The efficiency of mitochondrial electron transport chain is increased in the long‐lived mrg19 Saccharomyces cerevisiae

Integrity of mitochondrial functionality is a key determinant of longevity in several organisms. In particular, reduced mitochondrial ROS (mtROS) production leading to decreased mtDNA damage is believed to be a crucial aspect of longevity. The generation of low mtROS was thought to be due to low mitochondrial oxygen consumption. However, recent studies have shown that higher mitochondrial oxygen consumption could still result in low mtROS and contribute to longevity. This increased mitochondrial efficiency (i.e. low mtROS generated despite high oxygen consumption) was explained as a result of mitochondrial biogenesis, which provides more entry points for the electrons to the electron transport chain (ETC), thereby resulting in low mtROS production. In this study, we provide evidence for the existence of an alternative pathway to explain the observed higher mitochondrial efficiency in the long‐lived mrg19 mutant of Saccharomyces cerevisiae. Although we observe similar amounts of mitochondria in mrg19 and wild‐type (wt) yeast, we find that mrg19 mitochondria have higher expression of ETC components per mitochondria in comparison with the wt. These findings demonstrate that more efficient mitochondria because of increased ETC per mitochondria can also produce less mtROS. Taken together, our findings provide evidence for an alternative explanation for the involvement of higher mitochondrial activity in prolonging lifespan. We anticipate that similar mechanisms might also exist in eukaryotes including human.     

The Degradative Pathway of Gangliosides GM1 and GM2 in Neuro2a Cells by Sialidase

Abstract: Gangliosides GM1 [³H-labeled at the sphingosine (Sph) moiety] and GM2 [³H-labeled at the Sph or N -acetylgalactosamine (GalNAc) moiety] were administered to cultured Neuro2a cells for varying pulse (1–4 h) and chase (up to 4 h) periods, and their metabolic processing was followed. The main and earliest formed ³H-metabolites of [ Sph -³H]GM1 were GM2, asialo-GM1, asialo-GM2, and lactose-ceramide, and those of [ Sph -³H]GM2 were asialo-GM2 and lactose-ceramide. The asialo-GM1 and asialo-GM2 formed were isolated and chemically characterized. [³H]Asialo-GM2 was produced in identical amounts after treatment with equimolar [ Sph -³H]GM2 and [ GalNAc -³H]GM2. At low temperature or in the presence of chloroquine, the formation of all ³H-metabolites, including asialo-GM2 and asialo-GM1, was undetectable, indicating that ganglioside metabolic processing was an endocytosis- and lysosome-dependent process. These results demonstrate that in Neuro2a cells exogenous GM1 (and GM2) is mainly degraded through the pathway GM1 → GM2 → asialo-GM2 →→ Sph, with a minor fraction of GM1 undergoing degradation with the sequence GM1 → asialo-GM1 → asialo-GM2 →→ Sph. These findings are consistent with the hypothesis that Neuro2a cells contain a sialidase (likely of lysosomal nature) affecting ganglioside GM1 and GM2. The sialidase-mediated degradative pathway of GM1 and GM2 in Neuro2a cells might be related to the tumoral nature of these cells.     

Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.

Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). (3)H-Labeled Il(3)NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il(3)NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction. (3)H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function.     

Mitochondrial thioredoxin system: effects of TrxR2 overexpression on redox balance, cell growth, and apoptosis

Thioredoxin-2 (Trx2) is a mitochondrial protein-disulfide oxidoreductase essential for control of cell survival during mammalian embryonic development. This suggests that mitochondrial thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as compared with control cells. This growth alteration was not due to cytotoxic effects nor related to changes in basal mitochondrial transmembrane potential (DeltaPsi(m)), reactive oxygen species production, or to other mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx1, and glutathione whose levels were not affected by increased TrxR2 activity. In response to various apoptotic inducers, the extent of DeltaPsi(m) dissipation, reactive oxygen species induction, caspase activation, and loss of viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them against beta-amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey), or HeLa (human) cells revealed any differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-dependent apoptosis in these mammalian cells.     

