Gabriella Formiggini

Role of Mitochondria in Oxidative Stress and Aging

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production and found by combined use of specific inhibitors of Complex I that the one‐electron donor in the Complex to oxygen is a redox center located prior to the sites where three different types of coenzyme Q (CoQ) competitors bind, to be identified with an Fe‐S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short‐chain coenzyme Q analogues enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analogue idebenone is particularly effective, raising doubts about its safety as a drug. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by ROS as the primary cause of energy decline; in rat liver mitochondria, Complex I appears to be most affected by aging and to become strongly rate limiting for electron transfer. Mitochondrial energetics is also deranged in human platelets upon aging, as demonstrated by the decreased Pasteur effect (enhancement of lactate production by respiratory inhibitors). Cells counteract oxidative stress by antioxidants: CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT‐diaphorase are two such systems: likewise, they are overexpressed under oxidative stress conditions.

The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron–sulfur cluster N2

The mitochondrial respiratory chain is a powerful source of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles and found, by combined use of specific inhibitors of Complex I and by Coenzyme Q (CoQ) extraction from the particles, that the one‐electron donor in the Complex to oxygen is a redox center located prior to the binding sites of three different types of CoQ antagonists, to be identified with a Fe–S cluster, most probably N2 on the basis of several known properties of this cluster. Short chain CoQ analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective in promoting superoxide formation.

The Mitochondrial Production of Reactive Oxygen Species in Relation to Aging and Pathology

Abstract: Mitochondria are known to be strong producers of reactive oxygen species (ROS) and, at the same time, particularly susceptible to the oxidative damage produced by their action on lipids, proteins, and DNA. In particular, damage to mtDNA induces alterations to the polypeptides encoded by mtDNA in the respiratory complexes, with consequent decrease of electron transfer, leading to further production of ROS and thus establishing a vicious circle of oxidative stress and energetic decline. This deficiency in mitochondrial energetic capacity is considered the cause of aging and age‐related degenerative diseases. Complex I would be the enzyme most affected by ROS, since it contains seven of the 13 subunits encoded by mtDNA. Accordingly, we found that complex I activity is significantly affected by aging in rat brain and liver mitochondria as well as in human platelets. Moreover, due to its rate control over aerobic respiration, such alterations are reflected on the entire oxidative phosphorylation system. We also investigated the role of mitochondrial complex I in superoxide production and found that the one‐electron donor to oxygen is most probably the Fe‐S cluster N2. Short chain coenzyme Q (CoQ) analogues enhance ROS formation, presumably by mediating electron transfer from N2 to oxygen, both in bovine heart SMP and in cultured HL60 cells. Nevertheless, we have accumulated much evidence of the antioxidant role of reduced CoQ10 in several cellular systems and demonstrated the importance of DT‐diaphorase and other internal cellular reductases to reduce exogenous CoQ10 after incorporation.

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.

Mitochondrial production of oxygen radical species and the role of Coenzyme Q as an antioxidant.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), which is considered as the pathogenic agent of many diseases and of aging. We have investigated the role of complex I in superoxide radical production and found by the combined use of specific inhibitors of complex I that the one-electron donor to oxygen in the complex is a redox center located prior to the sites where three different types of Coenzyme Q (CoQ) competitors bind, to be identified with an Fe–S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain Coenzyme Q analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective, raising doubts on its safety as a drug. Cells counteract oxidative stress by antioxidants. CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems, likewise, they are overexpressed under oxidative stress conditions.

Control of oxidative phosphorylation by Complex I in rat liver mitochondria: implications for aging.

We compared NAD-dependent state 4 and state 3 respiration, NADH oxidation and Complex I specific activity in liver mitochondria from 4- and 30-month-old rats. All the activities examined were significantly decreased with aging. In both groups of animals, the flux control coefficients measured by rotenone titration indicated that Complex I is largely rate controlling upon NADH aerobic oxidation while, in state 3 respiration, it shares the control with other steps in the pathway. Moreover, we observed a trend wherein flux control coefficients of Complex I became higher with age. This indication was strengthened by examining the rotenone inhibition thresholds showing that Complex I becomes more rate controlling, over all the examined activities, during aging. Our results point out that age-related alterations of the mitochondrial functions are also present in tissues considered less prone to accumulate mitochondrial DNA mutations.

Structural and functional aspects of the respiratory chain of synaptic and nonsynaptic mitochondria derived from selected brain regions

Studies on brain mitochondria are complicated by the regional, cellular, and subcellular heterogeneity of the central nervous system. This study was performed using synaptic and nonsynaptic mitochondria obtained from cortex, hippocampus, and striatum of male Sprague-Dawley rats (3 months old). Ubiquinone content, detected by HPLC analysis, was about 1.5 nmol/mg protein with an approximate CoQ9/CoQ10 molecular ratio of 2:1. The activities of several respiratory chain complexes were also studied (succinate-cyt.c reductase, NADH-cyt.c reductase, succinate-DCIP, ubiquinol2-cyt.c reductase, and cytochrome oxidase), and generally found to be higher in mitochondria from cortex than from other regions. Study of the activities of some of these enzymes vs. 1/T (Arrhenius plots) showed a straight line with an activation energy between 7 and 10 kcal/mol in all the three areas considered. Only CoQ2H2-cyt.c reductase activity revealed a biphasic temperature dependence. Also anisotropy (as fluorescence polarization) of the hydrophobic probe DPH showed a deviation from linearity; the break points for both enzymatic activity and anisotropy were found at about 23–24°C.

Studies on the role of ubiquinone in the control of the mitochondrial respiratory chain.

This study examines the possible role of Coenzyme Q (CoQ, ubiquinone) in the control of mitochondrial electron transfer. The CoQ concentration in mitochondria from different tissues was investigated by HPLC. By analyzing the rates of electron transfer as a function of total CoQ concentration, it was calculated that, at physiological CoQ concentration NADH cytochrome c reductase activity is not saturated. Values for theoretical Vmax could not be reached experimentally for NADH oxidation, because of the limited miscibility of CoQ10 with the phospholipids. On the other hand, it was found that CoQ3 could stimulate alpha-glycerophosphate cytochrome c reductase over three-fold. Electron transfer being a diffusion-coupled process, we have investigated the possibility of its being subjected to diffusion control. A reconstruction study of Complex I and Complex III in liposomes showed that NADH cytochrome c reductase was not affected by changing the average distance between complexes by varying the protein: lipid ratios. The results of a broad investigation on ubiquinol cytochrome c reductase in bovine heart submitochondrial particles indicated that the enzymic rate is not diffusion-controlled by ubiquinol, whereas the interaction of cytochrome c with the enzyme is clearly diffusion-limited.

Inhibitor sensitivity of respiratory complex I in human platelets: A possible biomarker of ageing

NADH‐Coenzyme Q reductase was assayed in platelet mitochondrial membranes obtained from 19 pools of two venous blood samples from female young (19–30 years) individuals and 18 pools from aged ones (66–107 years). The enzyme activities were not significantly changed in the two groups, but a decrease of sensitivity to the specific inhibitor, rotenone, occurred in a substantial number of aged individuals. The results are in agreement with the predictions of the mitochondrial theory of ageing and may be used to develop a sensitive biomarker of the ageing process.

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.