Mitochondrial Bioenergetic Function Research Articles

Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3.

BackgroundSTAT3 is increasingly becoming known for its non-transcriptional regulation of mitochondrial bioenergetic function upon activation of its S727 residue (S727-STAT3). Lengthy mitochondrial dysfunction can lead to cell death. We tested whether an integrin-FAK-STAT3 signaling pathway we recently discovered regulates mitochondrial function and cell survival, and treatments thereof.MethodsCultured mouse brain bEnd5 endothelial cells were treated with integrin, FAK or STAT3 inhibitors, FAK siRNA, as well as integrin and STAT3 activators. STAT3 null cells were transfected with mutant STAT3 plasmids. Outcome measures included oxygen consumption rate for mitochondrial bioenergetics, Western blotting for protein phosphorylation, mitochondrial membrane potential for mitochondrial integrity, ROS production, and cell counts.ResultsVitronectin-dependent mitochondrial basal respiration, ATP production, and maximum reserve and respiratory capacities were suppressed within 4 h by RGD and αvβ3 integrin antagonist peptides. Conversely, integrin ligands vitronectin, laminin and fibronectin stimulated mitochondrial function. Pharmacological inhibition of FAK completely abolished mitochondrial function within 4 h while FAK siRNA treatments confirmed the specificity of FAK signaling. WT, but not S727A functionally dead mutant STAT3, rescued bioenergetics in cells made null for STAT3 using CRISPR-Cas9. STAT3 inhibition with stattic in whole cells rapidly reduced mitochondrial function and mitochondrial pS727-STAT3. Stattic treatment of isolated mitochondria did not reduce pS727 whereas more was detected upon phosphatase inhibition. This suggests that S727-STAT3 is activated in the cytoplasm and is short-lived upon translocation to the mitochondria. FAK inhibition reduced pS727-STAT3 within mitochondria and reduced mitochondrial function in a non-transcriptional manner, as shown by co-treatment with actinomycin. Treatment with the small molecule bryostatin-1 or hepatocyte growth factor (HGF), which indirectly activate S727-STAT3, preserved mitochondrial function during FAK inhibition, but failed in the presence of the STAT3 inhibitor. FAK inhibition induced loss of mitochondrial membrane potential, which was counteracted by bryostatin, and increased superoxide and hydrogen peroxide production. Bryostatin and HGF reduced the substantial cell death caused by FAK inhibition over a 24 h period.ConclusionThese data suggest that extracellular matrix molecules promote STAT3-dependent mitochondrial function and cell survival through integrin-FAK signaling. We furthermore show a new treatment strategy for cell survival using S727-STAT3 activators.

Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons

Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy.

The deleterious effect of cholesterol and protection by quercetin on mitochondrial bioenergetics of pancreatic β-cells, glycemic control and inflammation: In vitro and in vivo studies

Studying rats fed high cholesterol diet and a pancreatic β-cell line (Min6), we aimed to determine the mechanisms by which quercetin protects against cholesterol-induced pancreatic β-cell dysfunction and impairments in glycemic control. Quercetin prevented the increase in total plasma cholesterol, but only partially prevented the high cholesterol diet-induced alterations in lipid profile. Quercetin prevented cholesterol-induced decreases in pancreatic ATP levels and mitochondrial bioenergetic dysfunction in Min6 cells, including decreases in mitochondrial membrane potentials and coupling efficiency in the mitochondrial respiration (basal and maximal oxygen consumption rate (OCR), ATP-linked OCR and reserve capacity). Quercetin protected against cholesterol-induced apoptosis of Min6 cells by inhibiting caspase-3 and -9 activation and cytochrome c release. Quercetin prevented the cholesterol-induced decrease in antioxidant defence enzymes from pancreas (cytosolic and mitochondrial homogenates) and Min6 cells and the cholesterol-induced increase of cellular and mitochondrial oxidative status and lipid peroxidation. Quercetin counteracted the cholesterol-induced activation of the NFκB pathway in the pancreas and Min6 cells, normalizing the expression of pro-inflammatory cytokines. Quercetin inhibited the cholesterol-induced decrease in sirtuin 1 expression in the pancreas and pancreatic β-cells. Taken together, the anti-apoptotic, antioxidant and anti-inflammatory properties of quercetin, and its ability to protect and improve mitochondrial bioenergetic function are likely to contribute to its protective action against cholesterol-induced pancreatic β-cell dysfunction, thereby preserving glucose-stimulated insulin secretion (GSIS) and glycemic control. Specifically, the improvement of ATP-linked OCR and the reserve capacity are important mechanisms for protection of quercetin. In addition, the inhibition of the NFκB pathway is an important mechanism for the protection of quercetin against cytokine mediated cholesterol-induced glycemic control impairment. In summary, our data highlight cellular, molecular and bioenergetic mechanisms underlying quercetin's protective effects on β-cells in vitro and in vivo, and provide a scientifically tested foundation upon which quercetin can be developed as a nutraceutical to preserve β-cell function.

