Tiziana Cocco

Arachidonic acid interaction with the mitochondrial electron transport chain promotes reactive oxygen species generation.

A study has been carried out on the interaction of arachidonic acid and other long chain free fatty acids with bovine heart mitochondria. It is shown that arachidonic acid causes an uncoupling effect under state 4 respiration of intact mitochondria as well as a marked inhibition of uncoupled respiration. While, under our conditions, the uncoupling effect is independent of the fatty acid species considered, the inhibition is stronger for unsaturated acids. Experiments carried out with mitochondrial particles indicated that the arachidonic acid dependent decrease of the respiratory activity is caused by a selective inhibition of Complex I and III. It is also shown that arachidonic acid causes a remarkable increase of hydrogen peroxide production when added to mitochondria respiring with either pyruvate+malate or succinate as substrate. The production of reactive oxygen species (ROS) at the coupling site II was almost double than that at site I. The results obtained are discussed with regard to the impairment of the mitochondrial respiratory activity as occurring during the heart ischemia/reperfusion process.

The nuclear-encoded 18 kDa (IP) AQDQ subunit of bovine heart complex I is phosphorylated by the mitochondrial cAMP-dependent protein kinase

In bovine heart mitochondria a protein of M r 18 kDa, phosphorylated by mtPKA, is associated to the NADH‐ubiquinone oxidoreductase in the inner membrane and is present in purified preparation of this complex. The 18 kDa phosphoprotein has now been isolated and sequenced. It is identified as the 18 kDa (IP) AQDQ subunit of complex I, a protein of 133 amino acids with a phosphorylation consensus site RVS at position 129–131.

Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease

Mitochondrial dysfunction and oxidative stress occur in Parkinsons disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1αs target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.

Mitochondrial oxidative alterations following partial hepatectomy

Mitochondria, isolated from rat livers during the early phase of liver regeneration (7-24 h after partial hepatectomy), show: (i) decrease in the rate of ATP synthesis; (ii) increase of malondialdehyde and of oxidized protein production; (iii) decrease of the content of intramitochondrial glutathione and of protein thiols on mitochondrial proteins; (iv) increase of the glutathione bound to mitochondrial proteins by disulfide bonds. These observations suggest an increase of production of oxygen radicals in liver mitochondria, following partial hepatectomy, which can alter the function of the enzymes involved in the oxidative phosphorylation. Blue-native gel electrophoresis of rat liver mitochondria, isolated after partial hepatectomy, shows, during the early phase of liver regeneration (0-24 h after partial hepatectomy), a progressive decrease of the content of F0F1-ATP synthase complex. The amount of glutathione bound to the F0F1-ATP synthase, electroeluted from the blue-native gels, progressively increased during the early phase of liver regeneration. It is concluded that partial hepatectomy causes mitochondrial oxidative stress that, in turn, modifies proteins (such as F0F1-ATP synthase) involved in the mitochondrial oxidative phosphorylation.

Effect of dietary restriction and N-acetylcysteine supplementation on intestinal mucosa and liver mitochondrial redox status and function in aged rats.

The age-related changes of glutathione (GSH) levels and the effect of hypocaloric regimen and N-acetylcysteine (NAC) supplementation were investigated in intestinal mucosa and liver mitochondria of 28 months rats. Old rats exhibited lower proteins, GSH and protein sulphydrils (PSH) concentrations, higher GSH-peroxidase (GSH-Px) activity and protein carbonyl deposit, partial inhibition of succinate stimulated mitochondrial state III respiration and decreased mitochondrial nitrosothiols (RSNO) concentration. Lower electric potential and current intensity were found in the colonic mucosa. Old rats undergone hypocaloric regimen showed higher intestinal concentrations of GSH, lower oxidized protein accumulation and GSH-Px activity and higher mitochondrial RSNO levels. Mitochondrial state III respiration and intestinal transport were improved. NAC supplementation enhanced GSH and PSH levels in the ileal but not in the colonic mucosa, GSH and RSNO in liver mitochondria, while GSH-Px and protein carbonyls were decreased everywhere. Mitochondrial respiration ameliorated. In conclusion, ageing is characterized by a spread decrease of GSH concentrations, increased protein oxidation and decreased mitochondrial NO content. Hypocaloric diet ameliorated intestinal transport and, as well as NAC, was effective in enhancing GSH levels but at different extent according to the investigated districts. Both interventions reduced the age-associated increase of GSH-Px and protein carbonyls and improved mitochondrial respiration.

