Aging-dependent mitochondrial bioenergetic impairment in the skeletal muscle of NNT-deficient mice.

Overall health relies on features of skeletal muscle that generally decline with age, partly due to mechanisms associated with mitochondrial redox imbalance and bioenergetic dysfunction. Previously, aged mice genetically devoid of the mitochondrial NAD(P)+ transhydrogenase (NNT, encoded by the nicotinamide nucleotide transhydrogenase gene), an enzyme involved in mitochondrial NADPH supply, were shown to exhibit deficits in locomotor behavior. Here, by using young, middle-aged, and older NNT-deficient (Nnt-/-) mice and age-matched controls (Nnt+/+), we aimed to investigate how muscle bioenergetic function and motor performance are affected by NNT expression and aging. Mice were subjected to the wire-hang test to assess locomotor performance, while mitochondrial bioenergetics was evaluated in fiber bundles from the soleus, vastus lateralis and plantaris muscles. An age-related decrease in the average wire-hang score was observed in middle-aged and older Nnt-/- mice compared to age-matched controls. Although respiratory rates in the soleus, vastus lateralis and plantaris muscles did not significantly differ between the genotypes in young mice, the rates of oxygen consumption did decrease in the soleus and vastus lateralis muscles of middle-aged and older Nnt-/- mice. Notably, the soleus, which exhibited the highest NNT expression level, was the muscle most affected by aging, and NNT loss. Additionally, histology of the soleus fibers revealed increased numbers of centralized nuclei in older Nnt-/- mice, indicating abnormal morphology. In summary, our findings suggest that NNT expression deficiency causes locomotor impairments and muscle dysfunction during aging in mice.

Novel Role of CETP in Macrophages: Reduction of Mitochondrial Oxidants Production and Modulation of Cell Immune-Metabolic Profile.

Plasma cholesteryl ester transfer protein (CETP) activity diminishes HDL-cholesterol levels and thus may increase atherosclerosis risk. Experimental evidence suggests CETP may also exhibit anti-inflammatory properties, but local tissue-specific functions of CETP have not yet been clarified. Since oxidative stress and inflammation are major features of atherogenesis, we investigated whether CETP modulates macrophage oxidant production, inflammatory and metabolic profiles. Comparing macrophages from CETP-expressing transgenic mice and non-expressing littermates, we observed that CETP expression reduced mitochondrial superoxide anion production and H2O2 release, increased maximal mitochondrial respiration rates, and induced elongation of the mitochondrial network and expression of fusion-related genes (mitofusin-2 and OPA1). The expression of pro-inflammatory genes and phagocytic activity were diminished in CETP-expressing macrophages. In addition, CETP-expressing macrophages had less unesterified cholesterol under basal conditions and after exposure to oxidized LDL, as well as increased HDL-mediated cholesterol efflux. CETP knockdown in human THP1 cells increased unesterified cholesterol and abolished the effects on mitofusin-2 and TNFα. In summary, the expression of CETP in macrophages modulates mitochondrial structure and function to promote an intracellular antioxidant state and oxidative metabolism, attenuation of pro-inflammatory gene expression, reduced cholesterol accumulation, and phagocytosis. These localized functions of CETP may be relevant for the prevention of atherosclerosis and other inflammatory diseases.

Enhanced resistance to Ca2+-induced mitochondrial permeability transition in the long-lived red-footed tortoise Chelonoidis carbonaria.

The interaction between supraphysiological cytosolic Ca2+ levels and mitochondrial redox imbalance mediates the mitochondrial permeability transition (MPT). The MPT is involved in cell death, diseases and aging. This study compared the liver mitochondrial Ca2+ retention capacity and oxygen consumption in the long-lived red-footed tortoise (Chelonoidis carbonaria) with those in the rat as a reference standard. Mitochondrial Ca2+ retention capacity, a quantitative measure of MPT sensitivity, was remarkably higher in tortoises than in rats. This difference was minimized in the presence of the MPT inhibitors ADP and cyclosporine A. However, the Ca2+ retention capacities of tortoise and rat liver mitochondria were similar when both MPT inhibitors were present simultaneously. NADH-linked phosphorylating respiration rates of tortoise liver mitochondria represented only 30% of the maximal electron transport system capacity, indicating a limitation imposed by the phosphorylation system. These results suggested underlying differences in putative MPT structural components [e.g. ATP synthase, adenine nucleotide translocase (ANT) and cyclophilin D] between tortoises and rats. Indeed, in tortoise mitochondria, titrations of inhibitors of the oxidative phosphorylation components revealed a higher limitation of ANT. Furthermore, cyclophilin D activity was approximately 70% lower in tortoises than in rats. Investigation of critical properties of mitochondrial redox control that affect MPT demonstrated that tortoise and rat liver mitochondria exhibited similar rates of H2O2 release and glutathione redox status. Overall, our findings suggest that constraints imposed by ANT and cyclophilin D, putative components or regulators of the MPT pore, are associated with the enhanced resistance to Ca2+-induced MPT in tortoises.