The methyl donor S-adenosylmethionine prevents liver hypoxia and dysregulation of mitochondrial bioenergetic function in a rat model of alcohol-induced fatty liver disease

BackgroundMitochondrial dysfunction and bioenergetic stress play an important role in the etiology of alcoholic liver disease. Previous studies from our laboratory show that the primary methyl donor S-Adenosylmethionine (SAM) minimizes alcohol-induced disruptions in several mitochondrial functions in the liver. Herein, we expand on these earlier observations to determine whether the beneficial actions of SAM against alcohol toxicity extend to changes in the responsiveness of mitochondrial respiration to inhibition by nitric oxide (NO), induction of the mitochondrial permeability transition (MPT) pore, and the hypoxic state of the liver. MethodsFor this, male Sprague-Dawley rats were pair-fed control and alcohol-containing liquid diets with and without SAM for 5 weeks and liver hypoxia, mitochondrial respiration, MPT pore induction, and NO-dependent control of respiration were examined. ResultsChronic alcohol feeding significantly enhanced liver hypoxia, whereas SAM supplementation attenuated hypoxia in livers of alcohol-fed rats. SAM supplementation prevented alcohol-mediated decreases in mitochondrial state 3 respiration and cytochrome c oxidase activity. Mitochondria isolated from livers of alcohol-fed rats were more sensitive to calcium-mediated MPT pore induction (i.e., mitochondrial swelling) than mitochondria from pair-fed controls, whereas SAM treatment normalized sensitivity for calcium-induced swelling in mitochondria from alcohol-fed rats. Liver mitochondria from alcohol-fed rats showed increased sensitivity to NO-dependent inhibition of respiration compared with pair-fed controls. In contrast, mitochondria isolated from the livers of SAM treated alcohol-fed rats showed no change in the sensitivity to NO-mediated inhibition of respiration. ConclusionCollectively, these findings indicate that the hepato-protective effects of SAM against alcohol toxicity are mediated, in part, through a mitochondrial mechanism involving preservation of key mitochondrial bioenergetic parameters and the attenuation of hypoxic stress.

Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells.

Lung alveoli are lined by alveolar type (AT) 1 cells and cuboidal AT2 cells. The AT1 cells are likely to be exposed to cell-free hemoglobin (Hb) in multiple lung diseases; however, the role of Hb redox (reduction-oxidation) reactions and their precise contributions to AT1 cell injury are not well understood. Using mouse lung epithelial cells (E10) as an AT1 cell model, we demonstrate here that higher Hb oxidation states, ferric Hb (HbFe(3+)) and ferryl Hb (HbFe(4+)) and subsequent heme loss play a central role in the genesis of injury. Exposures to HbFe(2+) and HbFe(3+) for 24 hours induced expression of heme oxygenase (HO)-1 protein in E10 cells and HO-1 translocation in the purified mitochondrial fractions. Both of these effects were intensified with increasing oxidation states of Hb. Next, we examined the effects of Hb oxidation and free heme on mitochondrial bioenergetic function by measuring changes in the mitochondrial transmembrane potential and oxygen consumption rate. In contrast to HbFe(2+), HbFe(3+) reduced basal oxygen consumption rate, indicating compromised mitochondrial activity. However, HbFe(4+) exposure not only induced early expression of HO-1 but also caused mitochondrial dysfunction within 12 hours when compared with HbFe(2+) and HbFe(3+). Exposure to HbFe(4+) for 24 hours also caused mitochondrial depolarization in E10 cells. The deleterious effects of HbFe(3+) and HbFe(4+) were reversed by the addition of scavenger proteins, haptoglobin and hemopexin. Collectively, these data establish, for the first time, a central role for cell-free Hb in lung epithelial injury, and that these effects are mediated through the redox transition of Hb to higher oxidation states.

