Abstract
Presentation Description :
Dominant optic atrophy (DOA) is the commonest inherited optic neuropathy with an estimated prevalence of at least 1 in 30,000. A majority of patients with DOA carry a pathogenic variant in the nuclear gene OPA1, which encodes for a dynamin-like GTPase protein that participates in mitochondrial inner membrane fusion, mitochondrial DNA maintenance and bioenergetic function. Vision loss in DOA is driven by the loss of retinal ganglion cells (RGCs) and optic nerve degeneration. Until recently, however, developing a model to study DOA pathophysiology using human RGCs has been limited by inadequate access to patient tissue samples. To address this, our lab has developed a bank of OPA1-variant patient-derived induced pluripotent stem cells (iPSCs) with CRISPR-corrected isogenic controls. These were differentiated into RGCs using a 42-day two-dimensional protocol prior to molecular and functional assessment. The iPSC-RGCs develop long axonal projections and form a complex neuronal network. They express mature neuronal and RGC-associated markers when assessed by immunocytochemistry and quantitative PCR. Further analysis with single-cell RNA sequencing has revealed a heterogenous population of neurons that express established RGC markers, such as BRN3B, SNCG and RBPMS. Using fluorescent calcium indicators, we have investigated the role of OPA1 variants in the buffering of cytosolic calcium in RGCs in response to the release of Ca2+ from the endoplasmic reticulum and stimulation by excitatory neurotransmitters, such as glutamate. The generation of reactive oxygen species, cellular antioxidant defence mechanisms and susceptibility to apoptosis in OPA1-mutant and isogenic control RGCs was also investigated. Collectively, our data demonstrate the utility of iPSC-RGCs as a model of DOA and provide important insight into the molecular and functional phenotypes caused by OPA1 mutations in RGCs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.