occur in response to increased oxidative stress, decreases mitochondrial fission and contributes to a hyperfused mitochondrial network.This study has identified the Keap1-Nrf2 nexus and modulation of proteasomal activity as novel avenues to inhibit mitochondrial fission. These findings are important, because inhibiting mitochondrial fission is a promising therapeutic approach to restore the balance between fission and fusion, which is attractive for an increasing number of disorders linked to mitochondrial dysfunction. Antioxid. Redox Signal. 27, 1447-1459.

While Indium's toxicity to organs is realized, its effects on mitochondria are still under investigation. Mitochondrial permeability transition (MPT) is widely accepted in mitochondrial dysfunction approaches and its importance in metal-induced mitochondrial degradation has been proposed. Since mitochondria are respiratory organelles, their interaction with free In3+ is analyzed to access structural and functional changes. Spectral methods and multimode plate reader was used to detect mitochondrial swelling, membrane potential, membrane fluidity, and inner membrane permeability. Flow cytometry was employed to detect mitochondrial reactive oxygen species (ROS) generation and transmission electron microscopy to image mitochondria. And oxygen electrode was used to measure respiratory rate, microcalorimetry to monitor long-term real-time mitochondrial metabolism. In3+ at a concentration up to 1mM induces mitochondrial swelling, membrane depolarization and inhibits the protons transportation. In3+-induced mitochondrial swelling and membrane depolarization is protected by MPT inhibitors and -SH protectors, but the influence on protons transportation is not protected. In addition, In3+ is able to accelerate the ROS production and inhibit the electron transition and respiratory chain while it stimulates long-term metabolism. Our findings show that In3+ induces MPT by inhibiting the proton channels located in the inner mitochondrial membrane and by stimulating mitochondrial oxidative stress.

Mitochondrial fragmentation due to fission/fusion imbalance has often been linked to mitochondrial dysfunction and apoptosis in neurodegeneration. Conventionally, it is believed that once mitochondrial morphology shifts away from its physiological tubular form, mitochondria become defective and downstream apoptotic signaling pathways are triggered. However, our study shows that beta-amyloid (Aβ) induces morphological changes in mitochondria where they become granular-shaped and are distinct from fragmented mitochondria in terms of both morphology and functions. Accumulation of mitochondrial reactive oxygen species triggers granular mitochondria formation, while mitoTEMPO (a mitochondria-targeted superoxide scavenger) restores tubular mitochondrial morphology within Aβ-treated neurons. Interestingly, modulations of mitochondria fission and fusion by genetic and pharmacological tools attenuated not only the induction of granular mitochondria, but also mitochondrial superoxide levels in Aβ−treated neurons. Our study shows a reciprocal relationship between mitochondrial dynamics and reactive oxygen species and provides a new potential therapeutic target at early stages of neurodegenerative disease pathogenesis., Graphical abstract fx1, Highlights • Aβ induces granular mitochondria at early stages of neurodegeneration. • Mitochondrial ROS triggers aberrant mitochondrial dynamics & granular mitochondria. • Granular mitochondria is reversible and attenuated by mitoTEMPO. • Manipulation of mitochondrial dynamics abolishes formation of granular mitochondria, but also reduces super oxide levels in mitochondria. • Manipulation of mitochondrial dynamics reduces super oxide levels in mitochondria.

Camptothecin (CPT), a natural alkaloid extracted from Camptotheca acuminata Decne, exhibits potential insecticidal activities against various insect species. Our previous studies have showed that CPTs induced apoptosis in Spodoptera exigua Hubner cell line which is mediated preliminarily by the mitochondrial pathway. In this study, changes of mitochondrial morphologic and function were investigated to characterize mitochondrial responses in CPTs induced apoptosis. After incubation IOZCAS-Spex-II cells with CPT and HCPT, mitochondria exhibited obvious changes in the size, morphology and distribution, and ultrastructural alterations characterized by disruption of cristae and membrane. The typical characteristics of apoptosis, including chromatin condensation, nucleus shrivels, and cytoplasmic vacuoles were found. CPT and HCPT induced IOZCAS-Spex-II cell apoptosis accompanied with increased dramatically cytosolic Ca2+ and reduced mitochondrial membrane potential in the dose and time-dependent pattern. Cytochrome c release induced by CPT and HCPT was partially reduced in the presence of CsA, which suggested that the opening of mitochondrial permeability transition pore. Taken together, these results suggested the role of mitochondria in regulation of insect cell apoptosis, which provided the basic information for illustrating the apoptosis pathway in insects and for using reasonably CPTs to control insect pests.

There is a constant need for better stroke treatments. Neurons at the periphery of an ischemic stroke affected brain tissue remains metabolically active for several hours or days after stroke onset. They later undergo mitochondrion-mediated apoptosis. It has been found that inhibiting apoptosis in the peripheral ischemic neurons could be very effective in the prevention of stroke progression. During stroke associated apoptosis, cytosolic c-Jun N-terminal Kinases (JNKs) and Bcl-2 family proteins translocate towards mitochondria and promote cytochrome c release by interacting with the outer mitochondrion membrane associated proteins. This review provides an overview of the plausible interactions of the outer mitochondrial membrane Voltage Dependent Anion Channel, Bcl-2 family proteins and JNKs in cytochrome c release in the peripheral ischemic stroke associated apoptotic neurons. The review ends with a note on designing new anti-stroke treatments., Highlights • Penumbral cells undergo mitochondrion-mediated apoptosis during ischemic stroke. • Mechanisms of penumbral cell mitochondrion-mediated apoptosis are unknown. • VDAC, JNKs and Bcl-2 family proteins play important role in neuronal apoptosis. • The review addresses the putative role of above proteins in penumbral cell apoptosis.

The molecular mechanisms of hepatotoxicity after propofol anaesthesia have not been fully elucidated, although there is a relation with mitochondrial dysfunction. The action of propofol on mitochondrial hepatic functions in a rat model was evaluated by infusion for 4h with 25 and 62.5mg/kg/h propofol or 3.125ml/kg/h (vehicle). Liver mitochondrial respiratory rates were evaluated as well as mitochondrial transmembrane potential (ΔΨ), calcium fluxes, mitochondrial enzymatic activities (Complex I-V) and oxidative stress biomarkers (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, lipid peroxidation and the oxidised/reduced glutathione ratio). Biophysical interactions with membrane models were also performed. The mitochondrial transmembrane potential was decreased and the opening time of the mitochondrial permeability transition pore was slightly reduced for the highest dose. The activity of complex II was stimulated by propofol, which also causes fluctuations on some respiratory parameters, whereas the antioxidant system was affected in a nonspecific manner. Fluorescence quenching studies suggested that propofol is preferably located in deeper regions of the bilayer and has a high affinity to mitochondrial membranes. It is suggested that propofol interacts with liver mitochondrial membranes with mild modification in mitochondrial function.