Mitochondrial Pyruvate Carrier Subunits Are Essential for Pyruvate-Driven Respiration, Infectivity, and Intracellular Replication of Trypanosoma cruzi.

Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of Trypanosoma cruzi, the etiologic agent of Chagas disease. Endogenous tagging of T. cruziMPC1 (TcMPC1) and TcMPC2 with 3×c-Myc showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of TcMPC1 and TcMPC2 genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized TcMPC1-KO and TcMPC2-KO epimastigotes showed reduced O2 consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O2 consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca2+ uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. TcMPC1-KO and TcMPC2-KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in TcMPC-KOs. Overall, T. cruzi MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes.IMPORTANCETrypanosoma cruzi is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual T. cruziMPC1 (TcMPC1) and TcMPC2 knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.

Lack of mitochondrial NADP(H)-transhydrogenase expression in macrophages exacerbates atherosclerosis in hypercholesterolemic mice.

The atherosclerosis prone LDL receptor knockout mice (Ldlr-/-, C57BL/6J background) carry a deletion of the NADP(H)-transhydrogenase gene (Nnt) encoding the mitochondrial enzyme that catalyzes NADPH synthesis. Here we hypothesize that both increased NADPH consumption (due to increased steroidogenesis) and decreased NADPH generation (due to Nnt deficiency) in Ldlr-/- mice contribute to establish a macrophage oxidative stress and increase atherosclerosis development. Thus, we compared peritoneal macrophages and liver mitochondria from three C57BL/6J mice lines: Ldlr and Nnt double mutant, single Nnt mutant and wild-type. We found increased oxidants production in both mitochondria and macrophages according to a gradient: double mutant > single mutant > wild-type. We also observed a parallel up-regulation of mitochondrial biogenesis (PGC1a, TFAM and respiratory complexes levels) and inflammatory (iNOS, IL6 and IL1b) markers in single and double mutant macrophages. When exposed to modified LDL, the single and double mutant cells exhibited significant increases in lipid accumulation leading to foam cell formation, the hallmark of atherosclerosis. Nnt deficiency cells showed up-regulation of CD36 and down-regulation of ABCA1 transporters what may explain lipid accumulation in macrophages. Finally, Nnt wild-type bone marrow transplantation into LDLr-/- mice resulted in reduced diet-induced atherosclerosis. Therefore, Nnt plays a critical role in the maintenance of macrophage redox, inflammatory and cholesterol homeostasis, which is relevant for delaying the atherogenesis process.

MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.

The mitochondrial Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ concentrations ([Ca2+]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.IMPORTANCETrypanosoma 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 Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]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 [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.

Functional analysis and importance for host cell infection of the Ca2+-conducting subunits of the mitochondrial calcium uniporter of Trypanosoma cruzi.

We report here that Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses two unique paralogues of the mitochondrial calcium uniporter complex TcMCU subunit that we named TcMCUc and TcMCUd. The predicted structure of the proteins indicates that, as predicted for the TcMCU and TcMCUb paralogues, they are composed of two helical membrane-spanning domains and contain a WDXXEPXXY motif. Overexpression of each gene led to a significant increase in mitochondrial Ca2+ uptake, while knockout (KO) of either TcMCUc or TcMCUd led to a loss of mitochondrial Ca2+ uptake, without affecting the mitochondrial membrane potential. TcMCUc-KO and TcMCUd-KO epimastigotes exhibited reduced growth rate in low-glucose medium and alterations in their respiratory rate, citrate synthase activity, and AMP/ATP ratio, while trypomastigotes had reduced ability to efficiently infect host cells and replicate intracellularly as amastigotes. By gene complementation of KO cell lines or by a newly developed CRISPR/Cas9-mediated knock-in approach, we also studied the importance of critical amino acid residues of the four paralogues on mitochondrial Ca2+ uptake. In conclusion, the results predict a hetero-oligomeric structure for the T. cruzi MCU complex, with structural and functional differences, as compared with those in the mammalian complex.

