Real-time OXPHOS capacity analysis in wounded skin from diabetic mice: A pilot study.

INTRODUCTION: Diabetes mellitus (DM) impairs wound healing. The aim was to determine whether DM influences mitochondrial respiration in wounded skin (WS) and non-wounded skin (NWS), in a pre-clinical wound healing model of streptozotocin (STZ)-induced diabetes. METHODS: Six weeks after diabetes induction, two wounds were created in the back of C57BL/J6 mice. Using high-resolution respirometry (HRR), oxygen flux was measured, in WS and NWS, using two substrate-uncoupler-inhibitor titration protocols, at baseline (day 0), day 3 and 10 post-wounding, in STZ-DM and non-diabetic (NDM) mice. Flux control ratios for the oxidative phosphorylation (OXPHOS) capacity were calculated. RESULTS: A significant increase in mitochondrial respiration was observed in STZ-DM skin compared to control skin at baseline. The OXPHOS capacity was decreased in WS under diabetes at day 3 post-wounding (inflammation phase). However, at day 10 post-wounding (remodeling phase), the OXPHOS capacity was higher in WS from STZ-DM compared to NDM mice, and compared to NWS from STZ-DM mice. A significant relative contribution of pyruvate, malate and glutamate (PMG) oxidation to the OXPHOS capacity was observed in WS compared to NWS from STZ-DM mice, at day 10, while the relative contribution of fatty acid oxidation to the OXPHOS capacity was higher in NWS. The OXPHOS capacity is altered in WS from STZ-DM compared to NDM mice across the healing process, and so is the substrate contribution in WS and NWS from STZ-DM mice, at each time point. CONCLUSION: HRR may be a sensitive tool to evaluate the underlying mechanisms of tissue repair during wound healing.

Skeletal muscle mitochondrial correlates of critical power and W' in healthy active individuals.

The asymptote (critical power; CP) and curvature constant (W') of the hyperbolic power-duration relationship can predict performance within the severe-intensity exercise domain. However, the extent to which these parameters relate to skeletal muscle mitochondrial content and respiratory function is not known. Fifteen males (peak O2 uptake, 52.2 ± 8.7 mL kg-1 min-1; peak work rate, 366 ± 40 W; and gas exchange threshold, 162 ± 41 W) performed three to five constant-load tests to task failure for the determination of CP (246 ± 44 W) and W' (18.6 ± 4.1 kJ). Skeletal muscle biopsies were obtained from the vastus lateralis to determine citrate synthase (CS) activity, as a marker of mitochondrial content, and the ADP-stimulated respiration (P) and maximal electron transfer (E) through mitochondrial complexes (C) I-IV. The CP was positively correlated with CS activity (absolute CP, r = 0.881, P < 0.001; relative CP, r = 0.751, P = 0.001). The W' was not correlated with CS activity (P > 0.05). Relative CP was positively correlated with mass-corrected CI + IIE (r = 0.659, P = 0.038), with absolute CP being inversely correlated with CS activity-corrected CIVE (r = -0.701, P = 0.024). Relative W' was positively correlated with CS activity-corrected CI + IIP (r = 0.713, P = 0.021) and the phosphorylation control ratio (r = 0.661, P = 0.038). There were no further correlations between CP or W' and mitochondrial respiratory variables. These findings support the assertion that skeletal muscle mitochondrial oxidative capacity is positively associated with CP and that this relationship is strongly determined by mitochondrial content.

Impairment of substrate-mediated mitochondrial respiration in cardiac cells by chloroquine.

