Abstract
Purpose :
Reduction of intraocular pressure (IOP) remains the only treatment option for Primary Open-Angle Glaucoma. However, sufficient lowering of IOP is not always possible and not safe, and even with severe IOP lowering retinal ganglion cells (RGCs) continue to die. RGCs are highly energy demanding and disruption of mitochondrial homeostasis is often associated with optic neuropathies including glaucoma. Degradation of damaged mitochondria by lysosomes (mitophagy) is key for maintaining mitochondrial homeostasis which remains unexplored in the RGCs. Of note, mutation in the key mitophagy player Optineurin (OPTN-E50K) found among severe form of normal-tension glaucoma (NTG) patients.
Methods :
We used CRISPR engineered human pluripotent stem cell reporter lines and patient derived induced pluripotent stem cells (iPSCs) with E50K mutation to differentiate and immunopurify hRGCs. We used CCCP, a potent mitochondrial membrane uncoupler for inducing damage. We used qPCR for mitophagy gene expression, western blots for the measurement mitophagy master regulator LC3B activation, Parkin/Pink and OPTN activation in hRGCs.
Results :
Our results show in hRGCs mitophagy occur through the adaptor proteins which share a LC3-interacting and a ubiquitin-binding domain through which the mitophagy process is initiated. P62, NDP52, NBR1, and OPTN are among the mitophagy adaptor proteins. We found under mitochondrial damage, Parkin/Pink activation and corresponding LC3B and OPTN-E50K activations are defective. These suggest mitophagy defects in the glaucomatous E50K hRGCs with potential disruption of the mitochondrial homeostasis.
Conclusions :
Our study showed that the glaucomatous E50K mutation causes mitophagy defects as a potential source for hRGC death and implicates rescuing mitophagy defects as possible therapeutic strategy for glaucoma neuroprotection.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.