Consequences of OPTN mutation in HTM cells

Abstract

Purpose : In 2002, optineurin(OPTN) mutations were identified responsible for POAG. The OPTN protein contains an ubiquitin-binding domain(UBD) and an LC3-interacting region, which allows optineurin acting like a bridge to bring ubiquitinated cargos to the autophagosome via LC3. H486R is a mutation found in POAG patients and is located in the UBD region of OPTN. In this study, H486R of OPTN was mutated to investigate its functional impacts in human trabecular meshwork(TM-1) cells under elevated oxidative stress.

Methods : We established the TM-1 cells stably expressing H486R or Wild-type(WT) optineurin. WT and H486R cells were treated with or without 1 µM rotenone after knocking down the endogenous OPTN for two days. To evaluate oxidative stress levels, cells were incubated with H₂DCF-DA which is an intracellular ROS probe. The mean fluorescence of H₂DCF-DA represents intracellular oxidative stress. To quantify the number of damaged mitochondria, we used Mito-Tracker to measure the mitochondrial membrane potential. When mitochondria are damaged, the mitochondrial membrane would be disrupted which leads to leakage, resulting in a reduction of the Mito-Tracker fluorescence signal. PI staining was performed to investigate the apoptosis levels. Mean ± standard deviation of three independent experiments were analyzed by using t-test. P<0.05 was considered statistically significant.

Results : With or without rotenone treatment, H486R cells had significantly higher oxidative stress levels than WT cells(p<0.05). There was a 1.5 fold and 2.5 fold induction of intracellular oxidative stress in WT cells and H486R cells, respectively, after rotenone treatment(p<0.05). We also found H486R cells contained significantly more damaged mitochondria than WT cells after rotenone treatment(p<0.05). There was a 2.9 fold and 3.4 fold increment of damaged mitochondria in WT and H486R cells, respectively, after the rotenone treatment(p<0.05). After rotenone treatment, the apoptosis levels were significantly increased in both WT and H486R cells(p<0.05). Compared to the solvent control treatment, there was a 1.5 fold and 1.9 fold induction of apoptosis in WT-4 and H486R-2 cells, respectively, after the rotenone treatment(p<0.05).

Conclusions : Our results indicated H486R mutation in OPTN affected the cellular responses to oxidative stress damages in TM-1 cells. Further studies are needed to confirm what molecular functions of OPTN are impaired in the H486R OPTN expressing cells.

This is a 2020 ARVO Annual Meeting abstract.