Chloroquine (CQ) has a long clinical history as an anti-malarial agent and also being used for the treatment of other infections and autoimmune diseases. Recently, this lysosomotropic agent and its derivatives are also been tested as adjuncts alongside conventional anti-cancer treatments in combinatorial therapies. However, their reported cardiotoxicity tends to raise concern over their indiscriminate use. Even though the influence of CQ and its derivatives on cardiac mitochondria is extensively studied in disease models, their impact on cardiac mitochondrial respiration under physiological conditions remains inconclusive. In this study, we aimed to evaluate the impact of CQ on cardiac mitochondrial respiration using both in-vitro and in-vivo model systems. Using high-resolution respirometry in isolated cardiac mitochondria from male C57BL/6 mice treated with intraperitoneal injection of 10 mg/kg/day of CQ for 14 days, CQ was found to impair substrate-mediated mitochondrial respiration in cardiac tissue. In an in-vitro model of H9C2 cardiomyoblasts, incubation with 50 µM of CQ for 24 h disrupted mitochondrial membrane potential, produced mitochondrial fragmentation, decreased mitochondrial respiration and induced superoxide generation. Altogether, our study results indicate that CQ has a deleterious impact on cardiac mitochondrial bioenergetics which in turn suggests that CQ treatment could be an added burden, especially in patients affected with diseases with underlying cardiac complications. As CQ is an inhibitor of the lysosomal pathway, the observed effect could be an outcome of the accumulation of dysfunctional mitochondria due to autophagy inhibition.

Assessment of Mitochondrial Respiration During Hypothermic Storage of Liver Biopsies Following Normothermic Machine Perfusion.

Organ quality can be assessed prior to transplantation, during normothermic machine perfusion (NMP) of the liver. Evaluation of mitochondrial function by high-resolution respirometry (HRR) may serve as a viability assessment concept in this setting. Freshly collected tissue is considered as optimal sample for HRR, but due to technical and personnel requirements, more flexible and schedulable measurements are needed. However, the impact of cold storage following NMP before processing biopsy samples for mitochondrial analysis remains unknown. We aimed at establishing an appropriate storage protocol of liver biopsies for HRR. Wedge biopsies of 5 human livers during NMP were obtained and assessed by HRR. Analysis was performed after 0, 4, 8, and 12 h of hypothermic storage (HTS) in HTK organ preservation solution at 4°C. With HTS up to 4 h, mitochondrial performance did not decrease in HTS samples compared with 0 h (OXPHOS, 44.62 [34.75-60.15] pmol·s-1·mg wet mass-1 vs. 43.73 [40.69-57.71], median [IQR], p > 0.999). However, at HTS beyond 4 h, mitochondrial respiration decreased. We conclude that HTS can be safely applied for extending the biopsy measurement window for up to 4 h to determine organ quality, but also that human liver respiration degrades beyond 4 h HTS following NMP.

The Predictive Value of Graft Viability and Bioenergetics Testing Towards the Outcome in Liver Transplantation.

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (-0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation.

Cryopreserved placental biopsies maintain mitochondrial activity for high-resolution respirometry.

BACKGROUND: High-resolution respirometry (HRR) of human biopsies can provide useful metabolic, diagnostic, and mechanistic insights for clinical research and comparative medical studies. Fresh tissues analysis offers the potential best condition, the drawback being the need to use them shortly after dissection for mitochondrial respiratory experiments. The development of effective long-term storage protocols for biopsies that allow the assessment of key Electron Transport System (ETS) parameters at later stages is thus a major need. METHODS: We optimised a cryopreservation protocol that preserves mitochondrial membranes intactness, otherwise affected by direct tissue freezing. The protocol is based on a gradual freezing step from on-ice to liquid nitrogen and - 80 °C storage using a specific DMSO-based buffer. RESULTS: Placenta is a suitable tissue to design and test the effectiveness of long-term storage protocols being metabolically active foetal tissue with mitochondrial dysfunctions contributing to placental disease and gestational disorders. Here we designed and tested the effectiveness of the cryopreservation protocol using human placenta biopsies; we measured the ETS activity by HRR of placenta specimens comparing fresh, cryopreserved, and snap frozen conditions. CONCLUSIONS: By this protocol, Oxygen Consumption Rate (OCR) measurements of fresh and cryopreserved placental specimens are comparable whereas snap frozen procedure impairs mitochondrial activity.

