Elucidating the role of Retinal Pigment Epithelium (RPE)-specific ABCA4 in Stargardt disease

Abstract

Purpose : Recessive Stargardt disease (STGD1) is an inherited macular degeneration caused by mutations in ABCA4. Mice with a null mutation in the Abca4 gene are characterized by the deposition of bis-retinoids and lipofuscin pigments in the RPE, dysregulation of the complement system, and late-onset photoreceptor degeneration. ABCA4 is thought to be expressed exclusively in photoreceptor outer-segment (OS) discs, and to function as an inward-directed flippase for N-ret-PE, the conjugate of retinaldehyde and phosphatidylethanolamine. However, in dark-reared Abca4-/- mice with less OS retinaldehyde burden, RPE bis-retinoid accumulation is still seen, suggesting that RPE-specific ABCA4 plays a protective role. Here, we show that ABCA4 is also expressed in RPE endo-lysosomal membranes, where it performs a similar function as in OS discs.

Methods : We generated transgenic mice using a construct that contains the normal mouse Abca4 coding-region downstream of the RPE-specific promoter. We crossed this transgene onto the Abca4 null background to obtain a line that expresses ABCA4 in the RPE, but not in the retina (Abca4-Tg5 mice). ABCA4 expression was determined by qRT-PCR, immunoblotting, and immunohistochemistry. Retinoids were measured by HPLC. Photoreceptor degeneration was evaluated by spectral-domain optical coherence tomography and light microscopy of retina sections.

Results : We demonstrated ABCA4 expression in normal RPE cells from several sources by qRT-PCR and immunoblotting. To confirm these observations, we showed that Mertk-/- mice, which do not phagocytose photoreceptor OS, also contained the Abca4 mRNA and protein. Levels of bis-retinoids and fluorescent lipofuscin pigments in Abca4-Tg5 RPE were about two-fold lower when compared to non-transgenic Abca4-/- RPE. Further, Abca4-Tg5 mice exhibited photoreceptor preservation at one year, in contrast to age-matched non-transgenic Abca4-/- mice, which lost approximately 40% of photoreceptors.

Conclusions : ABCA4 is expressed in intracellular membranes of RPE cells, in addition to photoreceptor OS discs. The Abca4-Tg5 mouse shows reduced bisretinoid accumulation and photoreceptor preservation, suggesting an active role of ABCA4 in the RPE. This study explains the accumulation of bis-retinoids seen in dark reared Abca4-/- mice by demonstrating that RPE-specific ABCA4 plays a key role in recycling retinoids released from phagocytosed OS.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.