G.O.4 - Autosomal recessive mutations of GPR126 are responsible for severe arthrogryposis multiplex congenita

Abstract

ophthalmoplegia. The OPA1 protein is essential for normal mitochondrial fusion. In mouse DOA (such as OPA1Q285STOP), autophagy (recycling of spent cellular components) is dysregulated in retinal ganglion cells (RGCs), and mitophagy (mitochondrial recycling) has been implicated. Quantifying mitophagy is technically demanding. The hallmarks of mitophagy are organelles called autophagosomesis, co-localisating with, and then engulfing mitochondria. We validated and used two high throughput imaging systems [Imagestream (Amnis) and InCell analyser (GE)] to quantify mitophagy in fibroblasts. Co-localisation of mitochondria and autophagosomes was increased in fibroblasts in five patients from four families with severe OPA1 phenotypes indicating increased mitophagy. ImageStream also showed that basal mitophagy was increased when control cultures were depleted of Opa1 by siRNA. Western blotting confirmed increased basal autophagy and autophagic flux in the presence of activators. Increased mitochondrial fragmentation, mitophagy and failure of mitochondrial transport may together cause local depletion of mitochondria in critical regions of the affected neurons, such as axons or synapses. To investigate mitophagy in a DOA model we crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse harbouring red fluorescent mitochondria and green fluorescent autophagosomes. We showed increased co-localisation between mitochondria and autophagosomes in both cell types investigated, confirming increased mitophagy in this model. The role of mitophagy in mitochondrial disease remains controversial, but appears to be excessive and poorly selective in OPA1 mutants. We intend to use our model to identify drug modulators of mitophagy for treating OPA1 and other mitochondrial patients.