Sodium arsenite and arsenic trioxide differently affect the oxidative stress of lymphoblastoid cells: An intricate crosstalk between mitochondria, autophagy and cell death.

Although the toxicity of arsenic depends on its chemical forms, few studies have taken into account the ambiguous phenomenon that sodium arsenite (NaAsO2) acts as a potent carcinogen while arsenic trioxide (ATO, As2O3) serves as an effective therapeutic agent in lymphoma, suggesting that NaAsO2 and As2O3 may act via paradoxical ways to either promote or inhibit cancer pathogenesis. Here, we compared the cellular response of the two arsenical compounds, NaAsO2 and As2O3, on the Burkitt lymphoma cell model, the Epstein Barr Virus (EBV)-positive P3HR1 cells. Using flow cytometry and biochemistry analyses, we showed that a NaAsO2 treatment induces P3HR1 cell death, combined with drastic drops in ΔΨm, NAD(P)H and ATP levels. In contrast, As2O3-treated cells resist to cell death, with a moderate reduction of ΔΨm, NAD(P)H and ATP. While both compounds block cells in G2/M and affect their protein carbonylation and lipid peroxidation, As2O3 induces a milder increase in superoxide anions and H2O2 than NaAsO2, associated to a milder inhibition of antioxidant defenses. By electron microscopy, RT-qPCR and image cytometry analyses, we showed that As2O3-treated cells display an overall autophagic response, combined with mitophagy and an unfolded protein response, characteristics that were not observed following a NaAsO2 treatment. As previous works showed that As2O3 reactivates EBV in P3HR1 cells, we treated the EBV- Ramos-1 cells and showed that autophagy was not induced in these EBV- cells upon As2O3 treatment suggesting that the boost of autophagy observed in As2O3-treated P3HR1 cells could be due to the presence of EBV in these cells. Overall, our results suggest that As2O3 is an autophagic inducer which action is enhanced when EBV is present in the cells, in contrast to NaAsO2, which induces cell death. That's why As2O3 is combined with other chemicals, as all-trans retinoic acid, to better target cancer cells in therapeutic treatments.

Tafazzin Mutation Affecting Cardiolipin Leads to Increased Mitochondrial Superoxide Anions and Mitophagy Inhibition in Barth Syndrome.

Tafazzin is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of the tafazzin gene cause Barth syndrome, which is characterized by mitochondrial dysfunction and dilated cardiomyopathy, leading to premature death. However, the molecular mechanisms underlying the cause of mitochondrial dysfunction in Barth syndrome remain poorly understood. We again highlight the fact that the tafazzin deficiency is also linked to defective oxidative phosphorylation associated with oxidative stress. All the mitochondrial events are positioned in a context where mitophagy is a key element in mitochondrial quality control. Here, we investigated the role of tafazzin in mitochondrial homeostasis dysregulation and mitophagy alteration. Using a HeLa cell model of tafazzin deficiency, we show that dysregulation of tafazzin in HeLa cells induces alteration of mitophagy. Our findings provide some additional insights into mitochondrial dysfunction associated with Barth syndrome, but also show that mitophagy inhibition is concomitant with apoptosis dysfunction through the inability of abnormal mitochondrial cardiolipin to assume its role in cytoplasmic signal transduction. Our work raises hope that pharmacological manipulation of the mitophagic pathway together with mitochondrially targeted antioxidants may provide new insights leading to promising treatment for these highly lethal conditions.

Highlighting Curcumin-Induced Crosstalk between Autophagy and Apoptosis as Supported by Its Specific Subcellular Localization.

Curcumin, a major active component of turmeric (Curcuma longa, L.), is known to have various effects on both healthy and cancerous tissues. In vitro studies suggest that curcumin inhibits cancer cell growth by activating apoptosis, but the mechanism underlying the anticancer effect of curcumin is still unclear. Since there is a recent consensus about endoplasmic reticulum (ER) stress being involved in the cytotoxicity of natural compounds, we have investigated using Image flow cytometry the mechanistic aspects of curcumin's destabilization of the ER, but also the status of the lysosomal compartment. Curcumin induces ER stress, thereby causing an unfolded protein response and calcium release, which destabilizes the mitochondrial compartment and induce apoptosis. These events are also associated with secondary lysosomal membrane permeabilization that occurs later together with an activation of caspase-8, mediated by cathepsins and calpains that ended in the disruption of mitochondrial homeostasis. These two pathways of different intensities and momentum converge towards an amplification of cell death. In the present study, curcumin-induced autophagy failed to rescue all cells that underwent type II cell death following initial autophagic processes. However, a small number of cells were rescued (successful autophagy) to give rise to a novel proliferation phase.

