Mitochondrial Electron Transport Chain Activity Research Articles

Secreted VAPB/ALS8 Major Sperm Protein Domains Modulate Mitochondrial Localization and Morphology via Growth Cone Guidance Receptors

The VAPB/ALS8 major sperm protein domain (vMSP) is implicated in amyotrophic lateral sclerosis and spinal muscular atrophy, yet its function in the nervous system is not well understood. In Caenorhabditis elegans and Drosophila, the vMSP is cleaved from its transmembrane anchor and secreted in a cell type-specific fashion. We show that vMSPs secreted by neurons act on Lar-like protein-tyrosine phosphatase and Roundabout growth cone guidance receptors expressed in striated muscle. This signaling pathway promotes Arp2/3-dependent actin remodeling and mitochondrial localization to actin-rich muscle I-bands. C. elegans VAPB mutants have mitochondrial localization, morphology, mobility, and fission/fusion defects that are suppressed by Lar-like receptor or Arp2/3 inactivation. Hence, growth cone guidance receptor pathways that remodel the actin cytoskeleton have unanticipated effects on mitochondrial dynamics. We propose that neurons secrete vMSPs to promote striated muscle energy production and metabolism, in part through the regulation of mitochondrial localization and function.

Oxygen versus Reactive Oxygen in the Regulation of HIF-1α: The Balance Tips.

Hypoxia inducible factor (HIF) is known as the master regulator of the cellular response to hypoxia and is of pivotal importance during development as well as in human disease, particularly in cancer. It is composed of a constitutively expressed β subunit (HIF-1β) and an oxygen-regulated α subunit (HIF-1α and HIF-2α), whose stability is tightly controlled by a family of oxygen- and iron-dependent prolyl hydroxylase enzymes. Whether or not mitochondria-derived reactive oxygen species (ROS) are involved in the regulation of Hypoxia Inducible Factor-1α has been a matter of contention for the last 10 years, with equally compelling evidence in favor and against their contribution. A number of recent papers appear to tip the balance against a role for ROS. Thus, it has been demonstrated that HIF prolyl hydroxylases are unlikely to be physiological targets of ROS and that the increase in ROS that is associated with downregulation of Thioredoxin Reductase in hypoxia does not affect HIF-1α stabilization. Finally, the protein CHCHD4, which modulates cellular HIF-1α concentrations by promoting mitochondrial electron transport chain activity, has been proposed to exert its regulatory effect by affecting cellular oxygen availability. These reports are consistent with the hypothesis that mitochondria play a critical role in the regulation of HIF-1α by controlling intracellular oxygen concentrations.

The failure to extend lifespan via disruption of complex II is linked to preservation of dynamic control of energy metabolism

A decrease in mitochondrial electron transport chain (ETC) activity results in an extended lifespan in Caenorhabditis elegans. This longevity has only been reported when complexes I, III and IV genes are silenced, but not genes of complex II. We now have suppressed each complex II subunit in turn and have confirmed that in no case is lifespan extended. Animals with impaired complex II function exhibit similar metabolic changes to those observed following suppression of complexes I, III and IV genes, but the magnitude of the changes is smaller. Furthermore, an inverse correlation exists between mitochondrial membrane potential and ATP levels (r2=0.82), which strongly suggests that dynamic allocation of energy resources is maintained. In contrast, suppression of genes from complexes I, III and IV, results in a metabolic crisis with an associated stress response and loss of metabolic flexibility. Thus, the maintenance of a normal metabolism at a moderately decreased level does not alter normal lifespan, whereas metabolic crisis and induction of a stress response is linked to lifespan extension.

