Mitochondrial DNA background modifies the bioenergetics of NARP/MILS ATP6 mutant cells.

Mutations in the mitochondrial DNA (mtDNA) encoded subunit 6 of ATPase (ATP6) are associated with variable disease expression, ranging from adult onset neuropathy, ataxia and retinitis pigmentosa (NARP) to fatal childhood maternally inherited Leigh's syndrome (MILS). Phenotypical variations have largely been attributed to mtDNA heteroplasmy. However, there is often a discrepancy between the levels of mutant mtDNA and disease severity. Therefore, the correlation among genetic defect, bioenergetic impairment and clinical outcome in NARP/MILS remains to be elucidated. We investigated the bioenergetics of cybrids from five patients carrying different ATP6 mutations: three harboring the T8993G, one with the T8993C and one with the T9176G mutation. The bioenergetic defects varied dramatically, not only among different ATP6 mutants, but also among lines carrying the same T8993G mutation. Mutants with the most severe ATP synthesis impairment showed defective respiration and disassembly of respiratory chain complexes. This indicates that respiratory chain defects modulate the bioenergetic impairment in NARP/MILS cells. Sequencing of the entire mtDNA from the different mutant cell lines identified variations in structural genes, resulting in amino acid changes that destabilize the respiratory chain. Taken together, these results indicate that the mtDNA background plays an important role in modulating the biochemical defects and clinical outcome in NARP/MILS.

In vivo regulation of oxidative phosphorylation in cells harboring a stop-codon mutation in mitochondrial DNA-encoded cytochrome c oxidase subunit I.

The mechanisms that regulate oxidative phosphorylation in mammalian cells are largely unknown. To address this issue, cybrids were generated by fusing osteosarcoma cells devoid of mitochondrial DNA (mtDNA) with platelets from a patient with a stop-codon mutation in cytochrome c oxidase subunit I (COX I). The molecular and biochemical characteristics of cybrids harboring varying levels of mutated mitochondrial DNA were studied. We found a direct correlation between the levels of mutated COX I DNA and mutated COX I mRNA, whereas the levels of COX I total mRNA were unchanged. COX I polypeptide synthesis and steady-state levels were inversely proportional to mutation levels. Cytochrome c oxidase subunit II was reduced proportionally to COX I, indicating impairment in complex assembly. COX enzymatic activity was inversely proportional to the levels of mutated mtDNA. However, both cell respiration and ATP synthesis were preserved in cells with lower proportions of mutated genomes, with a threshold at approximately 40%, and decreased linearly with increasing mutated mtDNA. These results indicate that COX levels in mutated cells were not regulated at the transcriptional, translational, and post-translational levels. Because of a small excess of COX capacity, the levels of expression of COX subunits exerted a relatively tight control on oxidative phosphorylation.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects.

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulin-dependent diabetes mellitus) patients and age-matched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly increased in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Decreased Pasteur effect in platelets of aged individuals.

We have investigated the mitochondrial energy state in human platelets of young (19-30 years old) and aged individuals (65-87 years old) exploiting the Pasteur effect, i.e. stimulation of lactate production by incubation of the purified platelets with the mitochondrial respiratory chain inhibitor, antimycin A. This assay allows the determination of mitochondrial function with respect to glycolysis, and the ratio of mitochondrial adenosine triphosphate (ATP) to glycolytic ATP. A significant increase of basal, non-stimulated lactate production and decrease of the stimulation by antimycin A were observed in the older individuals, suggesting that the impairment of oxidative phosphorylation detectable in post-mitotic tissues of aged individuals can be observed also in easily collectable blood cells.

Mitochondrial bioenergetics in aging.

Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.

Tyrosine phosphorylation in type-1 diabetes by immunogold detection: an in vitro human aortic endothelial cell (HAEC) study in the presence of diabetic low density lipoproteins (LDL).

An immunomorphometric study of tyrosine phosphorylation was performed by the immunogold technique on cultured human aortic endothelial cells (HAEC) with a view to demonstrating their impaired signal transduction status, induced in vitro by incubation with low-density lipoproteins from the plasma of Type-1 diabetic patients. The results seem to sustain the hypothesis that extranuclear bioenergetic derangement induced by low-density lipoproteins from Type-1 diabetic patients may be associated with an up-regulation of the nuclear energetic machinery aimed at maintaining intracellular metabolic equilibrium. Our data demonstrate that phosphorylated tyrosine is a useful marker to monitor this metabolic condition.

Protective effect of exogenous coenzyme Q in rats subjected to partial hepatic ischemia and reperfusion.

In a surgical model of liver ischemia lipid peroxidation occurs, as shown by increase of lipid peroxidation end products, endogenous CoQ9 is oxidized and mitochondrial respiration is lowered; however, pre-treatment of the rats by i.p. injection of CoQ10 for 14 days normalizes the above parameters, presumably by way of the observed high extent of reduction of the incorporated quinone; moreover, liver homogenates of the CoQ10-treated rats are more resistant than those of non-treated rats to oxidative stress induced by an azido free radical initiator. This preliminary study suggests that CoQ10 pre-treatment can be of beneficial effect against oxidative damage during liver surgery transplantation.

Oxidative stress, antioxidant defences and aging.

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.

Coenzyme Q deficiency in mitochondria: kinetic saturation versus physical saturation.

The coenzyme Q (CoQ) concentration in the inner membrane of beef heart mitochondria is not kinetically saturating for NADH oxidation inasmuch as the K(m) of NADH oxidation for endogenous CoQ10 is in the mM range in membrane lipids. Using CoQ1 as an electron acceptor from complex I, we have found additional evidence that the high Km of NADH oxidase for CoQ is not an artifact due to the use of organic solvents in reconstitution studies. We have also obtained experimental evidence that CoQ concentration may be rendered more rate-limiting for NADH oxidation either by a decrease of CoQ content (as in liver regeneration or under an acute oxidative stress), or by a possible increase of the Km for CoQ, as in some mitochondrial diseases and ageing. The possibility of enhancing the rate of NADH oxidation by CoQ therapy is hindered by the fact that the CoQ concentration in mitochondria appears to be regulated by its mixability with the membrane phospholipids. Nevertheless CoQ10 incorporated into heart submitochondrial particles by sonication enhances NADH oxidation (but not succinate oxidation) up to twofold. Nontoxic CoQ homologs and analogs having shorter side-chains with respect to CoQ10 can be incorporated in the mitochondrial membrane without sonication, supporting an enhancement of NADH oxidation rate above 'physiological' values. It is worth investigating whether this approach can have a therapeutical value in vivo in mitochondrial bioenergetic disorders.