Explant cultures of Rpe65-/- mouse retina: a model to investigate cone opsin trafficking.

PURPOSE

In the absence of 11-cis retinal (e.g., Rpe65⁻/⁻), the chromophore for all pigments, cone opsins are mislocalized in vivo. Using the systemic application of 11-cis retinal, appropriate protein localization can be promoted. Here, we asked whether explant cultures of Rpe65⁻/⁻ mouse retina are amenable to screening retinoids for their ability to promote opsin trafficking.

METHODS

Retina-retinal pigment epithelium (RPE) cultures were prepared from 7-day-old Rpe65⁻/⁻ Rho⁻/⁻ or wild-type pups and cultured for 11 days. Explants were treated with retinoids throughout this period. Ultraviolet (UV)-opsin trafficking was analyzed by immunohistochemistry and quantitative image analysis, while its messenger RNA expression was examined by quantitative real-time PCR, and the interaction of retinoids with UV-opsin was probed in transducing-activation assays.

RESULTS

In wild-type explant cultures, UV-opsin was restricted to the outer segments, whereas in those derived from Rpe65⁻/⁻ Rho⁻/⁻ mice, opsin trafficking was impaired. In Rpe65⁻/⁻ Rho⁻/⁻ explants, administration of 11-cis retinal, 11-cis retinol or retinoic acid (RA) reversed the opsin trafficking phenotype. RA analogs designed to act by binding to the retinoic acid receptor or the retinoid X-receptor, however, had no effect. RA was shown to interact with the UV-cone opsin, demonstrated by its ability to effect ligand-dependent activation of transducin by UV-cone opsin. All compounds tested increased cone opsin messenger RNA expression.

CONCLUSIONS

Cone-opsin trafficking defects were replicated in Rpe65⁻/⁻ Rho⁻/⁻ retina-RPE cultures, and were reversed by 11-cis retinal treatment. Comparing the effects of different retinoids on their ability to promote UV-opsin trafficking to outer segments confirmed the critical role of agents that bind in the retinoid binding pocket. Retinoids that act as transcription factors, however, were ineffective. Thus, organ cultures may be a powerful low-throughput screening tool to identify novel compounds to promote cone survival.