Iron chelation by curcumin suppresses both curcumin-induced autophagy and cell death together with iron overload neoplastic transformation.

Iron overload, notably caused by hereditary hemochromatosis, is an excess storage of iron in various organs that causes tissue damage and may promote tumorigenesis. To manage that disorder, free iron depletion can be induced by iron chelators like deferoxamine that are of increasing interest also in the cancer field since iron stock could be a potent target for managing tumorigenesis. Curcumin, a well-known active substance extracted from the turmeric rhizome, destabilizes endoplasmic reticulum, and secondarily lysosomes, thereby increasing mitophagy/autophagy and subsequent apoptosis. Recent findings show that cells treated with curcumin also exhibit a decrease in ferritin, which is consistent with its chemical structure and iron chelating activity. Here we investigated how curcumin influences the intracellular effects of iron overload via Fe-nitriloacetic acid or ferric ammonium citrate loading in Huh-7 cells and explored the consequences in terms of antioxidant activity, autophagy, and apoptotic signal transduction. In experiments with T51B and RL-34 epithelial cells, we have found evidence that curcumin-iron complexation abolishes both curcumin-induced autophagy and apoptosis, together with the tumorigenic action of iron overload.

Hormetic effects of curcumin: What is the evidence?

Curcumin (diferuloylmethane), a component of the yellow powder prepared from the roots of Curcuma longa or Zingiberaceae (known as turmeric) is not only widely used to color and flavor food but also used as a pharmaceutical agent. Curcumin demonstrates anti-inflammatory, anticarcinogenic, antiaging, and antioxidant activity, as well as efficacy in wound healing. Notably, curcumin is a hormetic agent (hormetin), as it is stimulatory at low doses and inhibitory at high doses. Hormesis by curcumin could be also a particular function at low doses (i.e., antioxidant behavior) and another function at high dose (i.e., induction of autophagy and cell death). Recent findings suggest that curcumin exhibits biphasic dose-responses on cells, with low doses having stronger effects than high doses; examples being activation of the mitogen-activated protein kinase signaling pathway or antioxidant activity. This indicates that many effects induced by curcumin are dependent on dose and some effects might be greater at lower doses, indicative of a hormetic response. Despite the consistent occurrence of hormetic responses of curcumin in a wide range of biomedical models, epidemiological and clinical trials are needed to assess the nature of curcumin's dose-response in humans. Fortunately, more than one hundred clinical trials with curcumin and curcumin derivatives are ongoing. In this review, we provide the first comprehensive analysis supportive of the hormetic behavior of curcumin and curcumin derivatives.

Decreasing cytosolic translation is beneficial to yeast and human Tafazzin-deficient cells.

Cardiolipin (CL) optimizes diverse mitochondrial processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase Tafazzin (TAZ). Loss-of-function mutations in the TAZ gene are responsible for the Barth syndrome (BTHS), a rare X-linked cardiomyopathy, presumably because of a diminished OXPHOS capacity. Herein we show that a partial inhibition of cytosolic protein synthesis, either chemically with the use of cycloheximide or by specific genetic mutations, fully restores biogenesis and the activity of the oxidative phosphorylation system in a yeast BTHS model (taz1Δ). Interestingly, the defaults in CL were not suppressed, indicating that they are not primarily responsible for the OXPHOS deficiency in taz1Δ yeast. Low concentrations of cycloheximide in the picomolar range were beneficial to TAZ-deficient HeLa cells, as evidenced by the recovery of a good proliferative capacity. These findings reveal that a diminished capacity of CL remodeling deficient cells to preserve protein homeostasis is likely an important factor contributing to the pathogenesis of BTHS. This in turn, identifies cytosolic translation as a potential therapeutic target for the treatment of this disease.

Liver X Receptor exerts a protective effect against the oxidative stress in the peripheral nerve.