Abstract 1032: Characterizing the metabolic dependencies of rhabdomyosarcoma and Ewing's sarcoma cells

Abstract Sarcomas represent a diverse group of malignancies with unique molecular and pathological characteristics. In order to improve sarcoma treatment, a better understanding of the alterations associated with specific sarcoma subtypes is critically important. Renewed interest in the altered metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a novel therapeutic strategy. In this study, we have characterized the dependency of rhabdomyosarcoma (RMS) and Ewing's sarcoma (EWS) cells on key metabolic substrates by examining cell proliferation and bioenergetic properties under varying concentrations of glucose and glutamine. We utilized two alveolar RMS cell lines, one embryonal RMS cell line, and five EWS cell lines. While all cell lines tested were completely growth-inhibited by lack of glucose, individual RMS and EWS cell lines were differentially sensitive to removal of glutamine. Furthermore, glutamine deprivation significantly reduced mitochondrial bioenergetic function, as demonstrated by Seahorse metabolic flux analysis. The observed effects of glutamine deprivation on proliferation and mitochondrial bioenergetics were rescued by the re-introduction of glutamine into the culture media. Our findings suggest that metabolic dependencies of RMS and EWS cells should be further investigated in order to identify metabolic vulnerabilities that could be targeted for potential therapeutic benefit. Citation Format: Sameer Issaq, Lee Helman. Characterizing the metabolic dependencies of rhabdomyosarcoma and Ewing's sarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1032.

Novel subcellular localization of the DNA helicase Twinkle at the kinetochore complex during mitosis in neuronal-like progenitor cells

During mitosis, the kinetochore, a multi-protein structure located on the centromeric DNA, is responsible for proper segregation of the replicated genome. More specifically, the outer kinetochore complex component Ndc80/Hec1 plays a critical role in regulating microtubule attachment to the spindle for accurate sister chromatid segregation. In addition, DNA helicases play a key contribution for precise and complete disjunction of sister chromatids held together through double-stranded DNA catenations until anaphase. In this study, we focused our attention on the nuclear-encoded DNA helicase Twinkle, which functions as an essential helicase for replication of mitochondrial DNA. It regulates the copy number of the mitochondrial genome, while maintaining its integrity, two processes essential for mitochondrial biogenesis and bioenergetic functions. Although the majority of the Twinkle protein is imported into mitochondria, a small fraction remains cytosolic with an unknown function. In this study, we report a novel expression pattern of Twinkle during chromosomal segregation at distinct mitotic phases. By immunofluorescence microscopy, we found that Twinkle protein colocalizes with the outer kinetochore protein HEC1 as early as prophase until late anaphase in neuronal-like progenitor cells. Thus, our collective results have revealed an unexpected cell cycle-regulated expression pattern of the DNA helicase Twinkle, known for its role in mtDNA replication. Therefore, its recruitment to the kinetochore suggests an evolutionary conserved function for both mitochondrial and nuclear genomic inheritance.

Melatonin: A Potential Anti-Oxidant Therapeutic Agent for Mitochondrial Dysfunctions and Related Disorders

