D477G dominant mutation in RPE65 leads to decreased 11-cis-retinal generation and delayed dark-adaptation in knock-in mouse model of retinitis pigmentosa

Abstract

Purpose : RPE65, retinoid isomerase, is a key enzyme of the visual cycle in vertebrates. Most of the RPE65 mutations are recessive and leads to retinal dystrophies and blindness. Recently, a first dominant mutation D477G of RPE65 was identified to cause retinitis pigmentosa in patients; however, its pathogenic mechanism is unknown. To provide insight in the mechanism, we developed a knock-in (KI) mouse model for this mutation. The aim of this work was to investigate the impacts of D477G knock-in on the retinoid visual cycle and retinal structure and function.

Methods : Retinal morphology of the KI and wild-type (WT) control animals were evaluated by histology examination. Visual function was examined by electroretinography (ERG) at various ages (3-5 months). Expression levels of RPE65 in KI and WT mice were determined by western blotting. Retinoid profile recovery after bleaching was measured by HPLC. All-trans-[³H]-retinol was used as a substrate to measure the activity of RPE65 isomerase in mouse eyecups. The generated retinoids were extracted and analyzed by HPLC with a Radiomatic flow scintillation analyzer.

Results : No change in retinal morphology and opsin expression were observed in KI. Levels of RPE65 protein were lower in the eyecups of heterozygous KI (~65% of WT) and even lower in homozygous KI (~20% of WT), compared to that in WT. However, their scotopic and photopic ERG responses were comparable to that of wild-type mice. Levels of 11-cis-retinal in the fully dark-adapted heterozygous KI were similar to that in WT. After photobleaching, heterozygous KI generated lower levels of 11-cis-retinal than heterozygous KO at 15 and 30 minutes in the dark, suggesting that the presence of D477G impairs regeneration of 11-cis-retinal. Consistently, levels of all-trans-retinyl ester, the substrate of RPE65, were significantly higher in heterozygous KI, compared to WT and heterozygous KO. Furthermore, the heterozygous KI demonstrated slower ERG A-wave recovery following photobleaching compared to WT, suggesting a delayed dark-adaptation.

Conclusions : D477G functions as a dominant-negative mutant of RPE65 and decreases the regeneration of the chromophore. The knock-in mice provide a valuable model for understanding of the mechanism of pathogenesis of this mutant of RPE65 and for the development of therapy.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.