Peroxisome proliferator activator receptor gamma coactivator-1alpha (PGC-1α) improves motor performance and survival in a mouse model of amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects spinal cord and cortical motor neurons. An increasing amount of evidence suggests that mitochondrial dysfunction contributes to motor neuron death in ALS. Peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) is a principal regulator of mitochondrial biogenesis and oxidative metabolism.ResultsIn this study, we examined whether PGC-1α plays a protective role in ALS by using a double transgenic mouse model where PGC-1α is over-expressed in an SOD1 transgenic mouse (TgSOD1-G93A/PGC-1α). Our results indicate that PGC-1α significantly improves motor function and survival of SOD1-G93A mice. The behavioral improvements were accompanied by reduced blood glucose level and by protection of motor neuron loss, restoration of mitochondrial electron transport chain activities and inhibition of stress signaling in the spinal cord.ConclusionOur results demonstrate that PGC-1α plays a beneficial role in a mouse model of ALS, suggesting that PGC-1α may be a potential therapeutic target for ALS therapy.

Regulation of Yeast Chronological Life Span by TORC1 via Adaptive Mitochondrial ROS Signaling

Here we show that yeast strains with reduced target of rapamycin (TOR) signaling have greater overall mitochondrial electron transport chain activity during growth that is efficiently coupled to ATP production. This metabolic alteration increases mitochondrial membrane potential and reactive oxygen species (ROS) production, which we propose supplies an adaptive signal during growth that extends chronological life span (CLS). In strong support of this concept, uncoupling respiration during growth or increasing expression of mitochondrial manganese superoxide dismutase significantly curtails CLS extension in tor1Δ strains, and treatment of wild-type strains with either rapamycin (to inhibit TORC1) or menadione (to generate mitochondrial ROS) during growth is sufficient to extend CLS. Finally, extension of CLS by reduced TORC1/Sch9p-mitochondrial signaling occurs independently of Rim15p and is not a function of changes in media acidification/composition. Considering the conservation of TOR-pathway effects on life span, mitochondrial ROS signaling may be an important mechanism of longevity regulation in higher organisms.

Ultrafine Particulate Ferrous Iron and Anthracene Associations with Mitochondrial Dysfunction

The ultrafine size fraction of ambient particles (ultrafine particles [UFP], diameter < 100 nm) has been identified as being particularly potent in their adverse health effects, yet, the detailed mechanisms for why UFP display such distinctive toxicity are not well understood. In the present study, mitochondria were exposed to ambient UFP while monitoring mitochondrial electron transport chain (ETC) activity as a model system for biochemical toxicity. UFP samples were collected in rural and urban environments, and chemically characterized for trace metals, ferrous (Fe(II)) and easily reducible ferric (Fe(III)) iron, polycyclic aromatic hydrocarbons (PAHs), and surface constituents with X-ray photoelectron spectroscopy (XPS). Fixed doses of UFP (8 μg mL−1) inhibited mitochondrial ETC function compared to controls in 94% of the samples after the 20 min of exposure. Significant moderate to weak correlations exist between initial %ETC inhibition (0 – 10 min) and Fe(II) (R = 0.55, P = 0.03, N = 15), anthracene...

Intracellular fatty acids suppress β-adrenergic induction of PKA-targeted gene expression in white adipocytes

β-Adrenergic receptor (β-AR) activation elevates cAMP levels in fat cells and triggers both metabolic and transcriptional responses; however, the potential interactions between these pathways are poorly understood. This study investigated whether lipolysis affects β-AR-mediated gene expression in adipocytes. Acute β(3)-adrenergic receptor (β(3)-AR) stimulation with CL 316,243 (CL) increased expression of PKA-targeted genes PCG-1α, UCP1, and NOR-1 in mouse white fat. Limiting lipolysis via inhibition of hormone-sensitive lipase (HSL), a direct target of PKA, sharply potentiated CL induction of PCG-1α, UCP1, and NOR-1. CL also induced greater expression of PKA-targeted genes in white fat of HSL-null mice compared with wild-type littermates, further indicating that HSL activity limits PKA-mediated gene expression. Inhibiting HSL in 3T3-L1 adipocytes also potentiated the induction of PGC-1α, UCP1, and NOR-1 by β-AR activation, as did siRNA knockdown of adipose triglyceride lipase, the rate-limiting enzyme for lipolysis. Conversely, treatments that promote intracellular fatty acid accumulation suppressed induction of PGC-1α and UCP1 through β-AR stimulation. Analysis of β-adrenergic signaling indicated that excessive intracellular fatty acid production inhibits adenylyl cyclase activity and thereby reduces PKA signaling to the nucleus. Lastly, partially limiting lipolysis by inhibition of HSL increased the induction of oxidative gene expression and mitochondrial electron transport chain activity in white adipose tissue and facilitated fat loss in mice treated for 5 days with CL. Overall, our results demonstrate that fatty acids limit the upregulation of β-AR-responsive genes in white adipocytes and suggest that limiting lipolysis may be a novel means of enhancing β-AR signaling.

