Glaucomatous Optineurin-E50K mutation causes human retinal ganglion cell death by causing mitophagy defects

Abstract

Purpose : Reducing intraocular pressure (IOP) remains the only treatment for Primary Open-Angle Glaucoma but damage can continue despite IOP reduction. Understanding the molecular mechanism underlying retinal ganglion cell (RGC) death remains elusive as a major challenge is the limited availability of human RGCs. Aim of this study is to use human stem cell differentiated RGCs (hRGCs) with a glaucoma causing mutation to investigate cellular mechanisms and identify potential therapeutic targets.

Methods : We used CRISPR engineered human pluripotent stem cell reporter lines to differentiate and immunopurify hRGCs. For glaucoma modeling, we used patient derived induced pluripotent stem cells (iPSCs) and CRISPR mutated human embryonic stem cells (hESCs) with the Optineurin-E50K (OPTN-E50K) mutation, a widely accepted cause for a subpopulation of normal tension glaucoma. We used western blot for measuring mitophagy by LC3B lipidation and qPCR for mitophagy gene expression. Mitochondrial metabolic defects were analyzed using the Seahorse analyzer and RGC viability/apoptosis was assessed by fluorescence/luminescence-based assay. Statistical comparison between two independent groups was done by student’s t-test and among multiple groups by One-way Anova with Dunnett’s correction.

Results : Our study shows the glaucomatous OPTN-E50K mutation causes mitophagy defects in hRGCs. OPTN belongs to the mitophagy adaptor protein group which shares LC3 interacting (LIR) domain and ubiquitin binding domain (UBD) through which the mitophagy process is initiated. E50K hRGCs showed activation of other adaptor genes such as P62 and NDP52, possibly as a response to overcome the mitophagy defects. Mitophagy defects lead to the accumulation of damaged mitochondria, causing mitochondrial metabolism defects and cellular toxicity. Indeed, we observed reduced mitochondrial activity in the E50K hRGCs when compared to wild-type (Wt) cells. Mitophagy defects should lead to cellular toxicity in E50K hRGCs in presence of mitochondrial stress. Indeed, we observed increased cell death for the E50K hRGCs in presence of the mitochondrial toxic drug CCCP.

Conclusions : Our study showed that the glaucomatous E50K mutation causes mitophagy defects leading to hRGC death and implicates mitophagy pathway proteins as possible therapeutic target for OPTN associated glaucoma.

This is a 2021 ARVO Annual Meeting abstract.