Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy

Abstract

Purpose The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice. Methods Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ. Results Even though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss. Conclusions Mitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.

Rescue of the Congenital Hereditary Endothelial Dystrophy Mouse Model by Adeno-Associated Virus–Mediated Slc4a11 Replacement

“…Oxidative stress is likely the cause of the altered gene expression and paracellular permeability since quenching ROS can reduce corneal edema 6,29 . Consistent with this notion, we found that reintroduction of Slc4a11 reduces mitochondrial superoxide levels within the endothelium.…”

The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling

“…Therefore, the KO phenotype recapitulates CHED and indicates that the loss of SLC4A11 function is sufficient to cause endothelial dystrophy. Interestingly, oxidative stress attributable to the loss of SLC4A11 causes lysosomal and autophagy dysfunction [ 47 ], which could also be a consequence of ER stress. As described below in “SLC4A11 is a Mitochondrial Uncoupler”, the major source of oxidative stress in the SLC4A11 KO is from mitochondrial dysfunction.…”

“…In turn, this could produce ER stress and protein misfolding. If performed early, before there is significant cell loss and dysfunction, the mouse CHED model can be partially reversed by the delivery of the wild-type gene [ 48 ], or the systemic delivery of the antioxidant MitoQ that targets mitochondrial superoxide [ 47 ].…”

“…Damaged mitochondria will prompt mitophagy, part of the general autophagy system. Shyam et al [ 47 ] found that autophagy was activated in SLC4A11 KO MCEC as well as KO mouse endothelial tissue, relative to WT cells and fresh endothelial tissue. However, autophagy flux was aberrant due to reduced mass and poor functioning of lysosomes, the last step in the autophagy sequence where materials are degraded.…”

“…All of the SLC4A11 KO models found auditory deficits, leading to dysfunctional brain response and vestibular-evoked potential waveforms. Vestibular fibrocytes showed intracellular vacuolation [ 32 ] reminiscent of those seen in corneal endothelium [ 35 , 47 ]. Whether this is due to dysfunctional autophagy or lysosome production remains to be examined.…”