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Validating the RedMIT/GFP-LC3 mouse model by studying mitophagy in Autosomal Dominant Optic Atrophy due to the OPA1Q285STOP mutation

Diot, A, Agnew, T, Sanderson, J, Liao, C, Carver, J, Neves, R, Gupta, R, Guo, Y, Waters, Caroline, Seto, Sharon, Daniels, M, Dombi, E, Lodge, T, Morten, K, Williams, S, Enver, T, Iborra, FJ, Votruba, Marcela and Poulton, J 2018. Validating the RedMIT/GFP-LC3 mouse model by studying mitophagy in Autosomal Dominant Optic Atrophy due to the OPA1Q285STOP mutation. Frontiers in Cell and Developmental Biology 6 , 103. 10.3389/fcell.2018.00103

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Abstract

Background Autosomal dominant optic atrophy (ADOA) is usually caused by mutations in the essential gene, OPA1. This encodes a ubiquitous protein involved in mitochondrial dynamics, hence tissue specificity is not understood. Dysregulated mitophagy (mitochondria recycling) is implicated in ADOA, being increased in OPA1 patient fibroblasts. Furthermore, autophagy may be increased in retinal ganglion cells of the OPA1Q285STOP mouse model. Aims We developed a mouse model for studying mitochondrial dynamics in order to investigate mitophagy in ADOA. Methods We crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse, harboring red fluorescent mitochondria and green fluorescent autophagosomes. Colocalization between mitochondria and autophagosomes, the hallmark of mitophagy, was quantified in fluorescently labelled organelles in primary cell cultures, using two high throughput imaging methods (Imagestream (Amnis) and IN Cell Analyzer 1000 (GE Healthcare Life Sciences)). We studied colocalisation between mitochondria and autophagosomes in fixed sections using confocal microscopy. Results We validated our imaging methods for RedMIT/GFP-LC3 mouse cells, showing that colocalization of red fluorescent mitochondria and green fluorescent autophagosomes is a useful indicator of mitophagy. We showed that colocalization increases when lysosomal processing is impaired. Further, colocalization of mitochondrial fragments and autophagosomes is increased in cultures from the OPA1Q285STOP/RedMIT/GFP-LC3 mice compared to for RedMIT/GFP-LC3 control mouse cells that were wild type for OPA1. This was apparent in both mouse embryonic fibroblasts using IN Cell 1000 and in splenocytes using ImageStream imaging flow cytometer (Amnis). We confirmed that this represents increased mitophagic flux using lysosomal inhibitors. We also used microscopy to investigate the level of mitophagy in the retina from the OPA1Q285STOP/RedMIT/GFP-LC3 mice and the RedMIT/GFP-LC3 control mice. However, the expression levels of endogenous fluorescent proteins and the image signal-to-noise ratios precluded the detection of colocalisation so we were unable to show any difference in colocalization between these mice.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Optometry and Vision Sciences
Publisher: Frontiers Media
ISSN: 2296-634X
Date of First Compliant Deposit: 24 August 2018
Date of Acceptance: 13 August 2018
Last Modified: 28 Jun 2019 10:08
URI: http://orca.cf.ac.uk/id/eprint/114357

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