Crystalline silica particles cause rapid NLRP3-dependent mitochondrial depolarization and DNA damage in airway epithelial cells.

BACKGROUND: Respirable crystalline silica causes lung carcinomas and many thousand future cancer cases are expected in e.g. Europe. Critical questions are how silica causes genotoxicity in the respiratory epithelium and if new cases can be avoided by lowered permissible exposure levels. In this study we investigate early DNA damaging effects of low doses of silica particles in respiratory epithelial cells in vitro and in vivo in an effort to understand low-dose carcinogenic effects of silica particles. RESULTS: We find DNA damage accumulation already after 5-10 min exposure to low doses (5 µg/cm2) of silica particles (Min-U-Sil 5) in vitro. DNA damage was documented as increased levels of γH2AX, pCHK2, by Comet assay, AIM2 induction, and by increased DNA repair (non-homologous end joining) signaling. The DNA damage response (DDR) was not related to increased ROS levels, but to a NLRP3-dependent mitochondrial depolarization. Particles in contact with the plasma membrane elicited a Ser198 phosphorylation of NLRP3, co-localization of NLRP3 to mitochondria and depolarization. FCCP, a mitochondrial uncoupler, as well as overexpressed NLRP3 mimicked the silica-induced depolarization and the DNA damage response. A single inhalation of 25 µg silica particles gave a similar rapid DDR in mouse lung. Biomarkers (CC10 and GPRC5A) indicated an involvement of respiratory epithelial cells. CONCLUSIONS: Our findings demonstrate a novel mode of action (MOA) for silica-induced DNA damage and mutagenic double strand breaks in airway epithelial cells. This MOA seems independent of particle uptake and of an involvement of macrophages. Our study might help defining models for estimating exposure levels without DNA damaging effects.

High-concentration homocysteine inhibits mitochondrial respiration function and production of reactive oxygen species in neuron cells.

OBJECTIVE: Homocysteine plays critical roles in cellular redox homeostasis, and hyperhomocysteinemia has been associated with multiple diseases, including neurological disorders involving reactive oxygen species-inducing and pro-inflammatory effects of homocysteine that are related to mitochondria. This study investigated the role of homocysteine in regulating mitochondria of neuron cell lines. METHODS: Neuron cells were pre-treated with homocysteine, and then flow cytometry was used to detect reactive oxygen species production and mitochondrial membrane potential, while Seahorse XFp Mito stress assay was used to comprehensively analyze mitochondrial function. RESULTS: The experimental results showed that high-concentration homocysteine diminished carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone-stimulated oxygen consumption rate and mitochondrial spare respiration capacity in a time- and concentration-dependent manner, and homocysteine also reduced reactive oxygen species in cultured neuron cell lines while no changes in mitochondrial membrane potential were observed. CONCLUSION: These results indicate that homocysteine diminished mitochondrial respiration function in neuron cell lines mediated by its reactive oxygen species-reducing effects, which may underlie the association between hyperhomocysteinemia and human diseases.

Application of ECIS to Assess FCCP-Induced Changes of MSC Micromotion and Wound Healing Migration.

Electric cell-substrate impedance sensing (ECIS) is an emerging technique for sensitively monitoring morphological changes of adherent cells in tissue culture. In this study, human mesenchymal stem cells (hMSCs) were exposed to different concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) for 20 h and their subsequent concentration-dependent responses in micromotion and wound healing migration were measured by ECIS. FCCP disrupts ATP synthesis and results in a decrease in cell migration rates. To detect the change of cell micromotion in response to FCCP challenge, time-series resistances of cell-covered electrodes were monitored and the values of variance were calculated to verify the difference. While Seahorse XF-24 extracellular flux analyzer can detect the effect of FCCP at 3 µM concentration, the variance calculation of the time-series resistances measured at 4 kHz can detect the effect of FCCP at concentrations as low as 1 µM. For wound healing migration, the recovery resistance curves were fitted by sigmoid curve and the hill slope showed a concentration-dependent decline from 0.3 µM to 3 µM, indicating a decrease in cell migration rate. Moreover, dose dependent incline of the inflection points from 0.3 µM to 3 µM FCCP implied the increase of the half time for wound recovery migration. Together, our results demonstrate that partial uncoupling of mitochondrial oxidative phosphorylation reduces micromotion and wound healing migration of hMSCs. The ECIS method used in this study offers a simple and sensitive approach to investigate stem cell migration and its regulation by mitochondrial dynamics.

Maternal intake of trans-unsaturated or interesterified fatty acids during pregnancy and lactation modifies mitochondrial bioenergetics in the liver of adult offspring in mice.

