Sladen, Paul Edward; (2021) Investigating disease mechanisms in autosomal dominant optic atrophy with retinal ganglion cells derived from induced pluripotent stem cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy in the UK, characterised by the preferential loss of retinal ganglion cells (RGCs) and progressive blindness. 60-70 % of DOA patients harbour mutations in the OPA1 gene, encoding a mitochondrial protein that regulates mitochondrial morphology, bioenergetics and mitochondrial DNA (mtDNA) quality. Currently, DOA has no therapeutic options and the mechanisms driving RGC degeneration are poorly understood. In this study, a biobank of induced pluripotent stem cells iPSCs (iPSCs) encompassing the clinical and genetic DOA spectrum was created using patient-derived OPA1 mutant fibroblast cell lines, and CRISPR/Cas9 gene editing to generate isogenic cell lines. RGC differentiation was optimized and characterised in 2D and 3D in vitro methods, demonstrating expression of RGC-associated genes including BRN3B and ISL1. OPA1 mutant iPSCs showed no differentiation deficit compared to wild-type control cell lines, exhibiting comparable expression of all relevant markers. 2D-RGCs demonstrated enrichment of neuronal associated markers, including ELAVL3 and TAU, when compared to 3D retinal organoids. Phenotypic analysis demonstrated significant deficits in respiration, ATP production and increased mtDNA mutation in fibroblasts, iPSCs and 2D-RGCs compared to isogenic controls. Characterisation of mitochondrial stress through induction of stress associated gene expression demonstrated significant levels of upregulation in iPSCs, however, 3D- and 2D-RGCs exhibited fewer upregulated genes indicating that mitochondrial stress may be a cell type specific response. Importantly, correction of patient-derived iPSCs restored mitochondrial homeostasis, demonstrating that restoration of WT OPA1 expression is able to mitigate mutant associated phenotypes. Thus, an OPA1 mutant iPSC biobank has been established encompassing the clinical disease spectrum, enabling effective in vitro modelling to establish RGC specific disease mechanisms. OPA1 mutant RGCs demonstrate significant reductions in mitochondrial homeostasis, including reduced bioenergetic output and mtDNA quality. This work provides a platform for further investigation of OPA1-mediated disease mechanisms and therapeutic design.

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