Technical Report | Published:

Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo

Nature Medicine volume 20, pages 555560 (2014) | Download Citation

Abstract

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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Acknowledgements

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.

Author information

Author notes

    • Michael O Breckwoldt

    Present address: Department of Neuroradiology, University of Heidelberg, Heidelberg, Germany.

    • Martin Kerschensteiner
    •  & Thomas Misgeld

    These authors contributed equally to this work.

Affiliations

  1. Institute for Neuronal Cell Biology, Technical University Munich, Munich, Germany.

    • Michael O Breckwoldt
    • , Franz M J Pfister
    • , Petar Marinković
    • , Philip R Williams
    • , Monika S Brill
    • , Barbara Plomer
    • , Leanne Godinho
    •  & Thomas Misgeld
  2. Institute of Clinical Neuroimmunology, Ludwig-Maximilians University Munich, Munich, Germany.

    • Michael O Breckwoldt
    • , Franz M J Pfister
    • , Peter M Bradley
    • , Anja Schmalz
    • , Florence M Bareyre
    •  & Martin Kerschensteiner
  3. Graduate Center for Toxicology and Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky, USA.

    • Daret K St Clair
  4. Transgenic Core Facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

    • Ronald Naumann
  5. Max-Planck Institute of Neurobiology, Martinsried, Germany.

    • Oliver Griesbeck
  6. Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany.

    • Markus Schwarzländer
  7. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.

    • Florence M Bareyre
    • , Martin Kerschensteiner
    •  & Thomas Misgeld
  8. Division of Redox Regulation, German Cancer Research Center (DKFZ), Heidelberg, Germany.

    • Tobias P Dick
  9. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.

    • Thomas Misgeld
  10. Center for Integrated Protein Sciences (CIPS), Munich, Germany.

    • Thomas Misgeld

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Contributions

T.M., M.K., T.P.D. and M.O.B. conceived of the experiments. M.O.B., F.M.J.P., P.R.W., M.K. and T.M. performed imaging experiments and image analysis. F.M.B., L.G., T.P.D., O.G., M.S.B., B.P., R.N., D.K.S.C., F.M.J.P. and M.O.B. generated and characterized transgenic mouse lines. P.M.B. performed virus injections. A.S. generated AAV vectors. P.M. and F.M.J.P. conducted experiments in the ALS model. M.K., T.M., M.S. and M.O.B. interpreted the experiments and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Martin Kerschensteiner or Thomas Misgeld.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Table 1.

Videos

  1. 1.

    Parallel imaging of mitochondrial dynamics and glutathione redox potential.

    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.

  2. 2.

    Mitochondrial contraction and redox changes.

    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.

  3. 3.

    Mitochondrial contraction and pH changes.

    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.

  4. 4.

    Laser axotomy of a peripheral axon in a Thy1-mito-Grx1-roGFP2 mouse.

    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.

  5. 5.

    Calcium influx into mitochondria after peripheral laser axotomy.

    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).

  6. 6.

    Laser axotomy of a central axon in the spinal cord of a Thy1-mito-Grx1-roGFP2 mouse.

    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.

  7. 7.

    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.

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DOI

https://doi.org/10.1038/nm.3520

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