Ubiquinone Analogs: A Mitochondrial Permeability Transition Pore-Dependent Pathway to Selective Cell Death

International audienceBACKGROUND: Prolonged opening of the mitochondrial permeability transition pore (PTP) leads to cell death. Various ubiquinone analogs have been shown to regulate PTP opening but the outcome of PTP regulation by ubiquinone analogs on cell fate has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: The effects of ubiquinone 0 (Ub(0)), ubiquinone 5 (Ub(5)), ubiquinone 10 (Ub(10)) and decyl-ubiquinone (DUb) were studied in freshly isolated rat hepatocytes, cultured rat liver Clone-9 cells and cancerous rat liver MH1C1 cells. PTP regulation by ubiquinones differed significantly in permeabilized Clone-9 and MH1C1 cells from that previously reported in liver mitochondria. Ub(0) inhibited PTP opening in isolated hepatocytes and Clone-9 cells, whereas it induced PTP opening in MH1C1 cells. Ub(5) did not affect PTP opening in isolated hepatocytes and MH1C1 cells, but it induced PTP opening in Clone-9 cells. Ub(10) regulated PTP in isolated hepatocytes, whereas it did not affect PTP opening in Clone-9 and MH1C1 cells. Only DUb displayed the same effect on PTP regulation in the three hepatocyte lines tested. Despite such modifications in PTP regulation, competition between ubiquinones still occurred in Clone-9 and MH1C1 cells. As expected, Ub(5) induced a PTP-dependent cell death in Clone-9, while it did not affect MH1C1 cell viability. Ub(0) induced a PTP-dependent cell death in MH1C1 cells, but was also slightly cytotoxic in Clone-9 by an oxidative stress-dependent mechanism. CONCLUSIONS/SIGNIFICANCE: We found that various ubiquinone analogs regulate PTP in different ways depending on the cell studied. We took advantage of this unique property to develop a PTP opening-targeted strategy that leads to cell death specifically in cells where the ubiquinone analog used induces PTP opening, while sparing the cells in which it does not induce PTP opening

Mesure par microscopie confocale du métabolisme mitochondrial et du niveau énergétique cellulaire au cours d’épisodes de carences en substrats et/ou en oxygène

Mitochondria form an information hub at the center of the cellular metabolism because of its physiological role consisting in the porduction of ATP from the degradation of porducts stemming from our food through the OXPHOS process. However, changes in the functionnig of the mitochondria can be responsible for numerous diseases. Among the different foms of metabolic stress leading to mitchondrial dysfunctions, ischemia-reperfusion can be found in numerous pathological situations. This work aims at developing a methodological approach based on confocal microscopy and image analysis to dissect –at cell level- the consequences of metabolic stress induced by episodes of deprivation in substrata associated or not with hypoxia or anoxia. Having developed the program of image analysis based on the « tophat » method, two approaches were designed to vizualize and quantify the mitochondrial function. The first one, combining TMRM labelling with NADH fluorescence made it possible to highlight some differences in the response to the stress caused by ischemia-reperfusion at the level of the respiratory chain or concerning the PTP opening in the four cellular types that were tested : HMEC-1, INS1, RT112 or pirmary heaptocyes. The second approach consisted in testing the use of biosensors designed to follow the variations of ATP concentration (ATeam) or the activation of AMPK (AMPKAR). The experimental conditions established in this work did not allow us to validate their use.La mitochondrie est un carrefour d’informations au centre du fonctionnement cellulaire puisque son rôle physiologique consiste à récupérer l’énergie fournie par la dégradation des produits issus de notre alimentation pour produire de l’ATP, par le processus d’oxydation phosphorylante. Cependant, des altérations du fonctionnement de la mitochondrie peuvent être responsables de nombreuses pathologies. Parmi les stress métaboliques pouvant entraîner un dysfonctionnement mitochondrial, l’ischémie-reperfusion est un phénomène présent également dans de nombreuses situations pathologiques. L’objectif de ce travail consiste à développer une approche méthodologique basée sur la microscopie confocale et l’analyse d’images afin de décortiquer les conséquences cellulaires des stress métaboliques induits lors d’épisodes de privation de substrats associée ou non à une privation partielle ou totale d’oxygène. Après avoir mis au point le programme d’analyse d’images basée sur la méthode du « tophat », deux approches ont été développées pour visualiser et quantifier la fonction mitochondriale. La première, qui combine le marquage du TMRM et l’autofluorescence du NADH, a permis de mettre en évidence des différences de réponses au stress d’ischémie-reperfusion au niveau de la chaîne respiratoire ou de l’ouverture du PTP pour les quatre types cellulaires testés : HMEC-1, INS1, RT112 ou hépatocytes primaires. La seconde approche a consisté à tester l’utilisation de biosenseurs permettant de suivre les variations de concentration d’ATP (Ateam) ou d’activation de l’AMPK (AMPKAR). Les conditions expérimentales réalisées dans ce travail n’ont pas permis de valider leur utilisation

Protection of PC12 cells from cocaine-induced cell death by inhibiting mitochondrial permeability transition

