Bandyopadhyay Mausumi, Kono Masahiro, Rohrer Bärbel
Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA.
Mol Vis. 2013 May 29;19:1149-57. Print 2013.
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.
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.
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.
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.
在缺乏所有色素的发色团11 -顺式视黄醛（例如，Rpe65⁻/⁻）的情况下，视锥视蛋白在体内会出现定位错误。通过全身应用11 -顺式视黄醛，可以促进蛋白质的正确定位。在此，我们探讨了Rpe65⁻/⁻小鼠视网膜外植体培养物是否适合筛选具有促进视蛋白转运能力的类视黄醇。
从7日龄的Rpe65⁻/⁻ Rho⁻/⁻或野生型幼崽制备视网膜 - 视网膜色素上皮（RPE）培养物，并培养11天。在此期间，外植体用类视黄醇处理。通过免疫组织化学和定量图像分析来分析紫外线（UV） - 视蛋白的转运，同时通过定量实时PCR检测其信使RNA表达，并在转导激活试验中探究类视黄醇与UV - 视蛋白的相互作用。
在野生型外植体培养物中，UV - 视蛋白局限于外段，而在源自Rpe65⁻/⁻ Rho⁻/⁻小鼠的培养物中，视蛋白转运受损。在Rpe65⁻/⁻ Rho⁻/⁻外植体中，给予11 -顺式视黄醛、11 -顺式视黄醇或视黄酸（RA）可逆转视蛋白转运表型。然而，设计用于通过与视黄酸受体或类视黄醇X受体结合而起作用的RA类似物没有效果。RA被证明可与UV - 视锥视蛋白相互作用，这通过其影响UV - 视锥视蛋白对转导蛋白的配体依赖性激活的能力得以证明。所有测试的化合物均增加了视锥视蛋白信使RNA的表达。
在Rpe65⁻/⁻ Rho⁻/⁻视网膜 - RPE培养物中复制了视锥视蛋白转运缺陷，并通过11 -顺式视黄醛处理得以逆转。比较不同类视黄醇对其促进UV - 视蛋白转运至外段能力的影响，证实了在类视黄醇结合口袋中结合的物质的关键作用。然而，作为转录因子起作用的类视黄醇无效。因此，器官培养可能是一种强大的低通量筛选工具，用于鉴定促进视锥细胞存活的新型化合物。
