Abstract
Purpose :
Primary open angle glaucoma, regardless of IOP, leads to death of the retinal ganglion cells (RGCs), and ultimately, vision loss. The pathogenic mechanism of RGC loss is currently unknown. Herein, we investigated the role of oligomerization and aggregation of Optineurin (OPTN) in human donor tissue and in vitro cell culture.
Methods :
We analyzed OPTN oligomerization following oxidative stress in induced pluripotent stem cell derived RGCs and evaluated autophagy as a proxy marker for OPTN function. Oxidative stress was performed by culturing cells at physiological oxygen (5% O2, normoxia) and atmospheric oxygen (20% O2, hyperoxia) in a Biospherix hypoxia chamber for two weeks. Further, we used siRNA-mediated knockdown of OPTN to determine if reduced levels effected function. Using Alpha Fold 2 we predicted the structure of OPTN and its oligomeric forms, to determine if oligomerization blocks protein binding domains important for ubiquitination and autophagy. Finally, we evaluated OPTN oligomerization in human donor retinas with and without primary open angle glaucoma by Western blot, and aggregation in RGCs using immunohistochemistry.
Results :
Our results show that increased oxygen levels leads to a 100% increase in OPTN oligomerization. This leads to a 75% decrease in autophagy, as determined by LC3-II levels. Knockdown of OPTN using an siRNA approach resulted in 80% knockdown efficiency and a 150% decrease in autophagy. Alpha Fold 2 models of OPTN suggests that it is naturally a disordered protein composed of coiled regions. Upon oligomerization, the multiple OPTN proteins form a coiled-coil structure blocking most of the protein-binding domains. Western blot analysis of human POAG donor retina shows a 25-50% increase in oligomer formation with a 25-40% decrease in monomeric OPTN, relative to the age- and sex-matched normal controls. Further, immunohistochemistry reveals that OPTN has a diffuse cytoplasmic presence in normal human RGCs, suggesting monomeric OPTN, whereas the human POAG donors show aggregated OPTN with no diffuse staining.
Conclusions :
OPTN oligomerization and aggregation has the potential to be a disease causing mechanism in primary open angle glaucoma, similar to other neuropathies such as Alzheimer’s, Parkinson’s, and Huntington’s Disease. Although more functional information is needed regarding this process, it provides a potential therapeutic target without regard to the mutational status of the patient.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.