Can acetylcysteine ameliorate cisplatin-induced toxicities and oxidative stress without decreasing antitumor efficacy? A randomized, double-blind, placebo-controlled trial involving patients with head and neck cancer.

The protective antioxidant activity of acetylcysteine (NAC) against toxicity due to cisplatin has been reported in experimental models; however, its efficacy in patients has not been elucidated. The aim of this study was to investigate the possible protective effect of NAC on cisplatin-induced toxicity and the effect of NAC on clinical response and oxidative stress in patients treated for head and neck cancer. This was a randomized, double-blind, placebo-controlled trial conducted in patients receiving high-dose cisplatin chemotherapy concomitant to radiotherapy. Patients were randomly assigned to groups and received: (a) 600 mg NAC syrup, orally once daily at night for 7 consecutive days or (b) placebo, administered similarly to NAC. Nephro-, oto-, hepato-, myelo-, and gastrointestinal toxicities, clinical responses, and plasma and cellular markers of oxidative stress were evaluated. Fifty-seven patients were included (n = 28, NAC arm; and n = 29, placebo arm). A high prevalence of most types of toxicities was observed after cisplatin chemotherapy; however, the parameters were similar between the two groups. There was a predominance of partial response to treatment. In the cellular and plasmatic oxidative stress analyses, minor differences were observed. Overall, there was no statistically significant difference between the groups for all outcomes. These findings show that low-dose oral NAC does not protect patients with head and neck cancer from cisplatin-induced toxicities and oxidative stress. The antitumor efficacy of cisplatin was apparently not impaired by NAC.

Coenzyme Q10 or Creatine Counteract Pravastatin-Induced Liver Redox Changes in Hypercholesterolemic Mice.

Statins are the preferred therapy to treat hypercholesterolemia. Their main action consists of inhibiting the cholesterol biosynthesis pathway. Previous studies report mitochondrial oxidative stress and membrane permeability transition (MPT) of several experimental models submitted to diverse statins treatments. The aim of the present study was to investigate whether chronic treatment with the hydrophilic pravastatin induces hepatotoxicity in LDL receptor knockout mice (LDLr-/-), a model for human familial hypercholesterolemia. We evaluated respiration and reactive oxygen production rates, cyclosporine-A sensitive mitochondrial calcium release, antioxidant enzyme activities in liver mitochondria or homogenates obtained from LDLr-/- mice treated with pravastatin for 3 months. We observed that pravastatin induced higher H2O2 production rate (40%), decreased activity of aconitase (28%), a superoxide-sensitive Krebs cycle enzyme, and increased susceptibility to Ca2+-induced MPT (32%) in liver mitochondria. Among several antioxidant enzymes, only glucose-6-phosphate dehydrogenase (G6PD) activity was increased (44%) in the liver of treated mice. Reduced glutathione content and reduced to oxidized glutathione ratio were increased in livers of pravastatin treated mice (1.5- and 2-fold, respectively). The presence of oxidized lipid species were detected in pravastatin group but protein oxidation markers (carbonyl and SH- groups) were not altered. Diet supplementation with the antioxidants CoQ10 or creatine fully reversed all pravastatin effects (reduced H2O2 generation, susceptibility to MPT and normalized aconitase and G6PD activity). Taken together, these results suggest that 1- pravastatin induces liver mitochondrial redox imbalance that may explain the hepatic side effects reported in a small number of patients, and 2- the co-treatment with safe antioxidants neutralize these side effects.

Mangifera indica L. extract (Vimang®) reduces plasma and liver cholesterol and leucocyte oxidative stress in hypercholesterolemic LDL receptor deficient mice.

