Cell-specific roles of CRALBP in visual chromophore production and photoreceptor function revealed through conditional knockout mouse studies

Abstract

Purpose : Cellular retinaldehyde-binding protein (CRALBP), encoded by the RLBP1 gene, is an 11-cis-retinoid-binding protein expressed in both the retinal pigment epithelium (RPE) and Müller glia (MG). Prior studies on Rlbp1 global knockout mice as well as data from humans with loss-of-function RLBP1 mutations have demonstrated the critical role this protein plays in supporting visual cycle function. But the relative importance of the RPE and MG pools of CRALBP in supporting rod and cone photoreceptor function is incompletely defined. In this work, we developed mouse lines that enable selective, timed deletion of CRALBP in the RPE or MG allowing the physiological role(s) of this protein to be investigated.

Methods : Mouse models for Rlbp1 knockout in either RPE or MG were generated by crossing floxed Rlbp1 mice with RPE65^CreERT2 and GLAST^CreER mice. Cre activity was induced by IP injection of tamoxifen (Tam). Cell-specific CRALBP knockout was evaluated by IHC and western blotting. HPLC analysis of retinoid extracts from whole eyes was carried out to establish the kinetics of 11-cis-retinal recovery following exposure to intense light. Rod and cone functionality and dark adaptation were assessed by ERG using mice on Gnat1^+/+ or Gnat1^-/- backgrounds.

Results : Rlbp1 knockout efficiency was ~100% and ~85% in Tam-treated RPE and MG mouse lines. The V_max of visual chromophore recovery following a deep photobleach was slowed 15-fold in RPE-specific Rlbp1 KO mice concomitant with a delay in all-trans-retinyl ester clearance. Conversely, visual chromophore recovery rate was unaffected in MG-specific Rlbp1 KO mice. The recovery of cone sensitivity following a 95% pigment bleach was significantly suppressed in mice lacking CRALBP in the RPE. However, the dark recovery of rod sensitivity was not impacted by loss of CRALBP expression in the MG. Analysis of MG-specific CRALBP ablation on cone function is in progress.

Conclusions : RPE-specific ablation of CRALBP recapitulates the sluggish visual chromophore recovery observed in global Rlbp1 knockout mice. Importantly, the pool of CRALBP in the RPE is crucial for rapid and complete dark adaptation of cones. These observations suggest that the RPE visual cycle plays a significant role in the delivery of 11-cis-retinal to cone photoreceptors in addition to its role in visual chromophore supply to rods.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.