Mitochondria play a central role in cellular physiology. Besides their classic function of energy metabolism, mitochondria are involved in multiple cell functions, including energy distribution through the cell, energy/heat modulation, regulation of reactive oxygen species (ROS), calcium homeostasis, and control of apoptosis. Simultaneously, mitochondria are the main producer and target of ROS with the result that multiple mitochondrial diseases are related to ROS-induced mitochondrial injuries. Increased free radical generation, enhanced mitochondrial inducible nitric oxide synthase (iNOS) activity, enhanced nitric oxide (NO) production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pores have all been suggested as factors responsible for impaired mitochondrial function. Because of these, neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and aging, are caused by ROS-induced mitochondrial dysfunctions. Melatonin, the major hormone of the pineal gland, also acts as an anti-oxidant and as a regulator of mitochondrial bioenergetic function. Melatonin is selectively taken up by mitochondrial membranes, a function not shared by other anti-oxidants, and thus has emerged as a major potential therapeutic tool for treating neurodegenerative disorders. Multiple in vitro and in vivo experiments have shown the protective role of melatonin for preventing oxidative stress-induced mitochondrial dysfunction seen in experimental models of PD, AD, and HD. With these functions in mind, this article reviews the protective role of melatonin with mechanistic insights against mitochondrial diseases and suggests new avenues for safe and effective treatment modalities against these devastating neurodegenerative diseases. Future insights are also discussed.

The proinflammatory cytokine, interleukin-17A, augments mitochondrial function and neurite outgrowth of cultured adult sensory neurons derived from normal and diabetic rats

BackgroundDiabetic neuropathy comprises dying back of nerve endings that reflects impairment in axonal plasticity and regenerative nerve growth. Metabolic changes in diabetes can lead to a dysregulation of hormonal mediators, such as cytokines, that may constrain distal nerve fiber growth. Interleukin-17 (IL-17A), a proinflammatory and neurotropic cytokine produced by T-cells, was significantly reduced in sciatic nerve of streptozotocin (STZ)-diabetic rats. Thus we studied the effect of IL-17A on the phenotype of sensory neurons derived from age matched control or type 1 diabetic rats. The aims were to determine the ability of IL-17A to enhance neurite outgrowth in cultured sensory neurons, investigate the signaling pathways activated by IL-17A, study the role of mitochondria and mechanistically link to neurite outgrowth. ResultsIL-17A (10ng/ml; p<0.05) significantly and dose-dependently increased total neurite outgrowth in cultures of adult dorsal root ganglia (DRG) sensory neurons derived from both control and streptozotocin (STZ)-diabetic rats. This enhancement was mediated by IL-17A-dependent activation of extracellular-regulated protein kinase (ERK) and phosphoinositide-3 kinase (PI-3K) signal transduction pathways. Pharmacological blockade of one of these activated pathways triggered complete inhibition of neurite outgrowth. IL-17A augmented mitochondrial bioenergetic function of sensory neurons derived from control or diabetic rats and this was also mediated via ERK or PI-3K. IL-17A-dependent elevation of bioenergetic function was associated with augmented expression of proteins of the mitochondrial electron transport system complexes. ConclusionsIL-17A enhanced axonal plasticity through activation of ERK and PI-3K pathways and was associated with augmented mitochondrial bioenergetic function in sensory neurons.

Depletion of mitoferrins leads to mitochondrial dysfunction and impairment of adipogenic differentiation in 3T3-L1 preadipocytes

Dysregulation of iron homeostasis is a potential risk factor for type 2 diabetes mellitus (T2DM) and insulin resistance. Iron transported into mitochondria by mitoferrins is mainly utilized for the biosynthesis of iron–sulfur clusters, heme, and other cofactors. Recent studies revealed that mitochondrial dysfunction leads to impaired adipogenesis and insulin insensitivity in adipocytes. However, it is unknown whether mitochondrial iron import and iron status affect the biogenesis and function of mitochondria during adipogenic differentiation. In this study, we used double knockdown of mitoferrin 1 and mitoferrin 2 (Mfrn1/2) to investigate the role of mitochondrial iron homeostasis in mitochondrial bioenergetic function and adipogenic differentiation. The results showed that depletion of Mfrn1/2 in 3T3-L1 preadipocytes impaired the biosynthesis of iron–sulfur proteins in mitochondria due to a decrease in mitochondrial iron content. This was associated with a decrease in mitochondrial oxygen consumption rate and intracellular ATP level in adipocytes with Mfrn1/2 knockdown. Remarkably, Mfrn1/2 deficiency reduced the expression of adipogenic genes and lipid production during adipogenic differentiation. Moreover, insulin-induced glucose uptake and Akt phosphorylation at the Ser473 residue were decreased concurrently in adipocytes differentiated from 3T3-L1 preadipocytes after knockdown of Mfrn1/2. These findings suggest that dysregulation of mitochondrial iron metabolism elicited by knockdown of Mfrn1/2 results in mitochondrial dysfunction, which culminates in the compromise of differentiation and insulin insensitivity of adipocytes. This scenario may explain the recent findings that iron deficiency or alterations in iron metabolism are associated with the pathogenesis of T2DM.

Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV

MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5–10μM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols.

The mitochondrial-targeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats

Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats.To do this, twenty-four 6week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF+MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production.These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats.

Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism

One of the proposed mechanisms for tumor proliferation involves redox signaling mediated by reactive oxygen species such as superoxide and hydrogen peroxide generated at moderate levels. Thus, the antiproliferative and anti-tumor effects of certain antioxidants were attributed to their ability to mitigate intracellular reactive oxygen species (ROS). Recent reports support a role for mitochondrial ROS in stimulating tumor cell proliferation. In this study, we compared the antiproliferative effects and the effects on mitochondrial bioenergetic functions of a mitochondria-targeted cationic carboxyproxyl nitroxide (Mito-CP), exhibiting superoxide dismutase (SOD)-like activity and a synthetic cationic acetamide analog (Mito-CP-Ac) lacking the nitroxide moiety responsible for the SOD activity. Results indicate that both Mito-CP and Mito-CP-Ac potently inhibited tumor cell proliferation. Both compounds altered mitochondrial and glycolytic functions, and intracellular citrate levels. Both Mito-CP and Mito-CP-Ac synergized with 2-deoxy-glucose (2-DG) to deplete intracellular ATP, inhibit cell proliferation and induce apoptosis in pancreatic cancer cells. We conclude that mitochondria-targeted cationic agents inhibit tumor proliferation via modification of mitochondrial bioenergetics pathways rather than by dismutating and detoxifying mitochondrial superoxide.

Glutaredoxin 2a overexpression in macrophages promotes mitochondrial dysfunction but has little or no effect on atherogenesis in LDL-receptor null mice

AimsReactive oxygen species (ROS)-mediated formation of mixed disulfides between critical cysteine residues in proteins and glutathione, a process referred to as protein S-glutathionylation, can lead to loss of enzymatic activity and protein degradation. Since mitochondria are a major source of ROS and a number of their proteins are susceptible to protein-S-glutathionylation, we examined if overexpression of mitochondrial thioltranferase glutaredoxin 2a (Grx2a) in macrophages of dyslipidemic atherosclerosis-prone mice would prevent mitochondrial dysfunction and protect against atherosclerotic lesion formation. Methods and resultsWe generated transgenic Grx2aMacLDLR−/− mice, which overexpress Grx2a as an EGFP fusion protein under the control of the macrophage-specific CD68 promoter. Transgenic mice and wild type siblings were fed a high fat diet for 14 weeks at which time we assessed mitochondrial bioenergetic function in peritoneal macrophages and atherosclerotic lesion formation. Flow cytometry and Western blot analysis demonstrated transgene expression in blood monocytes and peritoneal macrophages isolated from Grx2aMacLDLR−/− mice, and fluorescence confocal microscopy studies confirmed that Grx2a expression was restricted to the mitochondria of monocytic cells. Live-cell bioenergetic measurements revealed impaired mitochondrial ATP turnover in macrophages isolated from Grx2aMacLDLR−/− mice compared to macrophages isolated from non-transgenic mice. However, despite impaired mitochondrial function in macrophages of Grx2aMacLDLR−/− mice, we observed no significant difference in the severity of atherosclerosis between wildtype and Grx2aMacLDLR−/− mice. ConclusionOur findings suggest that increasing Grx2a activity in macrophage mitochondria disrupts mitochondrial respiration and ATP production, but without affecting the proatherogenic potential of macrophages. Our data suggest that macrophages are resistant against moderate mitochondrial dysfunction and rely on alternative pathways for ATP synthesis to support the energetic requirements.

Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons.

