Carla Bovina

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.

Coenzyme Q content in synaptic and non-synaptic mitochondria from different brain regions in the ageing rat.

We investigated the Coenzyme Q (CoQ) content of different mitochondrial fractions [free mitochondria (FM), synaptic heavy (HM) and light mitochondria (LM)] from three brain areas (cortex, striatum, hippocampus) of rats at different ages. In rats from 2 to 26 months of age, we observed only small differences in total CoQ content (CoQ9 + CoQ10). In FM and LM fractions, values are very similar and appear to be much higher than in HM fractions. The CoQ10/CoQ9 ratios are much higher in brain mitochondria than in other organs, suggesting possible modifications of CoQ biosynthetic pathways in brain; nevertheless they appear to remain constant during ageing. CoQ9 and CoQ10 contents slowly decrease reaching their minimum in rats of 18 months of age, then increase in the older ages. Considering ageing as partially driven by a summation of free radical-mediated processes, we can hypothesize that damage occurring to biological structures in the first half of life might be followed by induction phenomena tending to re-establish the primitive levels of antioxidant molecules.

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.

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats

We investigated NADH oxidation in non‐synaptic and synaptic mitochondria from brain cortex of 4‐ and 24‐month‐old rats. The NADH oxidase activity was significantly lower in non‐synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone‐binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.