CCAR2 (cell cycle and apoptosis regulator protein 2; formerly DBC1, deleted in breast cancer 1) functions in diverse cellular processes including responses to genotoxic and metabolic stresses. However, its role in the mitochondrial stress response has not been fully elucidated. To investigate how CCAR2 regulates stress response, we purified CCAR2-containing complexes. Interestingly, the results revealed that CCAR2 localized to the mitochondria, and also bound Hsp60 (heat shock protein 60), a mitochondrial chaperone. The binding of CCAR2 to Hsp60 increased following rotenone-induced mitochondrial stress. The deficiencies in CCAR2 and Hsp60 also disrupted the mitochondrial membrane potential, thereby promoting apoptosis following mitochondrial stress. In summary, the CCAR2-Hsp60 complex promoted cell survival during mitochondrial stress-induced apoptosis. These data suggest that CCAR2 is critical for maintaining mitochondrial homeostasis in response to stress.

Vancomycin (VCM) is a first-line antibiotic for serious infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is one of the most complaint in VCM therapy. We previously reported that VCM induced apoptosis in a porcine proximal tubular epithelial cell line (LLC-PK1), in which mitochondrial complex I may generate superoxide, leading to cell death. In the present study, VCM caused production of mitochondrial reactive oxygen species and peroxidation of the mitochondrial phospholipid cardiolipin that was reversed by administration of the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). FCCP also significantly suppressed VCM-induced depolarization of the mitochondrial membrane and apoptosis. Moreover, the lipophilic antioxidant vitamin E and a mitochondria-targeted antioxidant, mitoTEMPO, also significantly suppressed VCM-induced depolarization of mitochondrial membrane and apoptosis, whereas vitamin C, n-acetyl cysteine, or glutathione did not provide significant protection. These findings suggest that peroxidation of the mitochondrial membrane cardiolipin mediated the VCM-induced production of intracellular reactive oxygen species and initiation of apoptosis in LLC-PK1 cells. Furthermore, regulation of mitochondrial function using a mitochondria-targeted antioxidant, such as mitoTEMPO, may constitute a potential strategy for mitigation of VCM-induced proximal tubular epithelial cell injury.

Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

During starvation, intra-mitochondrial sirtuins, NAD+ sensitive deacylating enzymes that modulate metabolic homeostasis and survival, directly adjust mitochondrial function to nutrient availability; concomitantly, mitochondria elongate to escape autophagic degradation. However, whether sirtuins also impinge on mitochondrial dynamics is still uncharacterized. Here we show that the mitochondrial Sirtuin 5 (Sirt5) is essential for starvation induced mitochondrial elongation. Deletion of Sirt5 in mouse embryonic fibroblasts increased levels of mitochondrial dynamics of 51kDa protein and mitochondrial fission protein 1, leading to mitochondrial accumulation of the pro-fission dynamin related protein 1 and to mitochondrial fragmentation. During starvation, Sirt5 deletion blunted mitochondrial elongation, resulting in increased mitophagy. Our results indicate that starvation induced mitochondrial elongation and evasion from autophagic degradation requires the energy sensor Sirt5.

Hsp60 is a typical mitochondrial protein in eukaryotes, and is involved in facilitating the correction of misfolded protein back into the correct conformation. Previous, we identified aspirin-induced HSPs in response to heat stress [1]. To investigate whether Hsp60 can protect against death under heat stress, we used lenti-siRNA to knock down the expression of Hsp60. When exposed to heat stress, more apoptosis was observed with increasing exposure to heat stress, while necrosis was not affected. Furthermore, heat stress induced the loss of mitochondrial membrane potential (ΔΨm) and a significant increase of reactive oxygen species (ROS) produced in mitochondria as measured by TMRE and MitoSOXTM red. The loss of ΔΨm indicated a change in inner mitochondrial function. Real-time Quantitative PCR was used to investigate the mechanism by detecting mRNA expression profile of the inner mitochondrial membrane, including CypD, ANT, and PIC. Results showed no differences between lenti-siRNA Hsp60 and control. However, bax in the cytoplasm translocated to mitochondrial during heat stress and regulated the permeability of outer mitochondrial membrane. We hypothesize that Hsp60 can protect cardiac myocytes against apoptosis involving in outer mitochondrial membrane not the inner mitochondrial membrane under heat stress.

Mitochondrial proteases ensure mitochondrial integrity and function after oxidative stress by providing mitochondrial protein quality control. However, the molecular mechanisms that regulate this basic biological function in eukaryotic cells remain largely unknown. Caveolin-1 is a scaffolding protein involved in signal transduction. We find that AFG3L2, a m-AAA type of mitochondrial protease, is a novel caveolin-1-interacting protein in vitro. We show that oxidative stress promotes the translocation of both caveolin-1 and AFG3L2 to mitochondria, enhances the interaction of caveolin-1 with AFG3L2 in mitochondria and stimulates mitochondrial protease activity in wild-type fibroblasts. Localization of AFG3L2 to mitochondria after oxidative stress is inhibited in fibroblasts lacking caveolin-1, which results in impaired mitochondrial protein quality control, an oxidative phosphorylation to aerobic glycolysis switch and reduced ATP production. Mechanistically, we demonstrate that a lack of caveolin-1 does not alter either mitochondrial number or morphology but leads to the cytoplasmic and proteasome-dependent degradation of complexes I, III, IV and V upon oxidant stimulation. Restoration of mitochondrial respiratory chain complexes in caveolin-1 null fibroblasts reverts the enhanced glycolysis observed in these cells. Expression of a mutant form of AFG3L2, which has reduced affinity for caveolin-1, fails to localize to mitochondria and promotes degradation of complex IV after oxidative stress. Thus, caveolin-1 maintains mitochondrial integrity and function when cells are challenged with free radicals by promoting the mitochondrial localization of m-AAA protease and its quality control functions.

