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Assessment of Mitochondrial Stress in Neurons: Proximity Ligation Assays to Detect Recruitment of Stress-Responsive Proteins to Mitochondria

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Cell Culture Techniques

Part of the book series: Neuromethods ((NM,volume 145))

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Abstract

Mitochondria are highly integrated organelles that must readily alter organelle physiology to adapt to the changing environment of neurons. Failure in the mechanisms regulating organelle adaptation and homeostasis manifests as perturbations in bioenergetics, Ca2+ buffering, and mitochondrial dynamics, which ultimately affect the integrity of organelle membranes, DNA, and proteins. Collectively, these anomalies in organelle function are referred to as mitochondrial stress. While elegant methods have been developed to measure fundamental mitochondrial physiology, only recently have new strategies emerged to investigate the regulatory mechanisms responsible for mitochondrial stress responses. The emergence of cytosolic, stress-responsive protein kinases and phosphatases demonstrates the importance of neuron-mitochondrial cross talk for regulating organelle health and quality. The magnitude of signaling cascades on the outer mitochondrial membrane (OMM) can greatly influence organelle form and function. Thus, interpreting OMM signaling events in the context of mitochondrial function is critical to understanding the role of stress-responsive protein kinases and phosphatases in health and disease.

In this chapter, we will provide a brief review of standard approaches to assess mitochondrial physiology and stress in neurons. The sources of neuronal mitochondria and the techniques used to measure bioenergetics, Ca2+ flux, organelle dynamics, radical production, and the integrity of fundamental organelle processes are discussed. The emphasis of the chapter pertains to methods that identify and validate the presence of stress-responsive signaling proteins (i.e., kinases and phosphatases) on the OMM in cultured neurons and fixed CNS tissues. We will describe our approach to proximity ligation assays for evaluating mitochondrial stress responses, specifically c-Jun N-terminal kinase (JNK) OMM signaling, in cells and brain sections.

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Acknowledgments

The authors would like to thank Florida International University and the Robert Stempel College of Public Health & Social Work for the start-up funds that supported the studies discussed in the chapter. MRS was supported by a RISE grant NIH/NIGMS R25 GM06134. The authors would also like to express their gratitude to Philip V. LoGrasso (formerly of the Scripps Research Institute) for the tissue samples and imaging data using the 6-OHDA model. The authors extend their appreciation to the members of the Chambers’ Lab who provided helpful comments during the preparation of the chapter.

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Authors and Affiliations

  1. Department of Environmental Health Sciences, Robert Stempel College of Public Health & Social Work, Florida International University, Miami, FL, USA

    Monica Rodriguez-Silva, Kristen T. Ashourian, Anthony D. Smith & Jeremy W. Chambers

  2. Biomolecular Sciences Institute, Florida International University, Miami, FL, USA

    Jeremy W. Chambers

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  1. Monica Rodriguez-Silva
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Correspondence to Jeremy W. Chambers .

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  1. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA

    Michael Aschner

  2. Department of Environment/STE 100, University of Washington, Seattle, WA, USA

    Lucio Costa

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Rodriguez-Silva, M., Ashourian, K.T., Smith, A.D., Chambers, J.W. (2019). Assessment of Mitochondrial Stress in Neurons: Proximity Ligation Assays to Detect Recruitment of Stress-Responsive Proteins to Mitochondria. In: Aschner, M., Costa, L. (eds) Cell Culture Techniques. Neuromethods, vol 145. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9228-7_6

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