Reactive oxygen species (ROS) modify proteins and lipids leading to deleterious outcomes. Thus, maintaining their homeostatic levels is vital. This study highlights the endogenous role of LXRs (LXRα and ß) in the regulation of oxidative stress in peripheral nerves. We report that the genetic ablation of both LXR isoforms in mice (LXRdKO) provokes significant locomotor defects correlated with enhanced anion superoxide production, lipid oxidization and protein carbonylation in the sciatic nerves despite the activation of Nrf2-dependant antioxidant response. Interestingly, the reactive oxygen species scavenger N-acetylcysteine counteracts behavioral, electrophysical, ultrastructural and biochemical alterations in LXRdKO mice. Furthermore, Schwann cells in culture pretreated with LXR agonist, TO901317, exhibit improved defenses against oxidative stress generated by tert-butyl hydroperoxide, implying that LXRs play an important role in maintaining the redox homeostasis in the peripheral nervous system. Thus, LXR activation could be a promising strategy to protect from alteration of peripheral myelin resulting from a disturbance of redox homeostasis in Schwann cell.

Synergistic cellular effects including mitochondrial destabilization, autophagy and apoptosis following low-level exposure to a mixture of lipophilic persistent organic pollutants.

Humans are exposed to multiple exogenous environmental pollutants. Many of these compounds are parts of mixtures that can exacerbate harmful effects of the individual mixture components. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is primarily produced via industrial processes including incineration and the manufacture of herbicides. Both endosulfan and TCDD are persistent organic pollutants which elicit cytotoxic effects by inducing reactive oxygen species generation. Sublethal concentrations of mixtures of TCDD and endosulfan increase oxidative stress, as well as mitochondrial homeostasis disruption, which is preceded by a calcium rise and, in fine, induce cell death. TCDD+Endosulfan elicit a complex signaling sequence involving reticulum endoplasmic destalilization which leads to Ca2+ rise, superoxide anion production, ATP drop and late NADP(H) depletion associated with a mitochondrial induced apoptosis concomitant early autophagic processes. The ROS scavenger, N-acetyl-cysteine, blocks both the mixture-induced autophagy and death. Calcium chelators act similarly and mitochondrially targeted anti-oxidants also abrogate these effects. Inhibition of the autophagic fluxes with 3-methyladenine, increases mixture-induced cell death. These findings show that subchronic doses of pollutants may act synergistically. They also reveal that the onset of autophagy might serve as a protective mechanism against ROS-triggered cytotoxic effects of a cocktail of pollutants in Caco-2 cells and increase their tumorigenicity.

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential.

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry-xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe's limitations.

Paraquat Induces Peripheral Myelin Disruption and Locomotor Defects: Crosstalk with LXR and Wnt Pathways.

AIMS: Paraquat (PQT), a redox-active herbicide, is a free radical-producing molecule, causing damage particularly to the nervous system; thus, it is employed as an animal model for Parkinson's disease. However, its impact on peripheral nerve demyelination is still unknown. Our aim is to decipher the influence of PQT-induced reactive oxygen species (ROS) production on peripheral myelin. RESULTS: We report that PQT provokes severe locomotor and sensory defects in mice. PQT elicited an oxidative stress in the nerve, resulting in an increase of lipid peroxidation and protein carbonylation, despite the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant defenses. We observed a dramatic disorganization of myelin sheaths in the sciatic nerves, dysregulation of myelin gene expression, and aggregation of myelin proteins, a hallmark of demyelination. PQT altered myelin gene expression via liver X receptor (LXR) signaling, a negative regulator of peripheral myelin gene expression through its dialog with the Wnt/ß-catenin pathway. PQT prevented ß-catenin binding on myelin gene promoters, resulting in the inhibition of Wnt/ß-catenin-dependent myelin gene expression. Wnt pathway activation by LiCl dampened the deleterious effects of PQT. LiCl blocked PQT-induced oxidative stress and reduced Schwann cell death. LiCl+PQT-treated mice had normal sensorimotor behaviors and a usual nerve structure. INNOVATION: We reveal that PQT damages the sciatic nerve by generating an oxidative stress, dysregulating LXR and Wnt/ß-catenin pathways. The activation of Wnt signaling by LiCl reduced the deleterious effects of PQT on the nerve. CONCLUSION: We demonstrate that PQT instigates peripheral nerve demyelinating neuropathies by enhancing ROS production and deregulating LXR and Wnt pathways. Stimulating Wnt pathway could be a therapeutic strategy for neuropathy treatment. Antioxid. Redox Signal. 27, 168-183.