The quality of dietary lipids in the maternal diet can programme the offspring to diseases in later life. We investigated whether the maternal intake of palm oil or interesterified fat, substitutes for trans-unsaturated fatty acids (FA), induces metabolic changes in the adult offspring. During pregnancy and lactation, C57BL/6 female mice received normolipidic diets containing partially hydrogenated vegetable fat rich in trans-unsaturated fatty acids (TG), palm oil (PG), interesterified fat (IG) or soyabean oil (CG). After weaning, male offspring from all groups received the control diet until day 110. Plasma glucose and TAG and liver FA profiles were ascertained. Liver mitochondrial function was accessed with high-resolution respirometry by measuring VO2, fluorimetry for detection of hydrogen peroxide (H2O2) production and mitochondrial Ca2+ uptake. The results showed that the IG offspring presented a 20 % increase in plasma glucose and both the IG and TG offspring presented a 2- and 1·9-fold increase in TAG, respectively, when compared with CG offspring. Liver MUFA and PUFA contents decreased in the TG and IG offspring when compared with CG offspring. Liver MUFA content also decreased in the PG offspring. These modifications in FA composition possibly affected liver mitochondrial function, as respiration was impaired in the TG offspring and H2O2 production was higher in the IG offspring. In addition, mitochondrial Ca2+ retention capacity was reduced by approximately 40 and 55 % in the TG and IG offspring, respectively. In conclusion, maternal consumption of trans-unsaturated and interesterified fat affected offspring health by compromising mitochondrial bioenergetics and lipid metabolism in the liver.

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model.

PURPOSE: Temporary and reversible downregulation of metabolism may improve the survival of tissues exposed to non-physiological conditions during transport, in vitro culture, and cryopreservation. The objectives of the study were to (1) optimize the concentration and duration of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP-a mitochondrial uncoupling agent) exposures for biopsies of domestic cat ovarian tissue and (2) examine the effects of FCCP pre-exposures on follicle integrity after tissue culture and/or cryopreservation. METHODS: Biopsies of cat ovarian tissue were first treated with various concentrations of FCCP (0, 10, 40, or 200 nM) for 10 or 120 min to determine the most suitable pre-exposure conditions. Based on these results, tissues were pre-exposed to 200 nM FCCP for 120 min for the subsequent studies on culture and cryopreservation. In all experiments and for each treatment group, tissue activity and integrity were measured by mitochondrial membrane potential (relative optical density of rhodamine 123 fluorescence), follicular viability (calcein assay), follicular morphology (histology), granulosa cell proliferation (Ki-67 immunostaining), and follicular density. RESULTS: Ovarian tissues incubated with 200 nM FCCP for 120 min led to the lowest mitochondrial activity (1.17 ± 0.09; P < 0.05) compared to control group (0 nM; 1.30 ± 0.12) while maintaining a constant percentage of viable follicles (75.3 ± 7.8 %) similar to the control group (71.8 ± 11.7 %; P > 0.05). After 2 days of in vitro culture, percentage of viable follicles (78.8 ± 8.9 %) in similar pre-exposure conditions was higher (P < 0.05) than in the absence of FCCP (61.2 ± 12.0 %) with percentages of morphologically normal follicles (57.6 ± 17.3 %) not different from the fresh tissue (70.2 ± 7.1 %; P > 0.05). Interestingly, percentages of cellular proliferation and follicular density were unaltered by the FCCP exposures. Based on the indicators mentioned above, the FCCP-treated tissue fragments did not have a better follicle integrity after freezing and thawing. CONCLUSIONS: Pre-exposure to 200 nM FCCP during 120 min protects and enhances the follicle integrity in cat ovarian tissue during short-term in vitro culture. However, FCCP does not appear to exert a beneficial or detrimental effect during ovarian tissue cryopreservation.

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Increased proton leak and SOD2 expression in myotubes from obese non-diabetic subjects with a family history of type 2 diabetes.

Muscle insulin resistance is linked to oxidative stress and decreased mitochondrial function. However, the exact cause of muscle insulin resistance is still unknown. Since offspring of patients with type 2 diabetes mellitus (T2DM) are susceptible to developing insulin resistance, they are ideal for studying the early development of insulin resistance. By using primary muscle cells derived from obese non-diabetic subjects with (FH+) or without (FH-) a family history of T2DM, we aimed to better understand the link between mitochondrial function, oxidative stress, and muscle insulin resistance. Insulin-stimulated glucose uptake and glycogen synthesis were normal in FH+ myotubes. Resting oxygen consumption rate was not different between groups. However, proton leak was higher in FH+ myotubes. This was associated with lower ATP content and decreased mitochondrial membrane potential in FH+ myotubes. Surprisingly, mtDNA content was higher in FH+ myotubes. Oxidative stress level was not different between FH+ and FH- groups. Reactive oxygen species content was lower in FH+ myotubes when differentiated in high glucose/insulin (25mM/150pM), which could be due to higher oxidative stress defenses (SOD2 expression and uncoupled respiration). The increased antioxidant defenses and mtDNA content in FH+ myotubes suggest the existence of compensatory mechanisms, which may provisionally prevent the development of insulin resistance.

