Abstract
Purpose :
Recent studies from Slc4a11-/- mice have identified glutamine-induced mitochondrial dysfunction[BJA1] as a significant contributor toward oxidative stress, impaired lysosomal function, aberrant autophagy, and cell death in this Congenital Hereditary Endothelial Dystrophy (CHED) model. Because lysosomes are derived from the endoplasmic reticulum (ER) – Golgi, we asked whether ER function is affected by the lack of Slc4a11.
Methods :
All experiments were conducted in 8 week old Slc4a11 WT and KO mice or in immortalized mouse WT and KO cell lines. Protein expressions were determined using the Simple Protein Wes® system. Real Time PCR analysis was conducted to amplify the transcripts of XBP1, unspliced-XBP1, spliced-XBP1, and β-actin. Fura-2AM was used to measure cytosolic calcium levels, and aggresomes were measured using a Proteostat Aggresome detection kit. In order to quench mitochondrial ROS, cells were treated with 2μM MitoQ for 16 hours.
Results :
In mouse Slc4a11-/- corneal endothelial tissue, we observed the presence of dilated ER and elevated expression of ER stress markers BIP and CHOP. Slc4a11 KO mouse corneal endothelial cells incubated with glutamine showed increased aggresome formation, BIP, and GADD, as well as less ER Ca2+ release as compared to WT. Treatment of Slc4a11 KO cells with the mitochondrial ROS quencher MitoQ restored ER Ca2+ release and relieved ER stress markers indicating that mitochondrial ROS induces ER stress in corneal endothelial cells.
Conclusions :
This study shows that in the presence of glutamine (in vivo or in vitro) the loss of Slc4a11 leads to ER stress that can be reversed by quenching mitochondrial ROS. Mitochondrial ROS also triggers lysosomal dysfunction with autophagy impairment in Slc4a11-/- corneal endothelial cells. Since lysosomes are derived from ER-Golgi, our findings suggest that ROS-induced ER stress is an important trigger for cellular dysfunction in the Slc4a11 KO model.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.