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


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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Figure 1: Thy1-mito-Grx1-roGFP2 mice allow quantification of the mitochondrial glutathione redox potential in situ.
Figure 2: Mitochondria undergo spontaneous, reversible contractions.
Figure 3: Mitochondrial contractions occur in response to physiological and pathological stressors.
Figure 4: Axotomy induces spreading mitochondrial oxidation and structural alterations caused by the influx of extracellular calcium.


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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

Authors and Affiliations



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.

Corresponding authors

Correspondence to Martin Kerschensteiner or Thomas Misgeld.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1. (PDF 25758 kb)

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. (AVI 2613 kb)

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. (AVI 5395 kb)

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. (AVI 3071 kb)

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. (AVI 6061 kb)

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). (AVI 3354 kb)

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. (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).

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