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FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy

Soutar, MPM; Kempthorne, L; Annuario, E; Luft, C; Wray, S; Ketteler, R; Ludtmann, MHR; (2019) FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy. Autophagy , 15 (11) pp. 2002-2011. 10.1080/15548627.2019.1603549. Green open access

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Abstract

Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from in vitro experiments involving the treatment of mammalian cells with high concentrations of mitochondrial uncouplers, such as CCCP. These chemicals have been shown to mediate off target effects, other than mitochondrial depolarization. A matter of controversy between mitochondrial physiologists and cell biologists is the discrepancy between concentrations of CCCP needed to activate mitophagy (usually >10 μM), when compared to the much lower concentrations used to depolarize mitochondria (<1 μM). Thus, there is an urgent need for optimizing the current methods to assess PINK1-PRKN mitophagy in vitro. In this study, we address the utilization of high CCCP concentrations commonly used to activate mitophagy. Combining live fluorescence microscopy and biochemistry, we show that the FBS/BSA in the cell culture medium reduces the ability of CCCP to induce PINK1 accumulation at depolarized mitochondria, subsequent PRKN recruitment and ubiquitin phosphorylation, and ultimately mitochondrial clearance. As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. These data unite mitochondrial physiology and mitophagy studies and are a first step toward a consensus on optimal experimental conditions for PINK1-PRKN mitophagy and mitochondrial physiology investigations to be carried out in parallel.

Type: Article
Title: FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy
Open access status: An open access version is available from UCL Discovery
DOI: 10.1080/15548627.2019.1603549
Publisher version: https://doi.org/10.1080/15548627.2019.1603549
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: BSA, CCCP, mitophagy, PINK1, PRKN
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Neurodegenerative Diseases
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10082623
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