Antioxidative Theranostic Iron Oxide Nanoparticles toward Brain Tumors Imaging and ROS Production.

Gliomas are the most common primary brain tumor in humans. To date, the only treatment of care consists of surgical removal of the tumor bulk, irradiation, and chemotherapy, finally resulting in a very poor prognosis due to the lack of efficiency in diagnostics. In this context, nanomedicine combining both diagnostic and magnetic resonance imaging (MRI) and therapeutic applications is a relevant strategy referred to theranostic. Magnetic nanoparticles (NP) are excellent MRI contrast agents because of their large magnetic moment, which induces high transverse relaxivity (r2) characteristic and increased susceptibility effect (T2*). NP can be also used for drug delivery by coating their surface with therapeutic molecules. Preliminary in vitro studies show the high potential of caffeic acid (CA), a natural polyphenol, as a promising anticancer drug due to its antioxidant, anti-inflammatory, and antimetastatic properties. In this study, the antioxidative properties of iron oxide NP functionalized with caffeic acid (γFe2O3@CA NP) are investigated in vitro on U87-MG brain cancer cell lines. After intravenous injection of these NP in mice bearing a U87 glioblastoma, a negative contrast enhancement was specifically observed on 11.7 T MRI images in cancerous tissue, demonstrating a passive targeting of the tumor with these nanoplatforms.

The iron component of particulate matter is antiapoptotic: A clue to the development of lung cancer after exposure to atmospheric pollutants?

The classification of outdoor air pollution as carcinogenic for humans strengthens the increasing concern about particulate matter (PM). We previously demonstrated that PM exposure produces an antiapoptotic effect resulting from polycyclic aromatic hydrocarbons (PAH) and water-soluble components. In this study, we investigated transition metallic compounds, particularly iron, in order to decipher their underlying molecular mechanisms that prevent apoptosis. Human bronchial epithelial cells were exposed for 4 h to different PM samples with established antiapoptotic effect (e.g. PM-AW) or not (e.g. PM-VS) or to their metallic components (Fe, Mn, Zn and Al) before apoptosis induction by the calcium ionophore A23187 or Staurosporine. PM-AW, Fe, Mn and Al significantly reduced induced apoptosis. The antiapoptotic effect of Fe was enhanced by benzo(a)pyrene, a typical PAH compound, but was totally reversed by the iron chelator, deferiprone. Furthermore, particles and iron triggered cellular ROS generation and prevented the depletion in glutathione levels observed during A23187-induced apoptosis. In contrast to benzo(a)pyrene, PM-AW and Fe rapidly activated NRF2, subsequently upregulated several target genes (HO1, NQO1 and GPX1) and modulated some genes which control cell death (BCL2, BAX and p53). The key role of the NRF2 pathway in the antiapoptotic effect mediated by Fe and PM was demonstrated using siRNA technology. Our results suggest that the iron component participates in the antiapoptotic effect of PM by activating a NRF2-dependent antioxidant process. As resisting to cell death is one of the hallmarks of cancer cells, these findings provide additional clues for understanding the development of lung cancer after atmospheric pollution exposure.

Barth Syndrome: From Mitochondrial Dysfunctions Associated with Aberrant Production of Reactive Oxygen Species to Pluripotent Stem Cell Studies.

Mutations in the gene encoding the enzyme tafazzin, TAZ, cause Barth syndrome (BTHS). Individuals with this X-linked multisystem disorder present cardiomyopathy (CM) (often dilated), skeletal muscle weakness, neutropenia, growth retardation, and 3-methylglutaconic aciduria. Biopsies of the heart, liver and skeletal muscle of patients have revealed mitochondrial malformations and dysfunctions. It is the purpose of this review to summarize recent results of studies on various animal or cell models of Barth syndrome, which have characterized biochemically the strong cellular defects associated with TAZ mutations. Tafazzin is a mitochondrial phospholipidlysophospholipid transacylase that shuttles acyl groups between phospholipids and regulates the remodeling of cardiolipin (CL), a unique inner mitochondrial membrane phospholipid dimer consisting of two phosphatidyl residues linked by a glycerol bridge. After their biosynthesis, the acyl chains of CLs may be modified in remodeling processes involving up to three different enzymes. Their characteristic acyl chain composition depends on the function of tafazzin, although the enzyme itself surprisingly lacks acyl specificity. CLs are crucial for correct mitochondrial structure and function. In addition to their function in the basic mitochondrial function of ATP production, CLs play essential roles in cardiac function, apoptosis, autophagy, cell cycle regulation and Fe-S cluster biosynthesis. Recent developments in tafazzin research have provided strong insights into the link between mitochondrial dysfunction and the production of reactive oxygen species (ROS). An important tool has been the generation of BTHS-specific induced pluripotent stem cells (iPSCs) from BTHS patients. In a complementary approach, disease-specific mutations have been introduced into wild-type iPSC lines enabling direct comparison with isogenic controls. iPSC-derived cardiomyocytes were then characterized using biochemical and classical bioenergetic approaches. The cells are tested in a "heart-on-chip" assay to model the pathophysiology in vitro, to characterize the underlying mechanism of BTHS deriving from TAZ mutations, mitochondrial deficiencies and ROS production and leading to tissue defects, and to evaluate potential therapies with the use of mitochondrially targeted antioxidants.

