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Protocols for Assessing Mitophagy in Neuronal Cell Lines and Primary Neurons

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Techniques to Investigate Mitochondrial Function in Neurons

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

Abstract

Mitochondria are organelles that regulate essential eukaryotic functions including generating energy, sequestering excess calcium, and modulating cell survival. In order for neurons to thrive, mitochondria have to be continuously replenished by maintaining autophagic-lysosomal mediated degradation of mitochondria (mitophagy) and mitochondrial biogenesis. While a plethora of image- and biochemical-based techniques have been developed for measuring autophagy (macroautophagy) in eukaryotic cells, the molecular toolbox for quantifying and assessing mitophagy in neurons continues to evolve. Compared to proliferating cells, quantifying mitophagy in neurons poses a technical challenge given that mitochondria are predominantly present in neurites (axons and dendrites) and are highly dynamic.

In this chapter, we provide a brief overview on mitophagy and provide a list of validated fluorescence- and biochemistry-based techniques used for assessing mitophagy in neuronal cells and primary neurons. Secondly, we provide comprehensive guidelines for interpreting steady-state levels of mitophagy and mitophagic flux in neurons using modern fluorescence- and biochemistry-based techniques. Finally, we provide a comprehensive list of common pitfalls to avoid when assessing mitophagy and offer practical solutions to overcome technical issues.

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Acknowledgments

The method development and the research data presented in this book chapter were supported by an NIH-NIGMS grant (GM103554) and by a University of Pittsburgh Pathology Post-doctoral Research Training Program Grant awarded to R.K.D. We give special thanks to Dr. Stefan Strack (Department of Pharmacology, University of Iowa College of Medicine) for graciously providing the mito-GFP and Flag-tagged Bβ2 constructs.

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Authors and Affiliations

  1. Department of Pharmacology, Reno School of Medicine, University of Nevada, 1664 N. Virginia St., Reno, NV, 89557, USA

    Ruben K. Dagda Ph.D. & Monica Rice

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  1. Ruben K. Dagda Ph.D.
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  2. Monica Rice
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Corresponding author

Correspondence to Ruben K. Dagda Ph.D. .

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Editors and Affiliations

  1. Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Stefan Strack

  2. Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Yuriy M. Usachev

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Dagda, R.K., Rice, M. (2017). Protocols for Assessing Mitophagy in Neuronal Cell Lines and Primary 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_13

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  • DOI: https://doi.org/10.1007/978-1-4939-6890-9_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6888-6

  • Online ISBN: 978-1-4939-6890-9

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