Functions of protein clearance pathways in corneal endothelial cells

Abstract

Purpose : Corneal endothelial cells are susceptible to increased oxidative stress and TGF-b signals. These stressors are causal in Fuchs Endothelial Corneal Dystrophy (FECD), a blinding disease with a global prevalence of 3-11%. In this proposal, we identified the relationship between the known stressors of corneal endothelium and their effects on protein clearance pathways. We also evaluated the impact of these treatments in causing cellular dysfunctions associated with FECD.

Methods : We performed Optical Coherence Tomography and in-vivo confocal microscopy of the cornea to assess corneal thickness and cell densities in 5-week and 20-week-old Col8a2 Q455K mice. We performed ex-vivo staining of the corneas to measure autophagy and proteasomal activities and nitrotyrosine staining to evaluate the levels of oxidative stress. For in-vitro experiments, bovine primary corneal endothelial cells (BCECs) in culture were treated with reactive oxygen species (ROS) inducer, proteasome, and autophagy activators and inhibitors for 24 hours. We evaluated protein expression changes using JESS immunoassay. Our analysis used antibodies against markers for protein clearance pathways, TGF-b signaling, endothelial-to-mesenchymal transition (EndoMT), ER stress, and apoptosis.

Results : In the 20-week-old Col8a2 Q455K animals, a decrease in corneal endothelial cell density and elevation in guttae numbers (Fig.1A, B, C) coincided with the decreased proteasomal activities (Fig.2) but elevated autophagy. Treatments with proteasomal inhibitor but not activator increased EndoMT markers - Snail (3.5-fold), and Zeb (0.5-fold), Apoptosis marker - cleaved caspase 3 (22-fold), and ER stress markers – Bip (0.8-fold), compared to the non-treated BCEC controls (Fig.3). TBHP, a known ROS inducer, also produced similar effects on primary corneal endothelial cells. Autophagy activation showed no significant changes to the protein expressions mentioned above.

Conclusions : Our data indicates that decreased proteasomal activities can lead to corneal endothelial dysfunctions similar to those observed in FECD. Future studies will determine if the activation of proteasomal functions can curb the phenotypic progressions.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1: Representative images and quantification of cell numbers and guttae.
Figure 2: Chymotrypsin activity in 5- and 20-week-old Col8a2 Q455K mice.
Figure 3: EndoMT, ER stress and Apoptosis markers in proteasomal inhibitor-treated BCEC.