44 results found
Lennicke C, Cochemé HM, 2021, Redox metabolism: ROS as specific molecular regulators of cell signaling and function, Molecular Cell, Vol: 81, Pages: 3691-3707, ISSN: 1097-2765
Lennicke C, Cocheme H, 2021, Redox regulation of the insulin signalling pathway, Redox Biology, Vol: 42, ISSN: 2213-2317
The peptide hormone insulin is a key regulator of energy metabolism, proliferation and survival. Binding of insulin to its receptor activates the PI3K/AKT signalling pathway, which mediates fundamental cellular responses. Oxidants, in particular H2O2, have been recognised as insulin-mimetics. Treatment of cells with insulin leads to increased intracellular H2O2 levels affecting the activity of downstream signalling components, thereby amplifying insulin-mediated signal transduction. Specific molecular targets of insulin-stimulated H2O2 include phosphatases and kinases, whose activity can be altered via redox modifications of critical cysteine residues. Over the past decades, several of these redox-sensitive cysteines have been identified and their impact on insulin signalling evaluated. The aim of this review is to summarise the current knowledge on the redox regulation of the insulin signalling pathway.
Lennicke C, dos Santos E, Cocheme HM, 2020, Sugar-induced dysregulation of purine metabolism impacts lifespan, Aging-US, Vol: 12, Pages: 24479-24480, ISSN: 1945-4589
Bjedov I, Cocheme HM, Foley A, et al., 2020, Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila, PLoS Genetics, Vol: 16, Pages: 1-33, ISSN: 1553-7390
Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention.
van Dam E, van Leeuwen LAG, Dos Santos E, et al., 2020, Sugar-induced obesity and insulin resistance are uncoupled from shortened survival in Drosophila, Cell Metabolism, Vol: 31, Pages: 710-725, ISSN: 1550-4131
High-sugar diets cause thirst, obesity, and metabolic dysregulation, leading to diseases including type 2 diabetes and shortened lifespan. However, the impact of obesity and water imbalance on health and survival is complex and difficult to disentangle. Here, we show that high sugar induces dehydration in adult Drosophila, and water supplementation fully rescues their lifespan. Conversely, the metabolic defects are water-independent, showing uncoupling between sugar-induced obesity and insulin resistance with reduced survival in vivo. High-sugar diets promote accumulation of uric acid, an end-product of purine catabolism, and the formation of renal stones, a process aggravated by dehydration and physiological acidification. Importantly, regulating uric acid production impacts on lifespan in a water-dependent manner. Furthermore, metabolomics analysis in a human cohort reveals that dietary sugar intake strongly predicts circulating purine levels. Our model explains the pathophysiology of high-sugar diets independently of obesity and insulin resistance and highlights purine metabolism as a pro-longevity target.
Lennicke C, Cochemé HM, 2020, Redox signalling and ageing: insights from Drosophila, Biochemical Society Transactions, Vol: 48, Pages: 367-377, ISSN: 0300-5127
Ageing and age-related diseases are major challenges for the social, economic and healthcare systems of our society. Amongst many theories, reactive oxygen species (ROS) have been implicated as a driver of the ageing process. As by-products of aerobic metabolism, ROS are able to randomly oxidise macromolecules, causing intracellular damage that accumulates over time and ultimately leads to dysfunction and cell death. However, the genetic overexpression of enzymes involved in the detoxification of ROS or treatment with antioxidants did not generally extend lifespan, prompting a re-evaluation of the causal role for ROS in ageing. More recently, ROS have emerged as key players in normal cellular signalling by oxidising redox-sensitive cysteine residues within proteins. Therefore, while high levels of ROS may be harmful and induce oxidative stress, low levels of ROS may actually be beneficial as mediators of redox signalling. In this context, enhancing ROS production in model organisms can extend lifespan, with biological effects dependent on the site, levels, and specific species of ROS. In this review, we examine the role of ROS in ageing, with a particular focus on the importance of the fruit fly Drosophila as a powerful model system to study redox processes in vivo.
Pryor R, Norvaisas P, Marinos G, et al., 2019, Host-microbe-drug-nutrient screen identifies bacterial effectors of metformin therapy, Cell, Vol: 178, Pages: 1299-1312.e29, ISSN: 0092-8674
Metformin is the first-line therapy for treating type-2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, impact the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signalling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modelling of the microbiota in metformin-treated type-2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies
Augustin H, McGourty K, Steinert JR, et al., 2017, Myostatin-like proteins regulate synaptic function and neuronal morphology, Development, Vol: 144, Pages: 2445-2455, ISSN: 0950-1991
Growth factors of the TGFβ superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. Here, we show that Myoglianin (MYO), the Drosophila homolog of myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. We also show that MYO acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth.