Characterization of reduced and oxidized dopamine and 3,4-dihydrophenylacetic acid, on brain mitochondrial electron transport chain activities

Loss of dopamine (DA) homeostasis may be a contributing factor to cell damage in Parkinson's disease (PD). Past studies showing deleterious effects of DA on mitochondrial function, however, have been inconsistent raising questions about mitochondria as a downstream target for DA. Issues such as the dopamine species i.e., reduced or oxidized, time of exposure and the effect of major metabolites such as 3,4-dihydrophenylacetic acid (DOPAC) may contribute to the disparate findings. The present study used isolated, lysed rat brain mitochondria to characterize the effects of oxidized or reduced DA and DOPAC on complex activities of the electron transport chain (ETC). Time of exposure and quantitation of reduced or oxidized catachols for DA and DOPAC were monitored for all experiments. Reduced DA and DOPAC with or without a 30 min preincubation had no affect on NADH oxidase activity which monitors the activities of complexes I, III and IV. Complex II activity was inhibited by reduced DA (≥ 500 μM), but not by reduced DOPAC and was significantly attenuated by SOD suggesting reactive oxygen species involvement. In contrast, fully oxidized DA and DOPAC dose dependently inhibited NADH oxidase, complex I and complex III activities with IC 50s in the 50–200 μM range. No preincubation was required for inhibition with the catechols when they were fully oxidized. Oxidized DA inhibited complex I only when exposure occurred during stimulated electron flow, suggesting covalent binding of quinones to proteins within active sites of the complex. In intact, well coupled mitochondria, extramitochondrial DA was shown to access the mitochondrial matrix in a dose, time and energy-dependent fashion. The findings suggest that many of the reported inconsistencies with regards to the effects of DA and DOPAC on ETC function can be attributed to the oxidized state of the catechol at the time of exposure. In addition, the findings provide possible downstream targets for DA that could contribute to the vulnerability of dopaminergic neurons in PD.

Mitochondrial plasticity in the anoxic turtle heart

Our objective was to assess mitochondrial respiration and electron transport chain (ETC) activities in permiabilised cardiac fibers from normoxic and anoxic (2 weeks) turtles (Trachemys scripta) acclimated to 5°C. All experiments on animals were conducted in conformance with the FASEB Principles. Mitochondrial maximum ADP stimulated respiration was measured with an Oroboros micro‐respirometer, and respiratory flux through complex I, II and IV of the ETC was assessed by pharmacological intervention. ADP affinity was determined by adding ADP in steps from 0.025 mM to a maximum of 1 mM. All preparations were tested at 5, 13 and 21°C. Anoxic acclimation led to significant reductions in maximum respiration rates through ETC complexes I, II and IV at all temperatures tested, and ADP affinity was significantly lower. State II respiration rates were significantly reduced in anoxic cardiac fibers, leading to a higher respiratory control ratio, which is indicative of a reduction in proton leak. Collectively, these results suggest a reduction in mitochondrial capacity contributes to overall hypometabolism in anoxic turtles. Since complex I of the ETC is a major site of reactive oxygen species generation, a downregulation of this protein may also help to prevent redox damage during anoxic recovery. This research was funded by The Killam Memorial Trust and The Natural Sciences and Educational Research Council of Canada.