During apoptosis mitochondria undergo cristae remodeling and fragmentation, but how the latter relates to outer membrane permeabilization and downstream caspase activation is unclear. Here we show that the mitochondrial fission protein Dynamin Related Protein (Drp) 1 participates in cytochrome c release by selected intrinsic death stimuli. While Bax, Bak double deficient (DKO) and Apaf1(-/-) mouse embryonic fibroblasts (MEFs) were less susceptible to apoptosis by Bcl-2 family member BID, H(2)O(2), staurosporine and thapsigargin, Drp1(-/-) MEFs were protected only from BID and H(2)O(2). Resistance to cell death of Drp1(-/-) and DKO MEFs correlated with blunted cytochrome c release, whereas mitochondrial fragmentation occurred in all cell lines in response to all tested stimuli, indicating that other mechanisms accounted for the reduced cytochrome c release. Indeed, cristae remodeling was reduced in Drp1(-/-) cells, potentially explaining their resistance to apoptosis. Our results indicate that caspase-independent mitochondrial fission and Drp1-dependent cristae remodeling amplify apoptosis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson’s disease-associated protein—parkin, which biochemically and genetically interacts with Drp1—in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division.

To uptake calcium ions of mitochondria is of significant functional connotation for cells, because calcium ions in mitochondria are involved in energy production, regulatory signals transfer, and mitochondrial permeability transition pore opening and even programmed cell death of apoptosis, further playing more roles in plant productivity and quality. Cytoplasmic calcium ions access into outer mitochondrial membrane (OMM) from voltage dependent anion-selective channel (VDAC) and were absorbed into inner mitochondrial membrane (IMM) by mitochondrial calcium uniporter (MCU), rapid mitochondrial calcium uptake (RaM) or mitochondrial ryanodine receptor (mRyR). Although both mitochondria and the mechanisms of calcium transport have been extensively studied, but there are still long-standing or even new challenges. Here we review the history and recent discoveries of the mitochondria calcium ions channel complex involved calcium assimilation, and discuss the role of calcium ions into mitochondria.

Mitochondria form a tubular network in mammalian cells, and the mitochondrial life cycle is determined by fission, fusion and autophagy. Dynamin-related protein 1 (Drp1) has a pivotal role in these processes because it alone is able to constrict mitochondria. However, the regulation and function of Drp1 have been shown to vary between cell types. Mitochondrial morphology affects mitochondrial metabolism and function. In pancreatic beta cells mitochondrial metabolism is a key component of the glucose-induced cascade of insulin secretion. The goal of the present study was to investigate the action of Drp1 in pancreatic beta cells. For this purpose Drp1 was down-regulated by means of shDrp1 in insulin-secreting INS1 cells and mouse pancreatic islets. In INS1 cells reduced Drp1 expression resulted in diminished expression of proteins regulating mitochondrial fusion, namely mitofusin 1 and 2, and optic atrophy protein 1. Diminished mitochondrial dynamics can therefore be assumed. After down-regulation of Drp1 in INS1 cells and spread mouse islets the initially homogenous mitochondrial network characterised by a moderate level of interconnections shifted towards high heterogeneity with elongated, clustered and looped mitochondria. These morphological changes were found to correlate directly with functional alterations. Mitochondrial membrane potential and ATP generation were significantly reduced in INS1 cells after Drp1down-regulation. Finally, a significant loss of glucose-stimulated insulin secretion was demonstrated in INS1 cells and mouse pancreatic islets. In conclusion, Drp1 expression is important in pancreatic beta cells to maintain the regulation of insulin secretion.

Previous study has demonstrated that mitochondrial-dependent apoptotic pathway is involved in the nephroprotective effect of puerarin (PU) against lead-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells. To further clarify how PU exerts its antiapoptotic effects, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and subsequent apoptotic events in the process of PU against Pb-induced cytotoxicity in rPT cells. The results showed that Pb-mediated mitochondrial permeability transition pore (MPTP) opening together with mitochondrial cytochrome c release, activations of caspase-9 and caspase-3, and subsequent poly-ADP-ribose polymerase (PARP) cleavage can be effectively blocked by the addition of PU. Simultaneously, upregulation and downregulation of Bcl-2 and Bax with increased Bcl-2/Bax ratio due to PU administration further alleviated Pb-induced mitochondrial apoptosis. Moreover, PU can reverse Pb-induced ATP depletion by restoring mitochondrial fragmentation to affect ATP production and by regulating expression levels of ANT-1 and ANT-2 to improve ATP transport. In summary, PU produced a significant protection against Pb-induced mitochondrial apoptosis in rPT cells by inhibiting MPTP opening to ameliorate the mitochondrial dysfunction.

After cholesterol is transported into the mitochondria of steroidogenic tissues, the first steroid, pregnenolone, is synthesized in adrenal and gonadal tissues to initiate steroid synthesis by catalyzing the conversion of pregnenolone to progesterone, which is mediated by the inner mitochondrial enzyme 3β-hydroxysteroid dehydrogenase 2 (3βHSD2). We report that the mitochondrial translocase Tom22 is essential for metabolic conversion, as its knockdown by small interfering RNA (siRNA) completely ablated progesterone conversion in both steroidogenic mouse Leydig MA-10 and human adrenal NCI cells. Tom22 forms a 500-kDa complex with mitochondrial proteins associated with 3βHSD2. Although the absence of Tom22 did not inhibit mitochondrial import of cytochrome P450scc (cytochrome P450 side chain cleavage enzyme) and aldosterone synthase, it did inhibit 3βHSD2 expression. Electron microscopy showed that Tom22 is localized at the outer mitochondrial membrane (OMM), while 3βHSD2 is localized at the inner mitochondrial space (IMS), where it interacts through a specific region with Tom22 with its C-terminal amino acids and a small amino acid segment of Tom22 exposed to the IMS. Therefore, Tom22 is a critical regulator of steroidogenesis, and thus, it is essential for mammalian survival.