Lipidomics of intact mitochondria by MALDI-TOF/MS

A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.

CORRELATION BETWEEN RAT LIVER REGENERATION AND MITOCHONDRIAL ENERGY METABOLISM

The time course of changes in mitochondrial energy metabolism during liver regeneration, following partial hepatectomy, is analyzed. For 24 h after surgical operation, a lag phase in the time course of the growth of liver is observed. In this period mitochondria showed a decrease of: (1) the respiratory control index; (2) the rate of oxidative phosphorylation; (3) the amount of immunodetected beta-F1 and F01-PVP subunits of F0F1-ATP synthase. No decrease, but instead a small increase in the content of mRNA for beta-F1 was observed in this phase. After this lag phase the growth of liver started, the content of mRNA for beta F1, as well as the level of immunodetected mitochondrial beta-F1 and F01-PVP subunits, increased and oxidative phosphorylation recovered. Analysis of the relative beta F1 protein/mRNA ratio indicates a decrease of beta F1 translational efficiency which remained low up to 72 h after partial hepatectomy and reached the same ratio of control at 96 h. It is concluded that the regenerating capability of rat liver is correlated with the efficiency of oxidative phosphorylation.

Activation of the cAMP cascade in human fibroblast cultures rescues the activity of oxidatively damaged complex I

A study of the relationship between cAMP/PKA-dependent phosphorylation and oxidative damage of subunits of complex I of the mitochondrial respiratory chain is presented. It is shown that, in fibroblast cultures, PKA-mediated phosphorylation of the NDUFS4 subunit of complex I rescues the activity of the oxidatively damaged complex. Evidence is presented showing that this effect is mediated by phosphorylation-dependent exchange of carbonylated NDUFS4 subunit in the assembled complex with the de novo synthesized subunit. These results indicate a potential use for β-adrenoceptor agonists in preventing/reversing the detrimental effects of oxidative stress in the mitochondrial respiratory system.

The β-adrenoceptor agonist isoproterenol promotes the activity of respiratory chain complex I and lowers cellular reactive oxygen species in fibroblasts and heart myoblasts

A study is presented on the effect of the β-adrenoceptor agonist isoproterenol on mitochondrial oxygen metabolism in fibroblast and heart myoblast cultures. Isoproterenol treatment of serum-limited fibroblasts and proliferating myoblasts results in the promotion of mitochondrial complex I activity and decrease of the cellular level of reactive oxygen species. These effects of isoproterenol are associated with cAMP-dependent phosphorylation of complex I subunit(s). Addition of okadaic acid, inhibitor of protein phosphatase(s), reverses the decline of complex I activity in serum-limited fibroblast cultures and activates the complex in proliferating myoblast cultures. The effects of isoproterenol on complex I activity and reactive oxygen species balance can contribute to the therapeutic effect of the drug.

Control of OXPHOS efficiency by complex I in brain mitochondria.

In the present work we have analysed the efficiency (P/O ratio) of energy production by oxidative phosphorylation (OXPHOS) in rat brain, liver and heart mitochondria. This study has revealed tissue-specific differences in the mean values of P/O ratios and ATP production rates. A marked dependence of the P/O ratio on the respiration rates has been observed with complex I (NADH:ubiquinone oxidoreductase), but not with complex II (succinate dehydrogenase) respiratory substrates. The physiological impact of the P/O variations with complex I substrates has been further confirmed by extending the analysis to brain mitochondria from three independent groups of animals utilized to study the effects of dietary treatments on the age-related changes of OXPHOS. The general site-specificity of the rate-dependent P/O variability indicates that the decoupling, i.e. decreased coupling between electron transfer and proton pumping, is likely to be mostly due to slip of mitochondrial complex I. These findings suggest an additional mechanism for the pivotal role played by the energy-conserving respiratory complex I in the physiological and adaptive plasticity of mitochondrial OXPHOS.