Christos Chinopoulos

Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

Mitochondria-produced reactive oxygen species (ROS) are thought to contribute to cell death caused by a multitude of pathological conditions. The molecular sites of mitochondrial ROS production are not well established but are generally thought to be located in complex I and complex III of the electron transport chain. We measured H2O2 production, respiration, and NADPH reduction level in rat brain mitochondria oxidizing a variety of respiratory substrates. Under conditions of maximum respiration induced with either ADP or carbonyl cyanide p-trifluoromethoxyphenylhydrazone,α-ketoglutarate supported the highest rate of H2O2 production. In the absence of ADP or in the presence of rotenone, H2O2 production rates correlated with the reduction level of mitochondrial NADPH with various substrates, with the exception of α-ketoglutarate. Isolated mitochondrial α-ketoglutarate dehydrogenase (KGDHC) and pyruvate dehydrogenase (PDHC) complexes produced superoxide and H2O2. NAD+ inhibited ROS production by the isolated enzymes and by permeabilized mitochondria. We also measured H2O2 production by brain mitochondria isolated from heterozygous knock-out mice deficient in dihydrolipoyl dehydrogenase (Dld). Although this enzyme is a part of both KGDHC and PDHC, there was greater impairment of KGDHC activity in Dld-deficient mitochondria. These mitochondria also produced significantly less H2O2 than mitochondria isolated from their littermate wild-type mice. The data strongly indicate that KGDHC is a primary site of ROS production in normally functioning mitochondria.

Calcium, mitochondria and oxidative stress in neuronal pathology

The interplay among reactive oxygen species (ROS) formation, elevated intracellular calcium concentration and mitochondrial demise is a recurring theme in research focusing on brain pathology, both for acute and chronic neurodegenerative states. However, causality, extent of contribution or the sequence of these events prior to cell death is not yet firmly established. Here we review the role of the alpha‐ketoglutarate dehydrogenase complex as a newly identified source of mitochondrial ROS production. Furthermore, based on contemporary reports we examine novel concepts as potential mediators of neuronal injury connecting mitochondria, increased [Ca2+]c and ROS/reactive nitrogen species (RNS) formation; specifically: (a) the possibility that plasmalemmal nonselective cationic channels contribute to the latent [Ca2+]c rise in the context of glutamate‐induced delayed calcium deregulation; (b) the likelihood of the involvement of the channels in the phenomenon of ‘Ca2+ paradox’ that might be implicated in ischemia/reperfusion injury; and (c) how ROS/RNS and mitochondrial status could influence the activity of these channels leading to loss of ionic homeostasis and cell death.

Depolarization of in situ mitochondria due to hydrogen peroxide-induced oxidative stress in nerve terminals: Inhibition of α-ketoglutarate dehydrogenase

Abstract: Mitochondrial membrane potential (Δ?m) was determined in intact isolated nerve terminals using the membrane potential‐sensitive probe JC‐1. Oxidative stress induced by H2O2 (0.1‐1 mM) caused only a minor decrease in Δ?m. When complex I of the respiratory chain was inhibited by rotenone (2 μM), Δ?m was unaltered, but on subsequent addition of H2O2, Δ?m started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in Δ?m when added after oligomycin (10 μM), an inhibitor of F0F1‐ATPase. H2O2 (0.1 or 0.5 mM) inhibited α‐ketoglutarate dehydrogenase and decreased the steady‐state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine Δ?m in the presence of H2O2: (a) The NADH level reduced owing to inhibition of α‐ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of Δ?m when the F0F1‐ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of Δ?m by F0F1‐ATPase. The results indicate that to maintain Δ?m in the nerve terminal during H2O2‐induced oxidative stress, both complex I and F0F1‐ATPase must be functional. Collapse of Δ?m could be a critical event in neuronal injury in ischemia or Parkinson’s disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.

Molecular mechanisms of cell death: central implication of ATP synthase in mitochondrial permeability transition

The term mitochondrial permeability transition (MPT) is commonly used to indicate an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Widespread MPT has catastrophic consequences for the cell, de facto marking the boundary between cellular life and death. MPT results indeed in the structural and functional collapse of mitochondria, an event that commits cells to suicide via regulated necrosis or apoptosis. MPT has a central role in the etiology of both acute and chronic diseases characterized by the loss of post-mitotic cells. Moreover, cancer cells are often relatively insensitive to the induction of MPT, underlying their increased resistance to potentially lethal cues. Thus, intense efforts have been dedicated not only at the understanding of MPT in mechanistic terms, but also at the development of pharmacological MPT modulators. In this setting, multiple mitochondrial and extramitochondrial proteins have been suspected to critically regulate the MPT. So far, however, only peptidylprolyl isomerase F (best known as cyclophilin D) appears to constitute a key component of the so-called permeability transition pore complex (PTPC), the supramolecular entity that is believed to mediate MPT. Here, after reviewing the structural and functional features of the PTPC, we summarize recent findings suggesting that another of its core components is represented by the c subunit of mitochondrial ATP synthase.

Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria

•  Within cells, mitochondria oxidize carbohydrates, and fatty and amino acids to use the released energy to form ATP, and in the process, they also generate reactive oxygen species. Their maximal rates are linked to the magnitude of the mitochondrial membrane potential. •  Here we derive a model of fluorescent potentiometric probe dynamics, and on these principles we introduce an absolute quantitative method for assaying mitochondrial membrane potential in millivolts in individual cultured cells. •  This is the first micro‐scale method to enable measurement of differences in mitochondrial membrane potential between cells with different properties, e.g. size, mitochondrial density and plasma membrane potential, including cases when plasma membrane potential fluctuates. •  Mitochondrial membrane potential in cultured rat cortical neurons is −139 mV at rest. In response to electrical stimulation of the cells, it is regulated between −108 mV and −158 mV by concerted increases in energy demand and metabolic activation.

Mitochondria deficient in complex I activity are depolarized by hydrogen peroxide in nerve terminals: relevance to Parkinson's disease.

Deficiency of complex I in the respiratory chain and oxidative stress induced by hydrogen peroxide occur simultaneously in dopaminergic neurones in Parkinsons disease. Here we demonstrate that the membrane potential of in situ mitochondria (ΔΨm), as measured by the fluorescence change of JC‐l (5,5′,6,6′‐tetrachloro‐1,1,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide), collapses when isolated nerve terminals are exposed to hydrogen peroxide (H2O2,100 and 500 µm) in combination with the inhibition of complex I by rotenone (5 nm−1 µm). H2O2 reduced the activity of complex I by 17%, and the effect of H2O2 and rotenone on the enzyme was found to be additive. A decrease in ΔΨm induced by H2O2 was significant when the activity of complex I was reduced to a similar extent as found in Parkinsons disease (26%). The loss of ΔΨm observed in the combined presence of complex I deficiency and H2O2 indicates that when complex I is partially inhibited, mitochondria in nerve terminals become more vulnerable to H2O2‐induced oxidative stress. This mechanism could be crucial in the development of bioenergetic failure in Parkinsons disease.

Mitochondria as ATP consumers in cellular pathology

ATP provided by oxidative phosphorylation supports highly complex and energetically expensive cellular processes. Yet, in several pathological settings, mitochondria could revert to ATP consumption, aggravating an existing cellular pathology. Here we review (i) the pathological conditions leading to ATP hydrolysis by the reverse operation of the mitochondrial F(o)F(1)-ATPase, (ii) molecular and thermodynamic factors influencing the directionality of the F(o)F(1)-ATPase, (iii) the role of the adenine nucleotide translocase as the intermediary adenine nucleotide flux pathway between the cytosol and the mitochondrial matrix when mitochondria become ATP consumers, (iv) the role of the permeability transition pore in bypassing the ANT, thereby allowing the flux of ATP directly to the hydrolyzing F(o)F(1)-ATPase, (v) the impact of the permeability transition pore on glycolytic ATP production, and (vi) endogenous and exogenous interventions for limiting ATP hydrolysis by the mitochondrial F(o)F(1)-ATPase.

Forward operation of adenine nucleotide translocase during F0F1-ATPase reversal: critical role of matrix substrate-level phosphorylation

In pathological conditions, F0F1‐ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F0F1‐ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F0F1‐ATPase reverses, and the membrane potential is maintained as long as matrix substrate‐level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal KATP channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS‐7 cells transfected with cytosolicor nuclear‐targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate‐level phosphorylation.—Chinopoulos, C, Gerencser, A A., Mandi, M., Mathe, K., Töröcsik, B., Doczi, J., Turiak, L., Kiss, G., Konràd, C, Vajda, S., Vereczki, V., Oh, R. J., Adam‐Vizi, V. Forward operation of adenine nucleotide translocase during F0F1‐ATPase reversal: critical role of matrix substrate‐level phosphorylation. FASEB J. 24, 2405–2416 (2010). www.fasebj.org

Succinate, an intermediate in metabolism, signal transduction, ROS, hypoxia, and tumorigenesis

Succinate is an important metabolite at the cross-road of several metabolic pathways, also involved in the formation and elimination of reactive oxygen species. However, it is becoming increasingly apparent that its realm extends to epigenetics, tumorigenesis, signal transduction, endo- and paracrine modulation and inflammation. Here we review the pathways encompassing succinate as a metabolite or a signal and how these may interact in normal and pathological conditions.(1).

A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT

A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg(2+)] reported by the membrane-impermeable 5K(+) salt of the Mg(2+)-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg(2+)] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.