Lysosome dependent mitophagy is critical for human RGC survival which is disrupted in glaucoma-associated Optineurin E50K mutation

Abstract

Purpose : Human retinal ganglion cells (hRGCs) are susceptible towards the mitochondrial gene mutations given their strong association with optic neuropathies such as optic atrophy, LHON and for a subset of glaucoma patients. Purpose of this study is to understand mitochondrial homeostasis pathway in hRGCs and cellular defects associated with the disease-causing mutations which may be critical for developing therapeutic strategy.

Methods : Here we have used human stem cell derived retinal ganglion cells to analyze mitochondrial degradation pathways and studied glaucoma associated Optineurin-E50K (OPTN^E50K) mutant for its effect on RGC viability, mitophagy and other cellular processes. OPTN functions as an adaptor for the mitophagy protein complex. We have used previously established BRN3B-P2A-tdTomato-P2A-THY1.2 reporter stem cell lines to study RGC degeneration. For cell death assay, we have developed a fluorescence plate reader-based assay which measures both RGC death and cellular apoptosis; for mitochondrial content, we have developed qPCR and flow-cytometry-based analysis. For OPTN associated glaucoma disease modeling we have used patient derived induced pluripotent stem cells (iPSCs) as well as CRISPR/Cas9 mediated OPTN^E50K mutagenesis in human embryonic stem cells (hESCs) followed by hRGC differentiation.

Results : Proteasomal and the lysosomal pathways are the two major pathways for maintaining cellular homeostasis and we first tested their role in maintaining cellular and mitochondrial homeostasis for hRGC survival. Using pharmacological inhibitors, we found endo-lysosomal pathway is critical for mitophagy and hRGC survival but not the proteasomal pathway. Furthermore, upon mitochondrial damage with the uncoupler CCCP, wild-type hRGCs degraded damaged mitochondria very efficiently while for OPTN^E50K such clearance was significantly reduced owing to increased cellular apoptosis. Measurements were done using 3-9 biological replicates for each condition and statistical analysis were done using standard error of mean (SEM) and student’s t-test.

Conclusions : Overall, our study suggests that the lysosome dependent mitophagy is critical for RGC survival and OPTN^E50K negatively affect such processes. These results may suggest the cause for RGC loss in the OPTN associated severe form of glaucoma.

This is a 2020 ARVO Annual Meeting abstract.