Ischaemia-reperfusion (IR) injury occurs when 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 generation of mitochondrial reactive oxygen species (ROS)1-5. Although mitochondrial ROS production in IR is established, it has generally been considered a non-specific response to reperfusion1,3. Here, we developed a comparative in vivo metabolomic analysis and unexpectedly identified widely conserved metabolic pathways responsible for mitochondrial ROS production during IR. We showed that selective accumulation of the citric acid cycle (CAC) 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 (SDH), which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. Upon reperfusion, the accumulated succinate is rapidly re-oxidised by SDH, driving extensive ROS generation by reverse electron transport (RET) at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo IR 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 IR injury. Furthermore, these findings reveal a novel pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation upon subsequent reperfusion is a potential therapeutic target to decrease IR injury in a range of pathologies.

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.

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 unclear1–6. 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.

Human mitochondrial DNA (mtDNA) shows extensive within population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic,metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation,insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

SummaryIncreased production of reactive oxygen species (ROS) has long been considered a cause of aging. However, recent studies have implicated ROS as essential secondary messengers. Here we show that the site of ROS production significantly contributes to their apparent dual nature. We report that ROS increase with age as mitochondrial function deteriorates. However, we also demonstrate that increasing ROS production specifically through respiratory complex I reverse electron transport extends Drosophila lifespan. Reverse electron transport rescued pathogenesis induced by severe oxidative stress, highlighting the importance of the site of ROS production in signaling. Furthermore, preventing ubiquinone reduction, through knockdown of PINK1, shortens lifespan and accelerates aging; phenotypes that are rescued by increasing reverse electron transport. These results illustrate that the source of a ROS signal is vital in determining its effects on cellular physiology and establish that manipulation of ubiquinone redox state is a valid strategy to delay aging.

SummaryHydrogen 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.

Neurons depend on oxidative phosphorylation for energy generation, whereas astrocytes do not, a distinctive feature that is essential for neurotransmission and neuronal survival. However, any link between these metabolic differences and the structural organization of the mitochondrial respiratory chain is unknown. Here, we investigated this issue and found that, in neurons, mitochondrial complex I is predominantly assembled into supercomplexes, whereas in astrocytes the abundance of free complex I is higher. The presence of free complex I in astrocytes correlates with the severalfold higher reactive oxygen species (ROS) production by astrocytes compared with neurons. Using a complexomics approach, we found that the complex I subunit NDUFS1 was more abundant in neurons than in astrocytes. Interestingly, NDUFS1 knockdown in neurons decreased the association of complex I into supercomplexes, leading to impaired oxygen consumption and increased mitochondrial ROS. Conversely, overexpression of NDUFS1 in astrocytes promoted complex I incorporation into supercomplexes, decreasing ROS. Thus, complex I assembly into supercomplexes regulates ROS production and may contribute to the bioenergetic differences between neurons and astrocytes.redox | brain | bioenergetics | lactate | glycolysis T he brain is a metabolically demanding organ (1) that requires tight cooperation between neurons and astrocytes (2). Astrocytes provide crucial metabolic and structural support (3, 4) and are key players in neurotransmission (5-7) and behavior (8). The status of many major redox couples in the brain is also regulated by astrocytes (9), through their high content of antioxidant compounds and enzymes (10) and by the constitutive stabilization of the master antioxidant transcriptional activator, nuclear factor erythroid 2-related factor 2 (Nrf2) (11). Thus, astrocytes are equipped to protect themselves when exposed to excess reactive oxygen species (ROS) (12) and reactive nitrogen species (13,14). Moreover, astrocytes also provide nearby neurons with protective antioxidant precursors through a cell-signaling mechanism involving glutamate receptor activation by neurotransmission (11,15,16). The tight coupling between astrocytes and neurons therefore helps in energy and redox metabolism during normal brain function.Intriguingly, the ATP used by neurons is supplied by oxidative phosphorylation, whereas most energy needs of astrocytes are met by glycolysis (17). In fact, the survival of neurons requires oxidative phosphorylation (18,19). The different energy metabolisms of the two cell types are closely coupled, with astrocytes releasing the glycolytic end product, lactate, which is used by neighboring neurons to drive oxidative phosphorylation (20)(21)(22). As the molecular mechanisms underlying the markedly different modes of ATP production in the two cell types are not understood, we investigated whether the organization of the mitochondrial respiratory chain in brain cells could contribute. Here, we report that the extent of supercomplex f...

Rationale: DNA damage is present in both genomic and mitochondrial DNA in atherosclerosis. However, whether DNA damage itself promotes atherosclerosis, or is simply a byproduct of the risk factors that promote atherosclerosis, is unknown. Objective:To examine the effect of DNA damage on atherosclerosis, we studied apolipoprotein (Apo)E ؊/؊ mice that were haploinsufficient for the protein kinase ATM (ataxia telangiectasia mutated), which coordinates DNA repair. Methods and Results: ATM