COMMENTARY AND VIEWS Comment on “mt-Keima detects PINK1-PRKN mitophagy in vivo with greater sensitivity than mito-QC” Ian G. Ganleya, Alexander J. Whitworthb, and Thomas G. McWilliamsc aMRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland, UK; bMRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK; cTranslational Stem Cell Biology and Metabolism, Research Programs Unit, Faculty of Medicine,University of Helsinki, Helsinki, Finland; dDepartment of Anatomy, Faculty of Medicine,University of Helsinki, Helsinki, Finland ARTICLE HISTORY Received 5 March 2021; Revised 15 March 2021; Accepted 19 March 2021 One aspect of selective autophagy that has garnered intense interest is mitochondrial autophagy (mitophagy), particularly PINK1-PRKN-mediated mitophagy as this has direct implica tions for Parkinson disease. However, initial studies of differ ent mitophagy reporters in this context have resulted in a perceived conflict of results/outcomes and conclusions. A recent article by Liu et al. attempts to reconcile these discordant findings by comparing side-by-side two mitophagy reporter systems, mt-Keima and mito-QC [1]. A direct com parison of the reporter systems is certainly warranted, and in the cell-based analyses used in Liu et al. that involve PRKN overexpression and flow cytometry, it is encouraging to see that mt-Keima produces a robust signal. However, the head line claim that mito-QC is insufficiently sensitive for monitor ing PINK1-PRKN-dependent mitophagy should be brought into balance. mito-QC has been used in multiple published studies by independent groups as part of a series of experi ments to clearly and reliably show increased mitophagy, either in the presence or absence of PINK1-PRKN, and under endo genous or overexpressed PRKN conditions. Readers are encouraged to examine some of the many published, well controlled and validated studies [2–22]. Additionally, Liu et al. repeated their own results demon strating that exhaustive exercise induces PINK1-dependent mitophagy in the heart of mt-Keima mice. This is very encouraging given the difficult reproducibility associated with this type of exhaustive exercise study. However, we would like to point out that comparisons between mito-QC and mt-Keima should be made cautiously and are somewhat akin to comparing apples to oranges. One compelling reason is because the models have been generated and maintained using different mouse strains (C57BL/6 j-ntac versus FVB/NJ for mt-Keima [12,23]). Compared to C57 lines, FVB mice are naturally hyperactive with circadian dysregulation and beha vioral defects [24–26], display neuroanatomical abnormalities [27], retinal neurodegeneration and blindness [28]. It is important to note that significant differences also exist between FVB and C57 lines in adapting to and entrainment for treadmill exercise paradigms [29], including mitochon drial differences in muscle tissues [30] and divergent cardiac physiology [31]. We would like to refer the reader to the article by Enriquez that highlights the importance of consid ering genetic backgrounds in order to meaningfully compare data [32]. Finally, we would like to address the notion by Liu et al. that McWilliams et al., 2018 and Lee et al., 2018 have gener ated controversy in terms of the PINK1-PRKN mitophagy pathway [7,13]. From our perspective there is no controversy that multiple mitophagy pathways exist. The main conclusion of these papers was that the PINK1-PRKN pathway did not regulate mitophagy under basal conditions. This is the exact same observation that Liu et al. demonstrate in their current study: in the absence of exhaustive exercise, mitophagy levels in heart tissues of mt-Keima mice remain constant