Coupling mitogenesis and mitophagy for longevity

Maintenance of mitochondrial function and energy homeostasis requires both generation of newly synthesized and elimination of dysfunctional mitochondria. Impaired mitochondrial function and excessive mitochondrial content are major characteristics of aging and several human pathophysiological conditions, highlighting the pivotal role of the coordination between mitochondrial biogenesis and mitophagy. However, the cellular and molecular underpinnings of mitochondrial mass homeostasis remain obscure. In our recent study, we demonstrate that DCT-1, the Caenorhabditis elegans homolog of mammalian BNIP3 and BNIP3L/NIX, is a key mediator of mitophagy promoting longevity under stress. DCT-1 acts downstream of the PINK-1-PDR-1/Parkin pathway and is ubiquitinated upon mitophagy-inducing conditions to mediate the removal of damaged mitochondria. Accumulation of damaged mitochondria triggers SKN-1 activation, which initiates a bipartite retrograde signaling pathway stimulating the coordinated induction of both mitochondrial biogenesis and mitophagy genes. Taken together, our results unravel a homeostatic feedback loop that allows cells to adjust their mitochondrial population in response to environmental and intracellular cues. Age-dependent decline of mitophagy both inhibits removal of dysfunctional or superfluous mitochondria and impairs mitochondrial biogenesis resulting in progressive mitochondrial accretion and consequently, deterioration of cell function.     

Modulation of mitochondria and NADPH oxidase function by the nitrate-nitrite-NO pathway in metabolic disease with focus on type 2 diabetes

Mitochondria play fundamental role in maintaining cellular metabolic homeostasis, and metabolic disorders including type 2 diabetes (T2D) have been associated with mitochondrial dysfunction. Pathophysiological mechanisms are coupled to increased production of reactive oxygen species and oxidative stress, together with reduced bioactivity/signaling of nitric oxide (NO). Novel strategies restoring these abnormalities may have therapeutic potential in order to prevent or even treat T2D and associated cardiovascular and renal co-morbidities. A diet rich in green leafy vegetables, which contains high concentrations of inorganic nitrate, has been shown to reduce the risk of T2D. To this regard research has shown that in addition to the classical NO synthase (NOS) dependent pathway, nitrate from our diet can work as an alternative precursor for NO and other bioactive nitrogen oxide species via serial reductions of nitrate (i.e. nitrate-nitrite-NO pathway). This non-conventional pathway may act as an efficient back-up system during various pathological conditions when the endogenous NOS system is compromised (e.g. acidemia, hypoxia, ischemia, aging, oxidative stress). A number of experimental studies have demonstrated protective effects of nitrate supplementation in models of obesity, metabolic syndrome and T2D. Recently, attention has been directed towards the effects of nitrate/nitrite on mitochondrial functions including beiging/browning of white adipose tissue, PGC-1α and SIRT3 dependent AMPK activation, GLUT4 translocation and mitochondrial fusion-dependent improvements in glucose homeostasis, as well as dampening of NADPH oxidase activity. In this review, we examine recent research related to the effects of bioactive nitrogen oxide species on mitochondrial function with emphasis on T2D.     

Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes

Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity.     

Is mitochondrial generation of reactive oxygen species a trigger for autophagy?

