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Although the development of mitochondrial therapies has largely focused on diseases caused by mutations in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins, it has been found that mitochondrial dysfunction also contributes to the pathology of many common disorders, including neurodegeneration, metabolic disease, heart failure, ischaemia-reperfusion injury and protozoal infections. Mitochondria therefore represent an important drug target for these highly prevalent diseases. Several strategies aimed at therapeutically restoring mitochondrial function are emerging, and a small number of agents have entered clinical trials. This Review discusses the opportunities and challenges faced for the further development of mitochondrial pharmacology for common pathologies.

H 2 S reversibly modifies low molecular weight (L MW SH) and protein (PrSH) thiols to form persulfides (RSS − ) and polysulfides (RS(S) n S − ) for antioxidant defence and regulation of activity. However, our understanding of the biological significance of these processes is hampered by our inability to quantify these modifications. We develop a sensitive LC-MS/MS procedure that traps the sulfur atom of H 2 S, and the terminal sulfur atom of RSS − and RS(S) n S − as diagnostic products in biological samples. In parallel, we also trap internal S atoms of RS(S) n S − , enabling quantification of H 2 S, RSS − and RS(S) n S − . L MW S(S) n S − and PrS(S) n S − are determined simultaneously in the same sample. Glutathione (GSH) is the most abundant L MW SH so we develop an orthogonal approach to quantify GSS − , enabling corroboration of L MW SS − measurements by sulfur atom trapping. We demonstrate in systems from proteins to ex vivo tissues how these approaches enable exploration of persulfidation in biological systems. Hydrogen sulfide (H 2 S) reversibly modifies low molecular weight and protein thiols to form persulfides (RSS − ) and polysulfides (RS(S)nS − ) for antioxidant defence and regulation of activity. Here, the authors report a sensitive LC-MS/MS procedure that separately traps and quantifies the sulfur atom of H 2 S, the terminal sulfur atom of RSS and RS(S)nS-, and the internal sulfur atoms of RS(S)nS − as diagnostic products in biological samples.

Iron is critical for life but toxic in excess because of iron-catalysed formation of pro-oxidants that cause tissue damage in a range of disorders. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) orchestrates cell-intrinsic protective antioxidant responses, while the peptide hormone hepcidin maintains systemic iron homoeostasis, but is pathophysiologically decreased in haemochromatosis and β-thalassaemia. Here, we show that Nrf2 is activated by iron-induced, mitochondria-derived pro-oxidants and drives bone morphogenetic protein 6 (Bmp6) expression in liver sinusoidal endothelial cells, which in turn increases hepcidin synthesis by neighbouring hepatocytes. In Nrf2 knockout mice, the Bmp6–hepcidin response to oral and parenteral iron is impaired, and iron accumulation and hepatic damage are increased. Pharmacological activation of Nrf2 stimulates the Bmp6–hepcidin axis, improving iron homoeostasis in haemochromatosis and counteracting the inhibition of Bmp6 by erythroferrone in β-thalassaemia. We propose that Nrf2 links cellular sensing of excess toxic iron to the control of systemic iron homoeostasis and antioxidant responses, and may be a therapeutic target for iron-associated disorders. Iron homoeostasis is tightly orchestrated to avoid toxic iron overload. Here Lim and colleagues show that iron excess activates Nrf2 via mitochondrial reactive oxygen species, enhancing the expression of Bmp6 in liver sinusoidal endothelial cells, which in turn promotes hepcidin expression by hepatocytes, decreasing systemic iron levels.

The role of hydrogen peroxide (H 2 O 2 ) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H 2 O 2 in vivo . Here we describe a method for assessing changes in H 2 O 2 within the mitochondrial matrix of living Drosophila . We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H 2 O 2 to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H 2 O 2 . To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography–tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H 2 O 2 in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H 2 O 2 production in mouse and cell culture studies.

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.

In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred indirectly due to the limitations of estimating ROS from in vitro methods. However, measuring ROS (hydrogen peroxide, H 2 O 2 ) content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H 2 O 2 levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H 2 O 2 levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings.

Drugs that modulate fundamental mechanisms of ageing offer the promise of substantially improving the health of ageing populations, but innovative approaches to identify and evaluate such ‘gerotherapeutics’ are needed.Drugs that modulate fundamental mechanisms of ageing offer the promise of substantially improving the health of ageing populations, but innovative approaches to identify and evaluate such ‘gerotherapeutics’ are needed.