van Leeuwen LAG, Hinchy EC, Murphy MP, et al., 2017, Click-PEGylation - a mobility shift approach to assess the redox state of cysteines in candidate proteins, Free Radical Biology & Medicine, Vol: 108, Pages: 374-382, ISSN: 1873-4596
The redox state of cysteine thiols is critical for protein function. Whereas cysteines play an important role in the maintenance of protein structure through the formation of internal disulfides, their nucleophilic thiol groups can become oxidatively modified in response to diverse redox challenges and thereby function in signalling and antioxidant defences. These oxidative modifications occur in response to a range of agents and stimuli, and can lead to the existence of multiple redox states for a given protein. To assess the role(s) of a protein in redox signalling and antioxidant defence, it is thus vital to be able to assess which of the multiple thiol redox states are present and to investigate how these alter under different conditions. While this can be done by a range of mass spectrometric-based methods, these are time-consuming, costly, and best suited to study abundant proteins or to perform an unbiased proteomic screen. One approach that can facilitate a targeted assessment of candidate proteins, as well as proteins that are low in abundance or proteomically challenging, is by electrophoretic mobility shift assays. Redox-modified cysteine residues are selectively tagged with a large group, such as a polyethylene glycol (PEG) polymer, and then the proteins are separated by electrophoresis followed by immunoblotting, which allows the inference of redox changes based on band shifts. However, the applicability of this method has been impaired by the difficulty of cleanly modifying protein thiols by large PEG reagents. To establish a more robust method for redox-selective PEGylation, we have utilised a Click chemistry approach, where free thiol groups are first labelled with a reagent modified to contain an alkyne moiety, which is subsequently Click-reacted with a PEG molecule containing a complementary azide function. This strategy can be adapted to study reversibly reduced or oxidised cysteines. Separation of the thiol labelling step from the PEG conjugation
Kerr F, Sofola-Adesakin O, Ivanov DK, et al., 2017, Direct Keap 1-Nrf2 disruption as a potential therapeutic target for Alzheimer's disease, PLoS Genetics, Vol: 13, ISSN: 1553-7390
Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target forthe prevention of neurodegenerative diseases, including Alzheimer’s disease (AD). CurrentNrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimentaleffects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerativeconditions may therefore direct the design of drugs targeted for the prevention ofthese diseases with minimal side-effects. Our study provides the first in vivo evidence thatspecific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity inresponse to the AD-initiating Aβ42 peptide, in correlation with Nrf2 activation. Comparatively,lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aβ42 toxicity by mechanismsindependent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain alsoprevented synaptotoxicity mediated by naturally-derived Aβ oligomers in mouse corticalneurons. Overall, our findings highlight Keap1 specifically as an efficient target for the reactivationof Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors forthe prevention of neurodegeneration in vivo.
Logan A, Pell VR, Shaffer KJ, et al., 2015, Assessing the mitochondrial membrane potential in cells and in vivo using targeted click chemistry and mass spectrometry, Cell Metabolism, Vol: 23, Pages: 379-385, ISSN: 1932-7420
The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by "click" chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo.
Speakman JR, Blount JD, Bronikowski AM, et al., 2015, Oxidative stress and life histories: unresolved issues and current needs, Ecology and Evolution, Vol: 5, Pages: 5745-5757, ISSN: 2045-7758
Menger KE, James AM, Cocheme HM, et al., 2015, Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster (vol 11, pg 1856, 2015), CELL REPORTS, Vol: 13, Pages: 1285-1285, ISSN: 2211-1247
Robb EL, Gawel JM, Aksentijevic D, et al., 2015, Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat, Free Radical Biology and Medicine, Vol: 89, Pages: 883-894, ISSN: 0891-5849
Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratorychain and plays a major role in pathological oxidative stress and redox signaling. While there are tools todetect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxideproduction within the mitochondrial matrix. This lack impedes progress, making it challenging to assessaccurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, wesynthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprisesa triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulatesselectively in the mitochondrial matrix driven by the membrane potential. Within the matrix,MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxideproduction within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondriaand cells in culture a thousand-fold more effectively than untargeted paraquat. MitoPQ wasalso more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables theselective generation of superoxide within mitochondria and is a useful tool to investigate the many rolesof mitochondrial superoxide in pathology and redox signaling in cells and in vivo.
Jameson VJA, Cocheme HM, Logan A, et al., 2015, Synthesis of triphenylphosphonium vitamin E derivatives as mitochondria-targeted antioxidants, Tetrahedron, Vol: 71, Pages: 8444-8453, ISSN: 1464-5416
A series of mitochondria-targeted antioxidants comprising a lipophilic triphenylphosphonium cation attached to the antioxidant chroman moiety of vitamin E by an alkyl linker have been prepared. The synthesis of a series of mitochondria-targeted vitamin E derivatives with a range of alkyl linkers gave compounds of different hydrophobicities. This work will enable the dependence of antioxidant defence on hydrophobicity to be determined in vivo.
Kinghorn KJ, Castillo-Quan JI, Bartolome F, et al., 2015, Loss of PLA2G6 leads to elevated mitochondrial lipid peroxidation and mitochondrial dysfunction, Brain, Vol: 138, Pages: 1801-1816, ISSN: 1460-2156
The PLA2G6 gene encodes a group VIA calcium-independent phospholipase A2 beta enzyme that selectively hydrolyses glycerophospholipids to release free fatty acids. Mutations in PLA2G6 have been associated with disorders such as infantile neuroaxonal dystrophy, neurodegeneration with brain iron accumulation type II and Karak syndrome. More recently, PLA2G6 was identified as the causative gene in a subgroup of patients with autosomal recessive early-onset dystonia-parkinsonism. Neuropathological examination revealed widespread Lewy body pathology and the accumulation of hyperphosphorylated tau, supporting a link between PLA2G6 mutations and parkinsonian disorders. Here we show that knockout of the Drosophila homologue of the PLA2G6 gene, iPLA2-VIA, results in reduced survival, locomotor deficits and organismal hypersensitivity to oxidative stress. Furthermore, we demonstrate that loss of iPLA2-VIA function leads to a number of mitochondrial abnormalities, including mitochondrial respiratory chain dysfunction, reduced ATP synthesis and abnormal mitochondrial morphology. Moreover, we show that loss of iPLA2-VIA is strongly associated with increased lipid peroxidation levels. We confirmed our findings using cultured fibroblasts taken from two patients with mutations in the PLA2G6 gene. Similar abnormalities were seen including elevated mitochondrial lipid peroxidation and mitochondrial membrane defects, as well as raised levels of cytoplasmic and mitochondrial reactive oxygen species. Finally, we demonstrated that deuterated polyunsaturated fatty acids, which inhibit lipid peroxidation, were able to partially rescue the locomotor abnormalities seen in aged flies lacking iPLA2-VIA gene function, and restore mitochondrial membrane potential in fibroblasts from patients with PLA2G6 mutations. Taken together, our findings demonstrate that loss of normal PLA2G6 gene activity leads to lipid peroxidation, mitochondrial dysfunction and subsequent mitochondrial membrane abnormalit
Menger KE, James AM, Cocheme HM, et al., 2015, Fasting, but not aging, dramatically alters the redox status of cysteine residues on proteins in Drosophila melanogaster, Cell Reports, Vol: 11, Pages: 1856-1865, ISSN: 2211-1247
Logan A, Cochemé HM, Li Pun PB, et al., 2013, Using exomarkers to assess mitochondrial reactive species in vivo, Biochimica et Biophysica Acta - General Subjects, Vol: 1840, Pages: 923-930, ISSN: 0006-3002
BACKGROUND: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. SCOPE OF REVIEW: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
Chouchani ET, Methner C, Nadtochiy SM, et al., 2013, Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I, Nature Medicine, Vol: 19, Pages: 753-759, ISSN: 1078-8956
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 unclear1,2,3,4,5,6. 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.
Cabreiro F, Au C, Leung KY, et al., 2013, Metformin Retards Aging in C. elegans by Altering Microbial Folate and Methionine Metabolism, Cell, Vol: 153, Pages: 228-239, ISSN: 1097-4172
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.
Kelso GF, Maroz A, Cocheme HM, et al., 2012, A Mitochondria-Targeted Macrocyclic Mn(II) Superoxide Dismutase Mimetic, CHEMISTRY & BIOLOGY, Vol: 19, Pages: 1237-1246, ISSN: 1074-5521
Cocheme HM, Logan A, Prime TA, et al., 2012, Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila, NATURE PROTOCOLS, Vol: 7, Pages: 946-958, ISSN: 1754-2189
Collins Y, Chouchani ET, James AM, et al., 2012, Mitochondrial redox signalling at a glance (vol 125, pg 801, 2012), JOURNAL OF CELL SCIENCE, Vol: 125, Pages: 1837-1837, ISSN: 0021-9533
Alic N, Andrews TD, Giannakou ME, et al., 2011, Genome-wide dFOXO targets and topology of the transcriptomic response to stress and insulin signalling, Molecular Systems Biology, Vol: 7, ISSN: 1744-4292
FoxO transcription factors, inhibited by insulin/insulin-like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild-type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo-independent effects. We demonstrate genome-wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf-12 nuclear hormone receptor.
Cocheme HM, Quin C, McQuaker SJ, et al., 2011, Measurement of H2O2 within Living Drosophila during Aging Using a Ratiometric Mass Spectrometry Probe Targeted to the Mitochondrial Matrix, CELL METABOLISM, Vol: 13, Pages: 340-350, ISSN: 1550-4131
James AM, Cocheme HM, Murai M, et al., 2010, Complementation of coenzyme Q-deficient yeast by coenzyme Q analogues requires the isoprenoid side chain, FEBS JOURNAL, Vol: 277, Pages: 2067-2082, ISSN: 1742-464X
Cocheme HM, Murphy MP, 2010, Can antioxidants be effective therapeutics?, CURRENT OPINION IN INVESTIGATIONAL DRUGS, Vol: 11, Pages: 426-431, ISSN: 1472-4472
Rodriguez-Cuenca S, Cocheme HM, Logan A, et al., 2010, Consequences of long-term oral administration of the mitochondria-targeted antioxidant MitoQ to wild-type mice, FREE RADICAL BIOLOGY AND MEDICINE, Vol: 48, Pages: 161-172, ISSN: 0891-5849
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