Pavel Ješina

Mitochondrial ATP synthase deficiency due to a mutation in the ATP5E gene for the F1 ε subunit

F1Fo-ATP synthase is a key enzyme of mitochondrial energy provision producing most of cellular ATP. So far, mitochondrial diseases caused by isolated disorders of the ATP synthase have been shown to result from mutations in mtDNA genes for the subunits ATP6 and ATP8 or in nuclear genes encoding the biogenesis factors TMEM70 and ATPAF2. Here, we describe a patient with a homozygous p.Tyr12Cys mutation in the epsilon subunit encoded by the nuclear gene ATP5E. The 22-year-old woman presented with neonatal onset, lactic acidosis, 3-methylglutaconic aciduria, mild mental retardation and developed peripheral neuropathy. Patient fibroblasts showed 60-70% decrease in both oligomycin-sensitive ATPase activity and mitochondrial ATP synthesis. The mitochondrial content of the ATP synthase complex was equally reduced, but its size was normal and it contained the mutated epsilon subunit. A similar reduction was found in all investigated F1 and Fo subunits with the exception of Fo subunit c, which was found to accumulate in a detergent-insoluble form. This is the first case of a mitochondrial disease due to a mutation in a nuclear encoded structural subunit of the ATP synthase. Our results indicate an essential role of the epsilon subunit in the biosynthesis and assembly of the F1 part of the ATP synthase. Furthermore, the epsilon subunit seems to be involved in the incorporation of subunit c to the rotor structure of the mammalian enzyme.

Diminished synthesis of subunit a (ATP6) and altered function of ATP synthase and cytochrome c oxidase due to the mtDNA 2 bp microdeletion of TA at positions 9205 and 9206

Dysfunction of mitochondrial ATPase (F1F(o)-ATP synthase) due to missense mutations in ATP6 [mtDNA (mitochondrial DNA)-encoded subunit a] is a frequent cause of severe mitochondrial encephalomyopathies. We have investigated a rare mtDNA mutation, i.e. a 2 bp deletion of TA at positions 9205 and 9206 (9205DeltaTA), which affects the STOP codon of the ATP6 gene and the cleavage site between the RNAs for ATP6 and COX3 (cytochrome c oxidase 3). The mutation was present at increasing load in a three-generation family (in blood: 16%/82%/>98%). In the affected boy with severe encephalopathy, a homoplasmic mutation was present in blood, fibroblasts and muscle. The fibroblasts from the patient showed normal aurovertin-sensitive ATPase hydrolytic activity, a 70% decrease in ATP synthesis and an 85% decrease in COX activity. ADP-stimulated respiration and the ADP-induced decrease in the mitochondrial membrane potential at state 4 were decreased by 50%. The content of subunit a was decreased 10-fold compared with other ATPase subunits, and [35S]-methionine labelling showed a 9-fold decrease in subunit a biosynthesis. The content of COX subunits 1, 4 and 6c was decreased by 30-60%. Northern Blot and quantitative real-time reverse transcription-PCR analysis further demonstrated that the primary ATP6--COX3 transcript is cleaved to the ATP6 and COX3 mRNAs 2-3-fold less efficiently. Structural studies by Blue-Native and two-dimensional electrophoresis revealed an altered pattern of COX assembly and instability of the ATPase complex, which dissociated into subcomplexes. The results indicate that the 9205DeltaTA mutation prevents the synthesis of ATPase subunit a, and causes the formation of incomplete ATPase complexes that are capable of ATP hydrolysis but not ATP synthesis. The mutation also affects the biogenesis of COX, which is present in a decreased amount in cells from affected individuals.

Sustained deficiency of mitochondrial complex I activity during long periods of survival after seizures induced in immature rats by homocysteic acid

Our previous work demonstrated the marked decrease of mitochondrial complex I activity in the cerebral cortex of immature rats during the acute phase of seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side) and at short time following these seizures. The present study demonstrates that the marked decrease ( approximately 60%) of mitochondrial complex I activity persists during the long periods of survival, up to 5 weeks, following these seizures, i.e. periods corresponding to the development of spontaneous seizures (epileptogenesis) in this model of seizures. The decrease was selective for complex I and it was not associated with changes in the size of the assembled complex I or with changes in mitochondrial content of complex I. Inhibition of complex I was accompanied by a parallel, up to 5 weeks lasting significant increase (15-30%) of three independent mitochondrial markers of oxidative damage, 3-nitrotyrosine, 4-hydroxynonenal and protein carbonyls. This suggests that oxidative modification may be most likely responsible for the sustained deficiency of complex I activity although potential role of other factors cannot be excluded. Pronounced inhibition of complex I was not accompanied by impaired ATP production, apparently due to excess capacity of complex I documented by energy thresholds. The decrease of complex I activity was substantially reduced by treatment with selected free radical scavengers. It could also be attenuated by pretreatment with (S)-3,4-DCPG (an agonist for subtype 8 of group III metabotropic glutamate receptors) which had also a partial antiepileptogenic effect. It can be assumed that the persisting inhibition of complex I may lead to the enhanced production of reactive oxygen and/or nitrogen species, contributing not only to neuronal injury demonstrated in this model of seizures but also to epileptogenesis.

