7-Dehydrocholesterol (7DHC)-containing lipoproteins provoke cytological and autophagic flux defects in RPE in Smith-Lemli-Opitz Syndrome (SLOS) models in vivo and in vitro

Abstract

Purpose : Defective autophagic flux occurs in the RPE in SLOS models. SLOS is caused by recessive mutations in DHCR7, the gene encoding 7DHC reductase, which catalyzes conversion of 7DHC to cholesterol (Chol). We hypothesize that the observed RPE pathology is provoked by 7DHC or 7DHC-derived oxysterols, made by the RPE/retina and/or imported on circulating 7DHC-containing lipoproteins. Here, we tested that hypothesis.

Methods : We generated two novel conditional loxP Dhcr7 allele knockout (KO) mouse lines, targeting either Exon 8 (encodes a sterol-sensing domain) or Exon 9 (encodes an NADPH binding site). Dhcr7 Exon 8 or Exon 9 were ablated separately in liver by mating with albumin (Alb)-Cre mice. A novel anti-DHCR7 polyclonal was developed against a unique peptide region encoded by Exon 9. Sterol/oxysterol profiles of liver and serum from KO vs. WT mice were assessed (LC/MS). Fixed eyes from WT and Dhcr7 ≥≧KO mice were processed for cross-sectional or whole mount analysis of retina/RPE. Rabbits were treated with AY9944 (a selective inhibitor of DHCR7; 50 mg/kg, 3 wk, 3x i.p./wk), and serum was prepared therefrom. Primary monkey RPE cells were cultured on transwell inserts, followed by treatment with basolateral media supplemented with serum from either control or AY9944-treated rabbits for 72 h. Oil Red-O staining was used to identify lipid droplets in ocular frozen sections.

Results : 7DHC/Chol ratios and 7DHC-specific oxysterol levels were significantly (p≦0.01, N≧4) increased in liver and serum from Dhcr7 Exon 8/9 KO mice (PN 3 mo), vs. WT controls. Anti-opsin and -GFAP immunostaining of retinas and RPE from KO vs. WT mice revealed congestion of opsin-containing phagosomes in the RPE, without obvious retinal gliosis, in KO mice. Basolateral treatment of primary RPE cells with medium supplemented with serum from AY9944-treated rabbits provoked lipid droplet accumulation (vs. control serum).

Conclusions : Increased 7DHC and oxysterols levels in serum and liver caused by liver-specific Dhcr7 ablation is sufficient to cause autophagic defects in the RPE in vivo, despite the RPE’s ability to synthesize Chol de novo. In vitro challenge of RPE cells with exogenous 7DHC-containing lipoproteins causes marked lipid droplet accumulation. Hence, RPE function and lipid homeostasis are highly sensitive to serum sterol/oxysterol content.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.