Barth syndrome: cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation.

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I+III2+IVn but also decreased amounts of individual complexes I and IV and supercomplexes I+III and III+IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid.

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria.

COUP-TFII controls mouse pancreatic ß-cell mass through GLP-1-ß-catenin signaling pathways.

BACKGROUND: The control of the functional pancreatic ß-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how ß-cell mass is determined. METHODOLOGY/PRINCIPAL FINDINGS: Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal ß-cell proliferation and apoptosis, this suggests decreased ß-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured ß-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the ß-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via ß-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced ß-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat ß-cells providing a feedback loop. CONCLUSIONS/SIGNIFICANCE: Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases ß-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the ß-catenin-dependent pathway and its expression is under the control of TCF7L2.

Mechanistic issues of the interaction of the hairpin-forming domain of tBid with mitochondrial cardiolipin.

BACKGROUND: The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated. PRINCIPAL FINDINGS: The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix alphaH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, alphaH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix alphaH6 to efficiently induce cytochrome c release and apoptosis. CONCLUSIONS: Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix alphaH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix alphaH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated.

Cross-talk between oxysterols and glucocorticoids: differential regulation of secreted phopholipase A2 and impact on oligodendrocyte death.

BACKGROUND: Oxysterols are oxidized forms of cholesterol. They have been shown to be implicated in cholesterol turnover, inflammation and in neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis. Glial cells are targets of oxysterols: they inhibit astrocyte proliferation after brain injury, and we have previously shown that 25-hydroxycholesterol (25OH) provokes oligodendrocyte apoptosis and stimulates the expression of sPLA2 type IIA (sPLA2-IIA), which has a protective effect. METHODOLOGY/PRINCIPAL FINDINGS: As glucocorticoids are well-known for their anti-inflammatory effects, our aim was to understand their direct effects on oxysterol-induced responses in oligodendrocytes (sPLA2-IIA stimulation and apoptosis). We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) abolishes the stimulation of sPLA2-IIA by 25-hydroxycholesterol (25-OH). This inhibition is mediated by the glucocorticoid receptor (GR), which decreases the expression of the oxysterol receptor Pregnane X Receptor (PXR) and interferes with oxysterol signaling by recruiting a common limiting coactivator PGC1alpha. Consistent with the finding that sPLA2-IIA can partially protect oligodendrocytes against oxysterol-triggered apoptosis, we demonstrate here that the inhibition of sPLA2-IIA by Dex accelerates the apoptotic phenomenon, leading to a shift towards necrosis. We have shown by atomic force microscopy and electron microscopy that 25-OH and Dex alters oligodendrocyte shape and disorganizes the cytoplasm. CONCLUSIONS/SIGNIFICANCE: Our results provide a new understanding of the cross-talk between oxysterol and glucocorticoid signaling pathways and their respective roles in apoptosis and oligodendrocyte functions.

Interaction of the alpha-helical H6 peptide from the pro-apoptotic protein tBid with cardiolipin.

BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices alphaH6 and alphaH7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c-releasing activity. In the present study, we focused on the interaction between the alphaH6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the alphaH6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of alphaH6 into the phospholipid monolayer. The modification of two charged amino acid residues in alphaH6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and alphaH6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that alphaH6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via alphaH6.

25-hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR.

In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 microM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)-OH (1 and 10 microM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24(S)-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 microM) and by LXR beta at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.