Abstract
Purpose :
Exposure of the retinal pigment epithelial cell line (ARPE19) to the oxidant sodium iodate (NaIO3) is a well-established in vitro model of retinal degeneration, including age related macular degeneration (AMD). Higher levels of iron in AMD retinas and a protective effect of an iron chelator has been reported before by Hadziahmetovic et al. The underlying mechanism of NaIO3-induced iron accumulation and retinal degeneration, however, remains unclear. Here, we explored the mechanism of iron accumulation in NaIO3-treated ARPE19 cells.
Methods :
ARPE19 cells were obtained from ATCC and used until passage 20. Cells were treated with 10 mM of NaIO3 for 24 h, and the lysates were evaluated for various proteins with specific antibodies by Western blotting (WB). Additionally, NaIO3-treated cells were co-immunostained with organelle-specific antibodies to confirm the localization of ferritin.
Results :
Immunoblotting of lysates from NaIO3 treated ARPE19 cells revealed increased expression of LC3II, a marker of impaired autophagosomal activity, and ferritin relative to untreated controls. Cathepsin-D, a lysosomal enzyme, was significantly reduced in NaIO3-treated cells. Immunocytochemistry revealed co-localization of ferritin with LAMP-1 in NaIO3-treated cells, indicating accumulation in lysosomes. Surprisingly, exosomal structures co-immunostaining for ferritin and LAMP-1 were released in the extracellular milieu.
Conclusions :
These data indicate that NaIO3 inhibits lysosomal degradation of ferritin by inhibiting cathepsin-D. Accumulated ferritin is subsequently released to the extracellular milieu in vesicular structures that react for the lysosomal marker LAMP-1. Thus, accumulation of iron and iron-mediated retinal toxicity by NaIO3 is mediated by accumulation of iron-rich ferritin and its release to neighboring cells. Since NaIO3 is used to induce retinal degeneration in mice, the role of ferritin and other substrates of cathepsin-D requires consideration.
This is a 2021 ARVO Annual Meeting abstract.