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

The role of mitochondria in osteoarthritis

Mitochondria are important regulators of cellular function and survival that may have a key role in aging-related diseases. Mitochondrial DNA (mtDNA) mutations and oxidative stresses are known to contribute to aging-related changes. Osteoarthritis (OA) is an aging-associated rheumatic disease characterized by articular cartilage degradation and elevated chondrocyte mortality. Articular cartilage chondrocytes survive and maintain tissue integrity in an avascular, low-oxygen environment. Recent ex vivo studies have reported mitochondrial dysfunction in human OA chondrocytes, and analyses of mitochondrial electron transport chain activity in these cells show decreased activity of Complexes I, II and III compared to normal chondrocytes. This mitochondrial dysfunction may affect several pathways that have been implicated in cartilage degradation, including oxidative stress, defective chondrocyte biosynthesis and growth responses, increased cytokine-induced chondrocyte inflammation and matrix catabolism, cartilage matrix calcification, and increased chondrocyte apoptosis. Mitochondrial dysfunction in OA chondrocytes may derive from somatic mutations in the mtDNA or from the direct effects of proinflammatory mediators such as cytokines, prostaglandins, reactive oxygen species and nitric oxide. Polymorphisms in mtDNA may become useful as biomarkers for the diagnosis and prognosis of OA, and modulation of serum biomarkers by mtDNA haplogroups supports the concept that mtDNA haplogroups may define specific OA phenotypes in the complex OA process.

Effect of Lead on Oxidative Stress, Na+K+ATPase Activity and Mitochondrial Electron Transport Chain Activity of the Brain of Clarias batrachus L.

The present invivo study was designed to elucidate the toxic effect of lead on oxidative stress, Na(+)K(+)ATPase and mitochondrial electron transport chain activity of the brain of Clarias batrachus. The fish were exposed to 10 and 20% of the derived 96 h LC(50) value, 37.8 and 75.6 mg L(-1), respectively, and sampled on 20, 40 and 60 days. Exposure of fish brain to lead demonstrated an increased production of reactive oxygen species, increased lipid peroxidation, loss of protein thiol groups in synaptosomal fraction with the decreased activity of Na(+)K(+)ATPase, partial inactivation of mitochondrial electron transport chain activity and energy depletion. However, no change in protein carbonyl content in synaptosomal fraction was observed due to lead exposure. Concluding the results of our investigation we suggest that lead exposure induces oxidative stress in the brain of Clarias batrachus and the decline in Na(+)K(+)ATPase activity was presumeably mediated by the combined action of lipid peroxidation and deficient mitochondrial electron transport chain activity.

Chromium III Exposure Inhibits Brain Na+K+ATPase Activity of Clarias batrachus L. Involving Lipid Peroxidation and Deficient Mitochondrial Electron Transport Chain Activity

The present study elucidated the role of lipid peroxidation and diminished mitochondrial electron transport chain activity in partial dysfunction of brain Na+K+ATPase of Clarias batrachus exposed to chromium III ions. The fish were exposed to 10% and 20% of the derived 96 h LC50 value, 5.69 mg/L and 11.38 mg/L, respectively, and sampled on 20, 40 and 60 days. Exposure to chromium III on fish brain demonstrated an increased lipid peroxidation, production of protein carbonyl and reactive oxygen species and loss of protein thiol groups in synaptosomal fraction with decreased activity of Na+K+ATPase, partial inactivation of mitochondrial electron transport chain activity and energy depletion.

Altered skeletal muscle insulin signaling and mitochondrial complex II-III linked activity in adult offspring of obese mice

We recently reported insulin resistance in adult offspring of obese C57BL/6J mice. We have now evaluated whether parameters of skeletal muscle structure and function may play a role in insulin resistance in this model of developmental programming. Obesity was induced in female mice by feeding a highly palatable sugar and fat-rich diet for 6 wk prior to pregnancy, and during pregnancy and lactation. Offspring of obese dams were weaned onto standard laboratory chow. At 3 mo of age, skeletal muscle insulin signaling protein expression, mitochondrial electron transport chain activity (ETC), muscle fiber type, fiber density, and fiber cross-sectional area were compared with that of offspring of control dams weaned onto the chow diet. Female offspring of obese dams demonstrated decreased skeletal muscle expression of p110beta, the catalytic subunit of PI3K (P < 0.01), as well as reduced Akt phosphorylation at Serine residue 473 compared with control offspring. Male offspring of obese dams demonstrated increased skeletal muscle Akt2 and PKCzeta expression (P < 0.01; P < 0.001, respectively). A decrease in mitochondrial-linked complex II-III was observed in male offspring of obese dams (P < 0.01), which was unrelated to CoQ deficiency. This was not observed in females. There were no differences in muscle fiber density between offspring of obese dams and control offspring in either sex. Sex-related alterations in key insulin-signaling proteins and in mitochondrial ETC may contribute to a state of insulin resistance in offspring of obese mice.