Tissue-specific mitochondrial toxicity of cigarette smoke concentrate: consequence to oxidative phosphorylation.

Cigarette smoke exposure is a well-known risk factor for developing numerous chronic health conditions, including pulmonary disease and cardiometabolic disorders. However, the cellular mechanisms mediating the toxicity of cigarette smoke in extrapulmonary tissues are still poorly understood. Therefore, the purpose of this study was to characterize the acute dose-dependent toxicity of cigarette smoke on mitochondrial metabolism by determining the susceptibility and sensitivity of mitochondrial respiration from murine skeletal (gastrocnemius and soleus) and cardiac muscles, as well as the aorta to cigarette smoke concentrate (CSC). In all tissues, exposure to CSC inhibited tissue-specific respiration capacity, measured by high-resolution respirometry, according to a biphasic pattern. With a break point of 451 ± 235 µg/mL, the aorta was the least susceptible to CSC-induced mitochondrial respiration inhibition compared with the gastrocnemius (151 ± 109 µg/mL; P = 0.008, d = 2.3), soleus (211 ± 107 µg/mL; P = 0.112; d = 1.7), and heart (94 ± 51 µg/mL; P < 0.001; d = 2.6) suggesting an intrinsic resistance of the vascular smooth muscle mitochondria to cigarette smoke toxicity. In contrast, the cardiac muscle was the most susceptible and sensitive to the effects of CSC, demonstrating the greatest decline in tissue-specific respiration with increasing CSC concentration (P < 0.001, except the soleus). However, when normalized to citrate synthase activity to account for differences in mitochondrial content, cardiac fibers' sensitivity to cigarette smoke inhibition was no longer significantly different from both fast-twitch gastrocnemius and slow-twitch soleus muscle fibers, thus suggesting similar mitochondrial phenotypes. Collectively, these findings established the acute dose-dependent toxicity of cigarette smoke on oxidative phosphorylation in permeabilized tissues involved in the development of smoke-related cardiometabolic diseases.NEW & NOTEWORTHY Despite numerous investigations into the mechanisms underlying cigarette smoke-induced mitochondrial dysfunction, no studies have investigated the tissue-specific mitochondrial toxicity to cigarette smoke. We demonstrate that, while aorta is least sensitive and susceptible to cigarette smoke-induced toxicity, the degree of cigarette smoke-induced toxicity in striated muscle depends on the tissue-specific mitochondrial content. We conclude that while the mitochondrial content influences cigarette smoke-induced toxicity in striated muscles, aorta is intrinsically protected against cigarette smoke-induced mitochondrial toxicity.

Caffeine improves mitochondrial function in PINK1B9-null mutant Drosophila melanogaster.

Mitochondrial dysfunction plays a central role in Parkinson's disease (PD) and can be triggered by xenobiotics and mutations in mitochondrial quality control genes, such as the PINK1 gene. Caffeine has been proposed as a secondary treatment to relieve PD symptoms mainly by its antagonistic effects on adenosine receptors (ARs). Nonetheless, the potential protective effects of caffeine on mitochondrial dysfunction could be a strategy in PD treatment but need further investigation. In this study, we used high-resolution respirometry (HRR) to test caffeine's effects on mitochondrial dysfunction in PINK1B9-null mutants of Drosophila melanogaster. PINK1 loss-of-function induced mitochondrial dysfunction in PINK1B9-null flies observed by a decrease in O2 flux related to oxidative phosphorylation (OXPHOS) and electron transfer system (ETS), respiratory control ratio (RCR) and ATP synthesis compared to control flies. Caffeine treatment improved OXPHOS and ETS in PINKB9-null mutant flies, increasing the mitochondrial O2 flux compared to untreated PINKB9-null mutant flies. Moreover, caffeine treatment increased O2 flux coupled to ATP synthesis and mitochondrial respiratory control ratio (RCR) in PINK 1B9-null mutant flies. The effects of caffeine on respiratory parameters were abolished by rotenone co-treatment, suggesting that caffeine exerts its beneficial effects mainly by stimulating the mitochondrial complex I (CI). In conclusion, we demonstrate that caffeine may improve mitochondrial function by increasing mitochondrial OXPHOS and ETS respiration in the PD model using PINK1 loss-of-function mutant flies.

