RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65-/- mice
RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65-/- mice
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DOI：
10.1167/iovs.05-0965
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发表时间：
2006-03-01
影响因子：
4.4
通讯作者：
Ma, JX
中科院分区：
文献类型：
--
作者：
Chen, Y;Moiseyev, Q;Ma, JX
目的.最近的体外证据表明，RPE 65是异构水解酶，其将全反式视黄酯转化为11-顺式视黄酸，11-顺式视黄酸是脊椎动物视色素的发色团。纯合子RPE 65敲除（Rpe 65（-/-））小鼠缺乏11-顺式类维生素A并具有早期视锥细胞变性。本研究的目的是确定RPE 65基因递送是否恢复Rpe 65（-/-）小鼠中的异构水解酶活性和内源性类维生素A的正常分布。将表达RPE 65的腺病毒（Ad-RPE 65）注射到Rpe 65（-/-）小鼠的视网膜下腔中。免疫组化和Western blot检测RPE 65的表达。在眼杯匀浆中体外测量异构水解酶活性。通过高效液相色谱法（HPLC）分析眼杯中的内源性类维生素A谱。实时荧光定量PCR检测光感受器特异性基因表达。视锥细胞变性是通过视锥细胞特异性染色和计数视网膜中的视锥细胞来确定的。来自Ad-RPE 65注射的高水平RPE 65表达在Rpe 65（-/-）小鼠的眼杯中产生稳健的异构水解酶活性，其水平与野生型（wt）小鼠中的水平相当。因此，RPE 65基因递送导致Rpe 65（-/-）小鼠中大量的11-顺式视黄醛。RPE 65基因递送防止了Rpe 65（-/-）小鼠中视锥特异性基因（包括视锥视蛋白和视锥转导蛋白α亚基）的下调。此外，AdRPE 65注射液还可预防Rpe 65（-/-）小鼠早期大量视锥细胞变性。PPE 65基因递送在Rpe 65（-/-）小鼠中产生异构水解酶活性并恢复类维生素A谱。11-顺式视黄醇的再生对于视锥细胞的存活是必不可少的。
PURPOSE. Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65(-/-)) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65(-/-) mice.METHODS. Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65(-/-) mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina.RESULTS. High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65(-/-) mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65(-/-) mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin alpha subunit in Rpe65(-/-) mice. Moreover, the AdRPE65 injection also prevented massive cone degeneration at early ages of Rpe65(-/-) mice.CONCLUSIONS. PPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65(-/-) mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.