Identification of RDH12 as the Protein that Allows Cones to Access the Retinal Visual Cycle

Abstract

Purpose : The Cone or Retinal Visual Cycle takes place in both RPE and Müller cells. We previously showed that in this pathway RGR opsin acts cooperatively with RDH10 as a redox photoisomerase complex to regenerate the cone visual chromophore 11-cis-retinaldehyde (11cRAL). Importantly, previous studies have shown cones can utilize either 11-cis-retinol (11cROL) or 11cRAL to regenerate cone visual pigments, while rods can only utilize 11cRAL to regenerate rhodopsin. These findings suggest that cones, but not rods, possess an unknown retinol dehydrogenase activity that oxidizes 11cROL to 11cRAL. The goal of this project was to identify the cone 11cROL-dehydrogenase (11cRDH).

Methods : The unknown 11cRDH was identified using published zebrafish single-cell RNA-seq data sets. We colloquially referred to the identified unnamed protein as “ZCRDH”. Immunoblotting and confocal immunocytochemistry (IHC) was performed using antisera against zebrafish ZCRDH and other markers. HEK-293T cells were transfected with ZCRDH and other highly homologous retinol dehydrogenases for use in retinol dehydrogenase activity assays. Retinoids were extracted using hexane and identified by high-performance liquid chromatography (HPLC). Pigment formation in photoreceptors was measured using microspectrophotometry (MSP).

Results : Confocal immunofluorescence analysis showed the ZCRDH protein was present in cone inner segments, but was not detectable in rod photoreceptors. Michaelis–Menten kinetic analysis revealed ZCRDH displayed strong specificity towards 11cROL as substrate versus all-trans-retinol (atROL). Visual retinoids measured in whole eyes enucleated from wild-type and zcrdh-/- zebrafish exhibited impaired regeneration of 11cRAL after bleaching. MSP analysis showed that loss of ZCRDH in zcrdh-/- zebrafish retinas resulted in absent recovery of M-cone and L-cone opsins following a 90% photobleach upon addition of 11cROL, while wild-type zebrafish showed robust recovery of the same photoreceptors under identical conditions. The human ortholog of ZCRDH is RDH12 based on high homology and similar enzymatic activity.

Conclusions : The 11cROL-dehydrogenase present in zebrafish cone photoreceptors is RDH12. Mutations in the human RDH12 gene cause recessive Leber congenital amaurosis, cone-rod dystrophy, and retinitis pigmentosa.

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

 

Hypothesized retinal visual cycle for cone photoreceptor visual chromophore regeneration.