Mitochondrial complex I inhibition in cerebral cortex of immature rats following homocysteic acid-induced seizures

The major finding of the present study concerns the marked decrease of respiratory chain complex I activity in the cerebral cortex of immature rats following seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side). This decrease was already evident during the acute phase of seizures (60-90 min after infusion) and persisted for at least 20 h after the seizures. It was selective for complex I since activities of complex II and IV and citrate synthase remained unaffected. Inhibition of complex I activity was not associated with changes in complex I content. Based on enhanced lipoperoxidation and decreased aconitase activity, it can be postulated that oxidative modification is most likely responsible for the observed inhibition. Mitochondrial respiration, as well as cortical ATP levels remained in the control range, apparently due to excess capacity of the complex I documented by energy thresholds. On the other hand, the enhanced production of reactive oxygen species by inhibited complex I was observed in mitochondria from HCA-treated animals. The decrease of complex I activity was substantially attenuated when animals were treated with substances providing an anticonvulsant effect and also with selected free radical scavengers. We can assume that inhibition of complex I may elicit enhanced formation of reactive oxygen species and contribute thus to neuronal injury demonstrated in this model.

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.

Mitochondrial Membrane Potential and ATP Production in Primary Disorders of ATP Synthase

Studies of fibroblasts with primary defects in mitochondrial ATP synthase (ATPase) due to heteroplasmic mtDNA mutations in the ATP6 gene, affecting protonophoric function or synthesis of subunit a, show that at high mutation loads, mitochondrial membrane potential ΔΨm at state 4 is normal, but ADP-induced discharge of ΔΨm is impaired and ATP synthesis at state 3-ADP is decreased. Increased ΔΨm and low ATP synthesis is also found when the ATPase content is diminished by altered biogenesis of the enzyme complex. Irrespective of the different pathogenic mechanisms, elevated ΔΨm in primary ATPase disorders could increase mitochondrial production of reactive oxygen species and decrease energy provision.

Antioxidant enzymes in cerebral cortex of immature rats following experimentally-induced seizures: upregulation of mitochondrial MnSOD (SOD2).

We have recently demonstrated the evidence of oxidative stress in brain of immature rats during seizures induced by dl‐homocysteic acid (dl‐HCA). The aim of the present study was to investigate the antioxidant defense mechanisms under these conditions. Seizures were induced in immature 12‐day‐old rats by bilateral icv infusion of dl‐HCA (600 nmol/side), and the activities of the main antioxidant enzymes were examined in supernatants of the cerebral cortex during the acute phase of seizures and at several periods of survival, up to 5 weeks, following these seizures. In control animals individual antioxidant enzymes revealed different changes during the studied postnatal period (PD 12 till PD 47). Total superoxide dismutase (SOD), CuZn SOD (SOD1), Mn SOD (SOD2) and glutathione peroxidase (GPX) activities were increasing while, catalase activity decreased and the activity of glutathione reductase (GR) remained unchanged. In HCA‐treated animals, the activity of total SOD, SOD1 and particularly SOD2 significantly increased at 20 h and 6 days of survival. Importantly, upregulation of SOD2 was also confirmed in mitochondria at the protein level by immunoblotting. The activities of other antioxidant enzymes including catalase and GPX did not significantly differ upon HCA treatment from the appropriate controls at any of the studied time intervals. The pronounced and selective upregulation of SOD2 points to enhanced ROS levels in the mitochondrial matrix. This may be associated with inhibition of respiratory chain complex I that we have demonstrated in our previous studies. The present findings suggest that oxidative stress occurring in the brain of immature rats during and following the seizures induced by dl‐HCA is apparently due to both the increased free radical production and the limited antioxidant defense.

