Abstract
Purpose :
Exfoliation syndrome (XFS) is an age-related systemic disease characterized by the deposition of large aggregated fibrillar material in the anterior segment of the eye. These aggregates are a major risk factor for the development of exfoliation glaucoma (XFG), a secondary open angle glaucoma, accounting for up to 50% of cases. Genome-Wide Association Studies identified that XFG is linked to variants in exon 1 of the lysyl oxidase-like 1 (LOXL1) gene. Here we investigated the functional role of LOXL1 protein variants in XFG disease pathology.
Methods :
Two nonsynonymous single nucleotide polymorphisms of LOXL1, Arg141Leu (rs1048661; G>T) and Gly153Asp (rs3825942; G>A) were used in this study. The propensity of LOXL1 to misfold/aggregate was studied using a b-lactamase tripartite fusion vector expressing different regions of LOXL1 in E coli and native-PAGE western blotting from cell lysates of primary human tenon fibroblast cells (hTFs) isolated from no glaucoma controls (NG) and XFG patients undergoing trabeculectomy. ER stress related experiments were performed using overexpression of LOXL1 risk variants and LOXL2 as control using lentiviral transductions in human corneal HTERT fibroblasts. The bioenergetic profiles of LOXL1 over-expressing cells and primary hTFs were measured using Seahorse XFe96. Oxygen consumption rate (OCR, mitochondrial respiration) and extracellular acidification rate (ECAR, glycolysis) were analyzed.
Results :
The N-terminus of LOXL1 was found to be an intrinsically disordered region (IDR) using a b-lactamase reporter assay. Native PAGE western blotting with primary hTFs demonstrated that LOXL1 protein is significantly aggregated (p=0.0243) compared to NG controls. LOXL1 double high-risk variant (R141/G153) over-expression induced ER stress as measured using BiP (p=0.0776) and oxidative stress proteins PDI (p=0.0491) and ERO1a (p=0.0372) compared to LOXL2 control. Bioenergetics experiments revealed that mitochondrial function parameters such as maximal respiration (p=0.0453), ATP production (p=0.0481) and spare respiratory capacity (p=0.036) are reduced in XFG cells compared to NG controls.
Conclusions :
LOXL1 has an unstructured N-terminal domain and aggregates at a higher propensity in XFG primary cells. In addition, LOXL1 variants induce ER-oxidative stress and impairs mitochondrial cellular bioenergetics, suggesting that LOXL1 protein is a causative factor in XFG pathogenesis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.