Correction to: Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila.

The article Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila, written by Dmytro V. Gospodaryov. Olha M. Strilbytska. Uliana V. Semaniuk. Natalia V. Perkhulyn. Bohdana M. Rovenko. Ihor S. Yurkevych. Ana G. Barata. Tobias P. Dick. Oleh V. Lushchak and Howard T. Jacobs, was originally published electronically on the publisher's internet portal on 20 November 2019 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on 27 January 2020 to © The Author(s) 2020 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The original article has been corrected.

A role for peroxiredoxins in H2O2- and MEKK-dependent activation of the p38 signaling pathway.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the cellular response to various stresses and its deregulation accompanies pathological conditions such as cancer and chronic inflammation. Hydrogen peroxide (H2O2) is a well-established activator of the p38 MAPK signaling pathway. However, the mechanisms of H2O2-induced p38 activation are not yet fully understood. In Drosophila cells, we find that H2O2-induced activation of p38 depends on the MAPK kinase kinase (MAP3K) Mekk1. In line with the emerging role of peroxiredoxins as H2O2 sensors and signal transmitters we observe an H2O2-dependent interaction between Mekk1 and the cytosolic peroxiredoxin of Drosophila, Jafrac1. In human cells, MEKK4 (the homologue of Mekk1) and peroxiredoxin-2 (Prx2) interact in a similar manner, suggesting an evolutionarily conserved mechanism. In both organisms, H2O2 induces transient disulfide-linked conjugates between the MAP3K and a typical 2-Cys peroxiredoxin. We propose that these conjugates represent the relaying of oxidative equivalents from H2O2 to the MAP3K and that the oxidation of Mekk1/MEKK4 leads to the downstream activation of p38 MAPK. Indeed, the depletion of cytosolic 2-Cys peroxiredoxins in human cells diminished H2O2-induced activation of p38 MAPK.

Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila.

Mitochondrial alternative NADH dehydrogenase (aNDH) was found to extend lifespan when expressed in the fruit fly. We have found that fruit flies expressing aNDH from Ciona intestinalis (NDX) had 17-71% lifespan prolongation on media with different protein-tocarbohydrate ratios except NDX-expressing males that had 19% shorter lifespan than controls on a high protein diet. NDX-expressing flies were more resistant to organic xenobiotics, 2,4-dichlorophenoxyacetic acid and alloxan, and inorganic toxicant potassium iodate, and partially to sodium molybdate treatments. On the other hand, NDX-expressing flies were more sensitive to catechol and sodium chromate. Enzymatic analysis showed that NDX-expressing males had higher glucose 6-phosphate dehydrogenase activity, whilst both sexes showed increased glutathione S-transferase activity.

In vivo imaging of H2O2 production in Drosophila.

H2O2 plays many roles in cellular physiology. Therefore, we need tools for quantitative detection of H2O2 in tissues and whole model organisms. We recently introduced a genetically encoded H2O2 sensor, roGFP2-Orp1, which couples the redox-sensitive green fluorescent protein 2 (roGFP2) to the yeast H2O2 sensor protein Orp1. Expression of cytosolic or mitochondrial roGFP2-Orp1 in Drosophila allows the measurement of physiologically relevant changes in H2O2 levels, with compartment-specific resolution. Here, we provide a detailed protocol for the relative quantitation of H2O2 levels in living larvae by real-time imaging. We also describe a dissection and fixation method that conserves the redox state of the probe and thus allows reliable measurements on fixed adult tissues. Finally, we give recommendations for image processing, analysis, and interpretation, highlighting issues that require attention to detail, to ensure accuracy and validity of results.

Exposing cells to H2O2: a quantitative comparison between continuous low-dose and one-time high-dose treatments.

Most studies investigating the influence of H2O2 on cells in culture apply nonphysiological concentrations over nonphysiological time periods (i.e., a one-time bolus that is metabolized in minutes). As an alternative, the glucose oxidase/catalase (GOX/CAT) system allows application of physiologically relevant H2O2 concentrations (300nM-10µM) over physiologically relevant time periods (up to 24h). Recent findings suggest that bolus and GOX/CAT treatments can lead to opposing cellular responses, thus warranting a quantitative comparison between the two approaches. First, we established a reaction-diffusion model that can predict the behavior of the GOX/CAT system with spatiotemporal resolution, thus aiding selection of optimal experimental conditions for its application. Measurements of H2O2 concentration in the cellular supernatant with the luminol/hypochlorite system were consistent with the predictions of the model. Second, we compared the impact of bolus and GOX/CAT treatments on cytosolic H2O2 levels over time. Intracellular H2O2 was monitored by the response of the thiol peroxidase Prx2 and the H2O2 sensor roGFP2-Orp1. We found that Prx2 rapidly and reversibly responds to submicromolar H2O2 levels and accurately reflects kinetic competition with cellular catalase. Our measurements reveal fundamental differences in the dynamic response of cellular H2O2 concentrations following either bolus or GOX/CAT treatments. Thus, different, or even opposing, biological outcomes from differing means of H2O2 delivery may be expected. Cellular responses induced by bolus treatment may not occur under GOX/CAT conditions, and vice versa.