Apolipoprotein E glycosylation and its role in Alzheimer's disease pathogenesis

载脂蛋白E糖基化及其在阿尔茨海默病发病机制中的作用

• 批准号: 10213478
• 负责人: Sarah Ann Flowers
• 金额: $ 53.12万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10213478
• 关键词: Address; Affect; Age; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer' s disease risk; Amyloid; Amyloid beta-Protein; Apolipoprotein E; Astrocytes; Binding; Biological Assay; Brain; Cell Line; Cells; Characteristics; Charge; Coculture Techniques; Data; Disease; Environment; Enzymes; Flowers; Genotype; Glycobiology; Glycoproteins; Health; Hepatocyte; Human; Induced pluripotent stem cell derived neurons; Late Onset Alzheimer Disease; Lipid Binding; Lipids; Lipoprotein Binding; Lipoprotein Receptor; Location; Measures; Methods; Microelectrodes; Modeling; Modification; Monosaccharides; Mutation; Nervous system structure; Neurons; Pathogenesis; Pathogenicity; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Plasma; Polysaccharides; Process; Property; Protein Glycosylation; Protein Isoforms; Risk; Role; Sampling; Sialyltransferases; Site; Skin; Source; Techniques; Technology; Testing; Tissues; Transferase; Work; abeta accumulation; base; cell type; genetic risk factor; glycoproteomics; glycosylation; glycosyltransferase; improved; induced pluripotent stem cell; novel; promoter; receptor binding; response; sialylation; stem cell differentiation; stem cells; sugar; tool

Abstract Despite the critical importance of the O-glycoprotein apolipoprotein E (APOE) on Alzheimer’s disease (AD) risk, with the APOE4 isoform increasing risk compared to APOE3 and APOE2 reducing it, the precise mechanism behind this remains elusive. We have characterized APOE O-glycosylation using our quantitative glycoproteomic targeted mass spectrometric approach showing that CSF and plasma APOE glycosylation differ greatly, particularly in the lipid binding domain. We have also developed a range of APOE binding assays to determine the impact of glycosylation on function. Our data using APOE from induced pluripotent stem cell (iPSC) derived cells shows that glycosylation alters APOE binding properties. We also know that transferases, the enzymes that add the monosaccharides to glycans, wane with age and sialyltransferases are reduced in AD and there are differences in APOE modifications between APOE3.3 and APOE4.4 human brains. Together, this makes it critical to fully understand APOE glycosylation. Thus, we hypothesize that APOE shows isoform-dependent glycosylation and that aberrant glycosylation alters APOE binding properties, exacerbating AD pathogenesis. We will use a range of glycobiology and iPSC techniques to address four Aims. Aim 1 will use normal APOE isogenic iPSC-derived astrocytes and hepatocytes, the main producers of APOE. We will characterize their APOE glycoprofiles and associated binding properties, to gain an understanding of tissue and isoform-specific APOE glycosylation differences and their functional impacts. Transferase expression will be also analyzed to further determine the mechanisms behind glycosylation differences. Aim 2 will compare astrocytes derived from healthy and AD APOE isogenic iPSCs and determine how AD alters APOE glycosylation; how this is altered between APOE isoforms, and how such changes impact APOE functions involved in AD pathogenesis. Lipoprotein, receptor and Aβ binding will be compared. Finally, we will compare the APOE glycoprofiles of AD and normal human brain samples to our iPSC data. Aim 3 will use normal APOE isogenic iPSCs to model aging and AD by two methods to determine which more closely resembles the APOE glycosylation of AD. First by reducing the specific sialyltransferase expression seen in aging and second by disrupting the Aβ environment by introducing a known APP mutation. This will elucidate how pathogenic glycosylation changes begin. Aim 4 will address if astrocytes with aberrant APOE glycosylation alter neuronal network activity and amyloid accumulation by co-culture with iPSC-derived neurons. Our micro-electrode array (MEA) analyses have shown that the APOE genotype of astrocytes affects neuronal networks. We will use MEAs and measures of Aβ accumulation to determine the effect of these aberrantly glycosylated cell lines on neurons. Ultimately we will have characterized normal and AD isoform-specific APOE glycosylation and defined its impact on APOE functions, especially those relating to AD pathogenesis. We will determine how aberrant APOE glycosylation is a promoter of Aβ accumulation, AD pathogenesis and neuron network degeneration.

摘要 尽管O-糖蛋白载脂蛋白E(APOE)对阿尔茨海默病(AD)风险至关重要， 与APOE3和APOE2相比，APOE4亚型的风险增加，确切的机制是 这背后的原因仍然难以捉摸。我们已经用我们的定量糖蛋白组学表征了APOE O-糖基化 靶向质谱法显示，脑脊液和血浆载脂蛋白E糖基化有很大不同， 尤其是在脂结合区域。我们还开发了一系列APOE结合分析来确定 糖基化对功能的影响。我们从诱导多能干细胞(IPSC)中提取的APOE数据 细胞表明，糖基化改变了APOE的结合特性。我们还知道转移酶，这些酶 将单糖添加到多糖中，随着年龄的增长而减弱，唾液酸转移酶在AD中减少，有 APOE3.3和APOE4.4人脑中APOE修饰的差异。加在一起，这就是它 对充分理解APOE糖基化至关重要。因此，我们假设APOE表现出异构体依赖性 糖基化和异常糖基化改变APOE结合特性，加重AD 发病机制。我们将使用一系列的糖生物学和IPSC技术来解决四个目标。AIM 1将使用 正常载脂蛋白E等基因的星形胶质细胞和肝细胞是产生载脂蛋白E的主要来源。我们会 表征它们的载脂蛋白E糖谱和相关的结合特性，以了解组织和 异构体特异性APOE糖基化差异及其功能影响。转移酶的表达也会 分析以进一步确定糖基化差异背后的机制。AIM 2将比较星形胶质细胞 来自健康和AD载脂蛋白E等基因的IPSCs，并确定AD如何改变APOE糖基化；这是如何 在载脂蛋白E亚型之间的变化，以及这种变化如何影响参与AD发病的载脂蛋白E功能。 将比较脂蛋白、受体和Aβ结合情况。最后，我们将比较AD的载脂蛋白E糖谱 和正常的人脑样本到我们的iPSC数据中。AIM 3将使用正常的APOE等基因IPSCs来模拟衰老 和AD通过两种方法确定哪一种更接近AD的APOE糖基化。先按 通过破坏Aβ环境降低衰老和二次所见的特异性唾液酸转移酶的表达 通过引入一种已知的应用程序突变。这将阐明致病糖基化变化是如何开始的。目标4 将讨论APOE糖基化异常的星形胶质细胞是否会改变神经元网络活动和淀粉样蛋白 通过与IPSC来源的神经元共培养积聚。我们的微电极阵列(MEA)分析显示 星形胶质细胞的APOE基因影响神经元网络。我们将使用MEAs和β的措施 以确定这些糖基化异常的细胞系对神经元的影响。最终我们会 表征了正常和AD异构体特异性的APOE糖基化，并确定了其对APOE的影响 功能，尤其是与阿尔茨海默病发病相关的功能。我们将确定载脂蛋白E糖基化的异常程度 Aβ积聚、AD发病及神经网络变性的促进剂