regardless of the presence or absence of PINK1; i.e., their basal level of mitophagy is independent of PINK1. We would also like to highlight work from the Goessling Lab that recently generated zebrafish models of mitophagy and compared Keima- and tandem mCherry-GFP-based reporters [33]. Here, the authors noted that both reporters faithfully monitored mitophagy and, additionally, the characterized mitophagy was determined to be independent of PINK1 and PRKN, instead requiring BNIP3. Thus, taking into account multiple publications from independent laboratories, using three popular animal models, we think that all demonstrate the same main conclusion, which is that the PINK1-PRKN pathway does not significantly contribute to basal mitophagy. We think that both reporters have merit and, under different scenarios, use of one type of reporter may be more advanta geous than the other. Due to loss of signal upon fixation, use of the mt-Keima mouse requires very strict time-dependent pre paration and analysis, which may not be achievable in some laboratories. This is where use of mito-QC may be preferable, particularly when immunohistochemistry is required to identify CONTACT Ian G. Ganley i.ganley@dundee.ac.uk MRC Protein Phosphorylation & Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland, UK; Alexander J. Whitworth a.whitworth@mrc-mbu.cam.ac.uk MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK; Thomas G. McWilliams thomas.mcwilliams@helsinki.fi Translational Stem Cell Biology & Metabolism Program, Research Programs Unit, Faculty of Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki AUTOPHAGY 2021, VOL. 17, NO. 12, 4477–4479 https://doi.org/10.1080/15548627.2021.1907269 © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://www.tandfonline.com https://crossmark.crossref.org/dialog/?doi=10.1080/15548627.2021.1907269&domain=pdf&date_stamp=2021-12-18 specific cell types or additional cellular components. Targeting mitophagy is likely to have genuine translational importance, most notably for neurodegenerative disorders, particularly given the 99.7% failure rate for drugs in this area [34]. Accordingly, despite the limitations of both reporter systems, a carefully considered discussion is essential. Both reporter systems have contributed to our understanding of physiological mitophagy and are well-positioned to continue unraveling the mysteries of mitophagy in health and disease. Disclosure statement No potential conflict of interest was reported by the authors. Funding This work was supported by a grant from the Medical Research Council UK (IGG: MC_UU_00018/2; AJW: MC_UU_00015/6). Research in TGM lab is funded by the Academy of Finland, Novo Nordisk Foundation, Sigrid Jusélius Foundation, Sydäntutkimussäätiö and the University of Helsinki. References [1] Liu YT, Sliter DA, Shammas MK, et al. Mt-Keima detects PINK1-PRKN mitophagy in vivo with greater sensitivity than mito-QC. Autophagy. 2021: 1–10. In Press:. Epub 2021/03/10. PubMed PMID: 33685343. DOI: 10.1080/15548627.2021.1896924. [2] Allen GF, Toth R, James J, et al. Loss of iron triggers PINK1/ Parkin-independent mitophagy. EMBO Rep. 2013;14 (12):1127–1135. Epub 2013/ 11/02. PubMed PMID: 24176932; PubMed Central PMCID: PMCPMC3981094 [3] Alsina D, Lytovchenko O, Schab A, et al. FBXL4 deficiency increases mitochondrial removal by autophagy. EMBO Mol Med. 2020;12(7):e11659. Epub 2020/ 06/12. PubMed PMID: 32525278; PubMed Central PMCID: PMCPMC7338799 [4] Cummins N, Tweedie A, Zuryn S, et al. Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria. Embo J. 2019; 38(3). Epub 2018/ 12/13. PubMed PMID: 30538104; PubMed Central PMCID: PMCPMC6356067. DOI: 10.15252/embj.201899360. [5] Goljanek-Whysall K, Soriano-Arroquia A, McCormick R, et al. miR-181a regulates p62/SQSTM1, parkin, and protein DJ-1 pro moting mitochondrial dynamics in skeletal muscle aging. Aging Cell. 2020;19(4):e13140. Epub 2020/ 04/16. PubMed PMID: 32291905; PubMed Central PMCID: PMCPMC7189996. [6] Hombrebueno JR, Cairns L, Dutton LR, et al. Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy. JCI Insight. 2019;4(23) Epub 2019/ 10/30. PubMed PMID: 31661466; PubMed Central PMCID: PMCPMC6962019. DOI: 10.1172/jci. insight.129760 [7] Lee JJ, Sanchez-Martinez A, Martinez Zarate A, et al. Basal mito phagy is widespread in drosophila but minimally affected by loss of Pink1 or parkin. J Cell Biol. 2018;217(5):1613–1622. Epub 2018/ 03/04. PubMed PMID: 29500189; PubMed Central PMCID: PMCPMC5940313 [8] Livingston MJ, Wang J, Zhou J, et al. Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys. Autophagy. 2019;15 (12):2142–2162. Epub 2019/ 05/09. PubMed PMID: 31066324; PubMed Central PMCID: PMCPMC6844514 [9] Marcassa E, Kallinos A, Jardine J, et al. Dual role of USP30 in controlling basal pexophagy and mitophagy. EMBO Rep. 2018;19 (7) Epub 2018/ 06/14. PubMed PMID: 29895712; PubMed Central PMCID: PMCPMC6030704 [10] Maremanda KP, Sundar IK, Rahman I. Protective role of mesenchymal stem cells and mesenchymal stem cell-derived exosomes in cigarette smoke-induced mitochondrial dysfunc tion in mice. Toxicol Appl Pharmacol. 2019;385:114788. Epub 2019/ 11/05. PubMed PMID: 31678243; PubMed Central PMCID: PMCPMC6894395 [11] McWilliams TG, Barini E, Pohjolan-Pirhonen R, et al. Phosphorylation of Parkin at serine 65 is essential for its activa tion in vivo. Open Biol. 2018;8(11) Epub 2018/ 11/09. PubMed PMID: 30404819; PubMed Central PMCID: PMCPMC6282074. DOI: 10.1098/rsob.180108 [12] McWilliams TG, Prescott AR, Allen GF, et al. mito-QC illumi nates mitophagy and mitochondrial architecture in vivo. J Cell Biol. 2016;214(3):333–345. Epub 2016/ 07/28. PubMed PMID: 27458135; PubMed Central PMCID: PMCPMC4970326 [13] McWilliams TG, Prescott AR, Montava-Garriga L, et al. Basal mitophagy occurs independently of PINK1 in mouse tissues of high metabolic demand. Cell Metab. 2018;27(2):439–49e5. Epub 2018/ 01/18. PubMed PMID: 29337137; PubMed Central PMCID: PMCPMC5807059 [14] McWilliams TG, Prescott AR, Villarejo-Zori B, et al. A comparative map of macroautophagy and mitophagy in the vertebrate eye. Autophagy. 2019;15(7):1296–1308. Epub 2019/ 02/23. PubMed PMID: 30786807; PubMed Central PMCID: PMCPMC6613837 [15] Montava-Garriga L, Singh F, Ball G, et al. Semi-automated quan titation of mitophagy in cells and tissues. Mech Ageing Dev. 2020;185:111196. Epub 2019/ 12/18. PubMed PMID: 31843465; PubMed Central PMCID: PMCPMC6961211 [16] Moskal N, Riccio V, Bashkurov M, et al. ROCK inhibitors upre gulate the neuroprotective Parkin-mediated mitophagy pathway. Nat Commun. 2020;11(1):88. Epub 2020/ 01/05. PubMed PMID: 31900402; PubMed Central PMCID: PMCPMC6941965 [17] Nisr RB, Shah DS, Ganley IG, et al. Proinflammatory NFkB signalling promotes mitochondrial dysfunction in skeletal muscle in response to cellular fuel overloading. Cell Mol Life Sci. 2019;76 (24):4887–4904. Epub 2019/ 05/19. PubMed PMID: 31101940; PubMed Central PMCID: PMCPMC6881256 [18] Prudovsky I, Carter D, Kacer D, et al. Tranexamic acid suppresses the release of mitochondrial DNA, protects the endothelial mono layer and enhances oxidative phosphorylation. J Cell Physiol. 2019;234(11):19121–19129. Epub 2019/ 04/04. PubMed PMID: 30941770; PubMed Central PMCID: PMCPMC6660401 [19] Rosignol I, Villarejo-Zori B, Teresak P, et al. The mito-QC repor ter for quantitative mitophagy assessment in primary retinal gang lion cells and experimental glaucoma models. Int J Mol Sci. 2020;21(5) Epub 2020/ 03/14. PubMed PMID: 32164182; PubMed Central PMCID: PMCPMC7084520 [20] Rusilowicz-Jones EV, Jardine J, Kallinos A, et al. USP30 sets a trigger threshold for PINK1-PARKIN amplification of mito chondrial ubiquitylation. Life Sci Alliance. 