Cardiovascular diseases are major causes of death worldwide. Beyond the classical cholesterol risk factor, other conditions such as oxidative stress are well documented to promote atherosclerosis. The Mangifera indica L. extract (Vimang®) was reported to present antioxidant and hypocholesterolemic properties. Thus, here we evaluate the effects of Vimang treatment on risk factors of the atherosclerosis prone model of familial hypercholesterolemia, the LDL receptor knockout mice. Mice were treated with Vimang during 2 weeks and were fed a cholesterol-enriched diet during the second week. The Vimang treated mice presented significantly reduced levels of plasma (15%) and liver (20%) cholesterol, increased plasma total antioxidant capacity (10%) and decreased reactive oxygen species (ROS) production by spleen mononuclear cells (50%), P < 0.05 for all. In spite of these benefits, the average size of aortic atherosclerotic lesions stablished in this short experimental period did not change significantly in Vimang treated mice. Therefore, in this study we demonstrated that Vimang has protective effects on systemic and tissue-specific risk factors, but it is not sufficient to promote a reduction in the initial steps of atherosclerosis development. In addition, we disclosed a new antioxidant target of Vimang, the spleen mononuclear cells that might be relevant for more advanced stages of atherosclerosis.

Facilitation of Ca2+ -induced opening of the mitochondrial permeability transition pore either by nicotinamide nucleotide transhydrogenase deficiency or statins treatment.

Mitochondrial redox imbalance and high Ca2+ uptake induce the opening of the permeability transition pore (PTP) that leads to disruption of energy-linked mitochondrial functions and triggers cell death in many disease states. In this review, we discuss the major results from our studies investigating the consequences of NAD(P)-transhydrogenase (NNT) deficiency, and of statins treatment for mitochondrial functions and susceptibility to Ca2+ -induced PTP. We highlight the aggravation of high fat diet-induced fatty liver disease in the context of NNT deficiency and the role of antioxidants in the prevention of statins toxicity to mitochondria.

The mitochondrial calcium uniporter complex in trypanosomes.

The presence of a conserved mechanism for mitochondrial calcium uptake in trypanosomatids was crucial for the molecular identification of the mitochondrial calcium uniporter (MCU), a long-sought channel present in most eukaryotic organisms. Since then, research efforts to elucidate the role of MCU and its regulatory elements in different biological models have multiplied. MCU is the pore-forming subunit of a multimeric complex (the MCU complex or MCUC) and its predicted structure in trypanosomes is simpler than in mammalian cells, lacking two of its subunits and probably possessing other unidentified components. MCU protein has been characterized in Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African and American trypanosomiasis, respectively. Contrary to its mammalian homolog, TbMCU was found to be essential for cell growth and survival, while its paralog MCUb is an essential protein in T. cruzi. These findings could be further exploited for chemotherapeutic purposes. The emergence of new molecular tools for the genetic manipulation of trypanosomatids has been determinant for the functional characterization of the MCUC components in these organisms. However, further research has to be done to determine the role of each component in intracellular calcium signaling and cell bioenergetics. In this mini-review we summarize the original results on mitochondrial calcium uptake in trypanosomes, how did they contribute to the molecular identification of the MCU, and the functional characterization of the MCUC subunits that has so far been studied in these peculiar eukaryotes.

Contribution to mitochondrial research in Brazil: 10th anniversary of the mitomeeting.

This commentary introduces the subject, the context and the history of the Brazilian annually held meeting on Mitochondrial Research by the occasion of its 10th anniversary. Mitomeetings gather people interested in all aspects of mitochondrial biology in diverse species, including protists, animals, plants, and fungi.

Spontaneous experimental atherosclerosis in hypercholesterolemic mice advances with ageing and correlates with mitochondrial reactive oxygen species.

Ageing and atherosclerosis are associated with oxidative stress. Mitochondrial redox function declines with ageing. Here we tested whether ageing LDL receptor knockout mice (LDLr-/-) develop spontaneous atherosclerosis and whether mitochondrial reactive oxygen species (mtROS) correlate with atherosclerosis. Compared with young mice, aged LDLr-/- mice exhibited 20-fold larger aortic lesion size, although the plasma cholesterol levels did not vary between age groups. The lesion sizes increased exponentially from 3 to 24months of age (r=0.92, p=0.0001) and were correlated with mtROS across the age range (r=0.81, p=0.0001). Thus, LDLr-/- mice develop spontaneous diet-independent atherosclerosis, that advances exponentially with ageing. We propose that age related increases in mtROS contribute to accelerate atherosclerosis development in hypercholesterolemic mice.

Cisplatin-induced human peripheral blood mononuclear cells' oxidative stress and nephrotoxicity in head and neck cancer patients: the influence of hydrogen peroxide.