Effects of hyperthermia and acidosis on mitochondrial production of reactive oxygen species.

Exercise is associated with the development of oxidative stress, but the specific source and mechanism of production of pro-oxidant chemicals during exercise has not been confirmed. We used equine skeletal muscle mitochondria to test the hypothesis that hyperthermia and acidosis affect mitochondrial oxygen consumption and production of reactive oxygen species (ROS). Skeletal muscle biopsies were obtained at rest, after an acute episode of fatiguing exercise, and after a 9-wk conditioning program to increase aerobic fitness. Mitochondrial oxygen consumption and ROS production were measured simultaneously using high-resolution respirometry. Both hyperthermia and acidosis increased nonphosphorylating (LEAK) respiration (5.8× and 3.0×, respectively, P < 0.001) and decreased efficiency of oxidative phosphorylation. The combined effects of hyperthermia and acidosis resulted in large decreases in phosphorylating respiration, further decreasing oxidative phosphorylation efficiency from 97% to 86% (P < 0.01). Increased aerobic fitness reduced the effects of acidosis on LEAK respiration. Hyperthermia increased and acidosis decreased ROS production (2× and 0.23×, respectively, P < 0.001). There was no effect of acute exercise, but an aerobic conditioning program was associated with increased ROS production during both nonphosphorylating and phosphorylating respiration. Hyperthermia increased the ratio of ROS production to O2 consumption during phosphorylating respiration, suggesting that high-temperature impaired transfer of energy through the electron transfer system despite relatively low mitochondrial membrane potential. These data support the role of skeletal muscle mitochondria in the development of exercise-induced oxidative stress, particularly during forms of exercise that result in prolonged hyperthermia without acidosis.NEW & NOTEWORTHY The results of this study provide evidence for the role of mitochondria-derived ROS in the development of systemic oxidative stress during exercise as well as skeletal muscle diseases such as exertional rhabdomyolysis.

Methylmalonic Acid Impairs Cell Respiration and Glutamate Uptake in C6 Rat Glioma Cells: Implications for Methylmalonic Acidemia.

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L-methylmalonyl-CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and observable symptoms during metabolic decompensation are encephalopathy, cerebral atrophy, coma, and seizures, which commonly appear in newborns. This study aimed to investigate the toxic effects of MMA in a glial cell line presenting astrocytic features. Astroglial C6 cells were exposed to MMA (0.1-10 mM) for 24 or 48 h and cell metabolic viability, glucose consumption, and oxygen consumption rate, as well as glutamate uptake and ATP content were analyzed. The possible preventive effects of bezafibrate were also evaluated. MMA significantly reduced cell metabolic viability after 48-h period and increased glucose consumption during the same period of incubation. Regarding the energy homeostasis, MMA significantly reduced respiratory parameters of cells after 48-h exposure, indicating that cell metabolism is compromised at resting and reserve capacity state, which might influence the cell capacity to meet energetic demands. Glutamate uptake and ATP content were also compromised after exposure to MMA, which can be influenced energy metabolism impairment, affecting the functionality of the astroglial cells. Our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease. Methylmalonic acid compromises mitochondrial functioning leading to reduced ATP production and reduces glutamate uptake by C6 astroglial cells.

Combined effects of salinity and intermittent hypoxia on mitochondrial capacity and reactive oxygen species efflux in the Pacific oyster, Crassostrea gigas.

