Expression of the retina-specific flippase, ABCA4, in epidermal keratinocytes

Narrative

We have demonstrated that a truncated isoform of the retina-specific protein, ABCA4 is expressed in human epidermal keratinocytes. This isoform is required for cell viability at late passage and as such can be used to interrogate the pathophysiology of novel ABCA4 mutations.

Introduction

ATP-binding cassette, sub-family A, member 4 (ABCA4, previously named ABCR for its retina-specific expression) is a transmembrane protein that is primarily localized to retinal photoreceptors¹ where it is responsible for flipping N-retinyldene-phosphatidylethanolamine, a key intermediate in the visual cycle, from the lumen to the cytoplasmic leaflet of photoreceptor outer segment disks^1,2. Mutations in ABCA4 cause a build-up of toxic retinoids resulting in a variety of retinal degenerative phenotypes, including Stargardt disease, cone-rod dystrophy and retinitis pigmentosa². To date over 400 different mutations in ABCA4 have been reported³. Since many of these variants are rare and non-exomic, their pathogenicity is often difficult to prove⁴. As the primary focus of our group is to develop gene and autologous cell replacement-based treatments for rare inherited retinal degenerative diseases, unambiguous identification of disease-causing mutations is essential.

The neural retina is inaccessible to molecular analysis in living patients. As such, to determine if newly identified mutations in retina-specific genes are disease causing, we use patient-specific induced pluripotent stem cell (iPSCs)-derived retinal neurons⁵. Typically, iPSCs are generated in our lab using patient-specific keratinocytes isolated from the epidermis of 3 mm punch biopsies. Briefly, the epidermis is separated from the underlying dermis via dispase incubation, and keratinocytes are liberated via trituration. Keratinocytes are subsequently maintained on collagen-coated culture plates in Epilife medium (Gibco; Life Technologies) supplemented with 1% human Keratinocyte Growth Supplement (Gibco). While generating iPSCs from patients suspected of having ABCA4-associated retinal disease, we noticed that primary keratinocyte proliferation was often slow and reprograming efficiencies were low compared to age-matched controls, suggesting that ABCA4 mutations affect keratinocyte physiology directly. In this study a series of western blot, immunocytochemistry, and cytometry experiments were performed to demonstrate that ABCA4 is expressed in human keratinocytes and that ABCA4 mutations alter cellular viability.

Results

To determine if the ABCA4 protein was expressed in keratinocytes, a Western blot comparing lysates isolated from normal human, bovine and murine retina, murine retina with Abca4 deletions, and human keratinocytes was performed. Keratinocytes from three different control individuals of three different ages expressed a truncated protein that corresponded in size to an alternatively spliced 70 kDa ABCA4 isoform (detected previously via rt-PCR⁴; (Figure 1A)). Immunocytochemical labeling using an antibody targeted against ABCA4 demonstrated a perinuclear pattern of labeling (Figure 1B–C). To determine if mutations in ABCA4 affect cellular proliferation and/or senescence, keratinocytes from three control, three retinal disease patients without ABCA4 mutations, and three patients with molecularly confirmed ABCA4-associated retinal disease were analyzed using propidium iodide and a Tali image based cytometer (Figure 1D). For this analysis, keratinocytes received fresh medium every other day and were passaged routinely once a week. Cell viability readings were assessed at each cell passage. A significant decrease in cell viability was detected between passages 6–9 in cells isolated from patients with ABCA4-associated disease as compared to normal and non-ABCA4 disease controls (Figure 1D). Finally, immunocytochemical staining of normal human skin shows robust ABCA4 expression throughout the keratinizing layer (Figure 1E).

Figure 1.ABCA4 is expressed in keratinocytes and human skin and is required for cell viability.

A) Western blot of human (Hmn Ret), bovine (Bov Ret) and murine (Ms Ret) whole retinal lysate, retinal lysate from two mouse strains lacking Abca4 (Abca4 KO and Japanese Fancy (JF1)) and keratinocytes from three individuals 32, 54 and 68 years of age (KTC 32, 54, and 68, respectively). Robust ABCA4 expression is observed at 250 kDa in human, cow and mouse retina, but is completely absent in each Abca4 knockout retina. Keratinocytes from each control individual express a 70 kDa isoform of ABCA4. B–C) Immunocytochemical labeling of control KTC 32 (B) and control KTC 54 (C) patient keratinocytes with an anti-ABCA4 antibody (red) and a filamentous actin stain (Phalloidin; green). A perinuclear pattern of ABCA4 labeling was detected. D) Keratinocyte viability was assessed at passages 6–9 by staining cells with propidium iodide. Keratinocytes assessed from three different individuals with molecularly confirmed mutations in ABCA4 (“ABCA4”) are much less viable compared to control keratinocytes (“control”) and cells from three different patients with non-ABCA4-associated retinal disease (“Non-ABCA4”). E) Immunocytochemical staining of control human skin with the same anti-ABCA4 antibody used in Figure 1A–C above showing ABCA4 labeling (green) throughout the keratinizing layer of the epidermis. Scale bars = 100 μm (B–C) and 40 μm (E).

Conclusions

Taken together, these data demonstrate that a truncated version of the retinal-specific transmembrane enzyme ABCA4 is expressed in epidermal keratinocytes and is required for cell viability at late passage. Although future studies will be needed to elucidate the exact function of ABCA4 in the skin, this finding may be useful for individuals attempting to determine the pathogenicity of novel mutations in the ABCA4 gene.

Materials and methods

Data availability

F1000Research: Dataset 1. Raw data for ‘Expression of the retina-specific flippase, ABCA4, in epidermal keratinocytes’, 10.5256/f1000research.8089.d114077⁸