Helena M. Cochemé

Complex I Is the Major Site of Mitochondrial Superoxide Production by Paraquat

Paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. This superoxide production causes extensive mitochondrial oxidative damage, however, there is considerable uncertainty over the mitochondrial sites of paraquat reduction and superoxide formation. Here we show that in yeast and mammalian mitochondria, superoxide production by paraquat occurs in the mitochondrial matrix, as inferred from manganese superoxide dismutase-sensitive mitochondrial DNA damage, as well as from superoxide assays in isolated mitochondria, which were unaffected by exogenous superoxide dismutase. This paraquat-induced superoxide production in the mitochondrial matrix required a membrane potential that was essential for paraquat uptake into mitochondria. This uptake was of the paraquat dication, not the radical monocation, and was carrier-mediated. Experiments with disrupted mitochondria showed that once in the matrix paraquat was principally reduced by complex I (mammals) or by NADPH dehydrogenases (yeast) to form the paraquat radical cation that then reacted with oxygen to form superoxide. Together this membrane potential-dependent uptake across the mitochondrial inner membrane and the subsequent rapid reduction to the paraquat radical cation explain the toxicity of paraquat to mitochondria.

Metformin Retards Aging in C. elegans by Altering Microbial Folate and Methionine Metabolism

Summary The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy—and also to its side effects, which include folate deficiency and gastrointestinal upset. PaperClip

Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I

Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.

Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology.

Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.

Interactions of mitochondria-targeted and untargeted ubiquinones with the mitochondrial respiratory chain and reactive oxygen species : Implications for the use of exogenous ubiquinones as therapies and experimental tools

Antioxidants, such as ubiquinones, are widely used in mitochondrial studies as both potential therapies and useful research tools. However, the effects of exogenous ubiquinones can be difficult to interpret because they can also be pro-oxidants or electron carriers that facilitate respiration. Recently we developed a mitochondria-targeted ubiquinone (MitoQ10) that accumulates within mitochondria. MitoQ10 has been used to prevent mitochondrial oxidative damage and to infer the involvement of mitochondrial reactive oxygen species in signaling pathways. However, uncertainties remain about the mitochondrial reduction of MitoQ10, its oxidation by the respiratory chain, and its pro-oxidant potential. Therefore, we compared MitoQ analogs of varying alkyl chain lengths (MitoQn, n = 3–15) with untargeted exogenous ubiquinones. We found that MitoQ10 could not restore respiration in ubiquinone-deficient mitochondria because oxidation of MitoQ analogs by complex III was minimal. Complex II and glycerol 3-phosphate dehydrogenase reduced MitoQ analogs, and the rate depended on chain length. Because of its rapid reduction and negligible oxidation, MitoQ10 is a more effective antioxidant against lipid peroxidation, peroxynitrite and superoxide. Paradoxically, exogenous ubiquinols also autoxidize to generate superoxide, but this requires their deprotonation in the aqueous phase. Consequently, in the presence of phospholipid bilayers, the rate of autoxidation is proportional to ubiquinol hydrophilicity. Superoxide production by MitoQ10 was insufficient to damage aconitase but did lead to hydrogen peroxide production and nitric oxide consumption, both of which may affect cell signaling pathways. Our results comprehensively describe the interaction of exogenous ubiquinones with mitochondria and have implications for their rational design and use as therapies and as research tools to probe mitochondrial function.

Mitochondria-Targeted Antioxidant MitoQ10 Improves Endothelial Function and Attenuates Cardiac Hypertrophy

Mitochondria are a major site of reactive oxygen species production, which may contribute to the development of cardiovascular disease. Protecting mitochondria from oxidative damage should be an effective therapeutic strategy; however, conventional antioxidants are ineffective, because they cannot penetrate the mitochondria. This study investigated the role of mitochondrial oxidative stress during development of hypertension in the stroke-prone spontaneously hypertensive rat, using the mitochondria-targeted antioxidant, MitoQ10. Eight-week-old male stroke-prone spontaneously hypertensive rats were treated with MitoQ10 (500 &mgr;mol/L; n=16), control compound decyltriphenylphosphonium (decylTPP; 500 &mgr;mol/L; n=8), or vehicle (n=9) in drinking water for 8 weeks. Systolic blood pressure was significantly reduced by ≈25 mm Hg over the 8-week MitoQ10 treatment period compared with decylTPP (F=5.94; P=0.029) or untreated controls (F=65.6; P=0.0001). MitoQ10 treatment significantly improved thoracic aorta NO bioavailability (1.16±0.03 g/g; P=0.002, area under the curve) compared with both untreated controls (0.68±0.02 g/g) and decylTPP-treated rats (0.60±0.06 g/g). Cardiac hypertrophy was significantly reduced by MitoQ10 treatment compared with untreated control and decylTPP treatment (MitoQ10: 4.01±0.05 mg/g; control: 4.42±0.11 mg/g; and decylTPP: 4.40±0.09 mg/g; ANOVA P=0.002). Total MitoQ10 content was measured in liver, heart, carotid artery, and kidney harvested from MitoQ10-treated rats by liquid chromatography-tandem mass spectrometry. All of the organs analyzed demonstrated detectable levels of MitoQ10, with comparable accumulation in vascular and cardiac tissues. Administration of the mitochondria-targeted antioxidant MitoQ10 protects against the development of hypertension, improves endothelial function, and reduces cardiac hypertrophy in young stroke-prone spontaneously hypertensive rats. MitoQ10 provides a novel approach to attenuate mitochondrial-specific oxidative damage with the potential to become a new therapeutic intervention in human cardiovascular disease.