Coastal environments commonly experience fluctuations in salinity and hypoxia-reoxygenation (H/R) stress that can negatively affect mitochondrial functions of marine organisms. Although intertidal bivalves are adapted to these conditions, the mechanisms that sustain mitochondrial integrity and function are not well understood. We determined the rates of respiration and reactive oxygen species (ROS) efflux in the mitochondria of oysters, Crassostrea gigas, acclimated to high (33 psu) or low (15 psu) salinity, and exposed to either normoxic conditions (control; 21% O2) or short-term hypoxia (24 h at <0.01% O2) and subsequent reoxygenation (1.5 h at 21% O2). Further, we exposed isolated mitochondria to anoxia in vitro to assess their ability to recover from acute (∼10 min) oxygen deficiency (<0.01% O2). Our results showed that mitochondria of oysters acclimated to high or low salinity did not show severe damage and dysfunction during H/R stress, consistent with the hypoxia tolerance of C. gigas. However, acclimation to low salinity led to improved mitochondrial performance and plasticity, indicating that 15 psu might be closer to the metabolic optimum of C. gigas than 33 psu. Thus, acclimation to low salinity increased mitochondrial oxidative phosphorylation rate and coupling efficiency and stimulated mitochondrial respiration after acute H/R stress. However, elevated ROS efflux in the mitochondria of low-salinity-acclimated oysters after acute H/R stress indicates a possible trade-off of higher respiration. The high plasticity and stress tolerance of C. gigas mitochondria may contribute to the success of this invasive species and facilitate its further expansion into brackish regions such as the Baltic Sea.

Bioenergetic and Cytokine Profiling May Help to Rescue More DCD Livers for Transplantation.

The majority of organs used for liver transplantation come from brain-dead donors (DBD). In order to overcome the organ shortage, increasingly donation after circulatory death (DCD) organs are also considered. Since normothermic machine perfusion (NMP) restores metabolic activity and allows for in-depth assessment of organ quality and function prior to transplantation, such organs may benefit from NMP. We herein compare the bioenergetic performance through a comprehensive evaluation of mitochondria by high-resolution respirometry in tissue biopsies and the inflammatory response in DBD and DCD livers during NMP. While livers were indistinguishable by perfusate biomarker assessment and histology, our findings revealed a greater impairment of mitochondrial function in DCD livers after static cold storage compared to DBD livers. During subsequent NMPs, DCD organs recovered and eventually showed a similar performance as DBD livers. Cytokine expression analysis showed no differences in the early phase of NMP, while towards the end of NMP, significantly elevated levels of IL-1ß, IL-5 and IL-6 were found in the perfusate of DCD livers. Based on our results, we find it worthwhile to reconsider more DCD organs for transplantation to further extend the donor pool. Therefore, donor organ quality criteria must be developed, which may include an assessment of bioenergetic function and cytokine quantification.

Impairment of mitochondrial respiration in platelets and placentas: a pilot study in preeclamptic pregnancies.

Preeclampsia (PE) is a major complication of pregnancy with partially elucidated pathophysiology. Placental mitochondrial dysfunction has been increasingly studied as major pathomechanism in both early- and late-onset PE. Impairment of mitochondrial respiration in platelets has recently emerged as a peripheral biomarker that may mirror organ mitochondrial dysfunction in several acute and chronic pathologies. The present study was purported to assess mitochondrial respiratory dys/function in both platelets and placental mitochondria in PE pregnancies. To this aim, a high-resolution respirometry SUIT (Substrate-Uncoupler-Inhibitor-Titration) protocol was adapted to assess complex I (glutamate + malate)- and complex II (succinate)-supported respiration. A decrease in all respiratory parameters (basal, coupled, and maximal uncoupled respiration) in peripheral platelets was found in preeclamptic as compared to healthy pregnancies. At variance, placental mitochondria showed a dichotomous behavior in preeclampsia in relation to the fetal birth weight. PE pregnancies with fetal growth restriction were associated with decreased in coupled respiration (oxidative phosphorylation/OXPHOS capacity) and maximal uncoupled respiration (electron transfer/ET capacity). At variance, these respiratory parameters were increased for both complex I- and II-supported respiration in PE pregnancies with normal weight fetuses. Large randomized controlled clinical studies are needed in order to advance our understanding of mitochondrial adaptive vs. pathological changes in preeclampsia.