International audienceCocaine abuse induces brain injury and neurodegeneration by a mechanism that has not yet been fully elucidated. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). In this work, we examined the involvement of the PTP in cocaine-induced toxicity in PC12 cell lines. We used two different PTP inhibitors -i.e. cyclosporin A (CsA) and metformin-to assess their ability to counteract the cocaine induced effects. We first observed that a 48 h exposure to cocaine strongly sensitized cells to calcium overload, as measured by the calcium retention capacity. CsA and metformin significantly decreased the cocaine-induced PTP opening sensitization. We next showed by confocal microscopy that cocaine induced a permanent PTP opening in intact living cells, a phenomenon characterized by the collapse of the mitochondrial membrane potential and the relocation of the NAD(P)H from the mitochondrial matrix to the cytosol. As expected, a cocaine-induced PTP opening was prevented by PTP inhibitors. Finally, a flow cytometry analysis revealed that cocaine induced cell death while CsA and metformin promoted cell survival. Our results demonstrate that cocaine induces PC12 cell death through a mechanism involving permanent PTP opening

Cytometric assessment of mitochondria using fluorescent probes.

International audienceMitochondria are most important organelles in the survival of eukaryotic aerobic cells because they are the primary producers of ATP, regulators of ion homeostasis or redox state, and producers of free radicals. The key role of mitochondria in the generation of primordial ATP for the survival and proliferation of eukaryotic cells has been proven by extensive biochemical studies. In this context, it is crucial to understand the complexity of the mitochondrial compartment and its functionality and to develop experimental tools allowing the assessment of its nature and its function and metabolism. This review covers the role of the mitochondria in the cell, focusing on its structure, the mechanism of the mitochondrial respiratory chain, the maintenance of the transmembrane potential and the production of reactive oxygen species. The main probes used for mitochondrial compartment monitoring are described. In addition, various applications using mitochondrial-specific probes are detailed to illustrate the potential of flow and image cytometry in the study of the mitochondrial compartment. This review contains a panel of tools to explore mitochondria and to help researchers design experiments, determine the approach to be employed, and interpret their results

Prerequisites for ubiquinone analogs to prevent mitochondrial permeability transition-induced cell death.

International audienceThe permeability transition pore (PTP) is a mitochondrial inner membrane channel involved in cell death. The inhibition of PTP opening has been proved to be an effective strategy to prevent cell death induced by oxidative stress. Several ubiquinone analogs are known to powerfully inhibit PTP opening with an effect depending on the studied cell line. Here, we have studied the effects of ubiquinone 0 (Ub(0)), ubiquinone 5 (Ub(5)) and ubiquinone 10 (Ub(10)) on PTP regulation, H(2)O(2) production and cell viability in U937 cells. We found that Ub(0) induced both PTP opening and H(2)O(2) production. Ub(5) did not regulate PTP opening yet induced H(2)O(2) production. Ub(10) potently inhibited PTP opening yet induced H(2)O(2) production. Both Ub(0) and Ub(5) induced cell death, whereas Ub(10) was not toxic. Moreover, Ub(10) prevented tert-butyl hydroperoxide-induced PTP opening and subsequent cell death. We conclude that PTP-inhibitor ubiquinone analogs are able to prevent PTP opening-induced cell death only if they are not toxic per se, which is the case when they have no or low pro-oxidant activity

Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism

International audienceAMP-activated protein kinase AMPK senses and regulates cellular energy state. AMPK activation by increasing AMP and ADP concentrations involves a conformational switch within the heterotrimeric complex. This is exploited here for the construction of a synthetic sensor of cellular energetics and allosteric AMPK activation, AMPfret. Based on engineered AMPK fused to fluorescent proteins, the sensor allows direct, real-time readout of the AMPK conformational state by fluorescence resonance energy transfer (FRET). AMPfret faithfully and dynamically reports the binding of AMP and ADP to AMPK γ-CBS sites, competed by Mg2+-free ATP. FRET signals correlate with activation of AMPK by allosteric mechanisms and protection from dephosphorylation, attributed here to specific CBS sites, but does not require activation loop phosphorylation. Moreover, AMPfret detects binding of pharmacological compounds to the AMPK α/β-ADaM site enabling activator screening. Cellular assays demonstrate that AMPfret is applicable in vivo for spatiotemporal analysis of energy state and allosteric AMPK activation

Low-level cadmium doses do not jeopardize the insulin secretion pathway of β-cell models until the onset of cell death

International audienceBackground: Cadmium is an inescapable environmental pollutant that permeates the food chain and has been debatably associated with diabetes in humans.Purpose and procedures: To probe the specific impact of low-level cadmium exposure on insulin production, largely sub-cytotoxic (50-500 nM) concentrations of cadmium chloride challenged the INS-1 and MIN6 rodent models of pancreatic β-cells for the longest possible time, up to 4 days, before sub-culturing.Main findings: The concentration of detectable oxidants, the pattern of the actin cytoskeleton, the translocation of β-catenin, the activity of protein phosphatases, calcium traffic, and the phosphorylation status of several key signaling nodes, such as AMP kinase and mitogen activated kinases including nuclear translocation of Extracellular signal-Regulated Kinase, were all insensitive to the applied very low cadmium doses. Accordingly, low-level cadmium exposure did not alter the insulin secretion ability, the functional hallmark of β-cells, before the onset of cell death.Conclusions: These data define an operational toxicological threshold for these cellular models of β-cells that should be useful to address insulin secretion and the diabetogenic effects of chronic low-level cadmium exposure in animal models and in humans