Mitochondrial dysfunction is one of the major contributors to neurodegenerative disorders including Parkinson disease. The mitochondrial permeability transition pore is a protein complex located on the mitochondrial membrane. Under cellular stress, the pore opens, increasing the release of pro-apoptotic proteins, and ultimately resulting in cell death. MicroRNA-7 (miR-7) is a small non-coding RNA that has been found to exhibit a protective role in the cellular models of Parkinson disease. In the present study, miR-7 was predicted to regulate the function of mitochondria, according to gene ontology analysis of proteins that are down-regulated by miR-7. Indeed, miR-7 overexpression inhibited mitochondrial fragmentation, mitochondrial depolarization, cytochrome c release, reactive oxygen species generation, and release of mitochondrial calcium in response to 1-methyl-4-phenylpyridinium (MPP(+)) in human neuroblastoma SH-SY5Y cells. In addition, several of these findings were confirmed in mouse primary neurons. Among the mitochondrial proteins identified by gene ontology analysis, the expression of voltage-dependent anion channel 1 (VDAC1), a constituent of the mitochondrial permeability transition pore, was down-regulated by miR-7 through targeting 3'-untranslated region of VDAC1 mRNA. Similar to miR-7 overexpression, knockdown of VDAC1 also led to a decrease in intracellular reactive oxygen species generation and subsequent cellular protection against MPP(+). Notably, overexpression of VDAC1 without the 3'-UTR significantly abolished the protective effects of miR-7 against MPP(+)-induced cytotoxicity and mitochondrial dysfunction, suggesting that the protective effect of miR-7 is partly exerted through promoting mitochondrial function by targeting VDAC1 expression. These findings point to a novel mechanism by which miR-7 accomplishes neuroprotection by improving mitochondrial health.

To investigate the biological effects of a 50-Hz magnetic field (MF) on mitochondrial permeability.Human amniotic epithelial cells were exposed to MF (50 Hz, 0.4 mT) for different durations. Mitochondrial permeability, mitochondrial membrane potential (ΔΨm), cytochrome c (Cyt-c) release and the related mechanisms were explored.Exposure to the MF at 0.4 mT for 60 min transiently induced mitochondrial permeability transition (MPT) and Cyt-c release, although there was no significant effect on mitochondrial membrane potential (ΔΨm). Other than decreasing the total Bcl-2 associated X protein (Bax) level, MF exposure did not significantly affect the levels of Bax and B-cell lymphoma-2 (Bcl-2) in mitochondria. In addition, cells exposed to the MF showed increased intracellular reactive oxidative species (ROS) levels and glycogen synthase kinase-3β (GSK-3β) dephosphorylation at 9 serine residue (Ser(9)). Moreover, the MF-induced MPT was attenuated by ROS scavenger (N-acetyl-L-cysteine, NAC) or GSK-3β inhibitor, and NAC pretreatment prevented GSK-3β dephosphorylation (Ser(9)) caused by MF exposure.MPT induced by MF exposure was mediated through the ROS/GSK-3β signaling pathway.

P53 is known as a transcription factor to control apoptotic cell death through regulating a series of target genes in nucleus. There is accumulating evidences show that p53 can directly induce cell apoptosis through transcription independent way at mitochondria. However, the mechanism by which p53 translocation into mitochondria in response to oxidative stress remains unclear. Here, glucose oxidase (GOX) was used to induce ROS generation in HepG2 cells and liver tissues of mice. The results showed that p53 was stabilized and translocated to mitochondria in a time and dose dependent manner after GOX exposure. Interestingly, as an inhibitor of mitochondrial permeability transition, cyclosporine A (CsA) was able to effectively reduce GOX mediated mitochondrial p53 distribution without influencing on the expression of p53 target genes including Bcl-2 and Bax. These indicated that CsA could just block p53 entering into mitochondria, but not affect p53-dependent transcription. Meanwhile, CsA failed to inhibit the ROS generation induced by GOX, which indicated that CsA had no antioxidant function. Moreover, GOX induced typical apoptosis characteristics including, mitochondrial dysfunction, accumulation of Bax and release of cytochrome C in mitochondria, accompanied with activation of caspase-9 and caspase-3. These processions were suppressed after pretreatment with CsA and pifithrin-μ (PFT-μ, a specific inhibitor of p53 mitochondrial translocation). In vivo, CsA was able to attenuate p53 mitochondrial distribution and protect mice liver against from GOX mediated apoptotic cell death. Taken together, these suggested that CsA could suppress ROS-mediated p53 mitochondrial distribution and cell apoptosis depended on its inhibition effect to mitochondrial permeability transition. It might be used to rescue the hepatic cell apoptosis in the patients with acute liver injury.

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity.

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak.

Although palmitoleic acid (C16:1) is associated with arrhythmias, and increases in an age-dependent matter, the effects of L-carnitine, which is essential for the transport of long-chain fatty acids into the mitochondria, are unclear. It has been postulated that L-carnitine may attenuate palmitate (C16:0)-induced mitochondrial dysfunction and the apoptosis of cardiomyocytes. The aim of this study was to elucidate the activity of L-carnitine in the prevention of the palmitoleic acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoleoyl-CoA-induced mitochondrial respiration was not accelerated by L-carnitine treatment, and this respiration was slightly inhibited by oligomycin, which is an inhibitor of ATP synthase. Despite pretreatment with L-carnitine, the mitochondrial membrane potential decreased and mitochondrial swelling was induced by palmitoleoyl-CoA. In the presence of a combination of L-carnitine and tiron, a free radical scavenger, there was attenuated mitochondrial swelling and cytochrome c release following palmitoleoyl-CoA treatment. We concluded that palmitoleic acid, but not palmitate, induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

Mitochondrial connexin 43 (Cx43) is important in cardioprotection by ischemic preconditioning; however, whether mitochondrial Cx43 is involved in mitochondrial dysfunction in the pathogenesis of dilated cardiomyopathy (DCM) remains to be elucidated. The present study was performed to investigate the changes in expression and the phosphorylation state of mitochondrial Cx43 in a rat model of DCM, and to determine whether the altered phosphorylation state of mitochondrial Cx43 was involved in mitochondrial dysfunction. A rat model of DCM was generated by daily oral administration of furazolidone (FZD) for 30 weeks. Reverse transcription polymerase chain reaction and western blot analysis revealed a decrease in the overall expression of Cx43, accompanied by reduced levels of serine 368‑phosphorylated‑Cx43 immunoreactivity in the myocardium and myocardial mitochondria. In addition, the mitochondrial membrane potential and the activities of cytochrome c oxidase, succinate dehydrogenase and protein kinase C (PKC) e were all significantly reduced compared with those of the control group. Phorbol‑12‑myristate‑13‑acetate (PMA), a specific PKC activator, partially reversed the FZD‑induced mitochondrial Cx43 dephosphorylation at serine 368 and mitochondrial dysfunction in the cardiomyocytes. However, pretreatment with 18β‑glycerrhetinic acid, a connexin channel inhibitor, eliminated the mitochondrial protective effect of PMA in the cardiomyocytes sparsely plated without cell to cell contact. These results suggested that dephosphorylation of mitochondrial Cx43 at serine 368, due to the suppression of PKCe activity, may be a novel mechanism for mitochondrial dysfunction in the pathogenesis of DCM.