Silencing of PINK1 expression affects mitochondrial DNA and oxidative phosphorylation in dopaminergic cells.

BackgroundMitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD). Impairment of the mitochondrial electron transport chain (ETC) and an increased frequency in deletions of mitochondrial DNA (mtDNA), which encodes some of the subunits of the ETC, have been reported in the substantia nigra of PD brains. The identification of mutations in the PINK1 gene, which cause an autosomal recessive form of PD, has supported mitochondrial involvement in PD. The PINK1 protein is a serine/threonine kinase localized in mitochondria and the cytosol. Its precise function is unknown, but it is involved in neuroprotection against a variety of stress signalling pathways.Methodology/Principal FindingsIn this report we have investigated the effect of silencing PINK1 expression in human dopaminergic SH-SY5Y cells by siRNA on mtDNA synthesis and ETC function. Loss of PINK1 expression resulted in a decrease in mtDNA levels and mtDNA synthesis. We also report a concomitant loss of mitochondrial membrane potential and decreased mitochondrial ATP synthesis, with the activity of complex IV of the ETC most affected. This mitochondrial dysfunction resulted in increased markers of oxidative stress under basal conditions and increased cell death following treatment with the free radical generator paraquat.ConclusionsThis report highlights a novel function of PINK1 in mitochondrial biogenesis and a role in maintaining mitochondrial ETC activity. Dysfunction of both has been implicated in sporadic forms of PD suggesting that these may be key pathways in the development of the disease.

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

The effect of unbound palmitic acid (PA) at plasma physiological concentration range on reactive oxygen species (ROS) production by cultured rat skeletal muscle cells was investigated. The participation of the main sites of ROS production was also examined. Production of ROS was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. PA increased ROS production after 1 h incubation. A xanthine oxidase inhibitor did not change PA-induced ROS production. However, the treatment with a mitochondrial uncoupler and mitochondrial complex III inhibitor decreased superoxide production induced by PA. The importance of mitochondria was also evaluated in 1 h incubated rat soleus and extensor digitorum longus (EDL) muscles. Soleus muscle, which has a greater number of mitochondria than EDL, showed a higher superoxide production induced by PA. These results indicate that mitochondrial electron transport chain is an important contributor for superoxide formation induced by PA in skeletal muscle. Results obtained with etomoxir and bromopalmitate treatment indicate that PA has to be oxidized to raise ROS production. A partial inhibition of superoxide formation induced by PA was observed by treatment with diphenylene iodonium, an inhibitor of NADPH oxidase. The participation of this enzyme complex was confirmed through an increase of p47(phox) phosphorylation after treatment with PA.

Quinone and oxyradical scavenging properties of N-acetylcysteine prevent dopamine mediated inhibition of Na+, K+-ATPase and mitochondrial electron transport chain activity in rat brain: Implications in the neuroprotective therapy of Parkinson's disease

Dopamine oxidation products such as H2O2 and reactive quinones have been held responsible for various toxic actions of dopamine, which have implications in the aetiopathogenesis of Parkinson's disease. This study has shown that N-acetylcysteine (0.25–1 mm) is a potent scavenger of both H2O2 and toxic quinones derived from dopamine and it further prevents dopamine mediated inhibition of Na+,K+-ATPase activity and mitochondrial respiratory chain function. The quinone scavenging ability of N-acetylcysteine is presumably related to its protective effect against dopamine mediated inhibition of mitochondrial respiratory chain activity. However, both H2O2 scavenging and quinone scavenging properties of N-acetylcysteine probably account for its protective effect against Na+,K+-ATPase inhibition induced by dopamine. The results have important implications in the neuroprotective therapy of sporadic Parkinson's disease since inactivation of mitochondrial respiratory activity and Na+,K+-ATPase may trigger intracellular damage pathways leading to the death of nigral dopaminergic neurons.

