Retinal ganglion cells harboring the glaucoma OPTN(E50K) mutation exhibit neurodegenerative phenotypes when derived from pluripotent stem cell-derived three-dimensional retinal organoids

Abstract

Purpose : Glaucoma is one of the leading causes of blindness due to the progressive damage to retinal ganglion cells (RGCs), disrupting the connection between the eye and the brain. While many factors lead to the loss of RGCs in glaucoma, models for studying common mechanisms of glaucomatous neurodegeneration are instructive for understanding the loss of RGCs. Human pluripotent stem cells (hPSCs) allow for the study of human diseases in vitro, particularly those mechanisms resulting in neurodegeneration.

Methods : To establish hPSC models of glaucoma, CRISPR/Cas9 gene editing was used to study a severe glaucoma mutation in Optineurin (OPTN), along with corresponding isogenic controls. From these hPSCs, RGCs were differentiated for the analysis of neurodegenerative phenotypes associated with glaucoma. Initially, phenotypic changes including neurite remodeling were analyzed, as well as alterations to the autophagy pathway due to this mutation. Subsequently, RNA-seq analyses were performed to identify transcriptional changes in OPTN(E50K) RGCs. Additionally, whole cell patch clamp was performed to identify functional consequences of this mutation.

Results : Retinal organoids differentiated from these hPSCs self-organized in a manner that mimicked the temporal and spatial aspects of human retinal development. With prolonged culture, OPTN(E50K) RGCs downregulated the expression of key transcription factors. Moreover, OPTN(E50K) RGCs demonstrated retraction of neurites and increased functional excitability in comparison of isogenic controls, suggesting excitotoxicity may play a role in RGC neurodegeneration. RNA sequencing revealed the downregulation of autophagy associated pathways in OPTN(E50K) RGCs, with further investigation of these autophagy deficits revealing LC3 accumulation in the RGC layer of retinal organoids correlated with the expression of activated Caspase-3.

Conclusions : hPSC-derived RGCs demonstrated multiple neurodegenerative phenotypes due to the OPTN(E50K) mutation, with these phenotypes associated with functional changes to these RGCs. The results of this study will allow for the exploration of precise cellular pathways leading to RGC death in glaucoma, as well as develop novel translational approaches for glaucoma including pharmacological screening and cell replacement therapies.

This is a 2020 ARVO Annual Meeting abstract.