Laszlo Tretter

Alpha-ketoglutarate dehydrogenase: a target and generator of oxidative stress

Alpha-ketoglutarate dehydrogenase (α-KGDH) is a highly regulated enzyme, which could determine the metabolic flux through the Krebs cycle. It catalyses the conversion of α-ketoglutarate to succinyl-CoA and produces NADH directly providing electrons for the respiratory chain. α-KGDH is sensitive to reactive oxygen species (ROS) and inhibition of this enzyme could be critical in the metabolic deficiency induced by oxidative stress. Aconitase in the Krebs cycle is more vulnerable than α-KGDH to ROS but as long as α-KGDH is functional NADH generation in the Krebs cycle is maintained. NADH supply to the respiratory chain is limited only when α-KGDH is also inhibited by ROS. In addition being a key target, α-KGDH is able to generate ROS during its catalytic function, which is regulated by the NADH/NAD+ ratio. The pathological relevance of these two features of α-KGDH is discussed in this review, particularly in relation to neurodegeneration, as an impaired function of this enzyme has been found to be characteristic for several neurodegenerative diseases.

Initiation of neuronal damage by complex I deficiency and oxidative stress in Parkinson's disease.

Oxidative stress and partial deficiencies of mitochondrial complex I appear to be key factors in the pathogenesis of Parkinsons disease. They are interconnected; complex I inhibition results in an enhanced production of reactive oxygen species (ROS), which in turn will inhibit complex I. Partial inhibition of complex I in nerve terminals is sufficient for in situ mitochondria to generate more ROS. H2O2 plays a major role in inhibiting complex I as well as a key metabolic enzyme, α-ketoglutarate dehydrogenase. The vicious cycle resulting from partial inhibition of complex I and/or an inherently higher ROS production in dopaminergic neurons leads over time to excessive oxidative stress and ATP deficit that eventually will result in cell death in the nigro-striatal pathway.

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.

Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals

In this study reactive oxygen species (ROS) generated in the respiratory chain were measured and the quantitative relationship between inhibition of the respiratory chain complexes and ROS formation was investigated in isolated nerve terminals. We addressed to what extent complex I, III and IV,respectively, should be inhibited to cause ROS generation. For inhibition of complex I, III and IV, rotenone, antimycin and cyanide were used, respectively, and ROS formation was followed by measuring the activity of aconitase enzyme. ROS formation was not detected until complex III was inhibited by up to 71 ± 4%, above that threshold inhibition, decrease in aconitase activity indicated an enhanced ROS generation. Similarly, threshold inhibition of complex IV caused anaccelerated ROS production. By contrast, inactivation of complex I to a small extent (16 ± 2%) resulted in a significant increase in ROS formation, and no clear threshold inhibition could be determined. However, the magnitude of ROS generated at complex I when it is completely inhibited is smaller than that observed when complex III or complex IV was fully inactivated. Our findings may add a novel aspect to the pathology of Parkinsons disease, showing that a moderate level of complex I inhibition characteristic in Parkinsons disease leads to significant ROS formation. The amount of ROS generated by complex I inhibition is sufficient to inhibit in situ the activity of endogenous aconitase.

Characteristics of α‐glycerophosphate‐evoked H2O2 generation in brain mitochondria

Characteristics of reactive oxygen species (ROS) production in isolated guinea‐pig brain mitochondria respiring on α‐glycerophosphate (α‐GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (ΔΨm) and released H2O2 in parallel with an increase in NAD(P)H fluorescence in the presence of α‐GP (5–40 mm). H2O2 formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H2O2 formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by α‐GP, but not by succinate. ROS under these conditions are most likely to be derived from α‐GP‐dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in α‐GP‐supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol‐oxidizing center (Qo) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of α‐GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to α‐GP‐dehydrogenase in mammalian brain mitochondria. ROS generation by α‐GP at complex III is evident only when this complex is inhibited by antimycin.

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).

