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

Alan Diot, Alex Hinks-Roberts, Tiffany Lodge, Chunyan Liao, Eszter Dombi, Karl Morten, Stefen Brady, Carl Fratter, Janet Carver, Rebecca Muir, Ryan Davis, Charlotte J Green, Iain Johnston, David Hilton-Jones, Carolyn Sue, Heather Mortiboys, Joanna Poulton

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)

Abstract

Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses.

Original languageEnglish
Pages (from-to)24-35
Number of pages12
JournalPharmacological Research
Volume100
Early online date18 Jul 2015
DOIs
Publication statusPublished - Oct 2015

Keywords

  • Mitophagy
  • Autophagy
  • Mitochondrial DNA
  • Metformin
  • Aging

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