Molecular Biology Of Outer Retina-specific Proteins

视网膜色素上皮（retinal pigment epithelium，RPE）在外层视网膜的发育和功能中起着关键作用。我们感兴趣的RPE的具体机制，在监管和功能水平。为此，我们一直在研究RPE 65的功能和调节，RPE 65是一种表达仅限于RPE的基因，其突变导致人类严重失明。基于RPE的维生素A视觉周期代谢的全反式视黄酯的11-顺式-视网膜的破坏似乎是Rpe65敲除小鼠表型的基础。因此，RPE 65的功能似乎与视色素再生中的关键酶视黄醇异构酶的功能相关。我们还继续研究β-胡萝卜素15，15 '-双加氧酶（β-CD）。β-CD与RPE 65密切相关，两者都是新出现的多样化类胡萝卜素裂解酶家族的成员。我们推测β-CD和RPE 65可能具有相似的作用机制。在过去的一年中，我们取得了以下进展：a）与野生型相比，一岁的Rpe65基因敲除小鼠显示出脂褐素荧光团（与年龄相关性黄斑变性相关的维生素A的不可逆副产物）的累积显著降低。这与RPE中视黄酯的过度积累平行，表明脂褐质荧光团的产生需要功能性视觉周期。B）使用双敲除小鼠（Rpe 65敲除与视紫红质敲除（纯视锥细胞反应）或环核苷酸门控通道α 3敲除（纯视杆细胞反应）杂交）的合作努力来研究在Rpe 65缺陷小鼠中观察到的降低的视网膜电图（ERG）反应的起源。与最初的结论相反，确定ERG实际上是由于严重脱敏的视杆光感受器而不是视锥光感受器。由于极端的发色团饥饿，Rpe65缺陷小鼠的视杆细胞中的视紫红质非常少，以至于它们感知到的光远远少于所给予的光，并且伪装成视锥细胞，对会抑制正常视杆细胞的强光刺激做出反应。这些发现也解释了人类患者中RPE 65相关视网膜营养不良的特征，并可能在治疗中有用。c）通过免疫荧光显微镜和RT-PCR，发现β-CD在小鼠视网膜内层中表达，而在小鼠RPE中不表达。各种RPE细胞系的RT-PCR显示在猴RPE细胞系中表达，但在3种人RPE细胞系中不表达。d）我们已经分析了β-CD基因的基因组结构，并开始在包括猴RPE、COS-7和Caco-2在内的多种细胞系中瞬时转染β-CD基因启动子-报告基因构建体，以分析β-CD基因的启动子功能。e）由视网膜特异性抗原（例如，抑制蛋白、IRBP和视紫红质）是人眼部炎性疾病如葡萄膜炎的模型。与LI合作，我们发现在用RPE特异性RPE 65抗原免疫的刘易斯大鼠中诱导了中度免疫病理应答。该疾病在后段更活跃，并且不影响前段到与arrestin诱导的EAU相同的程度。疾病从免疫动物向未免疫动物的连续转移表明该疾病是细胞介导的，与其他形式的EAU一样。f）通过AAV介导的基因转移拯救Rpe65敲除小鼠表型导致经处理的小鼠中对光的电生理和行为反应的敏感性增加。

The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE and mutations in which cause severe blindness in humans. Disruption of the RPE-based vitamin A visual cycle metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie the phenotype of the Rpe65 knockout mouse. The function of RPE65 thus appears to be associated with that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. We have also continued studies on beta-carotene 15,15'-dioxygenase (beta-CD). Beta-CD is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that beta-CD and RPE65 may share a similar mechanism of action. In the past year we have made the following progress: a) One year-old Rpe65 knockout mice show a dramatically lowered accumulation of lipofuscin fluorophores (an irreversible by-product of vitamin A associated with age-related macular degeneration) compared to wildtype. This, in parallel with an over-accumulation of retinyl esters in the RPE, shows that a functioning visual cycle is required for the generation of lipofuscin fluorophores. b) A collaborative effort using double knockout mice (Rpe65 knockout crossed with rhodopsin-knockout (pure cone response) or cyclic nucleotide gated channel alpha 3 knockout (pure rod response)) was used to study the origin of the reduced electroretinogram (ERG) response seen in Rpe65-deficient mice. In contrast to the original conclusion, it was determined that the ERG is actually due to severely desensitized rod photoreceptors rather than to cone photoreceptors. The rods in Rpe65-deficient mice, because of extreme chromophore starvation, have so little rhodopsin that they perceive far less light than is administered and, masquerading as cones, respond to bright light stimuli that would suppress normal rods. These findings also explain features of RPE65-associated retinal dystrophy in human patients and may be useful in therapy. c) By immunofluorescence microscopy and RT-PCR, beta-CD was found to be expressed in the mouse inner retina and not in the mouse RPE. RT-PCR of various RPE cell lines revealed expression in a monkey RPE cell line but not in 3 human RPE cell lines. d) We have analyzed the genomic structure of the beta-CD gene and are beginning transient transfection in a variety of cell lines, including monkey RPE, COS-7 and Caco-2, of beta-CD gene promoter-reporter constructs to analyze promoter function of the beta-CD gene. e) Experimental autoimmune uveoretinitis (EAU) induced by retina-specific antigens (e.g., arrestin, IRBP and rhodopsin) is a model for human ocular inflammatory diseases such as uveitis. In collaboration with LI, we have found that a moderate immunopathogenic response is induced in Lewis rats immunized with the RPE-specific RPE65 antigen. The disease is more active in the posterior segment and does not affect the anterior segment to the same extent as arrestin-induced EAU. Adoptive transfer of the disease from immunized to naive animals indicates that the disease is cell-mediated, like other forms of EAU. f) Rescue of the Rpe65 knockout mouse phenotype by AAV-mediated gene transfer has resulted in increased sensitivity of electrophysiological and behavioral responses to light in treated mice.