Impairment of Mitochondrial Respiration in Metabolic Diseases: An Overview.

Mitochondrial dysfunction has emerged as a central pathomechanism in the setting of obesity and diabetes mellitus, linking these intertwined pathologies that share insulin resistance as a common denominator. High-resolution respirometry (HRR) is a state-of-the-art research method currently used to study mitochondrial respiration and its impairment in health and disease. Tissue samples, cells or isolated mitochondria are exposed to various substrate-uncoupler-inhibitor-titration protocols, which allows the measurement and calculation of several parameters of mitochondrial respiration. In this review, we discuss the alterations of mitochondrial bioenergetics in the main dysfunctional organs that contribute to the development of the obese and diabetic phenotypes in both animal models and human subjects. Herein we review data regarding the impairment of oxidative phosphorylation as integrated mitochondrial function assessed by means of HRR. We acknowledge the critical role of this method in determining the alterations in oxidative phosphorylation occurring in the early stages of metabolic pathologies. We conclude that there is a mutual two-way relationship between mitochondrial dysfunction and insulin insensitivity that characterizes these diseases.

The C30-Modulation of Betulinic Acid Using 1,2,4-Triazole: A Promising Strategy for Increasing Its Antimelanoma Cytotoxic Potential.

Cancer, in all its types and manifestations, remains one of the most frequent causes of death worldwide; an important number of anticancer drugs have been developed from plants, fungi and animals, starting with natural compounds that were later derivatized in order to achieve an optimized pharmacokinetic/pharmacological profile. Betulinic acid is a pentacyclic triterpenic compound that was identified as an anticancer agent whose main advantage consists in its selective activity, which ensures the almost total lack of cytotoxic side effects. Conjugates of betulinic acid with substituted triazoles, scaffolds with significant pharmacological properties, were synthesized and tested as anticancer agents in order to achieve new therapeutic alternatives. The current paper aims to obtain a C30-1,2,4-triazole derivative of betulinic acid simultaneously acetylated at C3 whose biological activity was tested against RPMI melanoma cells. The compound revealed significant cytotoxic effects at the tested concentrations (2, 10 and 50 µΜ) by significantly decreasing the cell viability to 88.3%, 54.7% and 24.5%, respectively, as compared to the control. The compound's testing in normal HaCaT cells showed a lack of toxicity, which indicates its selective dose-dependent anticancer activity. The investigation of its underlying molecular mechanism revealed an apoptotic effect induced at the mitochondrial level, which was validated through high-resolution respirometry studies.

A Link between Mitochondrial Dysregulation and Idiopathic Autism Spectrum Disorder (ASD): Alterations in Mitochondrial Respiratory Capacity and Membrane Potential.

Autism spectrum disorder (ASD) is a neurological disorder triggered by various factors through complex mechanisms. Research has been done to elucidate the potential etiologic mechanisms in ASD, but no single cause has been confirmed. The involvement of oxidative stress is correlated with ASD and possibly affects mitochondrial function. This study aimed to elucidate the link between mitochondrial dysregulation and idiopathic ASD by focusing on mitochondrial respiratory capacity and membrane potential. Our findings showed that mitochondrial function in the energy metabolism pathway was significantly dysregulated in a lymphoblastoid cell line (LCL) derived from an autistic child (ALCL). Respiratory capacities of oxidative phosphorylation (OXPHOS), electron transfer of the Complex I and Complex II linked pathways, membrane potential, and Complex IV activity of the ALCL were analyzed and compared with control cell lines derived from a developmentally normal non-autistic sibling (NALCL). All experiments were performed using high-resolution respirometry. Respiratory capacities of OXPHOS, electron transfer of the Complex I- and Complex II-linked pathways, and Complex IV activity of the ALCL were significantly higher compared to healthy controls. Mitochondrial membrane potential was also significantly higher, measured in the Complex II-linked pathway during LEAK respiration and OXPHOS. These results indicate the abnormalities in mitochondrial respiratory control linking mitochondrial function with autism. Correlating mitochondrial dysfunction and autism is important for a better understanding of ASD pathogenesis in order to produce effective interventions.

