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Live Imaging of Mitochondrial ROS Production and Dynamic Redox Balance in Neurons

Part of the Neuromethods book series (NM,volume 123)

Abstract

Mitochondria are the most prominent cellular source of reactive oxygen species. As a by-product of cellular respiration, superoxide constantly escapes from the electron transport chain and is converted into other reactive oxygen and nitrogen species, which then may mediate downstream redox changes also in neighboring compartments and organelles. Such mitochondria-derived redox signals crucially contribute to the modulation of normal cell function but may as well cause random oxidative damage to various cellular constituents and evoke aberrant signaling. The resulting redox stress is considered to contribute to the onset and progression of various neuropathologies. Hence, there is a tremendous interest in mapping subcellular ROS levels as well as redox changes and to understand their spatiotemporal dynamics.

It is only since the development of genetically encoded fluorescent redox sensors that such analysis has become possible in a reliable manner. These advanced optical sensors overcome the severe disadvantages of oxidation -sensitive synthetic fluorescent dyes. For the first time they allow to monitor both reducing as well as oxidizing changes on the subcellular level, to decipher their detailed dynamics, and to quantify their very extent. In this chapter we summarize the properties of protein-based optical redox indicators and explain their superiority to synthetic dyes. Furthermore, we address the challenges of a proper and efficient delivery of the sensor-coding DNA, with a special emphasis on viral transduction and vector design. Finally, we give a detailed description of redox live-imaging applications in different neuronal preparations and point out to potential pitfalls.

Key words

  • Redox signaling
  • Oxidative stress
  • Reactive oxygen species (ROS)
  • Redox imaging
  • Optical redox probes
  • 2-photon microscopy
  • Ratiometric imaging
  • Viral vectors
  • Viral transduction

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Acknowledgments

We thank Belinda Kempkes for excellent technical assistance, and we are grateful to Professor S. James Remington, Institute of Molecular Biology, University of Oregon, Eugene OR USA, for making available to us the plasmids expressing roGFP1 redox-sensitive proteins. Our research was funded by the Cluster of Excellence and Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) and the International Rett Syndrome Foundation (IRSF, grant #2817) as well as by the University Medical Center Göttingen and the State of Lower Saxony (large scale equipment grant INST 1525/14-1 FUGG).

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Can, K., Kügler, S., Müller, M. (2017). Live Imaging of Mitochondrial ROS Production and Dynamic Redox Balance in Neurons. In: Strack, S., Usachev, Y. (eds) Techniques to Investigate Mitochondrial Function in Neurons. Neuromethods, vol 123. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6890-9_9

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  • DOI: https://doi.org/10.1007/978-1-4939-6890-9_9

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