ISOPRENOID METABOLISM IN THE RETINA

描述(申请人提供)：这个项目的长期目标是阐明视网膜中胆固醇和相关分子的生物学功能。RSH/Smith-Lemli-Opitz综合征(SLOS)涉及7-脱氢胆固醇(7DHC)向胆固醇的缺陷转化。这是在一系列多发性同源异常(MCA)综合征和合成胆固醇途径缺陷中首次发现的，估计它是第四种最常见的人类隐性疾病。SLOS有一种相关的视网膜变性，这是最近才发现的，但其背后的机制尚不清楚。初步研究表明，氧化脂质和蛋白质的参与，以及固醇代谢和其他途径之间的“代谢串扰”，可能是通过转录调节。用SLOS中存在缺陷的同一种酶的选择性抑制剂(AY9944)治疗大鼠，可以提供一种进行性视网膜变性的动物模型，影响视杆和视锥细胞。利用这个模型，与年龄和性别匹配的对照组大鼠相比，我们将采用三种不同的、互补的方法来阐明疾病的机制：1)微阵列分析(基因组学)将揭示SLOS大鼠与对照组大鼠的差异基因表达模式：通过实时荧光定量PCR和生化方法确定靶点。2)脂质组学将揭示正常和氧化脂质稳态水平的定量差异，而3)蛋白质组学将确定这些动物视网膜蛋白质的特定氧化修饰的数量和类型的差异。此外，将使用与细胞凋亡相关的标准方法来评估光感受器细胞死亡的机制。生物相容抗氧化剂(α-硫辛酸和EPC-K1)阻止脂肪和蛋白质氧化以及部分改善SLOS大鼠视网膜退化的能力将在正常和“光损伤”条件下进行评估。通过这种方式，将获得与SLOS相关的视网膜变性机制的基本新见解。这些研究也可能为使用抗氧化剂作为补充胆固醇的辅助手段提供支持，胆固醇补充是目前SLOS患者管理的治疗策略(如果不完美)。后者已被证明可以改善SLOS大鼠模型的光感受器功能。因此，该项目支持NEI的使命，即开发拯救视力的治疗方法，减少视力损伤和失明，并提高所有年龄段的人的生活质量。

DESCRIPTION (provided by applicant): The long-range goal of this project is to elucidate the biological functions of cholesterol and related molecules in the retina. The RSH/Smith-Lemli-Opitz Syndrome (SLOS), involves defective conversion of 7- dehydrocholesterol (7DHC) to cholesterol. The first discovered in a series of multiple congential anomalies (MCA) syndromes and anabolic cholesterol pathway defects, it is estimated to be the fourth most common human recessive disease. SLOS has an associated retinal degeneration, discovered only recently, but the mechanism underlying the degeneration is unknown. Initial studies suggest the involvement of oxidized lipids and proteins, as well as "metabolic cross-talk" between sterol metabolism and other pathways, likely via transcriptional regulation. Treating rats with a selective inhibitor (AY9944) of the same enzyme that is defective in SLOS affords an animal model that exhibits a progressive retinal degeneration affecting both rods and cones. Using this model, in comparison with age- and sex-matched control rats, we will employ three different, complimentary approaches to elucidating the disease mechanism: 1) Microarray analysis (genomics) will reveal differential gene expression patterns in SLOS rat vs: control retinas, confirming targets by real-time PCR and biochemical methods. 2) Lipidomics will reveal quantitative differences in the steady-state levels of normal and oxidized lipids, while 3) proteomics will identify differences in the amounts and types of specific oxidative modifications of retinal proteins in retinas of these animals. In addition, the mechanism of photoreceptor cell death will be assessed using standard methods relevant to apoptosis. The ability of biologically compatible antioxidants (alpha-lipoic acid and EPC-K1) to block lipid and protein oxidation and to partially ameliorate the retinal degenerationln in SLOS rats will be assessed, under both normal and "light-damage" conditions. In this way, fundamental new insights into the mechanism of retinal degeneration associated with SLOS will be obtained. These studies also may provide support for the use of antioxidants as adjuncts to cholesterol supplementation, the current (if imperfect) therapeutic strategy for SLOS patient management. The latter has been shown to improve photoreceptor function in the SLOS rat model. As such, this project supports the NEI's mission of developing sight-saving treatments, reducing visual impairment and blindness, and improving the quality of life for people of all ages.