Abstract
Purpose :
Two retinol dehydrogenases (RDH5, RDH10) catalyze oxidation of retinol to retinaldehyde. Double knockout (DKO) of RDH5 and RDH10 shows that some level of activity remains, suggesting redundancy. We discovered two enzymes, RDH epidermal 2 (RDHE2) and RDHE2-similar (RDHE2S) (collectively RDHEs) that display activity towards both all-trans and 11-cis-retinol. To test the hypothesis that RDHEs are essential for ocular function and complement other RDH activities, we made a DKO of both genes.
Methods :
C57Bl56/J mice were used to create RDHE2 and RDHE2S DKO mice using CRISPR-Cas9 technology. Phenotypes were confirmed by PCR and Western blotting. Mice were bred to 2nd generation and analyses were performed on both WT and DKO mice. Immunohistochemistry (IHC) was performed with Ki67, a proliferation marker, and Keratin-1, an epidermis-specific antibody using previously published protocols. Dark- and light-adapted electroretinography (ERG) was done on overnight dark-adapted mice (n=3) using standard protocol. Structural changes and thickness of the retinal layers were compared using OCT. A two-tailed paired t-test was used for statistical analysis.
Results :
Surprisingly, DKO mice exhibit anterior and posterior segment phenotypes. The eyelids appeared thicker and puffy as early as 1 mo., while corneal opacities were observed in older mice at 1 year. Staining of the anterior segment showed abnormal growth and vascularization in the corneal epithelium, evidenced by increased proliferation of basal epithelium (Ki67) and the presence of blood cells (H&E). IHC with keratin-1 antibodies showed differentiation of corneal epithelium into a skin-like epidermis. Functionally, we found a significant reduction in light response in both dark-adapted rod a- (WT, 527 ± 67 µV; DKO, 196 ± 58 µV; p < 0.05), and b-wave responses (WT, 1312 ± 167 µV; DKO, 359 ± 99 µV; p < 0.01) in DKO mice. Similarly, light adapted a- (WT, 350 ± 34 µV; DKO, 122 ± 43 µV; p < 0.01), and b-wave responses (WT, 532 ± 24 µV; DKO, 155 ± 49 µV; p < 0.01) were also significantly reduced in DKO mice. OCT analyses did not reveal any structural changes in the retina.
Conclusions :
Our results are consistent with a requirement for RDHE activity in both the anterior and posterior segments. In the anterior segments, RDHEs are required for proper development of the corneal epithelium and in the posterior segment they are required for retinal function.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.