A potent LXR (liver X receptor) agonist can restore lysosome-mediated clearance in RPE cells from a mouse model with AMD-like phenotype

Abstract

Purpose : LXR, a member of the nuclear receptor family of transcription factors regulates cholesterol, fatty acids and glucose homeostasis. There has been growing interest in LXR agonists as therapeutic targets for the treatment of human diseases since such agonists have shown promise in animal models of atherosclerosis, diabetes and Alzheimer’s disease. Here we investigated the effects of novel LXR agonists in the rescue of the lysosome clearance process in RPE cells from a mouse model with an age-related macular degeneration (AMD)-like phenotype.

Methods : Primary culture of RPE cells from Cryba1^fl/fl (WT) and Cryba1 KO (lacks the Cryba1 gene, which encodes βA3/A1-crystalin) mice with AMD-like phenotype were treated with LXR agonists. MTT assays were carried out to test the viability of Cryba1^fl/fl mouse RPE cells after 24-hour treatment with different agonists. Autophagy flux (the conversion of LC3-I to LC3-II) and p62 levels were determined by western analysis using antibodies to LC3 (for autophagy flux) and p62. LC3-flux ratios for the floxed, rescued, and KO RPE cells were determined in the presence and absence of chloroquine. Cryba1^fl/fl and Cryba1 cKO (RPE specific KO) mice were injected intravitreally with DMSO (vehicle) or LXR agonists; RPE flatmounts were analyzed by staining with phallodin, DAPI, and antibody to RPE65.

Results : An LXR agonist (RO4897308-000-007) was used for both in vitro autophagy studies and for in vivo rescue experiments at concentrations determined by the MTT assay. Western data showed increased autophagy flux and decreased levels of p62/SQSTM1 in primary RPE cells from Cryba1 KO mice that were treated with the agonist compared to those treated with vehicle only. Our pilot data showed that RPE flatmounts from Cryba1 cKO mice RPE flat mounts had reduced levels of RPE65 (vehicle only). However, mice injected with RO4897308-000-007 twice a week for 3 weeks following concentration determined from cell viability assays exhibit increased RPE65 expression.

Conclusions :
Our studies indicate that LXR agonists can act to protect or restore the lysosome-mediated clearance process in RPE cells. Lysosomal efficiency declines with age, a decline implicated in age-related diseases such as Parkinson’s and Huntington’s and more recently, AMD. We postulate that LXR agonists may offer an attractive strategy for prevention or delay of AMD progression.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.