Mitochondrial redox signals have a central role in neuronal physiology and disease. Here we describe a new optical approach to measure fast redox signals with single-organelle resolution in living mice that express genetically encoded redox biosensors in their neuronal mitochondria. Moreover, we demonstrate how parallel measurements with several biosensors can integrate these redox signals into a comprehensive characterization of mitochondrial function. This approach revealed that axonal mitochondria undergo spontaneous 'contractions' that are accompanied by reversible redox changes. These contractions are amplified by neuronal activity and acute or chronic neuronal insults. Multiparametric imaging reveals that contractions constitute respiratory chain–dependent episodes of depolarization coinciding with matrix alkalinization, followed by uncoupling. In contrast, permanent mitochondrial damage after spinal cord injury depends on calcium influx and mitochondrial permeability transition. Thus, our approach allows us to identify heterogeneity among physiological and pathological redox signals, correlate such signals to functional and structural organelle dynamics and dissect the underlying mechanisms.
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D'Autréaux, B. & Toledano, M.B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 8, 813–824 (2007).
Albrecht, S.C., Barata, A.G., Großhans, J., Teleman, A.A. & Dick, T.P. In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab. 14, 819–829 (2011).
Niethammer, P., Grabher, C., Look, A.T. & Mitchison, T.J. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459, 996–999 (2009).
Dickinson, B.C., Peltier, J., Stone, D., Schaffer, D.V. & Chang, C.J. Nox2 redox signaling maintains essential cell populations in the brain. Nat. Chem. Biol. 7, 106–112 (2011).
Guarente, L. Mitochondria—A nexus for aging, calorie restriction, and sirtuins? Cell 132, 171–176 (2008).
Lin, M.T. & Beal, M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 (2006).
Murphy, M.P. How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13 (2009).
Zorov, D.B., Filburn, C.R., Klotz, L.O., Zweier, J.L. & Sollott, S.J. Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes. J. Exp. Med. 192, 1001–1014 (2000).
Nunnari, J. & Suomalainen, A. Mitochondria: in sickness and in health. Cell 148, 1145–1159 (2012).
Mahad, D.J. et al. Mitochondrial changes within axons in multiple sclerosis. Brain 132, 1161–1174 (2009).
Nikić, I. et al. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat. Med. 17, 495–499 (2011).
Sullivan, P.G., Krishnamurthy, S., Patel, S.P., Pandya, J.D. & Rabchevsky, A.G. Temporal characterization of mitochondrial bioenergetics after spinal cord injury. J. Neurotrauma 24, 991–999 (2007).
Gutscher, M. et al. Real-time imaging of the intracellular glutathione redox potential. Nat. Methods 5, 553–559 (2008).
Misgeld, T., Kerschensteiner, M., Bareyre, F.M., Burgess, R.W. & Lichtman, J.W. Imaging axonal transport of mitochondria in vivo. Nat. Methods 4, 559–561 (2007).
Gurney, M.E. et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772–1775 (1994).
Kerschensteiner, M., Schwab, M.E., Lichtman, J.W. & Misgeld, T. In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat. Med. 11, 572–577 (2005).
Mank, M. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Methods 5, 805–811 (2008).
Wardman, P. Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic. Biol. Med. 43, 995–1022 (2007).
Belousov, V.V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3, 281–286 (2006).
Schwarzländer, M., Logan, D.C., Fricker, M.D. & Sweetlove, L.J. The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide 'flashes'. Biochem. J. 437, 381–387 (2011).
Hansen, J.M., Go, Y.-M. & Jones, D.P. Nuclear and mitochondrial compartmentation of oxidative stress and redox signaling. Annu. Rev. Pharmacol. Toxicol. 46, 215–234 (2006).
Dooley, C.T. et al. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279, 22284–22293 (2004).
Schwarzländer, M. et al. Mitochondrial 'flashes': a radical concept repHined. Trends Cell Biol. 22, 503–508 (2012).
Poburko, D., Santo-Domingo, J. & Demaurex, N. Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations. J. Biol. Chem. 286, 11672–11684 (2011).
Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011).
Wang, J.-Q. et al. Dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic DNA damage in Huntington disease. J. Biol. Chem. 288, 3070–3084 (2013).
Schinzel, A.C. et al. Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc. Natl. Acad. Sci. USA 102, 12005–12010 (2005).
Keller, J.N. et al. Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J. Neurosci. 18, 687–697 (1998).
Yanik, M.F. et al. Neurosurgery: functional regeneration after laser axotomy. Nature 432, 822 (2004).
Grienberger, C. & Konnerth, A. Imaging calcium in neurons. Neuron 73, 862–885 (2012).
Love, N.R. et al. Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration. Nat. Cell Biol. 15, 222–228 (2013).
Rieger, S. & Sagasti, A. Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin. PLoS Biol. 9, e1000621 (2011).
Guzman, J.N. et al. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 468, 696–700 (2010).
Roma, L.P. et al. Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1. Biochem. J. 441, 971–978 (2012).
Meyer, A.J. & Dick, T.P. Fluorescent protein-based redox probes. Antioxid. Redox Signal. 13, 621–650 (2010).
Schwarzländer, M. et al. Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory bioenergetics in Arabidopsis. Plant Cell 24, 1188–1201 (2012).
Santo-Domingo, J., Giacomello, M., Poburko, D., Scorrano, L. & Demaurex, N. OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange. EMBO J. 32, 1927–1940 (2013).
Wang, W. et al. Superoxide flashes in single mitochondria. Cell 134, 279–290 (2008).
Marinkovic, P., Godinho, L. & Misgeld, T. in Imaging in Neuroscience: a Laboratory Manual (eds. Yuste, R. & Konnerth A.) Ch. 22 (Cold Spring Harbor Laboratory Press, 2011).
