Explant cultures of Rpe65-/- mouse retina: a model to investigate cone opsin trafficking.
Mol Vis
; 19: 1149-57, 2013.
Article
em En
| MEDLINE
| ID: mdl-23734084
ABSTRACT
PURPOSE:
In the absence of 11-cis retinal (e.g., Rpe65â»/â»), 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 Rpe65â»/â» 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 Rpe65â»/â» Rhoâ»/â» 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 Rpe65â»/â» Rhoâ»/â» mice, opsin trafficking was impaired. In Rpe65â»/â» Rhoâ»/â» 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 Rpe65â»/â» Rhoâ»/â» 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas de Cultura de Órgãos
/
Retina
/
Cis-trans-Isomerases
/
Opsinas dos Cones
/
Modelos Biológicos
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Mol Vis
Ano de publicação:
2013
Tipo de documento:
Article