title: 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.
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.
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Alan
authors.initials: A
authors.lastname: Diot
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Hinks-Roberts
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Lodge
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Liao
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Eszter
authors.initials: E
authors.lastname: Dombi
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Karl
authors.initials: K
authors.lastname: Morten
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Stefen
authors.initials: S
authors.lastname: Brady
authors.affiliation: Oxford Medical Genetics Laboratory, Oxford University Hospitals NHS Trust, Churchill Hospital, Oxford, UK.
authors.forename: Carl
authors.initials: C
authors.lastname: Fratter
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Janet
authors.initials: J
authors.lastname: Carver
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Muir
authors.affiliation: Department of Neurogenetics, Kolling Institute of Medical Research, University of Sydney and Royal North Shore Hospital, Sydney, Australia.
authors.forename: Ryan
authors.initials: R
authors.lastname: Davis
authors.affiliation: Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK.
authors.forename: Charlotte J
authors.initials: CJ
authors.lastname: Green
authors.affiliation: Department of Mathematics, Imperial College London, London, UK.
authors.forename: Iain
authors.initials: I
authors.lastname: Johnston
authors.affiliation: Department of Clinical Neurology, John Radcliffe Hospital, Oxford, UK.
authors.forename: David
authors.initials: D
authors.lastname: Hilton-Jones
authors.affiliation: Department of Neurogenetics, Kolling Institute of Medical Research, University of Sydney and Royal North Shore Hospital, Sydney, Australia.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Sue
authors.affiliation: Department of Neuroscience, Sheffield Institute of Translational Neuroscience, University of Sheffield, Sheffield, UK.
authors.forename: Heather
authors.initials: H
authors.lastname: Mortiboys
authors.affiliation: Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford, UK. Electronic address: Joanna.poulton@obs-gyn.ox.ac.uk.
authors.forename: Joanna
authors.initials: J
authors.lastname: Poulton
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D001681:Biological Assay / Q000379:methods*; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008687:Metformin / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D063306:Mitophagy / Q000187:drug effects / Q000502:physiology; D055815:Young Adult
chemical_list: D004272:DNA, Mitochondrial; D008687:Metformin
keywords: Aging; Autophagy; Metformin; Mitochondrial DNA; Mitophagy; PubChem CID: 3-MA 1673; PubChem CID: Bafilomycin A1 2287; PubChem CID: Chloroquine 2719; PubChem CID: Colchicine 6167; PubChem CID: E64d 65663; PubChem CID: Metformin 4091; PubChem CID: Pepstatin A 16218527; PubChem CID: Phenanthroline 1318; PubChem CID: Rapamycin 5284616; PubChem CID: Trehalose 7427