Shaner, N.C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008).
Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using μManager. Curr. Protoc. Mol. Biol. 92, 14.20 (2010).
Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).
Klugmann, M. et al. AAV-mediated hippocampal expression of short and long Homer 1 proteins differentially affect cognition and seizure activity in adult rats. Mol. Cell. Neurosci. 28, 347–360 (2005).
Bareyre, F.M. et al. In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration. Proc. Natl. Acad. Sci. USA 108, 6282–6287 (2011).
Romanelli, E. et al. Cellular, subcellular and functional in vivo labeling of the spinal cord using vital dyes. Nat. Protoc. 8, 481–490 (2013).
Zhang, Y. et al. Anesthetics isoflurane and desflurane differently affect mitochondrial function, learning, and memory. Ann. Neurol. 71, 687–698 (2012).
Boscolo, A. et al. Early exposure to general anaesthesia disturbs mitochondrial fission and fusion in the developing rat brain. Anesthesiology 118, 1086–1097 (2013).
Yang, G., Pan, F., Parkhurst, C.N., Grutzendler, J. & Gan, W.B. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat. Protoc. 5, 201–208 (2010).
Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
We thank G. Heitmann, M. Adrian and K. Wullimann for technical assistance, D. Matzek, M. Budak, N. Budak and L. Marinković for animal husbandry and M. Krumbholz for help with statistical analysis. We thank M. Murphy (University of Cambridge) for the gift of MitoQ. We thank R. Campbell (University of Alberta) for the R-GECO1 plasmid, L. Looger (Howard Hughes Medical Institute Janelia Farm Research Campus) for GCaMP3 and N. Demaurex (University of Geneva Medical School) for mito-SypHer. Work in M.K.'s laboratory is financed through grants from the Deutsche Forschungsgemeinschaft (DFG; Sonderforschungsbereich 870 and Transregio 128), the German Federal Ministry of Research and Education (BMBF, Competence Network Multiple Sclerosis), the European Research Council (ERC) under the European Union's Seventh Framework Program (FP/2007-2013; ERC grant agreement no. 310932), the Hertie Foundation and the 'Verein Therapieforschung für MS-Kranke e.V.' T.M. is supported by the Institute of Advanced Studies (Technische Universität München), the Alexander von Humboldt Foundation, the Center for Integrated Protein Science (Munich, EXC 114), the DFG (SFB 596) and the DZNE (Munich). T.M.'s work on this project was further supported by the BMBF as part of ERA-Net '2-photon imaging'. F.M.B. is supported by the DFG (SFB 870) and an independent group leader award of the BMBF. F.M.B., M.K. and T.M. are supported by SyNergy (EXC 1010), and T.P.D., M.K. and T.M. are supported by the DFG Priority Program 1710. Work in D.K.S.C.'s laboratory is supported by grants from the US National Institutes of Health Ca 049797 and the Edward P. Evans Foundation. T.P.D. is supported by the DFG (SFB 938, SFB 1036) and the BMBF ('LungSys'). M.O.B. is recipient of a doctoral fellowship from the Gertrud Reemtsma Foundation (Max Planck Society) and is supported by the German National Academic Foundation. P.R.W. is supported by a postdoctoral fellowship from the Wings of Life Foundation and received previous support from the Human Frontier Science Program. M.O.B. and P.M. were supported by the Graduate School of Technische Universität München.
The authors declare no competing financial interests.
Supplementary Figures 1–7 and Supplementary Table 1. (PDF 25758 kb)
Mitochondrial movement and glutathione redox potential are shown in the 488-nm channel (top) and as pseudocolor-coded R/RDTT (bottom) in a triangularis sterni explant of a Thy1-mito-Grx1-roGFP2 mouse. A mitochondrial contraction is indicated by the white arrow. (AVI 2613 kb)
Changes of mitochondrial morphology (top, 488-nm channel) and glutathione redox potential (bottom, R/R0) during a mitochondrial contraction in a triangularis sterni explant of a Thy1-mito-Grx1-roGFP2 mouse. Long mitochondria have a 'pearl on a string'-like appearance during contractions. (AVI 5395 kb)
Changes of mitochondrial morphology (top, 408-nm channel) and pH (bottom, R/R0) during a mitochondrial contraction (arrow) in a triangularis sterni explant of a wild-type mouse injected with rAAV-mito-SypHer. (AVI 3071 kb)
Three parallel running axons in a triangularis explant of a Thy1-mito-Grx1-roGFP2 mouse. A laser lesion is induced in the middle axon, followed by spreading mitochondrial shape changes (top, 488-nm channel) and oxidation (bottom, R/R0). The muscle twitches when the lesion is induced. (AVI 6061 kb)
Induction of a laser lesion in a triangularis sterni explant derived from a Thy1-mitoTagRFP-t mouse injected with rAAV-mito-GCaMP3. The lesion induces spreading mitochondrial shape changes (top, 550-nm channel) and the uptake of calcium into mitochondria (bottom, R/R0). (AVI 3354 kb)
In vivo time-lapse of mitochondrial shape changes (top, 488-nm channel) and oxidation (bottom, R/R0) after laser axotomy in the spinal cord of a Thy1-mito-Grx1-roGFP2 mouse. (AVI 3219 kb)
In vivo time-lapse of axonal calcium influx following laser axotomy in the spinal cord of a Thy1-TN-XXL mouse.
Top channel shows YFP only, bottom R/R0. (AVI 3638 kb)
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Breckwoldt, M., Pfister, F., Bradley, P. et al. Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo. Nat Med 20, 555–560 (2014). https://doi.org/10.1038/nm.3520
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