Cholesterol distribution & regulation of AB generation

胆固醇分布

• 批准号: 6789329
• 负责人: DORA M KOVACS
• 金额: $ 32.87万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-23 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6789329
• 关键词: Alzheimer' s disease; CHO cells; amyloid proteins; cell membrane; cholesterol; cholesterol esters; confocal scanning microscopy; endopeptidases; genetically modified animals; intracellular transport; laboratory mouse; lipid metabolism; plasma; protein localization; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Recent studies indicate that cholesterol levels alter Ab generation in cells and transgenic animals. To define the role of cholesterol in APP processing, we chose a genetic approach and took advantage of cholesterol-mutant CHO cell lines. Two of the cell lines overproduce cholesterol, one (AC29) as membrane-bound free cholesterol (4-fold), the other (25RA) as cholesteryl-esters (6-fold). The third cell line, M19, is defective in the regulation of cholesterol biosynthesis and contains 90% less free cholesterol than control cells. We stably transfected these three cholesterol-mutant cells with APP751 or APP751/V7171 and found that Af3 generation is differentially regulated in accord with the intracellular distribution of free and esterified forms of cholesterol. Ab generation was found to specifically correlate with cholesteryl-ester levels, independently of free cholesterol. Specifically, Ab secretion was reduced by ~95% in both AC29751 and AC29751/V7171 cells (lacking cholesteryl-esters) as compared to control CH0751 cells. In contrast, high levels of cholesteryl-esters in 25RA cells were associated with significantly increased Ab generation. In accord with these data, ACAT inhibitors reduced Af3 generation. We also show a possible mechanism for decreased Ab production in presence of reduced cholesteryl-esters based on accelerated PSi degradation. We also found that sphingolipids, which are tightly associated with cholesterol in cholesterol rich-domains (rafts), are strong regulators of Ab generation. These findings suggest that down regulation of cholesteryl-ester formation e.g. by specific ACAT inhibitors or modulation of membrane cholesterol may serve as a novel therapeutic strategy for treating or preventing AD. The overarching goal of this proposal is to determine how intracellular cholesterol trafficking and cellular compartmentation affect Ab generation. Specific Aim 1 is targeted at elucidating the effect of reduced cholesteryl ester levels on the processing, maturation and trafficking of both APP and BACE in neuronal and non-neuronal cells and ACAT' mice. In Specific Aim 2, we will further explore the reduction of Ab levels in the presence of low levels of cholesteryl-esters, and particularly the potential role of PSi turnover. Specific Aim 3 will test whether A13 generation requires the structural integrity of cholesterol rafts, as assessed by targeted disruption or overproduction of cholesterol-rich domains. These studies should provide a clearer understanding of the molecular and biochemicalevents involved in cholesterol-dependent regulation of Ab generation.

描述（由申请人提供）：最近的研究表明，胆固醇水平改变了细胞和转基因动物的AB产生。为了定义胆固醇在应用程序加工中的作用，我们选择了一种遗传方法，并利用了胆固醇突变的CHO细胞系。其中两条细胞系过度生产胆固醇，一个（AC29）作为膜结合的胆固醇（4倍），另一个（25RA）为胆汁固醇​​ - 糖（6倍）。第三个细胞系M19在调节胆固醇生物合成的调节中有缺陷，并且比对照细胞含有比对照细胞少的自由胆固醇的90％。我们稳定地用App751或App751/V7171转染了这三种胆固醇突变细胞，发现AF3的产生与自由和酯化形式的胆固醇的细胞内分布有差异调节。发现AB的产生与胆固醇水平特别相关，与游离胆固醇无关。具体而言，与对照CH0751细胞相比，AC29751和AC29751/V7171细胞（缺少胆固醇 - 植物）的AB分泌均降低了约95％。相比之下，25RA细胞中高水平的胆汁胆固醇 - 与AB产生显着增加有关。根据这些数据，ACAT抑制剂减少了AF3的产生。我们还显示了基于加速的PSI降解降低的胆固醇 - 植物量减少的AB产生的可能机制。我们还发现，与胆固醇富含胆固醇密切相关的鞘脂脂是AB发电的强烈调节剂。这些发现表明，降低胆固醇 - 酯形成的调节，例如通过特定的ACAT抑制剂或膜胆固醇的调节可以作为治疗或预防AD的新型治疗策略。该提案的总体目标是确定细胞内胆固醇运输和细胞隔室如何影响AB的产生。具体目标1的目标是阐明胆固醇酯水平降低对神经元和非神经元细胞以及ACAT小鼠的处理，成熟和运输的影响。在特定的目标2中，我们将进一步探讨在胆固醇水平较低的情况下，尤其是PSI周转的潜在作用的AB水平的降低。特定的目标3将测试A13生成是否需要胆固醇筏的结构完整性，这是通过靶向破坏或过量生产胆固醇富含域的含量来评估的。这些研究应更清楚地了解与胆固醇依赖性AB产生调节有关的分子和生化效应。