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer's disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons.

Protective role of melatonin in mitochondrial dysfunction and related disorders.

Mitochondria are the powerhouse of the eukaryotic cell through their use of oxidative phosphorylation to generate ATP. Mitochondrial dysfunction is considered an important contributing factor in a variety of physiopathological situations such as aging, heart ischemia/reperfusion injury, diabetes and several neurodegenerative and cardiovascular diseases, as well as in cell death. Increased formation of reactive oxygen species, altered respiratory chain complexes activity and opening of the mitochondrial permeability transition pore have been suggested as possible factors responsible for impaired mitochondrial function. Therefore, preventing mitochondrial dysfunction could be an effective therapeutic strategy against cellular degenerative processes. Cardiolipin is a unique phospholipid located at the level of inner mitochondrial membrane where it plays an important role in mitochondrial bioenergetics, as well as in cell death. Cardiolipin abnormalities have been associated with mitochondrial dysfunction in a variety of pathological conditions and aging. Melatonin, the major secretory product of the pineal gland, is a well-known antioxidant agent and thus an effective protector of mitochondrial bioenergetic function. Melatonin was reported to prevent mitochondrial dysfunction from oxidative damage by preserving cardiolipin integrity, and this may explain, at least in part, the beneficial effect of this compound in mitochondrial physiopathology. In this article, mechanisms through which melatonin exerts its protective role in mitochondrial dysfunction and related disorders are reviewed.

Isotope-reinforced polyunsaturated fatty acids protect mitochondria from oxidative stress

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure–activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D1-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D2-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20–50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function.

Regulation of Vascular Tone, Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by DL-α-Lipoic Acid.

Hydrogen sulfide (H2S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H2S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H2S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H2S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and μmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H2S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3-1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H2S production depended on enzymatic activity, although at higher concentrations (1-3 mmol/L) there was also evidence for an additional nonenzymatic H2S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants DL-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H2S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H2S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.

Endangered species mitochondrial DNA loss as a mechanism of human disease

The biologically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor in over 160 enzyme activities involved in a number of metabolic pathways, including neurotransmitter synthesis and degradation. In humans, PLP is recycled from food and from degraded PLP-dependent enzymes in a salvage pathway requiring the action of pyridoxal kinase, pyridoxine 5'-phosphate oxidase and phosphatases. Once pyridoxal 5'-phosphate is made, it is targeted to the dozens different apoenzymes that need it as a cofactor. The regulation of the salvage pathway and the mechanism of addition of PLP to the apoenzymes are poorly understood and represent a very challenging research field. Severe neurological disorders, such as convulsions and epileptic encephalopathy, result from a reduced availability of pyridoxal 5'-phosphate in the cell, due to inborn errors in the enzymes of the salvage pathway or other metabolisms and to interactions of drugs with PLP or pyridoxal kinase. Multifactorial neurological pathologies, such as autism, schizophrenia, Alzheimer's disease, Parkinson's disease and epilepsy have also been correlated to inadequate intracellular levels of PLP.

Endangered species: mitochondrial DNA loss as a mechanism of human disease.

Human mitochondrial DNA (mtDNA) is a small maternally inherited DNA, typically present in hundreds of copies in a single human cell. Thus, despite its small size, the mitochondrial genome plays a crucial role in the metabolic homeostasis of the cell. Our understanding of mtDNA genotype-phenotype relationships is derived largely from studies of the classical mitochondrial neuromuscular diseases, in which mutations of mtDNA lead to compromised mitochondrial bioenergetic function, with devastating pathological consequences. Emerging research suggests that loss, rather than mutation, of mtDNA plays a major role across a range of prevalent human diseases, including diabetes mellitus, cardiovascular disease, and aging. Here, we examine the 'rules' of mitochondrial genetics and function, the clinical settings in which loss of mtDNA is an emerging pathogenic mechanism, and explore mtDNA damage and its consequences for the organellar network and cell at large. As extranuclear genetic material arrayed throughout the cell to support metabolism, mtDNA is increasingly implicated in a host of disease conditions, opening a range of exciting questions regarding mtDNA and its role in cellular homeostasis.