Abstract
Purpose :
Our goal is to understand how loss of the membrane protein Slc4a11 produces Congenital Hereditary Endothelial Dystrophy. In the current study, we ask if autophagy flux and lysosomal function are affected in Slc4a11 KO cells and determine whether mitochondrial ROS is responsible for any dysfunctions.
Methods :
All experiments were conducted in 8 week old Slc4a11 WT and KO mice or in immortalized mouse WT and KO cell lines. Western Blot analyses were conducted using traditional (SDS-PAGE) or Simple Protein Wes® system. In order to quench mitochondrial ROS, cells were treated with 2mM MitoQ for 16 hours and animals were subject to i.p injections of 0.068mg on alternate days for four weeks.
Results :
In Slc4a11 KO tissue, we observed an increase in autophagy substrate P62 (2.5±0.5) and reduced lysosomal proteins; vATPase (0.2±0.08), Cathepsin B (0.1±0.02), Cathepsin D (0.1±0.01), TFEB (0.1±0.01) relative to WT. With MitoQ treatment, we observed a significant improvement in lysosomal protein expression and decrease in P62 levels. MitoQ treatment in Slc4a11 KO animals also showed improvement in two main disease characteristics, corneal endothelial cell density (20±2.2% improvement) and decreased corneal edema (35±1.9% reduction).
Conclusions :
We show that mitochondrial ROS induced lysosomal dysfunction in Slc4a11 KO corneal endothelia. MitoQ treatment improved lysosomal function, autophagy, decreased corneal edema and improved corneal endothelial cell density.
This is a 2021 ARVO Annual Meeting abstract.