Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV |
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| Affiliation: | 1. Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research (STAR);2. Department of Biochemistry and Molecular Biology;3. Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, U.S.A;1. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA;2. National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA;1. Department of Nuclear Medicine, School of Allied Health Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India;2. Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India;1. A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation;2. International Laser Center, Lomonosov Moscow State University, Moscow, Russian Federation;3. Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russian Federation;4. Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow, Russian Federation;5. Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA;1. Bloomsbury Institute of Intensive Care Medicine, Division of Medicine, University College London, London, UK;2. Department of Cell and Development Biology, University College London, London, UK;1. Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA;2. University of Exeter Medical School, St. Luke’s Campus, Exeter, England, United Kingdom;3. Biosciences, College of Life and Environmental Science, University of Exeter, England, United Kingdom;1. Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland;2. Department of Biochemistry (286), RIMLS, RCMM, Radboudumc, Nijmegen, The Netherlands |
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| Abstract: | MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5–10 μM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols. |
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| Keywords: | Bioenergetics Seahorse Reactive oxygen species Respiration Dihydroethidium Hydroethidine MitoSOX |
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