The production of reactive oxygen species in intact isolated nerve terminals is independent of the mitochondrial membrane potential

Dependence on mitochondrial membrane potential (ΔΨm) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H2O2 generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250 nM) or DNP (50 mM) and then the rate of H2O2 production was followed. Neither of the uncouplers had a significant effect on the rate of H2O2 production induced by rotenone or antimycin. Inhibition of the F0F1-ATPase by oligomycin, which also eliminates ΔΨm in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H2O2 production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of ΔΨm. In addition, we detected a significant antimycin-induced H2O2 production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.

Early events in free radical-mediated damage of isolated nerve terminals: Effects of peroxides on membrane potential and intracellular Na+ and Ca2+ concentrations

Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na+ ([Na+]i) and intracellular Ca2+ ([Ca2+]i) concentrations, and basal glutamate release of synaptosomes. Both H2O2 and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na+]i over an incubation period of 15 min. A steady rise of the [Ca2+]i due to peroxides was also observed that was external Ca2+ dependent and detected only at an inwardly directed Ca2+ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H2O2. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.

The neuroprotective drug vinpocetine prevents veratridine-induced [Na+](i) and [Ca2+](i) rise in synaptosomes

THE effect of the neuroprotective drug, vinpocetine on the veratridine-evoked [Na+]i and [Ca2+]i rise in isolated nerve terminals was studied. Vinpocetine, in a pharmacologically relevant concentration range (0.4–10 μM)i reduced the increase of [Na+]i induced by veratridine (100 μM). The effect of the drug was concentration-dependent with 10 μM vinpocetine completely preventing the increase of [Na+]i. The [Ca2plus;]i rise in response to veratridine was also prevented by vinpocetine. In addition, the [Ca2+]i signal induced by depolarization with 20 mM K+ was reduced by vinpocetine (1–20 μM). This effect was not influenced by preincubation with 1 μM TTX and was also observed when Na+ was replaced by N-methyl glucamine in the medium. It is concluded that vinpocetine is capable of inhibiting voltage-dependent Na+ and Ca2+ channels, respectively, and these effects might contribute to the neuroprotection exerted by the drug.

Uncoupling is without an effect on the production of reactive oxygen species by in situ synaptic mitochondria

Earlier reports that generation of reactive oxygen species (ROS) by isolated mitochondria supported by succinate was sensitive to small changes in the mitochondrial membrane potential (ΔΨm) served as a basis for the concept of ‘mild uncoupling’ suggesting that a few millivolts decrease in ΔΨm would be beneficial in neuroprotection because of reducing the production of ROS by mitochondria. In this study, we tested whether ROS generation by in situ mitochondria, which function in a normal cytosolic environment and oxidize glucose‐derived physiological substrates, is also dependent on changes in ΔΨm. The release of H2O2 was measured by the Amplex red fluorescence assay in freshly prepared isolated nerve terminals, synaptosomes incubated in a glucose‐containing medium. ΔΨm was decreased by the uncoupler carbonyl cyanide‐p‐trifluoromethoxyphenyl‐hydrazon (FCCP) (10–200 nmol/L), which accelerated the oxygen consumption, decreased the NADH level and induced depolarization, as shown by the fluorescence indicator JC‐1, in in situ mitochondria. These changes were detected at already the smallest FCCP concentration. H2O2 generation, however, was found to be unaltered by FCCP at any of the applied concentration. Depolarization of mitochondria was also induced by veratridine (40 μmol/L), which enhances the cytosolic Na+ concentration and imposes an ATP demand in synaptosomes. The accelerated oxygen consumption and the small depolarization of in situ mitochondria by veratridine were not paralleled by any significant alteration in the ROS generation. These findings indicate that a basal ROS generation by in situ mitochondria is not sensitive to changes in ΔΨm challenging the rational of the ‘mild uncoupling’ theory for neuroprotection and suggest that the ΔΨm‐dependent characteristics of ROS generation is limited mainly to well‐coupled succinate‐supported isolated mitochondria.