Milena Merlo Pich

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 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 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.

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 DNA in platelets from aged subjects

This study aimed to assess platelets as a possible model for screening the accumulation of mitochondrial DNA mutations, particularly during normal ageing. For this purpose we isolated platelets from young and old donors selected by lack of systemic and haematological diseases. We studied the accumulation of a particular deletion (4977-bp deletion) that usually accumulates in an age-related manner in different post-mitotic tissues, such as brain, heart and skeletal muscle, and in some non-post-mitotic tissues (skin, liver). Using different primers, we failed to detect this particular species of deletion in platelets both from young and old individuals. However, we cannot exclude the presence of other species of deletions or point mutations affecting the mitochondrial DNA in platelets during the aging process.

Increased transcription of mitochondrial genes for Complex I in human platelets during ageing.

We studied the effect of ageing on the mRNA levels of mitochondria‐encoded polypeptides in human platelets. We used quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) to investigate the expression of selected cytochrome c oxidase (COX) genes (subunits I and III) and Complex I genes (subunits reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND)1 and ND5) in platelets from young and aged healthy subjects. Northern blot analysis confirmed the PCR results. COX I expression is higher than that of COX III in both young and aged platelets. A significant increase of transcripts for Complex I was found during ageing. On the contrary, the mRNA levels of the two COX subunits did not significantly vary during ageing.

Ubiquinol and a Coenzyme Q reducing system protect platelet mitochondrial function of transfusional buffy coats from oxidative stress

The conditions under which Coenzyme Q (CoQ) may protect platelet mitochondrial function of transfusional buffy coats from aging and from induced oxidative stress were investigated. The Pasteur effect, i.e. the enhancement of lactate production after inhibition of mitochondrial respiratory chain, was exploited as a marker of mitochondrial function as it allows to calculate the ratio of mitochondrial ATP to glycolytic ATP. Reduced CoQ 10 improves platelet mitochondrial function of transfusional buffy coats and protects the cells from induced oxidative stress. Oxidized CoQ is usually less effective, despite the presence, shown for the first time in this study, of quinone reductase activities in the platelet plasma membranes. The addition of a CoQ reducing system to platelets is effective in enhancing the protection of platelet mitochondrial function from the oxidative stress. The results support on one hand a possibility of protection of mitochondrial function in aging by exogenous CoQ intake, on the other a possible application in protection of transfusional buffy coats from storage conditions and oxidative deterioration.

Modes of coenzyme Q function in electron transfer

SummaryIn the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.

MITOCHONDRIAL DYSFUNCTION AND BRAIN DISORDERS

Summary According to the mitochondrial theory of aging, it is expected that in postmitotic tissues, such as nervous system, an accumulation of damage to mitochondrial DNA could lead to a progressive failure of mitochondrial function during senescence. NADH-Coenzyme Q reductase (Complex I) activity should be severely affected since 7 subunits of this enzymatic complex are encoded by mitochondrial DNA. We decided to investigate the modifications occurring to this particular complex during aging in two different neuronal populations from aged rats, using rotenone sensitivity as a functional parameter of the subunits encoded by mitochondrial DNA. In order to have an easy model to study mitochondrial modifications during senescence and in other neurodegenerative disorders, we also investigated mitochondrial membranes from platelets of young and old individuals. In all systems investigated, a decease of rotenone sensitivity in aging was observed.

Alterations of Electron Transfer and Energy Conservation in Mitochondrial Complex I (Nadh-Coenzyme Q Oxido-Reductase) in Aging

The aging process is characteristically associated with a progressive decline in physiological functions. This is likely related to a failure of mitochondrial energy production as a consequence of impairment of the mitochondria) oxidative phosphorylation system.