Loss of ABCA4 Function And Altered Lipidomic of Retinal Pigment Epithelium: A New Link To Stargardt Pathogenesis

Abstract

Purpose : Stargardt disease is inherited as an autosomal recessive trait caused by mutations in the ABCA4 gene, which lead to accelerated accumulation of lipofuscin in the retinal pigment epithelium (RPE) leading to RPE atrophy and photoreceptor cell death. There is currently inadequate information on the complexity of changes in lipid composition, homeostasis, metabolism, and lipid-mediated signaling of the RPE, leading to ABCA4 retinopathy. ABCA4 ^-/- mouse model has shown similar phenotypes of human ABCA4 retinopathy, particularly lipofuscin deposits in the RPE. To understand the role of ABCA4 in RPE lipid profile and its contribution to Stargardt disease pathogenesis, we used the ABCA4 ^-/- mouse model and developed Stargardt iPSC-derived RPE with complete loss of ABCA4 function as an in vitro disease model.

Methods : Lipid extraction was performed from collected RPE/choroid samples of 12-month-old wild type and ABCA4 ^-/- (C57BL/6J) mice. To validate the observed alterations in RPE/choroid's lipid profile obtained from ABCA4^-/- mice, fully characterized Stargardt iPSC (patient and two ABCA4^-/- iPSC lines) were differentiated into functionally validated RPE using a developmentally guided protocol. Stargardt- iRPE was cultured on semi-permeable membranes for six weeks to obtain a functionally mature and polarized monolayer tissue. For lipid extraction, cell medium and lysate from Stargardt- iRPE cells were collected after exposure to photoreceptor outer segment regimen (8 days). Lipid extracts were subjected to liquid chromatography–mass spectrometry (LC-MS/MS) for lipidomic analysis.

Results : LC-MS/MS analysis revealed altered RPE/choroid and Stargardt- iRPE lipid profile, including changes in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) molecular species and fatty acids and their derivatives with complete loss of ABCA4 function. RPE/choroid samples collected from ABCA4 ^-/- mice showed general trends of having less amounts of stable metabolites leading to elovanoid pathways and less abundance of VLC-PUFAs (FA32:6 n3 and FA34:6 n3) compared with the wild type.

Conclusions : Our results indicate that ABCA4 plays an essential role in RPE lipid profile, and mutations in the ABCA4 result in the changed RPE lipidomic contributing to Stargardt disease pathogenesis. This work provides an improved understanding of Stargardt disease mechanism.

This is a 2021 ARVO Annual Meeting abstract.