Autophagy is a conserved lysosomal degradation pathway that has been extensively studied in recent years. However, the mechanism of autophagy induction is still not clear. Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial mediated apoptosis is the production of reactive oxygen species (ROS). Recently, several studies have indicated that ROS may be also involved in induction of autophagy. ROS are molecules or ions that are formed by the incomplete one-electron reduction of oxygen, including superoxide (O2 (*-)), hydrogen peroxide (H2O2), hydroxyl radical ((*)OH), nitric oxide (NO), and peroxynitrite (ONOO-). Our recent studies provide strong evidences for the involvement of mitochondrially-generated ROS production in the induction of autophagy as determined by the formation of autophagosomes and autolysosomes. This was accomplished through treatment with mitochondrial toxins that inhibit the electron transport chain in transformed and cancer cells. In addition, we have determined that H2O2 and 2-methoxyestradiol (inhibitor of superoxide dismutases and electron transport chain) induce autophagy leading to cell death. In contrast, normal astrocytes fail to induce autophagy following treatment with mitochondrial toxins. Herein, we discuss several important points of our studies and provide a model for mitochondrially-induced autophagic cell death mediated by ROS.     

Hyperosmotic stress induces metacaspase- and mitochondria-dependent apoptosis in Saccharomyces cerevisiae

During the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Deltacyc7Delta and cyc3Delta, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c.     

Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins.

We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced [Pacuszka, T., & Fishman, P. H. (1990) J. Biol. Chem. 265, 7673-7668]. We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP. This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1. To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf). The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells. The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation. By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation. Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine. Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin. Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.     

Mitochondrial activity in different regions of the brain at the onset of streptozotocin-induced diabetes in rats

Diabetes affects a variety of tissues including the central nervous system; moreover, some evidence indicates that memory and learning processes are disrupted. Also, oxidative stress triggers alterations in different tissues including the brain. Recent studies indicate mitochondria dysfunction is a pivotal factor for neuron damage. Therefore, we studied mitochondrial activity in three brain regions at early type I—diabetes induction. Isolated mitochondria from normal hippocampus, cortex and cerebellum revealed different rates of oxygen consumption, but similar respiratory controls. Oxygen consumption in basal state 4 significantly increased in the mitochondria from all three brain regions from diabetic rats. No relevant differences were observed in the activity of respiratory complexes, but hippocampal mitochondrial membrane potential was reduced. However, ATP content, mitochondrial cytochrome c, and protein levels of β-tubulin III, synaptophysin, and glutamine synthase were similar in brain regions from normal and diabetic rats. In addition, no differences in total glutathione levels were observed between normal and diabetic rat brain regions. Our results indicated that different regions of the brain have specific metabolic responses. The changes in mitochondrial activity we observed at early diabetes induction did not appear to cause metabolic alterations, but they might appear at later stages. Longer-term streptozotocin treatment studies must be done to elucidate the impact of hyperglycemia in brain metabolism and the function of specific brain regions.     

Molecular characterization of mouse CREB3 regulatory factor in Neuro2a cells

We performed expression and functional analysis of mouse CREB3 regulatory factor (CREBRF) in Neuro2a cells by constructing several expression vectors. Overexpressed full-length (FL) CREBRF protein was stabilized by MG132; however, the intrinsic CREBRF expression in Neuro2a cells was negligible under all conditions. On the other hand, N- or C-terminal deletion of CREBRF influenced its stability. Cotransfection of CREBRF together with GAL4-tagged FL CREB3 increased luciferase reporter activity, and only the N-terminal region of CREBRF was sufficient to potentiate luciferase activity. Furthermore, this positive effect of CREBRF was also observed in cells expressing GAL4-tagged cleaved CREB3, although CREBRF hardly influenced the protein stability of NanoLuc-tagged cleaved CREB3 or intracellular localization of EGFP-tagged one. In conclusion, this study suggests that CREBRF, a quite unstable proteasome substrate, positively regulates the CREB3 pathway, which is distinct from the canonical ER stress pathway in Neuro2a cells.