Measurement of H2O2 within Living Drosophila during Aging Using a Ratiometric Mass Spectrometry Probe Targeted to the Mitochondrial Matrix

Summary Hydrogen peroxide (H2O2) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H2O2 in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H2O2 levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H2O2 to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H2O2 that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H2O2 with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H2O2 correlates with aging, it may not be causative.

Consequences of long-term oral administration of the mitochondria-targeted antioxidant MitoQ to wild-type mice.

The mitochondria-targeted quinone MitoQ protects mitochondria in animal studies of pathologies in vivo and is being developed as a therapy for humans. However, it is unclear whether the protective action of MitoQ is entirely due to its antioxidant properties, because long-term MitoQ administration may alter whole-body metabolism and gene expression. To address this point, we administered high levels of MitoQ orally to wild-type C57BL/6 mice for up to 28 weeks and investigated the effects on whole-body physiology, metabolism, and gene expression, finding no measurable deleterious effects. In addition, because antioxidants can act as pro-oxidants under certain conditions in vitro, we examined the effects of MitoQ administration on markers of oxidative damage. There were no changes in the expression of mitochondrial or antioxidant genes as assessed by DNA microarray analysis. There were also no increases in oxidative damage to mitochondrial protein, DNA, or cardiolipin, and the activities of mitochondrial enzymes were unchanged. Therefore, MitoQ does not act as a pro-oxidant in vivo. These findings indicate that mitochondria-targeted antioxidants can be safely administered long-term to wild-type mice.

Mitochondria‐Targeted Antioxidants in the Treatment of Disease

Mitochondrial oxidative damage is thought to contribute to a wide range of human diseases; therefore, the development of approaches to decrease this damage may have therapeutic potential. Mitochondria‐targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death have been developed. These compounds contain antioxidant moieties, such as ubiquinone, tocopherol, or nitroxide, that are targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondrial membrane potential, the cations are accumulated within the mitochondria inside cells. There, the conjugated antioxidant moiety protects mitochondria from oxidative damage. Here, we outline some of the work done to date on these compounds and how they may be developed as therapies.

Oxidative stress and life histories: unresolved issues and current needs

Abstract Life‐history theory concerns the trade‐offs that mold the patterns of investment by animals between reproduction, growth, and survival. It is widely recognized that physiology plays a role in the mediation of life‐history trade‐offs, but the details remain obscure. As life‐history theory concerns aspects of investment in the soma that influence survival, understanding the physiological basis of life histories is related, but not identical, to understanding the process of aging. One idea from the field of aging that has gained considerable traction in the area of life histories is that life‐history trade‐offs may be mediated by free radical production and oxidative stress. We outline here developments in this field and summarize a number of important unresolved issues that may guide future research efforts. The issues are as follows. First, different tissues and macromolecular targets of oxidative stress respond differently during reproduction. The functional significance of these changes, however, remains uncertain. Consequently there is a need for studies that link oxidative stress measurements to functional outcomes, such as survival. Second, measurements of oxidative stress are often highly invasive or terminal. Terminal studies of oxidative stress in wild animals, where detailed life‐history information is available, cannot generally be performed without compromising the aims of the studies that generated the life‐history data. There is a need therefore for novel non‐invasive measurements of multi‐tissue oxidative stress. Third, laboratory studies provide unrivaled opportunities for experimental manipulation but may fail to expose the physiology underpinning life‐history effects, because of the benign laboratory environment. Fourth, the idea that oxidative stress might underlie life‐history trade‐offs does not make specific enough predictions that are amenable to testing. Moreover, there is a paucity of good alternative theoretical models on which contrasting predictions might be based. Fifth, there is an enormous diversity of life‐history variation to test the idea that oxidative stress may be a key mediator. So far we have only scratched the surface. Broadening the scope may reveal new strategies linked to the processes of oxidative damage and repair. Finally, understanding the trade‐offs in life histories and understanding the process of aging are related but not identical questions. Scientists inhabiting these two spheres of activity seldom collide, yet they have much to learn from each other.