Mitochondrial dysfunction is implicated in age-related degenerative disorders such as Alzheimer's disease (AD). Maintenance of mitochondrial dynamics is essential for regulating mitochondrial function. Aβ oligomers (AβOs), the typical cause of AD, lead to mitochondrial dysfunction and neuronal loss. AβOs have been shown to induce mitochondrial fragmentation, and their inhibition suppresses mitochondrial dysfunction and neuronal cell death. Oxidative stress is one of the earliest hallmarks of AD. Cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by disrupting the antioxidant system, including Prx2. Cdk5 is also regarded as a modulator of mitochondrial fission; however, a precise mechanistic link between Cdk5 and mitochondrial dynamics is lacking. We estimated mitochondrial morphology and alterations in mitochondrial morphology-related proteins in Neuro-2a (N2a) cells stably expressing the Swedish mutation of amyloid precursor protein (APP), which is known to increase AβO production. We demonstrated that mitochondrial fragmentation by AβOs accompanies reduced mitofusin 1 and 2 (Mfn1/2) levels. Interestingly, the Cdk5 pathway, including phosphorylation of the Prx2-related oxidative stress, has been shown to regulate Mfn1 and Mfn2 levels. Furthermore, Mfn2, but not Mfn1, over-expression significantly inhibits the AβO-mediated cell death pathway. Therefore, these results indicate that AβO-mediated oxidative stress triggers mitochondrial fragmentation via decreased Mfn2 expression by activating Cdk5-induced Prx2 phosphorylation. Mitochondrial fragmentation induced by amyloid-beta oligomer (AβOs) which is generated from the Swedish mutation of amyloid precursor protein (APP) accompanies reduced Mfn1/2 levels. Interestingly, the Cdk5 pathway, including phosphorylation of the Prx2-related oxidative stress, has been shown to regulate Mfn1/2. Furthermore, Mfn2 over-expression significantly inhibits the AβO-mediated neuronal cells death pathway, but not Mfn1 over-expression. Therefore, these results indicate that AβO-mediated oxidative stress triggers mitochondrial fragmentation via decreased Mfn2 expression by activating Cdk5-induced Prx2 phosphorylation. ATP, adenosine triphosphate; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; Cdk5, Cyclin-dependent kinase; Cyt C, cytochrome C; Mfn2, mitofusin 2; Prx2, peroxiredoxin 2; ROS, reactive oxygen species.

Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.

Review focuses on the analysis of literature data and own results concerning properties of mitochondria Ca2+ transporting systems and their regulation. Three mechanisms for mitochondrial Ca2+ uptake are described: the electrogenic mitochondrial Ca2+ uniporter, a rapid mode of Ca2+ uptake (RaM) and ryanodine-sensitive Ca2+ channels (RyR). Two mechanisms of Ca2+ efflux from the mitochondrial matrix have been proposed: Na+-dependent and Na+-independent Ca2+ exchange. The mitochondrial permeability transition pore can also function as a Ca2+ efflux mechanism. Ca2+ accumulation in mitochondria resulted in an increase of mitochondrial membrane potential, ATP synthesis and activation of metabolites transport. The dissipation of mitochondrial membrane potential should prevent mitochondrial Ca2+ uptake. Our data concerning depolarizing effect of calmodulin antagonists and hyperpolarizing effect of calix[4]arenes C-136 and C-137 on the myometrium mitochondria can be applied on the demand of mitochondria membrane potential correction.

Pb2+ exposure in humans occurs mainly through air inhalation, food and water uptake which has been shown to be generally associated with numerous body functions such as the central and peripheral nervous systems, the red blood cells, the kidneys and the liver. It has been reported that the liver is the storage site and an important primary target in Pb2+ toxicity, and the hepatotoxicity of Pb2+ could be resulted from the impairment of the liver mitochondria. In this study, several mitochondrial dysfunctions following the addition of Pb2+ (10-160 μM) were investigated. We found that Pb2+ inhibited the enzyme activities of mitochondrial respiratory complexes and complex III was the major source of Pb2+-induced significant reactive oxygen species (ROS) formation. As a consequence, our results showed that Pb2+ induced significant progress in mitochondrial lipid peroxidation, adenosine triphosphate (ATP) consumption and glutathione (GSH) oxidation. On the other hand, Pb2+ induced marked changes in mitochondrial permeability transition (MPT) accompanied by mitochondrial swelling, mitochondrial membrane potential collapse, mitochondrial membrane fluidity decrease and cytochrome c (Cyt c) release. Additionally, several mitochondrial MPT inhibitors and chelators were utilized to determine the possible interaction sites of Pb2+ on mitochondria. In general, our data supported that the Pb2+-induced liver toxicity was a result of the disruptive effect on the mitochondrial respiratory complexes. This disruptive effect caused oxidative stress and MPT, which led to mitochondrial dysfunctions and even cell death signalling via mitochondrial permeability transition pore (MPTP) opening and Cyt c release.

Mitochondria play an important role in tissue ischemia and reperfusion (IR) injury, with energetic failure and the opening of the mitochondrial permeability transition pore being the major causes of IR-induced cell death. Thus, mitochondria are an appropriate focus for strategies to protect against IR injury. Two widely studied paradigms of IR protection, particularly in the field of cardiac IR, are ischemic preconditioning (IPC) and volatile anesthetic preconditioning (APC). While the molecular mechanisms recruited by these protective paradigms are not fully elucidated, a commonality is the involvement of mitochondrial K+ channel opening. In the case of IPC, research has focused on a mitochondrial ATP-sensitive K+ channel (mitoKATP), but, despite recent progress, the molecular identity of this channel remains a subject of contention. In the case of APC, early research suggested the existence of a mitochondrial large-conductance K+ (BK, big conductance of potassium) channel encoded by the Kcnma1 gene, although more recent work has shown that the channel that underlies APC is in fact encoded by Kcnt2. In this review, we discuss both the pharmacologic and genetic evidence for the existence and identity of mitochondrial K+ channels, and the role of these channels both in IR protection and in regulating normal mitochondrial function.

Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis. Published version

Chondrocytes that are embedded within the growth plate or the intervertebral disc are sensitive to environmental stresses, such as inflammation and hypoxia. However, little is known about the molecular signalling pathways underlining the hypoxia-induced mitochondrial dysfunction and apoptosis in chondrocytes. In the present study, we firstly examined the hypoxia-induced apoptosis, mitochondrial dysfunction and the activation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signalling in human chondrocyte cell line, C28/I2 and then investigated the regulatory role of RhoA, a well-recognized apoptosis suppressor, in such process, with gain-of-function strategy. Our results indicated that hypoxia induced apoptosis and inhibited CREB phosphprylation in chondrocytes, meanwhile, the dysfunctional mitochondria with up-regulated mitochondrial superoxide and reactive oxygen species (ROS) levels, whereas with a reduced mitochondrial membrane potential (MMP) and Complex IV activity were observed in the hypoxia-treated C28/I2 cells. However, the overexpressed RhoA blocked the hypoxia-mediated reduction in CREB phosphprylation and inhibited the apoptosis induction, along with an ameliorated mitochondrial function in the hypoxia-treated C28/I2 cells. In conclusion, the present study confirmed the reduced CREB phosphorylation, along with the apoptosis induction and mitochondrial dysfunction in the hypoxia-treated chondrocyte cells. And the overexpression of RhoA ameliorated the hypoxia-induced mitochondrial dysfunction and apoptosis via blocking the hypoxia-mediated reduction in CREB phosphorylation.

Signal transducer and activator of transcription 3 (STAT3) is associated with various physiological and pathological functions, mainly as a transcription factor that translocates to the nucleus upon tyrosine phosphorylation induced by cytokine stimulation. In addition, a small pool of STAT3 resides in the mitochondria, where it serves as a sensor for various metabolic stressors including reactive oxygen species (ROS). Mitochondrially localized STAT3 largely exerts its effects through direct or indirect regulation of the activity of the electron transport chain (ETC). It has been assumed that the amounts of STAT3 in the mitochondria are static. We showed that various stimuli, including oxidative stress and cytokines, triggered a signaling cascade that resulted in a rapid loss of mitochondrially localized STAT3. Recovery of the mitochondrial pool of STAT3 over time depended on phosphorylation of Ser727 in STAT3 and new protein synthesis. Under these conditions, mitochondrially localized STAT3 also became competent to bind to cyclophilin D (CypD). Binding of STAT3 to CypD was mediated by the amino terminus of STAT3, which was also important for reducing mitochondrial ROS production after oxidative stress. These results outline a role for mitochondrially localized STAT3 in sensing and responding to external stimuli.

Proteins of the B-cell lymphoma-2 (BCL-2) family control the intrinsic apoptosis pathway. The pro-apoptotic BCL-2 proteins BAX and BAK can commit a cell to its programmed death by permeabilizing the outer mitochondrial membrane (OMM) and subsequent initiation of the caspase cascade. Therefore, the activities of BAX and BAK are precisely controlled by a complex network of proteins inside and outside the BCL-2 family. Cells survive by constant control of dynamic translocation and retrotranslocation of BAX and BAK to the mitochondria and back into the cytosol. Recent insights into BAX/BAK shuttling, BCL-2 protein interactions, the role of BH3-only proteins in apoptosis signaling and the active BAX complex set the stage for the development of novel strategies in cancer therapy and the analysis of cellular predisposition to apoptosis.

Mitochondrial dynamics, including constant fusion and fission, play critical roles in maintaining mitochondrial morphology and function. Here, we report that developmentally regulated GTP-binding protein 2 (DRG2) regulates mitochondrial morphology by modulating the expression of the mitochondrial fission gene dynamin-related protein 1 (Drp1). shRNA-mediated silencing of DRG2 induced mitochondrial swelling, whereas expression of an shRNA-resistant version of DRG2 decreased mitochondrial swelling in DRG2-depleted cells. Analysis of the expression levels of genes involved in mitochondrial fusion and fission revealed that DRG2 depletion significantly decreased the level of Drp1. Overexpression of Drp1 rescued the defect in mitochondrial morphology induced by DRG2 depletion. DRG2 depletion reduced the mitochondrial membrane potential, oxygen consumption rate (OCR), and amount of mitochondrial DNA (mtDNA), whereas it increased reactive oxygen species (ROS) production and apoptosis. Taken together, our data demonstrate that DRG2 acts as a regulator of mitochondrial fission by controlling the expression of Drp1.

In this work, we explored the protective effect of DT-018, a danshensu and tetramethylpyrazine conjugate, on mitochondrial injury induced by tert-butylhydroperoxide (t-BHP) and its possible mechanisms of action. DT-018 effectively quenched intracellular and mitochondrial reactive oxygen species (ROS), preserved the mitochondrial function, restored the intracellular level of ATP and augmented mitochondrial membrane potential (Δψm) while suppressed mitochondrial swelling in isolated myocardial mitochondria. DT-018 increased the expression of MEF2D and PGC-1α as well as Nrf2 and Tfam in H9c2 cells. Transfection of MEF2D-siRNA and PGC-1α-siRNA down-regulated the protein expression of PGC-1α and partially abolished the cardioprotection of DT-018 in t-BHP injured cells. For the in vivo studies, administration of DT-018 significantly alleviated infarct area induced by ischemia and reperfusion injury. These observations demonstrated that the cardioprotective effect of DT-018 is mediated, at least in part, by preservation of mitochondrial integrity through up-regulation of MEF2D/PGC-1α pathway.