     

Insulin-like growth factor-induced signals activate mitochondrial respiration

From experiments with lower eukaryotes it is known that the metabolic rate and also the rate of aging are tightly controlled by the insulin-like growth factor (IGF)/insulin signal transduction pathway. The mitochondrial theory of aging implies that an increased metabolic rate leads to increased mitochondrial activity; increased production of reactive oxygen species due to these alterations would speed up the aging process. To address the question if mitochondrial activity is influenced by insulin/IGF signaling, we have established an experimental system to determine the influence of IGF-I-dependent signaling on mitochondrial function. We used DU145 prostate cancer cells, known for the intact IGF signal transduction pathway, to address the influence of IGF receptor activation on mitochondrial function by high-resolution respirometry. These experiments revealed that indeed mitochondrial function is regulated by IGF signaling, and up-regulation of respiration seems to require phosphoinositide 3-kinase/AKT signaling, but is independent of IGF effects on cell cycle progression. Collectively these data establish a regulatory cross-talk between insulin/IGF signal transduction and mitochondrial function, two major pathways implicated in controlling the rate of aging.     

Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway

Mitochondrial DNA (mtDNA) is located close to the respiratory chain, a major source of reactive oxygen species (ROS). This proximity makes mtDNA more vulnerable than nuclear DNA to damage by ROS. Therefore, the efficient repair of oxidative lesions in mtDNA is essential for maintaining the stability of the mitochondrial genome. A series of genetic and biochemical studies has indicated that eukaryotic cells, including the model organism Saccharomyces cerevisiae, use several alternative strategies to prevent mutagenesis induced by endogenous oxidative damage to nuclear DNA. However, apart from base excision repair (BER), no other pathways involved in the repair of oxidative damage in mtDNA have been identified. In this study, we have examined mitochondrial mutagenesis in S. cerevisiae cells which lack the activity of the Ogg1 glycosylase, an enzyme playing a crucial role in the removal of 8-oxoG, the most abundant oxidative lesion of DNA. We show that the overall frequency of the mitochondrial oligomycin-resistant (Olir) mutants is increased in the ogg1 strain by about one order of magnitude compared to that of the wild-type strain. Noteworthy, in the mitochondrial oli1 gene, G:C to T:A transversions are generated approximately 50-fold more frequently in the ogg1 mutant relative to the wild-type strain. We also demonstrate that the increased frequency of Olir mutants in the ogg1 strain is markedly reduced by the presence of plasmids encoding Msh1p, a homologue of the bacterial mismatch protein MutS, which specifically functions in mitochondria. This suppression of the mitochondrial mutator phenotype of the ogg1 strain seems to be specific, since overexpression of the mutant allele msh1-R813W failed to exert this effect. Finally, we also show that the increased frequency of Olir mutants arising in an msh1/MSH1 heterozygote grown in glucose-containing medium is further enhanced if the cells are cultivated in glycerol-containing medium, i.e. under conditions when the respiratory chain is fully active. Taken together, these results strongly suggest that MSH1-dependent repair represents a significant back-up to mtBER in the repair of oxidative damage in mtDNA.     

Zafirlukast promotes mitochondrial respiration by stimulating mitochondrial biogenesis in human bronchial epithelial cells

Lung diseases, including asthma, pose a serious global health issue. Loss of mitochondrial function and decreased mitochondrial biogenesis play pivotal roles in the initiation and progression of chronic lung diseases. Thus, maintaining mitochondrial function and homeostasis is an important treatment goal. Zafirlukast is a CysLTR1 antagonist that is widely used as an adjuvant treatment for asthma. In the present study, we investigated the effects of zafirlukast in vitro using human bronchial epithelial cells (BECs). We performed measurements of oxygen consumption and bioenergetics and found that zafirlukast increased mitochondrial respiration and biogenesis in human BECs as evidenced by increased mitochondrial mass and mtDNA/nDNA. Through real-time PCR and western blot analysis, we found that zafirlukast significantly increased the expression of PGC-1α, NRF1, and TFAM at both the mRNA and protein levels. Finally, we determined that these effects are mediated through CREB signaling and that inhibition of CREB with its specific inhibitor H89 abolished the effects of zafirlukast described above. Thus, zafirlukast might have potential in enhancing mitochondrial function by promoting mitochondrial biogenesis in human bronchial epithelial cells through upregulating the expression of PGC-1α and activating the CREB pathway.