Signs of early cellular dysfunction in multiple system atrophy.

AIMS: Multiple system atrophy (MSA) is a fatal neurodegenerative disease that belongs to the family of α-synucleinopathies. At post mortem examination, intracellular inclusions of misfolded α-synuclein are found in neurons and oligodendrocytes and are considered to play a significant role in the pathogenesis. However, the early steps of the disease process are unknown and difficult to study in tissue derived from end-stage disease. METHODS: Induced pluripotent stem cells (iPSCs) were generated from patients' and control skin fibroblasts and differentiated into NCAM-positive neural progenitor cells (NPCs). The mitochondrial morphology and function were assessed by immunocytochemistry and high resolution respirometry. The ability to cope with exogenous oxidative stress was tested by exposure to different doses of luperox. The expression of α-synuclein was studied by immunocytochemistry. RESULTS: We identified increased tubulation of mitochondria with preserved respiration profile in MSA-derived NPCs. Exposure of these cells to exogenous oxidative stress even at low doses, triggered an excessive generation of reactive oxygen species (ROS) and cleavage of caspase-3. MSA-derived NPCs did not present changed levels of SNCA gene expression nor intracellular aggregates of α-synuclein. However, we identified disease-related translocation of α-synuclein to the nucleus. CONCLUSIONS: Our results show early cellular dysfunction in MSA-derived NPCs. We identified changes in the redox homeostasis which are functionally compensated at baseline but cause increased susceptibility to exogenous oxidative stress. In addition, nuclear translocation of α-synuclein in MSA-derived NPCs supports an early cellular stress response which may precede the neurodegenerative process in this disorder.

Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease.

BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.

Mitochondrial dynamics and quality control are altered in a hepatic cell culture model of cancer cachexia.

Hepatic mitochondrial function loss is associated with cancer cachexia pathology in vivo. Here, we examined if hepatic mitochondrial defects observed in vivo in the cachexic liver also recapitulate during the in vitro treatment of mouse hepatocytes with tumor conditioned media. In vitro experiments were combined with proteome-wide expression analysis of cachexic liver tissue curated for mitochondrial dynamics and quality control proteins, to determine the fidelity of hepatic mitochondrial maladaptation in cancer cachexia pathology. AML12 hepatocytes were exposed to colon-26 (C26) and Lewis lung carcinoma (LLC) conditioned media for 6-72 h and assayed for cell viability, membrane potential, respiratory function, H2O2 production, total ROS/RNS, and mitochondrial dynamics and quality control proteins by immunoblotting. Liver tissue from cachexic C26 mice was analyzed by TMT-based quantitative proteomics for in vivo comparison. Cell viability, membrane potential, H2O2 production, total ROS/RNS, and respiration were decreased 48-72 h after exposure to C26 and/or LLC. Protein expression of treated hepatocytes and cachexic liver tissue showed altered mitochondrial dynamics and quality control, in a manner that suggests limited fusion and content mixing, but also impaired ability to fragment and clear damaged mitochondria. Two strategies to maintain mitochondrial health, therefore, may not be functioning sufficiently in the cachexic liver. Together these findings imply adverse effects of C26 and LLC exposure on hepatocyte health, due to impaired mitochondrial function and remodeling. Exposure of mouse hepatocytes to tumor conditioned media models aspects of cachexic liver mitochondria dysfunction in vivo and validates the importance of hepatic mitochondrial maladaptation in cancer cachexia pathology.

Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica.

Oxygen fluctuations are common in marine waters, and hypoxia-reoxygenation (H-R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h <0.01% O2) and subsequent reoxygenation (1.5 h 21% O2) compared with normoxic control animals (21% O2). We demonstrated that flux capacity of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased the oxygen flux capacity of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H-R stress, whereas control by the proton leak subsystem of LEAK respiration increased during hypoxia and returned to normoxic levels during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, whereas it was suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.