Abstract
Purpose :
Recessive Stargardt disease (STGD1) is an inherited maculopathy caused by mutations in the ABCA4 gene. In the retinal pigment epithelium (RPE), ABCA4 functions as a flippase to translocate retinaldehyde conjugated to phosphatidylethanolamine (PE) across endolysosomal membranes. Loss of ABCA4 results in RPE buildup of auto-fluorescent lipofuscin containing bisretinoids, lipids, and protein aggregates. PE is the second most abundant phospholipid in mammalian cells involved in membrane fusion. Interestingly, PE alone is a substrate for ABCA4, and required for lipidation of microtubule-associated protein light chain 3 (LC3) in the LC3-associated phagocytosis (LAP) pathway. Phagosomes marked by lipidated LC3 are degraded by lysosomes. Reduced LC3 levels in the RPE of Abca4-/- mice was previously reported. Here, we used iPSC-RPE from three STGD1 patients (H, J, and S) and normal (no ABCA4 mutations) to investigate the intersection of PE and LC3 in disease pathogenesis.
Methods :
PE distribution was visualized by confocal and super-resolution microscopy in iPSC-RPE using duramycin, a cyclic peptide that binds PE with high specificity. Lipid levels (PE and lyso-PE) were quantified by direct infusion-tandem mass spectrometry. LC3 levels were assessed by immunostaining. Outer-segment phagocytosis was evaluated by pulse-chase assay. Statistical significance was determined by Student’s t-test.
Results :
At 3-mo, normal iPSC-RPE showed clear PE labeling of plasma and internal membranes. In contrast, intracellular PE-aggregates and diminished plasma membrane labeling of PE is evidenced in STGD1 RPE (H, J, and S). At 7-mo, both PE and lyso-PE levels were ~80% higher in STGD1(H) vs normal RPE. All three STGD1 patient RPE cells showed reduced LC3 staining vs normal at 4-mo. Similar diminished LC3 profile is also observed in aged STGD1(H) RPE. Lastly, STGD1 RPE cells exhibit diminished outer-segment (OS) degradation vs normal.
Conclusions :
These studies provide a mechanistic explanation for endolysosomal impairment in STGD1 RPE cells. Dysfunctional ABCA4 in the STGD1 RPE causes intracellular PE accumulation. Further, changes in PE membrane composition in STGD1 RPE have negative consequences on LC3 lipidation and compromises endolysosomal efficiency in degrading OS material. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.