Cisplatin is a widely used antineoplastic agent in the treatment of head and neck cancer. However, it is highly nephrotoxic. Oxidative stress is the main mechanism responsible for cisplatin-induced nephrotoxicity. The aim of this study was to characterize cisplatin-induced nephrotoxicity, oxidative stress in peripheral blood mononuclear cells, and the relationship between them. Twenty-four patients were included in the study. Patients had their blood collected prior to cisplatin administration, and 5 and 20 days after initiating therapy, to assess renal function and to determine oxidative stress with MitoSOX™Red, H2DCF-DA, and Amplex® Red tests. Renal function was assessed by measuring serum creatinine, creatinine clearance, and blood urea nitrogen (BUN). Serum creatinine and creatinine clearance were used to grade nephrotoxicity using Common Terminology Criteria for Adverse Events (CTCAE) v4.0. Compared to baseline values, the mean BUN and serum creatinine increased 135 and 100%, respectively, 5 days after cisplatin infusion. Mean creatinine clearance showed a 43% decrease compared to baseline value. Non-statistically significant changes in superoxide anion (O 2•- ), hydrogen peroxide (H2O2), and general reactive oxygen species production occurred. A higher production of H2O2 was correlated with variation in serum creatinine, and was associated with higher grades for serum creatinine increases and creatinine clearance reductions. Linear regression analyses showed an association between H2O2 production and serum creatinine, creatinine clearance, and BUN levels. These results were observed for 5 days following cisplatin administration. In conclusion, H2O2 production was significantly related to changes in all renal parameters that were evaluated, following the cisplatin infusion.

Redox imbalance due to the loss of mitochondrial NAD(P)-transhydrogenase markedly aggravates high fat diet-induced fatty liver disease in mice.

The mechanisms by which a high fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunctions and redox imbalance. We hypothesized that a HFD would increase mitochondrial reliance on NAD(P)-transhydrogenase (NNT) as the source of NADPH for antioxidant systems that counteract NAFLD development. Therefore, we studied HFD-induced liver mitochondrial dysfunctions and NAFLD in C57Unib.B6 congenic mice with (Nnt+/+) or without (Nnt-/-) NNT activity; the spontaneously mutated allele (Nnt-/-) was inherited from the C57BL/6J mouse substrain. After 20 weeks on a HFD, Nnt-/- mice exhibited a higher prevalence of steatohepatitis and content of liver triglycerides compared to Nnt+/+ mice on an identical diet. Under a HFD, the aggravated NAFLD phenotype in the Nnt-/- mice was accompanied by an increased H2O2 release rate from mitochondria, decreased aconitase activity (a redox-sensitive mitochondrial enzyme) and higher susceptibility to Ca2+-induced mitochondrial permeability transition. In addition, HFD led to the phosphorylation (inhibition) of pyruvate dehydrogenase (PDH) and markedly reduced the ability of liver mitochondria to remove peroxide in Nnt-/- mice. Bypass or pharmacological reactivation of PDH by dichloroacetate restored the peroxide removal capability of mitochondria from Nnt-/- mice on a HFD. Noteworthy, compared to mice that were chow-fed, the HFD did not impair peroxide removal nor elicit redox imbalance in mitochondria from Nnt+/+ mice. Therefore, HFD interacted with Nnt mutation to generate PDH inhibition and further suppression of peroxide removal. We conclude that NNT plays a critical role in counteracting mitochondrial redox imbalance, PDH inhibition and advancement of NAFLD in mice fed a HFD. The present study provide seminal experimental evidence that redox imbalance in liver mitochondria potentiates the progression from simple steatosis to steatohepatitis following a HFD.

Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi.

To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3' end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3' end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.

Pravastatin Chronic Treatment Sensitizes Hypercholesterolemic Mice Muscle to Mitochondrial Permeability Transition: Protection by Creatine or Coenzyme Q10.