Status Epilepticus in Immature Rats Is Associated with Oxidative Stress and Mitochondrial Dysfunction

Epilepsy is a neurologic disorder, particularly frequent in infants and children where it can lead to serious consequences later in life. Oxidative stress and mitochondrial dysfunction are implicated in the pathogenesis of many neurological disorders including epilepsy in adults. However, their role in immature epileptic brain is unclear since there have been two contrary opinions: oxidative stress is age-dependent and does not occur in immature brain during status epilepticus (SE) and, on the other hand, evidence of oxidative stress in immature brain during a specific model of SE. To solve this dilemma, we have decided to investigate oxidative stress following SE induced in immature 12-day-old rats by three substances with a different mechanism of action, namely 4-aminopyridine, LiCl-pilocarpine or kainic acid. Fluoro-Jade-B staining revealed mild brain damage especially in hippocampus and thalamus in each of the tested models. Decrease of glucose and glycogen with parallel rises of lactate clearly indicate high rate of glycolysis, which was apparently not sufficient in 4-AP and Li-Pilo status, as evident from the decreases of PCr levels. Hydroethidium method revealed significantly higher levels of superoxide anion (by ∼60%) in the hippocampus, cerebral cortex and thalamus of immature rats during status. SE lead to mitochondrial dysfunction with a specific pronounced decrease of complex I activity that persisted for a long period of survival. Complexes II and IV activities remained in the control range. Antioxidant treatment with SOD mimetic MnTMPYP or peroxynitrite scavenger FeTPPS significantly attenuated oxidative stress and inhibition of complex I activity. These findings bring evidence that oxidative stress and mitochondrial dysfunction are age and model independent, and may thus be considered a general phenomenon. They can have a clinical relevance for a novel approach to the treatment of epilepsy, allowing to target the mechanisms which play a crucial or additive role in the pathogenesis of epilepsies in infants and children.

Alteration of structure and function of ATP synthase and cytochrome c oxidase by lack of Fo-a and Cox3 subunits caused by mitochondrial DNA 9205delTA mutation.

Mutations in the MT-ATP6 gene are frequent causes of severe mitochondrial disorders. Typically, these are missense mutations, but another typexa0is represented by the 9205delTA microdeletion, which removes the stop codon of the MT-ATP6 gene and affects the cleavage site in the MT-ATP8/MT-ATP6/MT-CO3 polycistronic transcript. This interferes with the processing of mRNAs for the Atp6 (Fo-a) subunit of ATP synthase and the Cox3 subunit of cytochrome c oxidase (COX). Two cases described so far presented with strikingly different clinical phenotypes-mild transient lactic acidosis or fatal encephalopathy. To gain more insight into the pathogenic mechanism, we prepared 9205delTA cybrids with mutation load ranging between 52 and 99% and investigated changes in the structure and function of ATP synthase and the COX. We found that 9205delTA mutation strongly reduces the levels of both Fo-a and Cox3 proteins. Lack of Fo-a alters the structure but not the content of ATP synthase, which assembles into a labile, ∼60xa0kDa smaller, complex retaining ATP hydrolytic activity but which is unable to synthesize ATP. In contrast, lack of Cox3 limits the biosynthesis of COX but does not alter the structure of the enzyme. Consequently, the diminished mitochondrial content of COX and non-functional ATP synthase prevent most mitochondrial ATP production. The biochemical effects caused by the 9205delTA microdeletion displayed a pronounced threshold effect above ∼90% mutation heteroplasmy. We observed a linear relationship between the decrease in subunit Fo-a or Cox3 content and the functional presentation of the defect. Therefore we conclude that the threshold effect originated from a gene-protein level.

Effect of Resveratrol on Oxidative Stress and Mitochondrial Dysfunction in Immature Brain during Epileptogenesis

The presence of oxidative stress in immature brain has been demonstrated during the acute phase of status epilepticus (SE). The knowledge regarding the long periods of survival after SE is not unequivocal, lacking direct evidence. To examine the presence and time profile of oxidative stress, its functional effect on mitochondria and the influence of an antioxidant treatment in immature rats during epileptogenesis, status epilepticus (SE) was induced in immature 12-day-old rats by Li-pilocarpine and at selected periods of the epileptogenesis; rat pups were subjected to examinations. Hydroethidine method was employed for detection of superoxide anion (O2.−), 3-nitrotyrosine (3-NT), and 4-hydroxynonenal (4-HNE) for oxidative damage of mitochondrial proteins and complex I activity for mitochondrial function. Natural polyphenolic antioxidant resveratrol was given in two schemes: “acute treatment,” i.p. administration 30xa0min before, 30 and 60xa0min after induction of SE and “full treatment” when applications continued once daily for seven consecutive days (25xa0mg/kg each dose). The obtained results clearly document that the period of epileptogenesis studied (up to 4xa0weeks) in immature brain is associated with the significant enhanced production of O2.−, the increased levels of 3-NT and 4-HNE and the persisting deficiency of complex I activity. Application of resveratrol either completely prevented or significantly reduced markers both of oxidative stress and mitochondrial dysfunction. The findings suggest that targeting oxidative stress in combination with current antiepileptic therapies may provide a benefit in the treatment of epilepsy.