AD-linked, toxic NH2 human tau affects the quality control of mitochondria in neurons.

Functional as well as structural alterations in mitochondria size, shape and distribution are precipitating, early events in progression of Alzheimer's Disease (AD). We reported that a 20-22kDa NH2-tau fragment (aka NH2htau), mapping between 26 and 230 amino acids of the longest human tau isoform, is detected in cellular and animal AD models and is neurotoxic in hippocampal neurons. The NH2htau -but not the physiological full-length protein- interacts with Aß at human AD synapses and cooperates with it in inhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange. Here we show that the NH2htau also adversely affects the interplay between the mitochondria dynamics and their selective autophagic clearance. Fragmentation and perinuclear mislocalization of mitochondria with smaller size and density are early found in dying NH2htau-expressing neurons. The specific effect of NH2htau on quality control of mitochondria is accompanied by (i) net reduction in their mass in correlation with a general Parkin-mediated remodeling of membrane proteome; (ii) their extensive association with LC3 and LAMP1 autophagic markers; (iii) bioenergetic deficits and (iv) in vitro synaptic pathology. These results suggest that NH2htau can compromise the mitochondrial biology thereby contributing to AD synaptic deficits not only by ANT-1 inactivation but also, indirectly, by impairing the quality control mechanism of these organelles.

Hyperammonaemia induces mitochondrial dysfunction and neuronal cell death.

Background & Aims: In cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging. Methods: To primary cocultures of astrocytes and neurons, low concentrations (1 and 5 µM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy. Results: Neuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP. Conclusions: Our results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development. Lay summary: The impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.

Supercomplex supercomplexes: Raison d'etre and functional significance of supramolecular organization in oxidative phosphorylation.

Following structural determination by recent advances in electron cryomicroscopy, it is now well established that the respiratory Complexes I-IV in oxidative phosphorylation (OXPHOS) are organized into supercomplexes in the respirasome. Nonetheless, the reason for the existence of the OXPHOS supercomplexes and their functional role remains an enigma. Several hypotheses have been proposed for the existence of these supercomplex supercomplexes. A commonly-held view asserts that they enhance catalysis by substrate channeling. However, this - and other views - has been challenged based on structural and biophysical information. Hence, new ideas, concepts, and frameworks are needed. Here, a new model of energy transfer in OXPHOS is developed on the basis of biochemical data on the pure competitive inhibition of anionic substrates like succinate by the classical anionic uncouplers of OXPHOS (2,4-dinitrophenol, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and dicoumarol), and pharmacological data on the unique site-selective, energy-linked inhibition of energy conservation pathways in mitochondria induced by the guanidine derivatives. It is further found that uncouplers themselves are site-specific and exhibit differential selectivity and efficacy in reversing the inhibition caused by the Site 1/Complex I or Site 2/Complexes II-III-selective guanidine derivatives. These results lead to new vistas and sufficient complexity in the network of energy conservation pathways in the mitochondrial respiratory chain that necessitate discrete points of interaction with two classes of guanidine derivatives and uncoupling agents and thereby separate and distinct energy transfer pathways between Site 1 and Site 2 and the intermediate that energizes adenosine triphosphate (ATP) synthesis by Complex V. Interpretation based on Mitchell's single-ion chemiosmotic theory that postulates only a single energy pool is inadequate to rationalize the data and account for the required complexity. The above results and available information are shown to be explained by Nath's two-ion theory of energy coupling and ATP synthesis, involving coupled movement of succinate anions and protons, along with the requirement postulated by the theory for maintenance of homeostasis and ion translocation across the energy-transducing membrane of both succinate monoanions and succinate dianions by Complexes I-V in the OXPHOS supercomplexes. The new model of energy transfer in mitochondria is mapped onto the solved structures of the supercomplexes and integrated into a consistent model with the three-dimensional electron microscope computer tomography visualization of the internal structure of the cristae membranes in mammalian mitochondria. The model also offers valuable insights into diseased states induced in type 2 diabetes and especially in Alzheimer's and other neurodegenerative diseases that involve mitochondrial dysfunction.

