Validating INCell 1000 as a novel quantitative assay of mitophagy
Validating INCell 1000 as a novel quantitative assay of mitophagy: combining high content fluorescence analysis with mitochondrial DNA mutant load to identify novel pharmacological modulators of mitophagy.
The m.3243A>G mutation is one of the commonest mtDNA mutations, causing oligosymptomatic presbycusis or diabetes rather than mitochondrial myopathy in most cases. Mutant mtDNA co-exists with wild type (heteroplasmy), with the mutant load determining severity. Mitophagy (recycling of damaged mitochondria) is likely to be an important determinant of disease progression in such heteroplasmic mtDNA disease. Documenting mitophagy is technically demanding, however we have validated a technique using high throughput fluorescence microscopy and LC3-II (an autophagy marker) to examine changes in mitophagy. We reasoned that mitophagy might be apparent when heteroplasmic mtDNA mutants are lost during culture conditions requiring oxidative metabolism.
Metformin has been in therapeutic use since 1958 as a hypoglycemic agent, however only recently have the other potential applications of the drug been realised. Through activation of the AMPK signalling pathway, metformin is thought to have widespread uses as a neuro-protective, cardio-protective and tumour suppressive agent (Liu, Tang et al. 2014), (Johnson, Simpson et al. 2005). The novel uses for the drug led us to examine the effect on mitophagy.