Permeabilization of the outer mitochondrial membrane is an integral step in apoptosis. The resulting release of pro-apoptotic signaling proteins leads to cell destruction through activation of the cysteine-aspartic protease (caspase) cascade. However, the mechanism of outer mitochondrial membrane (OMM) permeabilization remains unclear. It was recently shown that cytochrome c can induce pore formation in cardiolipin-containing phospholipid membranes, leading to large dextran and protein permeability. In this work, the interaction of cytochrome c with cardiolipin-containing phospholipid vesicles, serving as models of the OMM, is investigated to probe cytochrome c-induced permeability. Lipid vesicles having either a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixed-DPPC/cardiolipin membrane and containing a membrane-impermeable Raman tracer 3-nitrobenzenesulfonate (3-NBS) were optically trapped, translated into a solution containing cytochrome c, and monitored for 3-NBS leakage. Cytochrome-correlated leakage was observed only in cardiolipin-containing vesicles. Structural changes observed in the Raman spectra during permeabilization indicated acyl chain disordering along with decreased intensity of the cardiolipin cis-double-bond stretching modes. When the vesicle-associated cytochrome c Raman spectrum is compared with a spectrum in buffer, heme-resonance bands are absent, indicating loss of Met-80 coordination. To verify selective interactions of cytochrome c with cardiolipin, these experiments were repeated where the DPPC acyl chains were deuterated (D62-DPPC), allowing spectral resolution of the DPPC acyl chain response from that of cardiolipin. Interestingly, D62-DPPC acyl chains were unaffected by cytochrome c accumulation, while cardiolipin showed major changes in acyl chain structure. These results suggest that cytochrome-induced permeabilization proceeds through selective interaction of cytochrome c with cardiolipin, resulting in protein unfolding, where the unfolded form interacts with cardiolipin acyl chains within the bilayer to induce permeability.

MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria., Highlights • MLN64 overexpression induces an increase in mitochondrial cholesterol content. • MLN64 protein expression is increased in NPC cells. • Down-regulation of MLN64 restores mitochondrial membrane potential and superoxide levels in NPC cells. • MLN64 overexpression produces mitochondrial dysfunction., Graphical abstract fx1

Due to the high metabolic demands of the placental tissue during gestation, we decide to analyzed the mitochondrial bioenergetic functions in the human term placenta. Different mitochondrial morphological parameters, membrane potential and cardiolipin content were determined by flow cytometry. Oxygen uptake, hydrogen peroxide production and cytochrome P450 content, were also measured. Some apoptotic mitochondrial proteins were also analyzed by western blot. Two isolated mitochondrial fractions were observed: large/heavy and small/light with different functional characteristics. Oxygen uptake showed a respiratory control (RC) of 3.4±0.3 for the heavy mitochondria, and 1.1±0.4 for light mitochondria, indicating a respiratory dysfunction in the light fraction. Good levels of polarization were detected in the heavy fraction, meanwhile the light population showed a collapsed ΔΨm. Increased levels of cytochrome P450, higher levels of hydrogen peroxide, and low cardiolipin content were described for the light fraction. Three pro-apoptotic proteins p53, Bax, and cytochrome c were found increased in the heavy mitochondrial fraction; and deficient in the light fraction. The heavy mitochondrial fraction showed an improved respiratory function. This mitochondrial fraction, being probably from cytotrophoblast cells showed higher content of proteins able to induce apoptosis, indicating that these cells can effectively execute an apoptotic program in the presence of a death stimulus. Meanwhile the light and small organelles probably from syncytiotrophoblast, with a low oxygen metabolism, low level ofΔΨm, and increased hydrogen peroxide production, may not actively perform an apoptotic process due to their deficient energetic level. This study contributes to the characterization of functional parameters of human placenta mitochondria in order to understand the oxygen metabolism during the physiological process of gestation. Fil: Bustamante, Jaunita. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Ramírez Vélez, Robinson. Universidad del Valle. Facultad de Medicina; Colombia Fil: Czerniczyniec, Analia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Cicerchia, Daniela Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Aguilar de Plata, Ana C.. Universidad del Valle. Facultad de Medicina; Colombia Fil: Lores Arnaiz, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina

Aims: Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Innovation: Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863–879.

Mutations in SURF1 (surfeit locus protein 1) COX (cytochrome c oxidase) assembly protein are associated with Leigh's syndrome, a human mitochondrial disorder that manifests as severe mitochondrial phenotypes and early lethality. In contrast, mice lacking the SURF1 protein ( Surf1 −/−) are viable and were previously shown to have enhanced longevity and a greater than 50% reduction in COX activity. We measured mitochondrial function in heart and skeletal muscle, and despite the significant reduction in COX activity, we found little or no difference in ROS (reactive oxygen species) generation, membrane potential, ATP production or respiration in isolated mitochondria from Surf1−/− mice compared with wild-type. However, blood lactate levels were elevated and Surf1−/− mice had reduced running endurance, suggesting compromised mitochondrial energy metabolism in vivo . Decreased COX activity in Surf1−/− mice is associated with increased markers of mitochondrial biogenesis [PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α) and VDAC (voltage-dependent anion channel)] in both heart and skeletal muscle. Although mitochondrial biogenesis is a common response in the two tissues, skeletal muscle has an up-regulation of the UPRMT (mitochondrial unfolded protein response) and heart exhibits induction of the Nrf2 (nuclear factor-erythroid 2-related factor 2) antioxidant response pathway. These data are the first to show induction of the UPRMT in a mammalian model of decreased COX activity. In addition, the results of the present study suggest that impaired mitochondrial function can lead to induction of mitochondrial stress pathways to confer protective effects on cellular homoeostasis. Abbreviations: ARE, antioxidant response element; CHOP, C/EBP (CCAAT/enhancer-binding protein)-homologous protein; ClpP, caseinolytic peptidase; COX, cytochrome c oxidase; DCIP, dichlorophenol-indophenol; DIPPMPO, 5-(di-isopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; ETC, electron transport chain; 18FDG, 2-[18F]fluoro-2-deoxy-D-glucose; HO-1, haem oxygenase 1; Hsp60, heat-shock protein 60; MURE, mitochondrial unfolded response element; Nrf2, nuclear factor-erythroid 2-related factor 2; PET, positron-emission tomography; PGC-1α, peroxisome-proliferator-activated receptor γ co-activator 1α; RCR, respiratory control ratio; ROS, reactive oxygen species; SOD, superoxide dismutase; SURF1, surfeit locus protein 1; SUV, standardized uptake value; TFAM, mitochondrial transcription factor A; Trx, thioredoxin; UPRMT, mitochondrial unfolded protein response; VDAC, voltage-dependent anion channel

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.