Curcumin pre-treatment may protect against mitochondrial damage in LRRK2-mutant Parkinson's disease and healthy control fibroblasts.

Mitochondrial dysfunction has been proposed as one of the pathobiological underpinnings in Parkinson's disease. Environmental stressors, such as paraquat, induce mitochondrial dysfunction and promote reactive oxygen species production. Targeting oxidative stress pathways could prevent mitochondrial dysfunction and thereby halt the neurodegeneration in Parkinson's disease. Since curcumin is touted as an antioxidant and neuroprotective agent, the aim of this study was to investigate if curcumin is a suitable therapy to target mitochondrial dysfunction in Parkinson's disease using a paraquat-toxicity induced model in fibroblasts from LRRK2-mutation positive Parkinson's disease individuals and healthy controls. The fibroblasts were exposed to five treatment groups, (i) untreated, (ii) curcumin only, (iii) paraquat only, (iv) pre-curcumin group: with curcumin for 2hr followed by paraquat for 24hr and (v) post-curcumin group: with paraquat for 24hr followed by curcumin for 2hr. Mitochondrial function was determined by measuring three parameters of mitochondrial respiration (maximal respiration, ATP-associated respiration, and spare respiratory capacity) using the Seahorse XFe96 Extracellular Flux Analyzer. As expected, paraquat effectively disrupted mitochondrial function for all parameters. Pre-curcumin treatment improved maximal and ATP-associated respiration whereas, post-curcumin treatment had no effect. These findings indicate that curcumin may be most beneficial as a pre-treatment before toxin exposure, which has implications for its therapeutic use. These promising findings warrant future studies testing different curcumin dosages, exposure times and curcumin formulations in larger sample sizes of Parkinson's disease and control participants.

Proteomic and mitochondrial adaptations to early-life stress are distinct in juveniles and adults.

Exposure to early-life stress (ELS) increases risk for poor mental and physical health outcomes that emerge at different stages across the lifespan. Yet, how age interacts with ELS to impact the expression of specific phenotypes remains largely unknown. An established limited-bedding paradigm was used to induce ELS in mouse pups over the early postnatal period. Initial analyses focused on the hippocampus, based on documented sensitivity to ELS in humans and various animal models, and the large body of data reporting anatomical and physiological outcomes in this structure using this ELS paradigm. An unbiased discovery proteomics approach revealed distinct adaptations in the non-nuclear hippocampal proteome in male versus female offspring at two distinct developmental stages: juvenile and adult. Gene ontology and KEGG pathway analyses revealed significant enrichment in proteins associated with mitochondria and the oxidative phosphorylation (OXPHOS) pathway in response to ELS in female hippocampus only. To determine whether the protein adaptations to ELS reflected altered function, mitochondrial respiration (driven through complexes II-IV) and complex I activity were measured in isolated hippocampal mitochondria using a Seahorse X96 Flux analyzer and immunocapture ELISA, respectively. ELS had no effect on basal respiration in either sex at either age. In contrast, ELS increased OXPHOS capacity in juvenile males and females, and reduced OXPHOS capacity in adult females but not adult males. A similar pattern of ELS-induced changes was observed for complex I activity. These data suggest that initial adaptations in juvenile hippocampus due to ELS were not sustained in adults. Mitochondrial adaptations to ELS were also exhibited peripherally by liver. Overall, the temporal distinctions in mitochondrial responses to ELS show that ELS-generated adaptations and outcomes are complex over the lifespan. This may contribute to differences in the timing of appearance of mental and physical disturbances, as well as potential sex differences that influence only select outcomes.

Exploration of the chondrosarcoma metabolome; the mTOR pathway as an important pro-survival pathway.

