Shepard, Zachary1,2; Greiner, Mark1,2; Skeie, Jess1,2; Eggleston, Tim1,2; Shevalye, Hanna2
1Carver College of Medicine, Department of Ophthalmology, University of Iowa; 2Iowa Lions Eye Bank
Purpose: Fuchs endothelial corneal dystrophy (FECD) is a progressive disease that causes premature death of corneal endothelial (CE) cells. Despite the global impact of FECD as the most common disorder of the corneal endothelium, relatively little is known about the mechanisms contributing to disease progression. Based on preliminary data demonstrating that iron-dependent lipid peroxidation mediates cell death in FECD, we hypothesized that FECD progression is mediated by alterations in ferritinophagy, a selective form of autophagy that contributes to ferritin degradation. This triggers labile iron overload, reactive oxygen species accumulation, lipid peroxidation, and cell death. The presence of ferritinophagy can be tracked through RNA and protein quantification of cellular markers NCOA4, LC3, and ferritin. We also hypothesized that ultraviolet-A (UV-A) light exposure, which has been demonstrated to contribute to both FECD progression and ferroptosis, participates in altered ferritinophagy.
Methods: We utilized control and FECD cells from both cultured immortal cell lines at the Iowa Lions Eye Bank. For human samples, we utilized patient derived DMEK peels (collected at time of FECD surgery) and healthy donor tissues. Cells were treated with various doses of UV-A and antimycin A (AMA) to induce oxidative damage. Protein and RNA markers of ferritinophagy were extracted and analyzed using PCR and microfluidics western blot to quantify NCOA4 and LC3 cellular levels. Additionally, treated cells were plated on coverslips, stained via immunohistochemistry, and analyzed using confocal microscopy to further determine cellular changes that may be associated with ferritinophagy in FECD.
Results: Immortalized cell lines affected with FECD demonstrated increased protein levels of the ferritinophagy markers NCOA4 and LC3 when compared to control cells, indicating that iron accumulation contributed to the cell death characteristic of FECD. This increase of NCOA4 protein levels in FECD was further validated in donor samples of diseased and healthy cells. UV-A exposure further increased the protein and mRNA levels of NCOA4 and LC3 present in FECD cell cultures, highlighting the effect of UV-A on ferritinophagy in FECD. Finally, on immunohistochemistry, ferritin and LC3 were noted to colocalize in a non-punctate pattern within the nuclei of cells with FECD.
Conclusions: Our results reinforce the role of ferritinophagy in FECD by demonstrating an increase of ferritinophagy markers NCOA4 and LC3 in FECD cells, both from immortalized cell lines and from donor samples. Additionally, this study provides a mechanism for the contribution of UV exposure to disease development, furthering the current understanding of how FECD contributes to cell death and vision loss. Lastly, based on our immunohistochemistry studies, our results indicate a potential mechanism that cells with FECD enact to protect against nuclear oxidative damage from Fenton reactions.
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