Ana S.H. Costa

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS

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). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. 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.

Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications

Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m5dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N6-methyldeoxyadenosine (m6dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m6dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.

Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef

Summary Critical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism.

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Mitochondrial Protein Lipoylation and the 2-Oxoglutarate Dehydrogenase Complex Controls HIF1α Stability in Aerobic Conditions

Summary Hypoxia-inducible transcription factors (HIFs) control adaptation to low oxygen environments by activating genes involved in metabolism, angiogenesis, and redox homeostasis. The finding that HIFs are also regulated by small molecule metabolites highlights the need to understand the complexity of their cellular regulation. Here we use a forward genetic screen in near-haploid human cells to identify genes that stabilize HIFs under aerobic conditions. We identify two mitochondrial genes, oxoglutarate dehydrogenase (OGDH) and lipoic acid synthase (LIAS), which when mutated stabilize HIF1α in a non-hydroxylated form. Disruption of OGDH complex activity in OGDH or LIAS mutants promotes L-2-hydroxyglutarate formation, which inhibits the activity of the HIFα prolyl hydroxylases (PHDs) and TET 2-oxoglutarate dependent dioxygenases. We also find that PHD activity is decreased in patients with homozygous germline mutations in lipoic acid synthesis, leading to HIF1 activation. Thus, mutations affecting OGDHC activity may have broad implications for epigenetic regulation and tumorigenesis.

Extracellular vesicles are independent metabolic units with asparaginase activity.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. While it has been shown that cells can traffic metabolic enzymes via EVs much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Both our metabolomics and functional analyses revealed that EVs harbour L-asparaginase activity catalysed by the enzyme Asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC-derived EVs traffic ASRGL1. Our results demonstrate for the first time that NSC EVs function as independent, extracellular metabolic units able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Mammalian Circadian Period, But Not Phase and Amplitude, Is Robust Against Redox and Metabolic Perturbations

M.P. was supported by the Dutch Cancer Foundation (KWF, BUIT-2014-6637) and EMBO (ALTF-654-2014). J.S.O. was supported by the Medical Research Council (MC_UP_1201/4) and the Wellcome Trust (093734/Z/10/Z).

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation

Summary Neural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells

&NA; Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3&bgr; (GSK3&bgr;) at mitochondria‐endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK‐I) to the voltage‐dependent anion channel (VDAC) on mitochondria. Binding of HK‐I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon‐&ggr; (IFN‐&ggr;) in memory T cells. Subcellular organization of mTORC2‐AKT‐GSK3&bgr; at mitochondria‐ER contact sites, promoting HK‐I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function. Graphical Abstract Figure. No caption available. HighlightsmTORC2, AKT, and GSK3&bgr; are present at mitochondria‐ER contact sites of CD8+ T cellsmTORC2‐activated AKT inhibits GSK3&bgr; in nascent activated memory CD8+ T cellsGSK3&bgr; inhibition enables binding of HK‐I to VDAC, promoting pyruvate oxidationPyruvate oxidation is required for rapid generation of IFN‐&ggr; in memory T cells &NA; How glucose metabolism enables rapid acquisition of effector function in memory CD8+ T cells remains poorly understood. Bantug et al. demonstrate that mitochondria‐endoplasmic reticulum contact sites are signaling hubs that enable the metabolic reprogramming required for rapid CD8+ T cell recall responses.

Metabolomic Profiling in Acute ST‐Segment–Elevation Myocardial Infarction Identifies Succinate as an Early Marker of Human Ischemia–Reperfusion Injury

Background Ischemia–reperfusion injury following ST‐segment–elevation myocardial infarction (STEMI) is a leading determinant of clinical outcome. In experimental models of myocardial ischemia, succinate accumulation leading to mitochondrial dysfunction is a major cause of ischemia–reperfusion injury; however, the potential importance and specificity of myocardial succinate accumulation in human STEMI is unknown. We sought to identify the metabolites released from the heart in patients undergoing primary percutaneous coronary intervention for emergency treatment of STEMI. Methods and Results Blood samples were obtained from the coronary artery, coronary sinus, and peripheral vein in patients undergoing primary percutaneous coronary intervention for acute STEMI and in control patients undergoing nonemergency coronary angiography or percutaneous coronary intervention for stable angina or non‐STEMI. Plasma metabolites were analyzed by targeted liquid chromatography and mass spectrometry. Metabolite levels for coronary artery, coronary sinus, and peripheral vein were compared to derive cardiac and systemic release ratios. In STEMI patients, cardiac magnetic resonance imaging was performed 2 days and 6 months after primary percutaneous coronary intervention to quantify acute myocardial edema and final infarct size, respectively. In total, 115 patients undergoing acute STEMI and 26 control patients were included. Succinate was the only metabolite significantly increased in coronary sinus blood compared with venous blood in STEMI patients, indicating cardiac release of succinate. STEMI patients had higher succinate concentrations in arterial, coronary sinus, and peripheral venous blood than patients with non‐STEMI or stable angina. Furthermore, cardiac succinate release in STEMI correlated with the extent of acute myocardial injury, quantified by cardiac magnetic resonance imaging. Conclusion Succinate release by the myocardium correlates with the extent of ischemia.