van der Spuy Jacqueline, Munro Peter M G, Luthert Philip J, Preising Markus N, Bek Toke, Heegaard Steffen, Cheetham Michael E
Division of Pathology, Institute of Ophthalmology, University College London, London, United Kingdom.
Mol Vis. 2005 Jul 22;11:542-53.
An unusual retinal vascular morphology in an enucleated eye from a patient with Leber congenital amaurosis (LCA) has been associated with a mutation in AIPL1. The AIPL1 protein is expressed in the pineal gland and retinal photoreceptors. In the retina, AIPL1 is expressed in both developing cone and rod photoreceptors, but it is restricted to rod photoreceptors in the adult human retina. Therefore, this study was conducted to determine the photoreceptor phenotype in this LCA patient to determine if photoreceptors were differentially affected.
Additional genetic screening was performed and the consequences of the H82Y amino acid substitution characterized in an in vitro assay of NUB1 modulation. The morphology of the photoreceptors was examined by light and electron microscopy. Immunohistochemistry and immunofluorescent confocal microscopy was performed using a range of retinal photoreceptor markers.
Transfection of the H82Y mutant AIPL1 in SK-N-SH cells revealed a normal subcellular localization and solubility but resulted in an increased ability of AIPL1 to redistribute GFP-NUB1 to the cytoplasm and resolve NUB1 fragment inclusion formation. Morphologically, the LCA retina appeared to be cone-dominant with a single layer of cone-like cells remaining in the central retina. Photoreceptor outer segments were absent and the surviving residual inner segments were severely shortened. Severe degeneration of the LCA retina was associated with upregulation of glial fibrillary acidic protein (GFAP). No signal was detected for AIPL1, rhodopsin, or L/M and S cone opsins in the LCA retina. Double labeling with peanut agglutinin (PNA) and wheat germ agglutinin (WGA) supported a cone-dominant phenotype for the surviving photoreceptors in the LCA retina, as did double labeling for cone arrestin, and rod and cone recoverin. The cone arrestin signal was restricted to the residual photoreceptor inner segments and was not detected in the cell bodies, axons, or axon terminals of the surviving photoreceptors. Recoverin immunoreactivity was most intense in the residual photoreceptor inner segments.
The phenotype in this patient suggests that although AIPL1 is required for the development of normal rod and cone photoreceptor function, it might only be essential for rod and not cone survival in the adult.
来自一名莱伯先天性黑蒙（LCA）患者的摘除眼球中出现的一种不寻常的视网膜血管形态与AIPL1基因突变有关。AIPL1蛋白在松果体和视网膜光感受器中表达。在视网膜中，AIPL1在发育中的视锥和视杆光感受器中均有表达，但在成人视网膜中仅限于视杆光感受器。因此，本研究旨在确定该LCA患者的光感受器表型，以确定光感受器是否受到不同程度的影响。
进行了额外的基因筛查，并在NUB1调节的体外试验中对H82Y氨基酸取代的后果进行了表征。通过光学显微镜和电子显微镜检查光感受器的形态。使用一系列视网膜光感受器标记物进行免疫组织化学和免疫荧光共聚焦显微镜检查。
在SK-N-SH细胞中转入H82Y突变型AIPL1显示出正常的亚细胞定位和溶解性，但导致AIPL1将GFP-NUB1重新分布到细胞质并解决NUB1片段包涵体形成的能力增强。形态学上，LCA视网膜似乎以视锥为主，中央视网膜中仅残留单层视锥样细胞。光感受器外节缺失，存活的残留内节严重缩短。LCA视网膜的严重变性与胶质纤维酸性蛋白（GFAP）的上调有关。在LCA视网膜中未检测到AIPL1、视紫红质或L/M和S视锥视蛋白的信号。用花生凝集素（PNA）和麦胚凝集素（WGA）进行双重标记支持LCA视网膜中存活的光感受器以视锥为主的表型，视锥抑制蛋白、视杆和视锥恢复蛋白的双重标记也是如此。视锥抑制蛋白信号仅限于残留的光感受器内节，在存活的光感受器的细胞体、轴突或轴突终末未检测到。恢复蛋白免疫反应性在残留的光感受器内节中最为强烈。
该患者的表型表明，尽管AIPL1是正常视杆和视锥光感受器功能发育所必需的，但它可能仅对成人视杆而非视锥的存活至关重要。
