In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Overview

Journal J Vis Exp

Specialties Biology
General Medicine

Date 2017 Dec 30

PMID 29286376

Citations 17

Authors

Evandro F Fang

Konstantinos Palikaras

Nuo Sun

Elayne M Fivenson

Ryan D Spangler

Jesse S Kerr

Stephanie A Cordonnier

Yujun Hou

Eszter Dombi

Henok Kassahun

Nektarios Tavernarakis

Joanna Poulton

Hilde Nilsen

Vilhelm A Bohr

Affiliations

Soon will be listed here.

Abstract

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Citing Articles

Circadian rhythms in stem cells and their therapeutic potential.

Putthanbut N, Su P, Lee J, Borlongan C Stem Cell Res Ther. 2025; 16(1):85.

PMID: 39988679 PMC: 11849187. DOI: 10.1186/s13287-025-04178-9.

Phosphorylation of Optineurin by protein kinase D regulates Parkin-dependent mitophagy.

Weil R, Laplantine E, Attailia M, Oudin A, Curic S, Yokota A iScience. 2024; 27(12):111384.

PMID: 39669425 PMC: 11634986. DOI: 10.1016/j.isci.2024.111384.

Spautin-1 promotes PINK1-PRKN-dependent mitophagy and improves associative learning capability in an alzheimer disease animal model.

Yi J, Wang H, Lu G, Zhang H, Wang L, Li Z Autophagy. 2024; 20(12):2655-2676.

PMID: 39051473 PMC: 11587853. DOI: 10.1080/15548627.2024.2383145.

Combined use of multiparametric high-content-screening and in vitro circadian reporter assays in neurotoxicity evaluation.

Park Y, Kang H, Kang S, Ku H, Zarbl H, Fang M Arch Toxicol. 2024; 98(5):1485-1498.

PMID: 38483585 PMC: 10965668. DOI: 10.1007/s00204-024-03686-6.

Mitochondrial-Derived Vesicles: The Good, the Bad, and the Ugly.

Picca A, Guerra F, Calvani R, Coelho-Junior H, Landi F, Bucci C Int J Mol Sci. 2023; 24(18).

PMID: 37762138 PMC: 10531235. DOI: 10.3390/ijms241813835.

References

Palikaras K, Lionaki E, Tavernarakis N . Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature. 2015; 521(7553):525-8. DOI: 10.1038/nature14300. View

Schiavi A, Maglioni S, Palikaras K, Shaik A, Strappazzon F, Brinkmann V . Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans. Curr Biol. 2015; 25(14):1810-22. DOI: 10.1016/j.cub.2015.05.059. View

Sun N, Malide D, Liu J, Rovira I, Combs C, Finkel T . A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima. Nat Protoc. 2017; 12(8):1576-1587. DOI: 10.1038/nprot.2017.060. View

Kerr J, Adriaanse B, Greig N, Mattson M, Cader M, Bohr V . Mitophagy and Alzheimer's Disease: Cellular and Molecular Mechanisms. Trends Neurosci. 2017; 40(3):151-166. PMC: 5341618. DOI: 10.1016/j.tins.2017.01.002. View

Shadel G, Horvath T . Mitochondrial ROS signaling in organismal homeostasis. Cell. 2015; 163(3):560-9. PMC: 4634671. DOI: 10.1016/j.cell.2015.10.001. View

Sargsyan A, Cai J, Fandino L, Labasky M, Forostyan T, Colosimo L . Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor. Sci Rep. 2015; 5:12397. PMC: 4517063. DOI: 10.1038/srep12397. View

Rosado C, Mijaljica D, Hatzinisiriou I, Prescott M, Devenish R . Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast. Autophagy. 2007; 4(2):205-13. DOI: 10.4161/auto.5331. View

Rubinsztein D, Shpilka T, Elazar Z . Mechanisms of autophagosome biogenesis. Curr Biol. 2012; 22(1):R29-34. DOI: 10.1016/j.cub.2011.11.034. View

McWilliams T, Prescott A, Allen G, Tamjar J, Munson M, Thomson C . mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J Cell Biol. 2016; 214(3):333-45. PMC: 4970326. DOI: 10.1083/jcb.201603039. View

10.

Fang E, Scheibye-Knudsen M, Brace L, Kassahun H, SenGupta T, Nilsen H . Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction. Cell. 2014; 157(4):882-896. PMC: 4625837. DOI: 10.1016/j.cell.2014.03.026. View

11.

Lazarou M, Sliter D, Kane L, Sarraf S, Wang C, Burman J . The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature. 2015; 524(7565):309-314. PMC: 5018156. DOI: 10.1038/nature14893. View

12.

Diot A, Hinks-Roberts A, Lodge T, Liao C, Dombi E, Morten K . A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA. Pharmacol Res. 2015; 100:24-35. DOI: 10.1016/j.phrs.2015.07.014. View

13.

Eiyama A, Okamoto K . Assays for Mitophagy in Yeast. Methods Mol Biol. 2017; 1567:337-347. DOI: 10.1007/978-1-4939-6824-4_20. View

14.

Menzies F, Fleming A, Rubinsztein D . Compromised autophagy and neurodegenerative diseases. Nat Rev Neurosci. 2015; 16(6):345-57. DOI: 10.1038/nrn3961. View

15.

Sun N, Youle R, Finkel T . The Mitochondrial Basis of Aging. Mol Cell. 2016; 61(5):654-666. PMC: 4779179. DOI: 10.1016/j.molcel.2016.01.028. View

16.

Fang E, Scheibye-Knudsen M, Chua K, Mattson M, Croteau D, Bohr V . Nuclear DNA damage signalling to mitochondria in ageing. Nat Rev Mol Cell Biol. 2016; 17(5):308-21. PMC: 5161407. DOI: 10.1038/nrm.2016.14. View

17.

Katayama H, Kogure T, Mizushima N, Yoshimori T, Miyawaki A . A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol. 2011; 18(8):1042-52. DOI: 10.1016/j.chembiol.2011.05.013. View

18.

Palikaras K, Tavernarakis N . Assessing Mitochondrial Selective Autophagy in the Nematode Caenorhabditis elegans. Methods Mol Biol. 2017; 1567:349-361. DOI: 10.1007/978-1-4939-6824-4_21. View

19.

Sun N, Yun J, Liu J, Malide D, Liu C, Rovira I . Measuring In Vivo Mitophagy. Mol Cell. 2015; 60(4):685-96. PMC: 4656081. DOI: 10.1016/j.molcel.2015.10.009. View

20.

Menzies F, Moreau K, Puri C, Renna M, Rubinsztein D . Measurement of autophagic activity in mammalian cells. Curr Protoc Cell Biol. 2012; Chapter 15:Unit 15.16. DOI: 10.1002/0471143030.cb1516s54. View