Differential Modes of Cell Death in Mouse Models of Fuchs Dystrophy Harboring Homozygous L450W and Q455K Mutations in the Col8a2 Gene

Abstract

Purpose: : Fuchs endothelial corneal dystrophy (FECD) is a leading cause of progressive vision loss, and the only effective treatment is corneal transplantation. Knock-in mice harboring homologous missense mutations, L450W or Q455K in the Col8a2 gene, closely recapitulate the disease and provide a useful animal model to study early disease pathogenesis and potential therapies.

Methods: : In vivo endothelial cell (EC) counts were imaged and analyzed by confocal microscopy. Ultrastructural analysis of ECs was performed using transmission electron microscopy (TEM). Quantitative (q)RT-PCR profiling of 91 autophagy-related genes was performed on total RNA from stripped ECs.

Results: : In vivo clinical confocal microscopy of Col8a2^L450W/L450W mice at 20, 40, and 80wks reveal a 10% (p<.05), 27% (p<.0001), and 41% (p<.0001) decrease in ECs, respectively, compared to age-matched WT mice. In comparison, Col8a2^Q455K/Q455K mice show a 27% (p<.0001), 41% (p<.0001), and 75% (p<.0001) decrease in ECs, respectively, compared to age-matched WT mice. TEM reveals dilated rough endoplasmic reticulum (RER) in both the Col8a2^L450W/L450W and Col8a2^Q455K/Q455K mice at 20wks; however, by 40wks, the dilated RER of the Col8a2^Q455K/Q455K mice persists while the Col8a2^L450W/L450W mice show significantly less RER, which is replaced by dilated vacuoles with partially degraded organelles inside. In addition, TEM of Col8a2^Q455K/Q455K mice reveals accumulation of abnormal mitochondria marked for degradation. qRT-PCR of Col8a2^Q455K/Q455K mice shows a 23.1-fold (p<.05) increase in Eif2ak3 levels and a 16.3-fold (p<.05) increase in mTOR. Contrastingly, profiling of Col8a2^L450W/L450W mice reveal a downward trend in Eif2ak3 and mTOR (p>.05) expression.

Conclusions: : Both Col8a2^L450W/L450W and Col8a2^Q455K/Q455K mice exhibit the hallmarks of FECD - progressive EC loss and increased numbers of guttae; however, the Col8a2^Q455K/Q455K mice have earlier disease onset and greater than double the cell death rate. Ultrastructural differences coupled with gene expression analysis suggest that impaired autophagy may play a role in the delayed turnover of stressed ER and mitochondria. Future studies testing mTOR inhibitors to stimulate autophagy may be a valuable therapeutic approach to restoring functional autophagy and delaying progressive EC death.

Keywords: cornea: endothelium • gene/expression • microscopy: electron microscopy