Mitochondria are the most important sensor for apoptosis. Extracellular adenosine is well reported to induce apoptosis of tumor cells. Here we found that extracellular adenosine suppresses the cell growth by induction of apoptosis in BEL-7404 liver cancer cells, and identified a novel mechanism that extracellular adenosine triggers apoptosis by increasing Reactive Oxygen Species (ROS) production and mitochondrial membrane dysfunction in the cells. We observed that adenosine increases ROS production, activates c-Caspase-8 and -9 and Caspase effectors, c-Caspase-3 and c-PARP, induces accumulation of apoptosis regulator Bak, decreases Bcl-xL and Mcl-1, and causes the mitochondrial membrane dysfunction and the release of DIABLO, Cytochrome C, and AIF from mitochondria to cytoplasm in the cells; ROS inhibitor, NAC significantly reduces adenosine-induced ROS production; it also shows the same degree of blocking adenosine-induced loss of mitochondrial membrane potential (MMP) and apoptosis. Our study first observed that adenosine increases ROS production in tumor cells and identified the positive feedback loop for ROS-mediated mitochondrial membrane dysfunction which amplifies the death signals in the cells. Our findings indicated ROS production and mitochondrial dysfunction play a key role in adenosine-induced apoptosis of 7404 cells.

Dynamin-related membrane remodeling proteins regulate mitochondrial morphology by mediating fission and fusion. Although mitochondrial morphology is considered an important factor in maintaining mitochondrial function, a direct mechanistic link between mitochondrial morphology and function has not been defined. We report here a previously unrecognized cellular process of transient contraction of the mitochondrial matrix. Importantly, we found that this transient morphological contraction of mitochondria is accompanied by a reversible loss or decrease of inner membrane potential. Fission deficiency greatly amplified this phenomenon, which functionally exhibited an increase of inner membrane proton leak. We found that electron transport activity is necessary for the morphological contraction of mitochondria. Furthermore, we discovered that silencing the inner membrane-associated dynamin optic atrophy 1 (OPA1) in fission deficiency prevented mitochondrial depolarization and decreased proton leak without blocking mitochondrial contraction, indicating that OPA1 is a factor in coupling matrix contraction to mitochondrial depolarization. Our findings show that transient matrix contraction is a novel cellular mechanism regulating mitochondrial activity through the function of the inner membrane dynamin OPA1.

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2 / Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2 / Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.

Portulacerebroside A (PCA) is a novel cerebroside compound isolated from Portulaca oleracea L. The present results showed that PCA significantly inhibited cell viability time- and dose-dependently, elicited chromatin aggregation and fragmented nuclei, and enhanced the percentage of apoptotic cells. Furthermore PCA remarkably increased the protein phosphorylation of p38 MAPK and JNK, disrupted mitochondrial membrane penetrability, and triggered the release of mitochondrial cytochrome c and AIF to cytosol. Treatment with PCA also evidently activated caspase-9 and caspase-3 but not caspase-8. These results suggest that PCA-induced apoptosis is associated with activation of the p38 MAPK- and JNK-triggered mitochondrial death pathway. PCA possibly has the high potential for clinical applications in treating liver cancer and improving cancer chemotherapy.

Mitochondria produce the majority of cellular energy through the process of oxidative phosphorylation and play a central role in regulating the functionality and survival of eukaryotic cells. Under physiological stress, mitochondrial membrane permeabilization results in the release of apoptogenic material such as cytochrome c in the cytoplasm, which thereby initiates caspase activation and the consequent cell death. In our present study, we screened a series of compounds for their ability to inhibit mitochondrial membrane permeabilization and to prevent cytochrome c release during the endoplasmic reticulum stress in cultured pancreatic β-cells. Three benzofuran-based macrocyclic small molecules, that is, 2.4c, c104, and c108, were found to restore the depolarization of mitochondrial membrane potential and to prevent the release of cytochrome c from mitochondria. Interestingly, the acyclic precursor of 2.4c (i.e., 2.3c) did not show any effect, whereas the macrocyclic derivative obtained by utilizing ring-closing metathesis as the “stitching technology” led to this function. The macrocyclic architecture seems to play a crucial role in presenting various functional moieties in the right orientation to observe this effect.

Peroxiredoxin 3 (PRX3), a typical 2-Cys peroxiredoxin located exclusively in the mitochondrial matrix, is the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide, a byproduct of cellular respiration originating from the mitochondrial electron transport chain. Mitochondrial oxidants are produced in excess in cancer cells due to oncogenic transformation and metabolic reorganization, and signals through FOXM1 and other redox-responsive factors to support a hyper-proliferative state. Over-expression of PRX3 in cancer cells has been shown to counteract oncogene-induced senescence and support tumor cell growth and survival making PRX3 a credible therapeutic target. Using malignant mesothelioma (MM) cells stably expressing shRNAs to PRX3 we show that decreased expression of PRX3 alters mitochondrial structure, function and cell cycle kinetics. As compared to control cells, knockdown of PRX3 expression increased mitochondrial membrane potential, basal ATP production, oxygen consumption and extracellular acidification rates. shPRX3 MM cells failed to progress through the cell cycle compared to wild type controls, with increased numbers of cells in G2/M phase. Diminished PRX3 expression also induced mitochondrial hyperfusion similar to the DRP1 inhibitor mdivi-1. Cell cycle progression and changes in mitochondrial networking were rescued by transient expression of either catalase or mitochondrial-targeted catalase, indicating high levels of hydrogen peroxide contribute to perturbations in mitochondrial structure and function in shPRX3 MM cells. Our results indicate that PRX3 levels establish a redox set point that permits MM cells to thrive in response to increased levels of mROS, and that perturbing the redox status governed by PRX3 impairs proliferation by altering cell cycle-dependent dynamics between mitochondrial networking and energy metabolism., Graphical abstract, Highlights • Knockdown of PRX3 in malignant mesothelioma cells increases mitochondrial oxidants. • Knockdown of PRX3 induces mitochondrial fusion and an increase in G2/M cells. • Overexpression of catalase or mito-catalase rescues G2/M cell cycle block and mitochondrial defects.