Abstract
Purpose :
Rare damaging mutations in CYP39A1, coding for 24-hydroxycholesterol 7α-hydroxylase, were previously reported to double the risk of pseudoexfoliation (PEX) syndrome and PEX glaucoma. Expression of CYP39A1, which is involved in cholesterol clearance by conversion of 24S-hydroxycholesterol (24S-OHC) to downstream intermediates, was significantly reduced in ocular tissues of PEX patients compared to controls. Because the role of CYP39A1 in PEX pathogenesis has not been clarified, we investigated the cellular effects of CYP39A1 deficiency mimicking reduced enzymatic activity.
Methods :
CYP39A1 mRNA and protein expression was analyzed in ocular tissues of eyes with PEX syndrome/glaucoma and age-matched control eyes. Cultured human nonpigmented ciliary epithelial (hNPE), trabecular meshwork (hTM) and retinal pigment epithelial (hRPE) cells were exposed to 1-25 µM 24S-OHC or 2 µM all-trans retinoic acid (ATRA) for 48 hours, or subjected to siRNA-mediated knockdown of CYP39A1. 24S-OHC levels were measured in CYP39A1-silenced cell lysates as well as in aqueous humor and serum samples of patients with PEX syndrome, PEX glaucoma without and with CYP39A1 mutations, and age-matched controls using gas chromatography-mass spectrometry.
Results :
Reduced CYP39A1 expression was observed in all ocular tissues of PEX compared to control eyes. Aqueous humor and serum levels of 24S-OHC and 24S-OHC/cholesterol ratios were significantly higher in PEX glaucoma patients without and with CYP39A1 mutations compared to controls. Silencing CYP39A1 protein expression by ~85% resulted in a ~2-fold accumulation of 24S-OHC within cells. Both CYP39A1 deficiency and exposure to sub-lethal concentrations of 24S-OHC induced generation of free radicals and transcriptional deregulation of genes related to inflammation (IL6, IL8, TNF), oxidative stress (SOD2, NFKB2), barrier function (TJP1-3), matrix metabolism (ELN, LOXL1, LTBP1-2), cholesterol metabolism (ABCA1, ABCG1, LXRA) and retinoic acid signaling (STRA6, ALDH1A1, CRABP2, RARA), which reflect key aspects of PEX pathogenesis. The deleterious effects of 24S-OHC could be partially prevented by physiologic concentrations of ATRA.
Conclusions :
The findings suggest that CYP39A1 deficiency and cellular accumulation of 24S-OHC can trigger substantial transcriptional changes and disturbances in cellular homeostasis, highlighting a novel role of CYP39A1 in PEX pathogenesis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.