Vera Adam-Vizi

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.

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.

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.

Calcium and Mitochondrial Reactive Oxygen Species Generation: How to Read the Facts

A number of recent discoveries indicate that abnormal Ca2+ signaling, oxidative stress, and mitochondrial dysfunction are involved in the neuronal damage in Alzheimers disease. However, the literature on the interactions between these factors is controversial especially in the interpretation of the cause-effect relationship between mitochondrial damage induced by Ca2+ overload and the production of reactive oxygen species (ROS). In this review, we survey the experimental observations on the Ca2+-induced mitochondrial ROS production, explain the sources of controversy in interpreting these results, and discuss the different molecular mechanisms underlying the effect of Ca2+ on the ROS emission by brain mitochondria.

Role of sodium channel inhibition in neuroprotection: Effect of vinpocetine

Vinpocetine (ethyl apovincaminate) discovered during the late 1960s has successfully been used in the treatment of central nervous system disorders of cerebrovascular origin for decades. The increase in the regional cerebral blood flow in response to vinpocetine administration is well established and strengthened by new diagnostical techniques (transcranial Doppler, near infrared spectroscopy, positron emission tomography). The latest in vitro studies have revealed the effect of the compound on Ca(2+)/calmodulin dependent cyclic guanosine monophosphate-phosphodiesterase 1, voltage-operated Ca(2+) channels, glutamate receptors and voltage dependent Na(+)-channels; the latest being especially relevant to the neuroprotective action of vinpocetine. The good brain penetration profile and heterogenous brain distribution pattern (mainly in the thalamus, basal ganglia and visual cortex) of labelled vinpocetin were demonstrated by positron emission tomography in primates and man. Multicentric, randomized, placebo-controlled clinical studies proved the efficacy of orally administered vinpocetin in patients with organic psychosyndrome. Recently positron emission tomography studies have proved that vinpocetine is able to redistribute regional cerebral blood flow and enhance glucose supply of brain tissue in ischemic post-stroke patients.

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.

Release of acetylcholine from rat brain synaptosomes by various agents in the absence of external calcium ions.

The relationship between 86Rb+ distribution across synaptosomal membrane and [14C]acetylcholine (ACh) release have been studied in a rat brain cortex synaptosomal preparation using K+, ouabain and veratridine depolarization. Decrease in membrane potential, approximated from the 86Rb+ distribution, is accompanied by an increase in [14C]ACh release, but the extent of the increase at a certain depolarization is dependent on how the depolarization is induced. A substantial depolarization by K+ is necessary to enhance ACh release, as compared to ouabain and veratridine where only a slight depolarization is accompanied by an increase in ACh release. In Ca2+‐free, EGTA‐containing medium ouabain and veratridine can also increase [14C]ACh release. The relationship between membrane potential and ACh release is very similar in the presence of ouabain and veratridine both in Ca2+‐containing and Ca2+‐free medium. The effect of ouabain and veratridine on the Na‐K exchange pump is different; ouabain can completely abolish Na‐K‐ATPase activity and 86Rb+ uptake of synaptosomes, whereas veratridine does not seem to influence the activity of the pump. m‐Chloro‐carbonylcianid phenyl hydrazon (50‐500 nM) increases [14C]ACh release in a concentration‐dependent manner without a considerable change of membrane potential or Na‐K pump activity. The Ca2+ ionophore A 23187 induces a substantial increase in [14C]ACh release in the absence of external Ca2+. In this case neither Na‐K pump activity nor membrane potential of synaptosomes is changed. A possible role of intracellular Ca2+ mobilization as a consequence of increased intracellular Na+ concentration in some depolarization‐induced transmitter release is discussed.

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.