Abstract
Purpose :
Oculocutaneous albinism (OCA) is a genetically heterogenous group of conditions that affect pigmentation and vision. Previous studies have suggested a correlation between the extent of pigmentation loss and vision defects, suggesting shared affected pathways in different forms of albinism. We have already described the in vitro characterization of RPE monolayers derived from individuals affected with OCA1A and OCA2 forms of oculocutaneous albinism (OCA_RPE). Our present work was designed to study the biochemical conversion of all-trans retinol to 11-cis retinal in the visual cycle pathway of the OCA_RPE.
Methods :
Isogenic pairs of induced pluripotent stem cells (iPSCs) lines were generated for OCA1A and OCA2 using CRISPR/Cas9 system. The iPSCs were differentiated to form RPE monolayers and expression of proteins involved in the visual cycle was studied using immuno-fluorescence staining and Western blot. RPE monolayers were treated with all-trans retinol (atRol) on the apical side overnight, lysed on the second day by sonication, and mixed with hydroxylamine buffer and 1 mL ethanol. Retinoids were extracted by two 4 mL volumes of hexane. The solvent was evaporated under a gentle stream of argon at 37°C and the dried samples re-dissolved in mobile phase. Retinoids in samples and standards were analyzed by LiChrospher Si-60 normal phase HPLC. Spectral data were acquired over the range of 250–400 nm. Absorbance was monitored at 350 nm for all-trans and 11-cis retinal oximes and at 325 nm for retinyl esters and atRol.
Results :
We detected the presence of the visual cycle enzymes LRAT, RPE65 and RDH5, and of the retinoid-binding partners RBP1 and CRALBP in both control and OCA_RPE monolayers. Extracts of RPE cells treated with atRol exhibited peaks for retinyl esters and 11-cis retinal oximes, suggesting uptake and processing of atRol. In DMSO-treated control_RPE samples, the peak for retinyl esters was significantly smaller and 11-cis retinal oximes were not detectable. Supplementation of apical atRol to OCA1A and OCA2_RPE and their isogenic control_RPE revealed no significant differences in uptake and conversion of atRol to retinyl esters.
Conclusions :
Enzymes and retinoid-binding proteins involved in the visual cycle are expressed in albino RPE monolayers at levels comparable to control pigmented RPE. OCA_RPE are as efficient as pigmented RPE in uptake and processing of retinoids.
This is a 2020 ARVO Annual Meeting abstract.