A metabolomics study on the ischaemic heart identifies succinate as a metabolite that drives the production of reactive oxygen species and contributes to ischaemia-reperfusion injury; pharmacological inhibition of succinate accumulation ameliorates ischaemia-reperfusion injury in a mouse model of heart attack and a rat model of stroke. Succinate a heart breaker In this metabolomics study of the ischaemic heart, Michael Murphy and colleagues identify a metabolite that drives the production of reactive oxygen species and contributes to ischaemia reperfusion injury. They show that succinate is a conserved metabolic signature of ischaemia in several tissues. Succinate accumulates during ischaemia due to a reversal of the enzyme succinate dehydrogenase. Upon reperfusion the accumulated succinate is rapidly oxidized and drives reactive oxygen species production through reverse electron transport at mitochondrial complex I. Pharmacological blockade of succinate accumulation ameliorates ischaemia reperfusion injury in mouse models of heart attack and stroke. Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS) 1 , 2 , 3 , 4 , 5 . Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion 1 , 3 . Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo , while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Nitric oxide donors protect from myocardial ischemia-reperfusion injury, but the underlying mechanisms have been unclear. Edward T Chouchani et al . uncover the molecular target of such donors, a cysteine residue in a subunit of complex I of the mitochondrial respiratory chain, and suggest that this cysteine residue has a general role in regulating complex I activity and modulating ischemia-reperfusion injury. 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 1 , 2 , 3 , 4 , 5 , 6 . 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.

Summary Many animals experience periods of food shortage in their natural environment. It has been hypothesised that the metabolic responses of animals to naturally‐occurring periods of food deprivation may have long‐term negative impacts on their subsequent life‐history. In particular, reductions in energy requirements in response to fasting may help preserve limited resources but potentially come at a cost of increased oxidative stress. However, little is known about this trade‐off since studies of energy metabolism are generally conducted separately from those of oxidative stress. Using a novel approach that combines measurements of mitochondrial function with in vivo levels of hydrogen peroxide (H2O2) in brown trout (Salmo trutta), we show here that fasting induces energy savings in a highly metabolically active organ (the liver) but at the cost of a significant increase in H2O2, an important form of reactive oxygen species (ROS). After a 2‐week period of fasting, brown trout reduced their whole‐liver mitochondrial respiratory capacities (state 3, state 4 and cytochrome c oxidase activity), mainly due to reductions in liver size (and hence the total mitochondrial content). This was compensated for at the level of the mitochondrion, with an increase in state 3 respiration combined with a decrease in state 4 respiration, suggesting a selective increase in the capacity to produce ATP without a concomitant increase in energy dissipated through proton leakage. However, the reduction in total hepatic metabolic capacity in fasted fish was associated with an almost two‐fold increase in in vivo mitochondrial H2O2 levels (as measured by the MitoB probe). The resulting increase in mitochondrial ROS, and hence potential risk of oxidative damage, provides mechanistic insight into the trade‐off between the short‐term energetic benefits of reducing metabolism in response to fasting and the potential long‐term costs to subsequent life‐history traits. Foreign Language Résumé Les restrictions alimentaires sont courantes dans le milieu naturel et peuvent impacter de nombreux animaux. Il a été émis l'hypothèse que les animaux, face à ces épisodes de restriction alimentaire, mettaient en place des réponses métaboliques pouvant affecter leurs histoires de vie future. En particulier, si une diminution des besoins énergétiques lors du jeûne peut contribuer à préserver les réserves de l'animal cela peut néanmoins entraîner une augmentation du stress oxydant. Ce type de compromis n'a toutefois pas encore été démontré car l'étude du métabolisme énergétique est généralement réalisée séparément de celle du stress oxydant. Par une nouvelle approche combinant des mesures du fonctionnement mitochondrial et des niveaux in vivo de peroxyde d'hydrogène (H2O2) chez la truite commune (Salmo trutta), nous montrons ici que le jeûne entraîne une économie d'énergie dans un tissu métaboliquement très actif tel que le foie, mais au coût d'une augmentation significative en H2O2, une forme majeure des espèces réactives de l'oxygène. Après deux semaines de jeûne, les truites communes ont réduit leurs capacités respiratoires mitochondriales (état 3, état 4 et l'activité de la cytochrome c oxydase) principalement du fait d'une réduction de la taille du foie (et donc du nombre total de mitochondries). Une compensation a été observée au niveau de la mitochondrie. Cela se traduit par une augmentation de la respiration en état 3 et une diminution concomitante de celle en état 4, suggérant une augmentation sélective des capacités de production de l'ATP sans augmentation parallèle de l'énergie dissipée par la fuite de protons. La diminution des capacités métaboliques du foie chez les poissons à jeun était associée in vivo à des niveaux quasiment doubles de H2O2 mitochondriaux (mesurés par la sonde MitoB). Cette augmentation en espèces réactives de l'oxygène dans les mitochondries, avec son risque inhérent de dommages oxydatifs, apporte une vision mécanistique du compromis entre les bénéfices énergétiques à court terme d'une réduction métabolique en réponse au jeûne et les possibles coûts à long terme sur leurs traits histoires de vie futurs. A plain language summary is available for this article. Plain Language Summary