BACKGROUND: Chondrosarcomas are malignant cartilage-producing tumors showing mutations and changes in gene expression in metabolism related genes. In this study, we aimed to explore the metabolome and identify targetable metabolic vulnerabilities in chondrosarcoma. METHODS: A custom-designed metabolic compound screen containing 39 compounds targeting different metabolic pathways was performed in chondrosarcoma cell lines JJ012, SW1353 and CH2879. Based on the anti-proliferative activity, six compounds were selected for validation using real-time metabolic profiling. Two selected compounds (rapamycin and sapanisertib) were further explored for their effect on viability, apoptosis and metabolic dependency, in normoxia and hypoxia. In vivo efficacy of sapanisertib was tested in a chondrosarcoma orthotopic xenograft mouse model. RESULTS: Inhibitors of glutamine, glutathione, NAD synthesis and mTOR were effective in chondrosarcoma cells. Of the six compounds that were validated on the metabolic level, mTOR inhibitors rapamycin and sapanisertib showed the most consistent decrease in oxidative and glycolytic parameters. Chondrosarcoma cells were sensitive to mTORC1 inhibition using rapamycin. Inhibition of mTORC1 and mTORC2 using sapanisertib resulted in a dose-dependent decrease in viability in all chondrosarcoma cell lines. In addition, induction of apoptosis was observed in CH2879 after 24 h. Treatment of chondrosarcoma xenografts with sapanisertib slowed down tumor growth compared to control mice. CONCLUSIONS: mTOR inhibition leads to a reduction of oxidative and glycolytic metabolism and decreased proliferation in chondrosarcoma cell lines. Although further research is needed, these findings suggest that mTOR inhibition might be a potential therapeutic option for patients with chondrosarcoma.

Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway.

Over recent decades, many studies have reported that hypocrellin A (HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy (PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation (iTRAQ) 2D liquid chromatography with tandem mass spectrometric (LC-MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species (ROS) generations. Further studies showed that PDT treatment with 0.08 µmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome c, and activation of caspase-3, -9, and -7. Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.

Uncoupling of the Electron Transport Chain Compromises Mitochondrial Oxidative Phosphorylation and Exacerbates Stroke Outcomes.

OBJECTIVE: Mitochondrial dysfunction is known to be implicated in stroke, but the complex mechanisms of stroke have led to few stroke therapies. The present study to disrupted mitochondrial oxidative phosphorylation through a known electron transport chain (ETC) uncoupler, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP). Analyzing the resulting neurological deficits as well as infarct volume could help determine the role of mitochondria in stroke outcome and determine whether uncoupling the ETC could potentially be a strategy for new stroke therapies. The objective of this study was to determine the effects of uncoupling electron flow on mitochondrial oxidative phosphorylation and stroke infarction. METHODS: Cerebral endovascular cells (CECs) were treated with various concentrations of FCCP, and bioenergetics were measured. For the stroke mouse model, FCCP (1 mg/kg, i.p) or vehicle was administered followed by 1-hour transient middle cerebral artery occlusion (tMCAO). Infarct volume was measured after a 23-hour reperfusion, and triphenyl tetrazolium chloride (TTC) staining was used to assess infarct volume. RESULTS: FCCP significantly decreased basal respiration, ATP turnover, maximal respiration, and spare capacity when the concentration of FCCP was greater than 1000 nM. The mice pretreated with FCCP had a significantly increased infarct volume within the cortex, striatum, and total hemisphere. Mice receiving FCCP had a significantly increased neurological deficit score compared to the vehicle. CONCLUSIONS: FCCP compromised mitochondrial oxidative phosphorylation in CECs in a dose-dependent manner. Uncoupling the electron transport chain with FCCP prior to tMCAO exacerbated stroke infarction in mice.

Noninvasive diagnostics of mitochondrial disorders in isolated lymphocytes with high resolution respirometry.

BACKGROUND: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. METHODS: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses. RESULTS: Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p 50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations. CONCLUSION: Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes. GENERAL SIGNIFICANCE: The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.

Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity.

Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR) was significantly increased whereas extracellular acidification rate (ECAR), a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2 (•-) level compared to cells in the glucose model. An antimycin A (AA) dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a galactose media model and are consequently more susceptible to mitochondrial toxicants.

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control.

The 18-kDa TSPO (translocator protein) localizes on the outer mitochondrial membrane (OMM) and participates in cholesterol transport. Here, we report that TSPO inhibits mitochondrial autophagy downstream of the PINK1-PARK2 pathway, preventing essential ubiquitination of proteins. TSPO abolishes mitochondrial relocation of SQSTM1/p62 (sequestosome 1), and consequently that of the autophagic marker LC3 (microtubule-associated protein 1 light chain 3), thus leading to an accumulation of dysfunctional mitochondria, altering the appearance of the network. Independent of cholesterol regulation, the modulation of mitophagy by TSPO is instead dependent on VDAC1 (voltage-dependent anion channel 1), to which TSPO binds, reducing mitochondrial coupling and promoting an overproduction of reactive oxygen species (ROS) that counteracts PARK2-mediated ubiquitination of proteins. These data identify TSPO as a novel element in the regulation of mitochondrial quality control by autophagy, and demonstrate the importance for cell homeostasis of its expression ratio with VDAC1.

Inhibition of autophagy and glycolysis by nitric oxide during hypoxia-reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons.

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated.