Mechanisms of regulation of mitochondrial metabolism in trypanosomes are not completely understood. Here we present evidence that the Trypanosoma brucei mitochondrial calcium uniporter (TbMCU) is ...essential for the regulation of mitochondrial bioenergetics, autophagy and cell death, even in the bloodstream forms that are devoid of a functional respiratory chain and oxidative phosphorylation. Localization studies reveal its co-localization with MitoTracker staining. TbMCU overexpression increases mitochondrial Ca(2+) accumulation in intact and permeabilized trypanosomes, generates excessive mitochondrial reactive oxygen species (ROS) and sensitizes them to apoptotic stimuli. Ablation of TbMCU in RNAi or conditional knockout trypanosomes reduces mitochondrial Ca(2+) uptake without affecting their membrane potential, increases the AMP/ATP ratio, stimulates autophagosome formation and produces marked defects in growth in vitro and infectivity in mice, revealing its essentiality in these parasites. The requirement of TbMCU for proline and pyruvate metabolism in procyclic and bloodstream forms, respectively, reveals its role in regulation of mitochondrial bioenergetics.
NADPH is the reducing agent for mitochondrial H2O2 detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes ...to an elevated mitochondrial NADPH/NADP+ ratio. This enzyme catalyzes the reduction of NADP+ at the expense of NADH oxidation and H+ reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-NntMUT) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-NntMUT mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-NntW) and from B6J-NntMUT mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-NntMUT mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H2O2 release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca2+-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-NntMUT mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-NntW mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-NntMUT mice in biomedical research should not be overlooked.
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•The NNT enzyme is a source of NADPH in the mitochondrial matrix.•C57BL/6J mice strain lacks NNT activity due to a Nnt gene mutation.•C57BL/6J mitochondria are unable to properly metabolize exogenous peroxide.•Intact mitochondria from C57BL/6J mice exhibit several redox abnormalities.•The widespread use of C57BL/6J mice in biomedical research should be reassessed.
Aim: Atherosclerosis is responsible for high morbidity and mortality rates around the world. Local arterial oxidative stress is involved in all phases of atherosclerosis development. Mitochondria is ...a relevant source of the oxidants, particularly under certain risky conditions, such as hypercholesterolemia. The aim of this study was to test whether lowering the production of mitochondrial oxidants by induction of a mild uncoupling can reduce atherosclerosis in hypercholesterolemic LDL receptor knockout mice. Methods: The mice were chronically treated with very low doses of DNP (2,4-dinitrophenol) and metabolic, inflammatory and redox state markers and atherosclerotic lesion sizes were determined.Results: The DNP treatment did not change the classical atherosclerotic risk markers, such as plasma lipids, glucose homeostasis, and fat mass, as well as systemic inflammatory markers. However, the DNP treatment diminished the production of mitochondrial oxidants, systemic and tissue oxidative damage markers, peritoneal macrophages and aortic rings oxidants generation. Most importantly, development of spontaneous and diet-induced atherosclerosis (lipid and macrophage content) were significantly decreased in the DNP-treated mice. In vitro, DNP treated peritoneal macrophages showed decreased H2O2 production, increased anti-inflammatory cytokines gene expression and secretion, increased phagocytic activity, and decreased LDL-cholesterol uptake.Conclusions: These findings are a proof of concept that activation of mild mitochondrial uncoupling is sufficient to delay the development of atherosclerosis under the conditions of hypercholesterolemia and oxidative stress. These results promote future approaches targeting mitochondria for the prevention or treatment of atherosclerosis.
In the first part of this review, we summarize basic mitochondrial bioenergetics concepts showing that mitochondria are critical regulators of cell life and death. Until a few decades ago, ...mitochondria were considered to play essential roles only in respiration, ATP formation, non-shivering thermogenesis and a variety of metabolic pathways. However, the concept presented by Peter Mitchell regarding coupling between electron flow and ATP synthesis through the intermediary of a H+ electrochemical potential leads to the recognition that the proton-motive force also regulates a series of relevant cell signalling processes, such as superoxide generation, redox balance and Ca2+ handling. Alterations in these processes lead to cell death and disease states. In the second part of this review, we discuss the role of mitochondrial dysfunctions in the specific context of hypercholesterolemia-induced atherosclerosis. We provide a literature analysis that indicates a decisive role of mitochondrial redox dysfunction in the development of atherosclerosis and discuss the underlying molecular mechanisms. Finally, we highlight the potential mitochondrial-targeted therapeutic strategies that are relevant for atherosclerosis.
Mitochondrially generated reactive oxygen species are involved in a myriad of signaling and damaging pathways in different tissues. In addition, mitochondria are an important target of reactive ...oxygen and nitrogen species. Here, we discuss basic mechanisms of mitochondrial oxidant generation and removal and the main factors affecting mitochondrial redox balance. We also discuss the interaction between mitochondrial reactive oxygen and nitrogen species, and the involvement of these oxidants in mitochondrial diseases, cancer, neurological, and cardiovascular disorders.
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•T. cruzi IP3 receptor (TcIP3R) is located in acidocalcisomes of different stages.•TcIP3R is involved in Ca2+ release from acidocalcisomes and mitochondrial integrity.•Ablation of ...TcIP3R was done using CRISPR/Cas9 gene editing.•TcIP3R-KO trypanosomes have reduced mitochondrial O2 consumption.•TcIP3R-KO trypanosomes have increased AMP/ATP ratio and autophagy.
In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIP3R) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIP3R is a Ca2+ release channel gated by IP3 when expressed in DT40 cells knockout for all vertebrate IP3 receptors, and is required for Ca2+ uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O2 consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIP3R by CRISPR/Cas9 genome editing caused: a) a reduction in O2 consumption rate and citrate synthase activity; b) decreased mitochondrial Ca2+ transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIP3R overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIP3R-mediated acidocalcisome Ca2+ release on cell bioenergetics in T. cruzi.
Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report ...here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP3R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP3R, endogenously tagged TcIP3R showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.
Metformin is a widely used antidiabetic drug whose anticancer effects, mediated by the activation of AMP-activated protein kinase (AMPK) and reduction of mTOR signaling, have become noteworthy. ...Chemotherapy produces genotoxic stress and induces p53 activity, which can cross-talk with AMPK/mTOR pathway. Herein, we investigate whether the combination of metformin and paclitaxel has an effect in cancer cell lines.
Human tumors were xenografted into severe combined immunodeficient (SCID) mice and the cancer cell lines were treated with only paclitaxel or only metformin, or a combination of both drugs. Western blotting, flow cytometry, and immunohistochemistry were then used to characterize the effects of the different treatments.
The results presented herein show that the addition of metformin to paclitaxel leads to quantitative potentialization of molecular signaling through AMPK and a subsequent potent inhibition of the mTOR signaling pathway. Treatment with metformin and paclitaxel resulted in an increase in the number of cells arrested in the G(2)-M phase of the cell cycle, and decreased the tumor growth and increased apoptosis in tumor-bearing mice, when compared with individual drug treatments.
We have provided evidence for a convergence of metformin and paclitaxel induced signaling at the level of AMPK. This mechanism shows how different drugs may cooperate to augment antigrowth signals, and suggests that target activation of AMPK by metformin may be a compelling ally in cancer treatment.
Mitochondria possess a Ca2+ transport system composed of separate Ca2+ influx and efflux pathways. Intramitochondrial Ca2+ concentrations regulate oxidative phosphorylation, required for cell ...function and survival, and mitochondrial redox balance, that participates in a myriad of signaling and damaging pathways. The interaction between Ca2+ accumulation and redox imbalance regulates opening and closing of a highly regulated inner membrane pore, the membrane permeability transition pore (PTP). In this review, we discuss the regulation of the PTP by mitochondrial oxidants, reactive nitrogen species, and the interactions between these species and other PTP inducers. In addition, we discuss the involvement of mitochondrial redox imbalance and PTP in metabolic conditions such as atherogenesis, diabetes, obesity and in mtDNA stability.
•Mitochondria possess Ca2+ influx and efflux pathways.•Mitochondrial Ca2+ regulates oxidative phosphorylation and redox balance.•Opening of mitochondrial permeability transition pore (PTP) is regulated by oxidants.•Mitochondrial Ca2+ accumulation and redox imbalance regulate PTP.•PTP is involved in metabolic disorders such as atherogenesis, diabetes, and obesity.
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Trypanosoma cruzi
is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with ...the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca
2+
sensors and gatekeepers of the MCU, preventing Ca
2+
uptake under resting conditions and favoring it at high cytosolic Ca
2+
concentrations (Ca
2+
cyt
). Using the CRISPR/Cas9 technique, we generated
TcMICU1
and
TcMICU2
knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low Ca
2+
cyt
but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.
The mitochondrial Ca
2+
uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used
Trypanosoma cruzi
to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca
2+
uptake.
T. cruzi
MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated
TcMICU1
and
TcMICU2
knockout (-KO) cell lines. Ablation of either
TcMICU1
or
TcMICU2
showed a significantly reduced mitochondrial Ca
2+
uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca
2+
concentrations (Ca
2+
cyt
), as occurs with their mammalian orthologs.
TcMICU1
-KO and
TcMICU2
-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.
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