Retinoid recycling is impaired in Stargardt iPSC-derived RPE

Abstract

Purpose : Mutations in the ABCA4 gene are responsible for Stargardt disease (STGD). Previously, we demonstrated that ABCA4 is expressed in the retinal pigment epithelium (RPE), in addition to the photoreceptor outer segments (OS). We have proposed that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis. In this study, we used induced pluripotent stem cell (iPSC) derived RPE from a STGD patient with a heterozygous variant (c.3386G>T; p.R1129L) to test the role of ABCA4 in RPE cells and gain insights into the pathogenesis of STGD. This mutation resides in nucleotide binding domain 1 (NBD1) of ABCA4 and was reported to be associated with reduced protein yield and ATP-binding capacity.

Methods : Fibroblasts collected from a STGD patient and an unaffected human control were reprogrammed and differentiated into RPE cells using standard protocols. Cellular morphology was studied by light and electron microscopy. RPE-specific proteins were evaluated by immunocytochemistry and immunoblotting. Phagocytosis assay was done using bovine photoreceptor OS following a pulse-chase approach. Vitamin A uptake and recycling of retinoids were evaluated after incubating the cells with free all-trans-retinol (atROL) or rhodopsin-containing bovine photoreceptor OS. Retinoids were extracted into hexane from the media and cells and analyzed by HPLC.

Results : Morphologically, STGD iPSC-RPE cells were polarized and showed structural features similar of control cells. By immunocytochemistry, STGD and control iPSC-RPE cells expressed ABCA4, RPE65, and LRAT. Both control and STGD iPSC-RPE cells degraded ~60% of the internalized OS. Control and STGD iPSC-RPE cells processed the atROL comparably and released similar levels of 11-cis-retinaldehyde in the media. However, the STGD iPSC-RPE cells showed significantly lower level of all-trans-retinyl palmitate vs control cells, suggesting impaired recycling of retinaldehyde from the phagocytosed rhodopsin with reduced ABCA4-flippase activity due to the ABCA4 mutation.

Conclusions : (1) STGD iPSC-derived RPE cells undergo similar differentiation and maturation steps as control. (2) ABCA4 is expressed in the iPSC-derived RPE cells, validating this system to study the function of ABCA4 in the RPE. (3) STGD iPSC-RPE cells, carrying a mutation in the NBD1 of the ABCA4 protein, exhibit decreased recycling of the retinaldehyde from phagocytosed OS.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.