Localization of ABCA4 in the Retinal Pigment Epithelium and its implications for Stargardt Disease

Abstract

Purpose : Recessive Stargardt disease (STGD1) is an inherited macular degeneration caused by mutations in ABCA4, a membrane protein thought to be exclusively expressed in photoreceptor (PR) outer-segment (OS) discs. Loss of ABCA4 results in retinal pigment epithelium (RPE) deposition of bisretinoid-lipofuscin and late-onset PR degeneration. Preliminary studies showed that ABCA4 is additionally expressed in the RPE. Here, we sought to further investigate the expression and function of ABCA4 in RPE cells.

Methods : ABCA4 transcript was identified by RNAscope in situ hybridization (ISH) assay in human donor eyes, in human fetal (hf) RPE cultured cells, and in wild-type (WT) and Abca4^-/- mouse eyes. ABCA4 protein was evaluated, by immunoblotting, immuno-electron microscopy (EM), and immunofluorescence (IF) in WT and Abca4^-/- mice, in Mertk^-/- mice which do not phagocytose PR-OS, and in hfRPE cells which never contact PRs. ABCA4 co-localization with endo-lysosomal markers was assessed by IF in mouse and human RPE sections. ABCA4 was functionally evaluated in a transgenic (Tg) mouse line that expresses ABCA4 in the RPE, but not in retina. Ocular bisretinoids were measured by HPLC. Retinal morphology, lipofuscin and autofluorescence were assessed by light, electron, and confocal microscopy, respectively.

Results : By ISH of human and WT mouse eyes, ABCA4 mRNA was clearly visualized in both PRs and RPE. ABCA4 mRNA was also detected in hfRPE cells. Notably, ABCA4 mRNA was absent in Abca4^-/- eyes. ABCA4 expression was confirmed in Mertk^-/- RPE and hfRPE cells by IF. Immuno-EM detection of ABCA4 in human RPE showed an intracellular membrane distribution. By IF, ABCA4 appears to co-localize with endo-lysosomal structures in human and murine RPE. Compared to Abca4^-/- mice, Tg mice with only RPE-expressed ABCA4 (on the Abca4^-/- background) had less RPE bisretinoid-lipofuscin and greater PR preservation, suggesting an active role for ABCA4 in the RPE.

Conclusions : ABCA4 is expressed in the RPE within endo-lysosomal membranes, where it may serve an important role in protecting PRs. Our studies suggest that in the RPE, ABCA4 performs a similar function as in PR-OS: to facilitate recycling of retinaldehydes released from rhodopsin proteolysis, and to prevent the buildup of toxic bisretinoids. This finding introduces a novel, cell-autonomous pathway in the pathophysiology of STGD1 with significant therapeutic implications.

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