Statins are efficient cholesterol-lowering medicines utilized worldwide. However, 10% of patients suffer from adverse effects specially related to skeletal muscle function. Pro- or anti-oxidant effects of statins have been reported. Here we hypothesized that statins induce muscle mitochondrial oxidative stress leading to mitochondrial permeability transition (MPT) which may explain statin muscle toxicity. Thus, our aims were to investigate the effects of statin chronic treatment on muscle mitochondrial respiration rates, MPT and redox state indicators in the context of hypercholesterolemia. For this purpose, we studied muscle biopsies of the hypercholesterolemic LDL receptor knockout mice (LDLr-/-) treated with pravastatin during 3 months. Plantaris, but not soleus muscle of treated mice showed significant inhibition of respiration rates induced by ADP (-14%), oligomycin (-20%) or FCCP (-40%). Inhibitions of respiratory rates were sensitive to EGTA (Ca2+ chelator), cyclosporin A (MPT inhibitor), ruthenium red (inhibitor of mitochondria Ca2+ uptake) and coenzyme Q10 (antioxidant), indicating that pravastatin treatment favors Ca2+ induced MPT. Diet supplementation with creatine (antioxidant) also protected treated mice against pravastatin sensitization to Ca2+ induced MPT. Among several antioxidant enzymes analyzed, only catalase activity was increased by 30% in plantaris muscle of pravastatin treated mice. Oxidized lipids, but not proteins biomarkers were identified in treated LDLr-/- plantaris muscle. Taken together, the present results suggest that chronic pravastatin administration to a model of familial hypercholesterolemia promotes mitochondrial dysfunctions in plantaris muscle that can be counteracted by antioxidants administered either in vitro (CoQ10) or in vivo (creatine). Therefore, we propose that inhibition of muscle mitochondrial respiration by pravastatin leads to an oxidative stress that, in the presence of calcium, opens the permeability transition pore. This mitochondrial oxidative stress caused by statin treatment also signals for cellular antioxidant system responses such as catalase upregulation. These results suggest that the detrimental effects of statins on muscle mitochondria could be prevented by co-administration of a safe antioxidant such as creatine or CoQ10.

Lack of XPC leads to a shift between respiratory complexes I and II but sensitizes cells to mitochondrial stress.

Genomic instability drives tumorigenesis and DNA repair defects are associated with elevated cancer. Metabolic alterations are also observed during tumorigenesis, although a causal relationship between these has not been clearly established. Xeroderma pigmentosum (XP) is a DNA repair disease characterized by early cancer. Cells with reduced expression of the XPC protein display a metabolic shift from OXPHOS to glycolysis, which was linked to accumulation of nuclear DNA damage and oxidants generation via NOX-1. Using XP-C cells, we show that mitochondrial respiratory complex I (CI) is impaired in the absence of XPC, while complex II (CII) is upregulated in XP-C cells. The CI/CII metabolic shift was dependent on XPC, as XPC complementation reverted the phenotype. We demonstrate that mitochondria are the primary source of H2O2 and glutathione peroxidase activity is compromised. Moreover, mtDNA is irreversibly damaged and accumulates deletions. XP-C cells were more sensitive to the mitochondrial inhibitor antimycin A, an effect also prevented in XPC-corrected cells. Our results show that XPC deficiency leads to alterations in mitochondrial redox balance with a CI/CII shift as a possible adaptation to lower CI activity, but at the cost of sensitizing XP-C cells to mitochondrial oxidative stress.

Increased glutathione levels contribute to the beneficial effects of hydrogen sulfide and inducible nitric oxide inhibition in allergic lung inflammation.

OBJECTIVE: The interaction between nitric oxide (NO) and hydrogen sulfide (H2S) in the airways could have significant implications for the pathogenesis and therapeutic effects of both on lung diseases. In this study we investigated whether the beneficial effects of H2S on asthma could be comparable to that inhibition of inducible NO synthase (iNOS). METHODS: Female BALB/C mice sensitized with ovalbumin (OVA) received either the H2S donor sodium hydrosulfide (NaHS, 14µmol/kg) or the iNOS inhibitor 1400W (1mg/kg), 30min before each OVA challenge during six days. On the first, second and sixth days, the leucocyte infiltration in lung parenchyma and bronchoalveolar lavage was evaluated. The aconitase activity (a sensor of O2 formation) and lipid peroxidation, as well as levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined in the lung tissues. RESULTS: OVA-challenge caused a significant and time-dependent increase in the eosinophil number in the airways, which was accompanied by a significant decrease of aconitase activity and GSH/GSSG ratio along with enhanced lipid peroxidation in the lungs. Treatment with NaHS or 1400W significantly attenuated the airways eosinophilia that was paralleled by an increase in aconitase activity and decrease of lipid peroxidation. NaHS or 1400W treatments also reversed the decreased GSH/GSSG ratio seen after OVA-challenge. CONCLUSIONS: The present study shows for the first time that the increased GSH/GSSG ratio caused by either H2S supplementation or iNOS-inhibition is a potential mechanism protecting airways against oxidative stress and inflammatory lung diseases.