Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE

Abstract

Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, and analyze conditions that lead to ABCA1 dysregulation in induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs). Our results indicate that the risk-conferring alleles rs1883025 (C) and rs2740488 (A) in ABCA1 are associated with increased ABCA1 mRNA and protein levels and reduced efficiency of cholesterol efflux from the RPE. Hypoxia, an environmental risk factor for AMD, reduced expression of ABCA1 and increased intracellular lipid accumulation. Treatment with a liver X receptor (LXR) agonist led to an increase in ABCA1 expression and reduced lipid accumulation. Our data strengthen the homeostatic role of cholesterol efflux in the RPE and suggest that increasing cellular cholesterol export by stimulating ABCA1 expression might lessen lipid load, improving RPE survival and reducing the risk of developing AMD.

Keywords: ABCA1; LXR agonist; age-related macular degeneration; hypoxia; iPSC-RPE; lipid accumulation; reverse cholesterol transport.

Figure 1

Generation, differentiation and analysis of ABCA1-deficient iRPEs. (A) Immunofluorescence staining for ZO-1 (red) in 4-week cultured iRPEs. Nuclei were stained with DAPI (blue). Scale bar = 100 μm. (B) Bright-field microscopy of iRPEs. (C) Ct values of RPE marker genes BEST1, OTX2, RLBP1 and RPE65 in iRPEs and parental iPSCs obtained by qPCR. Values shown are means ± SD (n = 3). Unpaired Student’s t-test. **** p < 0.0001. (D) Sequence alignment of parts of exon 14 (top) and exon 46 (bottom) of ABCA1-deficient iRPE cell clones and parental line. (E) Western blot of ABCA1 protein levels in ABCA1-deficient iRPE cell lines and parental cell line after 16 h of stimulation with 1 μM LXR agonist. Actin was detected as loading control. (F) Relative expression of ABCA1 mRNA in ABCA1-deficient iRPE cell lines and parental cell line normalized to ACTB. Values shown are means ± SD (n = 3). One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001. (G) Relative expression of ABCG1 mRNA in ABCA1-deficient iRPE cell lines and parental cell line normalized to ACTB. Values shown are means ± SD (n = 3). One-way ANOVA with Tukey’s post hoc test. ** p < 0.01; *** p < 0.001. (H) Cholesterol efflux assay in iRPEs in the presence of ApoAI and/or LXR agonist (LXR ag). Values shown are means ± SD (n = 3). Two-way ANOVA with Bonferroni post hoc test vs. DMSO + BSA control. **** p < 0.0001. (I) Bright-field (BF) microscopy and Nile Red fluorescence microscopy (overview and close-up view marked with white squares) of 4-week cultured ABCA1-deficient iRPE cell lines and parental cell line. Relative fluorescence was quantified and is shown as mean ± SD (n = 8). One-way ANOVA with Tukey’s post hoc test. * p < 0.05, **** p < 0.0001.

Figure 2

Basal ABCA1 expression and function in patient-derived iRPEs. (A) Representation of patient-derived iPSC lines and genotypes harboring polymorphisms in ABCA1 that are associated with decreased or increased risk for AMD development. (B) Relative expression of ABCA1 mRNA in patient-derived iRPEs under basal conditions normalized to RPL28 and decreased risk group. (C) Western blot analysis of ABCA1 levels in patient-derived iRPEs under basal conditions. Actin was detected as loading control. ABCA1 expression was quantified and normalized to actin. (D,E) Relative expression of ABCG1 (D) and NR1H3 (E) mRNA in patient-derived iRPEs under basal conditions normalized to RPL28 and decreased risk group. (F) Cholesterol efflux in patient-derived iRPEs after direct cell labeling and in the presence of ApoAI. (G) Cholesterol efflux in patient-derived iRPEs after phagocytosis of BODIPY-cholesterol-loaded POSs and in the presence of ApoAI. (H) Quantification of phagocytosed POSs per nuclei in patient-derived iRPEs. Values shown are means ± SD (n = 3). * p < 0.05; *** p < 0.001. Unpaired Student’s t-test. (I) Representative fluorescence microscopy images of phagocytosed FITC-labeled POSs (green) and staining for ZO-1 (red) and DAPI (blue) of increased risk cell line IPS19-00096. Scale bar = 100 μm.

Figure 3

LXR agonist-stimulated ABCA1 expression and function in patient-derived iRPEs. Patient-derived iRPE lines were stimulated for 16 h with LXR agonist and are relative to unstimulated cells shown in Figure 2. (A) Expression of ABCA1 mRNA normalized to RPL28 and relative to unstimulated cells shown in Figure 2B. (B) Western blot analysis of ABCA1 protein levels. Actin was detected as loading control. ABCA1 expression was quantified and normalized to actin. (C) Cholesterol efflux assay after direct cell labeling and in the presence of ApoAI. (D) Cholesterol efflux assay after phagocytosis of BODIPY-cholesterol-loaded POSs and in the presence of ApoAI. Data are relative to unstimulated cells shown in Figure 2F,G, respectively. Data are presented as means ± SD (n = 3). Unpaired Student’s t-test. * p < 0.05.

Figure 4

Gene expression and cholesterol efflux under hypoxia in patient-derived iRPEs. (A–E) Relative expression of (A) ABCA1, (B) SREBP-1C, (C) NR1H3, (D) ADM and (E) PDK1 mRNA in patient-derived iRPEs after 3 days under 4% O₂ (hypoxia) or 21% O₂ (normoxia). All data were normalized to RPL28 and are presented as means ± SD (n = 3). Two-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. (F) Cholesterol efflux assay in patient-derived iRPEs under 4% O₂ (hypoxia) or 21% O₂ (normoxia). Values shown are means ± SD (n = 3). Two-way ANOVA with Tuckey post hoc test.

Figure 5

ABCA1 expression and function under hypoxia in iRPEs. (A–C) Relative expression of (A) ABCA1 and NR1H3, (B) ADM and (C) PDK1 mRNA in iRPEs before and after 1, 2 and 3 days incubation at 4% O₂ (hypoxia). All data were normalized to RPL28 and are presented as means ± SD (n = 3). One-way ANOVA with Tukey’s post hoc test to day 0. * p < 0.05; ** p < 0.01; ⁺⁺⁺ p < 0.001; ****/⁺⁺⁺⁺ p < 0.0001. (D) Relative expression of Abca1 in the RPE/choroid of mice kept for 11 weeks at 14% O₂ (hypoxia) or in normoxic control conditions. Data are presented as means ± SD (n ≥ 5) and were normalized to Actb. Unpaired Student’s t-test. * p < 0.05. (E) Cholesterol efflux in the presence of ApoAI from normoxic or hypoxic (3 d at 4% O₂) iRPEs after direct labeling and stimulation by LXR agonist as indicated. Data are presented as means ± SD (n = 6). One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001. (F) Bright-field (BF) microscopy and Nile red fluorescence microscopy (overview and close-up view marked with white squares) of iRPEs after 3 days at normoxic or hypoxic (4% O₂) conditions in the presence of ApoAI (all samples) and LXR agonist (as indicated). (G) Relative fluorescence of cells labeled with Nile red after 3 days of incubation in normoxic or hypoxic (4% O₂) conditions in the presence of ApoAI. LXR agonist (indicated) was added to stimulate ABCA1 expression. Values shown are means ± SD (n = 12 images per condition). One-way ANOVA with Tukey’s post hoc test. * p < 0.05. **** p < 0.0001.