ISOPRENOID METABOLISM IN THE RETINA

描述（由申请人提供）：该项目的长期目标是阐明胆固醇和相关分子在视网膜中的生物学功能。RSH/Smith-Lemli-Opitz综合征（SLOS）涉及7-脱氢胆固醇（7 DHC）转化为胆固醇的缺陷。在一系列多先天性异常（MCA）综合征和合成代谢胆固醇途径缺陷中首次发现，估计是人类第四大常见隐性疾病。SLOS具有相关的视网膜变性，最近才发现，但变性的机制尚不清楚。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”，可能通过转录调控。用SLOS缺陷的相同酶的选择性抑制剂（AY 9944）处理大鼠提供了表现出影响视杆细胞和视锥细胞的进行性视网膜变性的动物模型。使用该模型，与年龄和性别匹配的对照大鼠相比，我们将采用三种不同的互补方法来阐明疾病机制：1）微阵列分析（基因组学）将揭示SLOS大鼠与对照视网膜中的差异基因表达模式，通过实时PCR和生物化学方法确认靶标。2)脂质组学将揭示正常脂质和氧化脂质的稳态水平的定量差异，而3）蛋白质组学将鉴定这些动物视网膜中视网膜蛋白的特定氧化修饰的量和类型的差异。此外，将使用与细胞凋亡相关的标准方法评估感光细胞死亡的机制。将在正常和“光损伤”条件下评估生物相容性抗氧化剂（α-硫辛酸和EPC-K1）阻断脂质和蛋白质氧化并部分改善SLOS大鼠视网膜变性的能力。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胆固醇补充剂提供支持，这是SLOS患者管理的当前（如果不完美）治疗策略。后者已被证明可以改善SLOS大鼠模型中的光感受器功能。因此，该项目支持NEI的使命，即开发拯救视力的治疗方法，减少视力障碍和失明，提高所有年龄段人群的生活质量。

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.