2020;3(8) Epub 2020/ 07/09. PubMed PMID: 32636217; PubMed Central PMCID: PMCPMC7362391. DOI: 10.26508/lsa.202000768 [21] Zhao JF, Rodger CE, Allen GFG, et al. HIF1alpha-dependent mitophagy facilitates cardiomyoblast differentiation. Cell Stress. 2020;4(5):99–113. Epub 2020/ 05/19. PubMed PMID: 32420530; PubMed Central PMCID: PMCPMC7212530. [22] Yamada T, Dawson TM, Yanagawa T, et al. SQSTM1/p62 pro motes mitochondrial ubiquitination independently of PINK1 and PRKN/parkin in mitophagy. Autophagy. 2019;15(11):2012–2018. Epub 2019/ 07/25. PubMed PMID: 31339428; PubMed Central PMCID: PMCPMC6844492 [23] Sun N, Yun J, Liu J, et al. Measuring in vivo mitophagy. Mol Cell. 2015;60(4):685–696. Epub 2015/ 11/10. PubMed PMID: 26549682; PubMed Central PMCID: PMCPMC4656081. [24] Eltokhi A, Kurpiers B, Pitzer C. Behavioral tests assessing neu ropsychiatric phenotypes in adolescent mice reveal strain- and sex-specific effects. Sci Rep. 2020;10(1):11263. Epub 2020/ 07/11. PubMed PMID: 32647155; PubMed Central PMCID: PMCPMC7347854. 4478 I. G. GANLEY ET AL. https://doi.org/10.1080/15548627.2021.1896924 https://doi.org/10.15252/embj.201899360 https://doi.org/10.1172/jci.insight.129760 https://doi.org/10.1172/jci.insight.129760 https://doi.org/10.1098/rsob.180108 https://doi.org/10.26508/lsa.202000768 [25] Farley SJ, McKay BM, Disterhoft JF, et al. Reevaluating hippocampus-dependent learning in FVB/N mice. Behav Neurosci. 2011;125(6):871–878. Epub 2011/ 11/30. PubMed PMID: 22122148; PubMed Central PMCID: PMCPMC3246014. [26] Pugh PL, Ahmed SF, Smith MI, et al. A behavioural character isation of the FVB/N mouse strain. Behav Brain Res. 2004;155 (2):283–289. Epub 2004/ 09/15. PubMed PMID: 15364488. [27] Mineur YS, Crusio WE. Behavioral and neuroanatomical charac terization of FVB/N inbred mice. Brain Res Bull. 2002;57 (1):41–47. Epub 2002/ 02/06. PubMed PMID: 11827736 [28] Chang B, Hawes NL, Hurd RE, et al. Retinal degeneration mutants in the mouse. Vision Res. 2002;42(4):517–525. Epub 2002/ 02/21. PubMed PMID: 11853768 [29] Gibb AA, McNally LA, Riggs DW, et al. FVB/NJ mice are a useful model for examining cardiac adaptations to treadmill exercise. Front Physiol. 2016;7:636. Epub 2017/ 01/10. PubMed PMID: 28066267; PubMed Central PMCID: PMCPMC5174104. [30] Singh S, Periasamy M, Bal NC. Strain-specific differences in muscle Ca(2+) transport and mitochondrial electron transport chain proteins between FVB/N and C57BL/6J mice. J Exp Biol. 2021; 224(Pt2). Epub 2020/ 12/04. PubMed PMID: 33268531; PubMed Central PMCID: PMCPMC7823165. DOI: 10.1242/ jeb.238634 [31] Shah AP, Siedlecka U, Gandhi A, et al. Genetic background affects function and intracellular calcium regulation of mouse hearts. Cardiovasc Res. 2010;87(4):683–693. Epub 2010/ 04/24. PubMed PMID: 20413651. [32] Enriquez JA. Mind your mouse strain. Nat Metab. 2019;1(1):5–7. Epub 2019/ 01/01. PubMed PMID: 32694816. [33] Wrighton PJ, Shwartz A, Heo JM, et al. Quantitative intravital imaging in zebrafish reveals in vivo dynamics of physiological-stress-induced mitophagy. J Cell Sci. 2021;134(4) Epub 2021/ 02/05. PubMed PMID: 33536245 [34] Cummings J, Feldman HH, Scheltens P. The “rights” of precision drug development for Alzheimer’s disease. Alzheimers Res Ther. 2019;11(1):76. Epub 2019/ 09/01. PubMed PMID: 31470905; PubMed Central PMCID: PMCPMC6717388 AUTOPHAGY 4479 https://doi.org/10.1242/jeb.238634 https://doi.org/10.1242/jeb.238634 Disclosure statement Funding References