Fig 2: ABCA4 is expressed in the RPE of RPE-Abca4-Tg/Abca4-/- mice. (A) Representative immunoblots of retina and RPE homogenates from BALB/c, Abca4-/-, and RPE-Abca4-Tg/Abca4-/- mice (all albino) reacted with antisera against ABCA4 or a-tubulin. Total protein load was 10 µg for neural retina and 25 µg for RPE/eyecup homogenates. (B) Levels of ABCA4 protein in Abca4-/- and RPE-Abca4-Tg/Abca4-/- homogenates were normalized to a-tubulin and presented as relative to wild-type BALB/c levels; n = 7 6-mo-old mice per group. (C) Representative confocal images of retinal sections from BALB/c (Left), Abca4-/- (Center), and RPE-Abca4-Tg/Abca4-/- (Right) mice. ABCA4 immunoreactivity (red) in the RPE-Abca4-Tg/Abca4-/- shows specificity mainly for RPE. The OS layer of both Abca4-/- and RPE-Abca4-Tg/Abca4-/- mice is not stained by the ABCA4 antibody. DAPI nuclear staining is shown in blue. (Scale bars, 10 µm.) n = 3 1-y-old mice per group.

Fig 3: Photoreceptors are preserved in RPE-Abca4-Tg/Abca4-/- vs. Abca4-/- mice. (A) Representative retina images from 1-y-old albino mice acquired by light microscopy. (Scale bars, 20 µm.) (B) Total numbers of photoreceptor nuclei were counted per 100-µm2 cell area. Note the increased number of cells in the ONL of RPE-Abca4-Tg/Abca4-/- mice compared with Abca4-/- mice indicating partial rescue of photoreceptor degeneration. Data are presented as mean ± SD; n = 5–9 mice per group; RPE-Abca4-Tg/Abca4-/- vs. Abca4-/-, *P = 0.0319; Abca4-/- vs. BALB/c, **P < 0.0001; and RPE-Abca4-Tg/Abca4-/- vs. BALB/c, P = 0.0061.

Fig 4: (A–C) Abca4 mRNA and protein is expressed in RPE cells. In situ hybridization using the RNAscope assay with an Abca4-specific probe on human cadaveric ocular sections (A), mouse retina sections (B), and hfRPE cells in culture (C). Note the intense chromogenic reactivity (red punctate staining, indicated by the black arrows) for Abca4 mRNA in outer nuclear layer (ONL) and inner segments (IS) of the photoreceptor cells and in RPE cells of human (A) and wild-type BALB/c sections (B, Left). This reactivity is absent in Abca4-/- tissue (B, Right). Red punctate staining (white arrows) corresponding to ABCA4 mRNA is also observed in hfRPE cultured cells (C). CC, choriocapillaris; INL, inner nuclear layer. (Scale bars, 20 µm.) (D) ABCA4 immunohistochemistry (red fluorescence) on retina sections from pigmented wild-type (129/Sv), Mertk-/-, and Abca4-/- mice. Note that ABCA4 immunoreactivity is seen in the RPE and OS of 129/Sv mice and in the RPE but not in the OS (indicated by white asterisk) of Mertk-/- mice but is not seen in the retina section from an Abca4-/- mouse. The white arrows indicate retinal detachment. Cell nuclei are stained with DAPI (blue). (Scale bars, 10 µm.) (E) Representative immunoblots for ABCA4 protein using neural retina and RPE/eyecup homogenates loaded as a fraction of one mouse eye per lane, as indicated. The RNAscope assay (A–C) was done with two human cadaveric eyes, three cultured hfRPE cells of different donor eyes, and n = 3 mice (5-mo-old) of each genotype; Immunohistochemistry experiments (D) were repeated three times with n = 3 5-mo-old mice per group. The immunoblotting experiment (E) was done in duplicates varying the fraction of the homogenate corresponding to one mouse eye (n = 4 mice for each experiment).

Fig 5: Proposed function of ABCA4 in the endolysosomal membranes of RPE. (A) Normal RPE cell. 11cRAL released during proteolysis of rhodopsin within a phagolysosome condenses with PE on the luminal surface to form 11-cis-N-retinylidene-phosphatidylethanolamine (11c-N-ret-PE), which undergoes isomerization to form a mixture of all-trans (at) and 11c-N-ret-PEs. Both N-ret-PE isomers are flipped by ABCA4 to the cytoplasmic surface, where hydrolysis of N-ret-PE is driven by mass action through binding of 11cRAL by cellular retinaldehyde-binding protein (CRALBP) or the reduction of atRAL to atROL by retinol dehydrogenase type 11 (RDH11). The atROL is processed by the RPE visual cycle through esterification by lecithin retinol acyltransferase (LRAT) to yield an all-trans-retinyl ester such as all-trans-retinyl palmitate (atRP), isomerization by RPE65 to yield 11-cis-retinol (11cROL), and oxidation by retinol dehydrogenase type 5 (RDH5) to yield 11cRAL, which binds to CRALBP. 11cRAL leaves the RPE cell to regenerate visual pigments in the adjacent photoreceptor OS. (B) Abca4-/- mutant RPE cell. The lack of ABCA4 in RPE endolysosomes of Abca4-/- mice or STGD1 patients causes delayed clearance of retinaldehydes and hence higher concentrations of both free retinaldehydes and N-ret-PE. This leads to secondary condensation of atRAL or 11cRAL with N-ret-PE to form bisretinoids.