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会改变细胞和转基因动物中抗体的产生。为了确定胆固醇在 APP 加工中的作用，我们选择了遗传方法并利用了胆固醇突变的 CHO 细胞系。其中两种细胞系过量产生胆固醇，一种 (AC29) 作为膜结合的游离胆固醇（4 倍），另一种 (25RA) 作为胆固醇酯（6 倍）。第三种细胞系 M19 在胆固醇生物合成调节方面存在缺陷，其游离胆固醇含量比对照细胞少 90%。我们用 APP751 或 APP751/V7171 稳定转染这三种胆固醇突变细胞，发现 Af3 的生成根据游离和酯化形式胆固醇的细胞内分布受到差异性调节。研究发现抗体生成与胆固醇酯水平特别相关，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（缺乏胆固醇酯）的 Ab 分泌减少约 95%。相反，25RA 细胞中高水平的胆固醇酯与抗体生成的显着增加相关。根据这些数据，ACAT 抑制剂减少了 Af3 的产生。我们还展示了在存在还原胆固醇酯的情况下基于 PSi 加速降解而减少抗体产生的可能机制。我们还发现，鞘脂与富含胆固醇的结构域（筏）中的胆固醇密切相关，是抗体生成的强大调节剂。这些发现表明胆固醇酯形成的下调，例如通过特定的 ACAT 抑制剂或调节膜胆固醇可以作为治疗或预防 AD 的新治疗策略。该提案的首要目标是确定细胞内胆固醇运输和细胞区室如何影响抗体的产生。具体目标 1 旨在阐明降低胆固醇酯水平对神经元和非神经元细胞以及 ACAT 小鼠中 APP 和 BACE 的加工、成熟和运输的影响。在具体目标 2 中，我们将进一步探讨在低水平胆固醇酯存在的情况下抗体水平的降低，特别是 PSi 周转的潜在作用。具体目标 3 将测试 A13 生成是否需要胆固醇筏的结构完整性，通过有针对性地破坏或过量产生富含胆固醇的结构域来评估。这些研究应该可以让我们更清楚地了解抗体生成的胆固醇依赖性调节所涉及的分子和生化事件。