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: 10601040
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10601040
• 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; Early Diagnosis; Environment; Enzymes; Flowers; Genotype; Glycobiology; Glycoproteins; Health; Hepatocyte; Human; Induced pluripotent stem cell derived neurons; Late Onset Alzheimer Disease; Lipid Binding; Lipids; Lipoprotein Binding; Lipoproteins; Location; Measures; Methods; Microelectrodes; Modeling; Modification; Monosaccharides; Mutation; Nervous System; Neurons; Pathogenesis; Pathogenicity; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Plasma; Polysaccharides; Process; Property; Protein Isoforms; Proteins; Risk; Role; Sampling; Sialyltransferases; Site; Skin; Source; Techniques; Technology; Testing; Tissues; Transferase; Work; abeta accumulation; apolipoprotein E-3; apolipoprotein E-4; cell type; genetic risk factor; glycoproteomics; glycosylation; glycosyltransferase; improved; induced pluripotent stem cell; novel; promoter; receptor; receptor binding; response; sialylation; 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APOE 3和APOE 2降低风险相比，APOE 4亚型增加风险， 这背后仍然难以捉摸。我们使用定量糖蛋白质组学方法表征了APOE O-糖基化 靶向质谱方法显示CSF和血浆APOE糖基化差异很大， 特别是在脂质结合结构域中。我们还开发了一系列APOE结合试验，以确定 糖基化对功能的影响。我们的数据使用来自诱导多能干细胞（iPSC）的APOE， 细胞显示糖基化改变APOE结合特性。我们还知道转移酶， 添加单糖到聚糖，随着年龄的增长和唾液酸转移酶在AD中减少， APOE 3.3和APOE 4.4人脑之间APOE修饰的差异。加在一起， 这对全面了解APOE糖基化至关重要。因此，我们假设APOE表现出亚型依赖性， 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糖生物学和iPSC技术来解决四个目标。目标1将使用 正常APOE同基因iPSC衍生的星形胶质细胞和肝细胞，APOE的主要生产者。我们将 表征其APOE糖谱和相关结合特性，以了解组织和 同种型特异性APOE糖基化差异及其功能影响。转移酶表达也将是 分析以进一步确定糖基化差异背后的机制。目标2将比较星形胶质细胞 来源于健康和AD APOE同基因iPSC，并确定AD如何改变APOE糖基化;这是如何 在APOE亚型之间发生改变，以及这些变化如何影响AD发病机制中涉及的APOE功能。 将比较脂蛋白、受体和Aβ结合。最后，我们将比较AD患者的APOE糖表达谱， 和正常人脑样本进行对比目标3将使用正常的APOE同基因iPSC来模拟衰老 和AD的APOE糖基化，以确定哪种更接近AD的APOE糖基化。首先通过 减少衰老中观察到的特异性唾液酸转移酶表达，其次通过破坏Aβ环境 通过引入已知的APP突变。这将阐明致病性糖基化变化如何开始。目标4 将解决是否具有异常APOE糖基化的星形胶质细胞改变神经元网络活性和淀粉样蛋白 通过与iPSC衍生的神经元共培养来积累。我们的微电极阵列（MEA）分析表明， 星形胶质细胞的APOE基因型影响神经网络。我们将使用多边环境协定和Aβ的措施， 累积以确定这些异常糖基化的细胞系对神经元的影响。最终我们会 已经表征了正常和AD亚型特异性APOE糖基化，并确定了其对APOE的影响 功能，特别是与AD发病机制有关的功能。我们将确定异常的APOE糖基化是如何 Aβ蓄积、AD发病机制和神经元网络变性的促进剂。