Intracellular Localization of Optineurin in Response to Oxidative Stress in Rgc-5 Cells

Abstract

Purpose:: A role for optineurin has been suggested in the neuroprotection of cells against insults, including oxidative stress. In fibroblasts, cytoplasmic optineurin has been reported to translocate to the nucleus following stress. Disruption of this nuclear translocation augments the oxidative stress-induced cell death. Our previous results demonstrated the high expression of optineurin in retinal ganglion cells (RGCs) of the rat retina and in the RGC-5 cell line, suggesting the possibility of a similar role for optineurin in RGCs.

Methods:: RGC-5 cells were cultured in DMEM/F12 with 10% FBS and pen/strept. Prior to treatment, cells were differentiated for 24 hours with 1 µM staurosporine. Differentiated RGC-5 cells were treated with H₂O₂ for 24 hours. Reactive oxygen species production was measured using an H₂DCF fluorescence assay following H₂O₂ treatment. Cell viability was measured using the MTT assay. Immunocytochemistry was performed to examine the expression of optineurin in RGC-5 cells. Nuclear translocation of the optineurin protein was examined with immunocytohemistry using antisera against optineurin and the nuclear dye Yo-Pro-1 and antisera directed against optineurin and the Golgi protein TGN-1. Western immunoblot analysis was also performed on nuclear and cytoplasmic fractions of H₂O₂ treated RGC-5 cells to examine the nuclear translocation of optineurin due to oxidative stress.

Results:: An EC₅₀ of 9.5x10^-4 M H₂O₂ was determined by measuring RGC-5 cell viability using the MTT assay after 24-hour exposure to increasing concentrations of H₂O₂. H₂DCF fluorescence increased in a concentration dependent manner after exposure to H₂O₂. Immunocytochemical analysis determined if there was an H₂O₂ concentration-dependent nuclear translocation of optineurin from the Golgi apparatus in the cytosol to the nucleus of differentiated RGC-5 cells. Western immunoblot analysis was used to examine a shift in optineurin protein expression from the cytosolic protein fraction to the nuclear protein fraction following treatment of RGC-5 cells with H₂O₂.

Conclusions:: Differentiated RGC-5 cells are susceptible to H₂O₂ in a concentration-dependent manner and result in release of reactive oxygen species and cell death. In response to H₂O₂-mediated stress, optineurin can translocate from the cytosol to the nucleus. Similar to those previously reported in other cell types, including embryonic fibroblast cells and this suggests a mechanism for the neuroprotective functions of optineurin. These results provide evidence for an important role for optineurin on RGC survival.

Keywords: cell survival • oxidation/oxidative or free radical damage • protective mechanisms