Mitochondrial Fusion Protects against Neurodegeneration in the Cerebellum

Mutations in the mitochondrial fusion gene Mfn2 cause the human neurodegenerative disease Charcot-Marie-Tooth type 2A. However, the cellular basis underlying this relationship is poorly understood. By removing Mfn2 from the cerebellum, we established a model for neurodegeneration caused by loss of mitochondrial fusion. During development and after maturity, Purkinje cells require Mfn2 but not Mfn1 for dendritic outgrowth, spine formation, and cell survival. In vivo, cell culture, and electron microscopy studies indicate that mutant Purkinje cells have aberrant mitochondrial distribution, ultrastructure, and electron transport chain activity. In fibroblasts lacking mitochondrial fusion, the majority of mitochondria lack mitochondrial DNA nucleoids. This deficiency provides a molecular mechanism for the dependence of respiratory activity on mitochondrial fusion. Our results show that exchange of mitochondrial contents is important for mitochondrial function as well as organelle distribution in neurons and have important implications for understanding the mechanisms of neurodegeneration due to perturbations in mitochondrial fusion.

The Role of Autophagy in Mitochondria Maintenance:Characterization of Mitochondrial Functions in Autophagy-Deficient S. cerevisiae Strains

Autophagy is a lysosome-dependent cellular degradation process. Organisms bearing deletions of the essential autophagy genes exhibit various pathological conditions, including cancer in mammals and shortened life span in C. elegans. The direct cause for these phenotypes is not clear. Here we used yeast as a model system to characterize the cellular consequence of ATG (autophagy-related) gene deletions. We found that the atg mutant strains, atg1∆, atg6∆, atg8∆ and atg12∆, showed defects related to mitochondrial biology. These strains were unable to degrade mitochondria in stationary culture. In non-fermentable medium, which requires mitochondrial oxidative phosphorylation for survival, these atg strains showed a growth defect with an increased cell population at the G1 phase of the cell cycle. The cells had lower oxygen consumption rates and reduced mitochondrial electron transport chain activities. Under these growth conditions, the atg strains had lower mitochondrial membrane potential. In addition, these mutants generated higher levels of reactive oxygen species (ROS) and they were prone to accumulate dysfunctional mitochondria. This study clearly indicates that an autophagy defect has a functional impact on various aspects of mitochondrial functions and suggests a critical role of autophagy in mitochondria maintenance.

The enigmatic contribution of mitochondrial function in photosynthesis

Considerable cumulative evidence has accrued suggesting a vital role for mitochondrial function in optimizing photosynthesis. Both pharmacological approaches using respiratory inhibitors and reverse genetic approaches have recently underscored the high degree of interconnection between photosynthesis and respiration--the major pathways of energy production which are largely confined to the plastid and mitochondria, respectively. Here recent studies into the nature of these interactions are reviewed, with particular focus on (i) the recently described link between the mitochondrial electron transport chain activity, ascorbate biosynthesis, and photosynthesis; and (ii) the contribution of mitochondrial metabolism to the photorespiratory process. Whilst there is increasing evidence of a role for ascorbate in co-ordinating the rates of respiration and photosynthesis, some data are presented here for plants grown under extreme environmental conditions that suggest that this relationship is not absolute. It thus seems likely that interactions between these compartments are perhaps more numerous and complicated than previously thought. This observation suggests that although the elucidation of the genetic bases of both photorespiration and the Wheeler-Smirnoff pathway of ascorbate biosynthesis has recently been completed, much further research is probably necessary in order to understand fully how energy metabolism is co-ordinated in the illuminated leaf.