ABCA1 activity in the RPE is unnecessary for RPE reverse cholesterol transport (RCT) and AMD pathophysiology

Abstract

Purpose : Age-related Macular degeneration (AMD) is the leading cause of blindness amongst older adults in the industrialized world. Late-stage characteristics of AMD progression include retinal extracellular deposits (drusen). Drusen characteristics combined with genome-wide association studies (GWAS) has linked lipid deposits to reverse cholesterol transport genes (APOE, CETP and ABCA1). Pathophysiological examination of ATP binding cassette reporter A1 (abca1) gene was assessed in across different modalities (human tissue, iPSC-RPE and zebrafish) to determine its contribution in maintaining retinal homeostasis via RCT.

Methods : Gene expression of ABCA1 and ApoA1 on human donor tissue and iPSC-RPE were examined by qPCR (n=3). Bulk RNAseq examined transcript changes in key RCT genes on donor retinas across different stages of disease progression. RNAscope probes (ACDBio) were designed against abca1 transcripts with appropriate mismatch controls. Neutral lipid stain with oil-red O on 10um cryo-sections of abca1 KO and wild type (WT) eyes (N= 5). Two siRNAs knocked down abca1 in iPSC-RPE cells to assess abca1 contribution to cholesterol efflux (n=3). Samples were analyzed with the cholesterol efflux kit (ab196985) and compared to non-targeting control siRNAs. Histological analysis of ABCA1 protein using anti-ABCA1 (Invitrogen-MA516026) on human donor retinas (AMD1 vs AMD3).

Results : Key RCT genes are expressed in iPSC-RPE and donor retinas; with disease progression negatively effecting RCT genes expression. Abca1 RNAscope probes identified transcripts in the RPE zebrafish. Knockout of abca1 in zebrafish (n=5) found increased neutral lipids In the basal RPE(OrO stain) compared to wild type. No evidence of retinal degeneration accompanied lipid accumulation in the knockout fish. Polarized iPSC-RPE cells found cholesterol efflux to be apical and not disrupted by siRNA KD of ABCA1. Histological analysis of ABCA1 protein expression in human donor retinas was below detection in the RPE, sparse in the choroid and expression was largely unchanged compared to disease progressed (AMD1 vs AMD3) eyes.

Conclusions : Our results suggest ABCA1 plays a minimal role in RPE cholesterol efflux; and the contribution of abca1 SNPs to AMD is likely negligible. We hypothesize that other key RCT genes play more integral roles in cholesterol homeostasis within the retina.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.