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: 10380786
• 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/10380786
• 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.

抽象的 尽管O-糖蛋白载脂蛋白E（APOE）对阿尔茨海默氏病（AD）风险至关重要，但 与APOE3相比，与APOE3相比，APOE4同工型增加了风险，而APOE2降低了精度机制 背后仍然难以捉摸。我们使用定量糖蛋白质组学表征了APOE O-糖基化 靶向质谱方法表明CSF和等离子体APOE糖基化良好不同， 特别是在脂质结合域中。我们还开发了一系列APOE绑定测定法以确定 糖基化对功能的影响。我们的数据使用诱导多能干细胞（IPSC）的APOE得出 细胞表明糖基化会改变APOE结合特性。我们也知道转移，酶 将单糖添加到聚糖中，随着年龄的增长而减弱，AD降低了，并且有 APOE3.3和APOE4.4人类大脑之间APOE修饰的差异。在一起，这使它 完全了解ApoE糖基化的至关重要。这，我们假设APOE显示同工型依赖性 糖基化和异常糖基化改变了APOE结合特性，加剧AD 发病。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 正常的APOE等源性IPSC衍生的星形胶质细胞和肝细胞，APOE的主要生产商。我们将 表征其ApoE糖胶和相关的结合特性，以了解组织和 同工型特异性的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。 AIM 2将比较星形胶质细胞 源自健康和AD APOE等源性IPSC，并确定AD如何改变ApoE糖基化；如何 在APOE同工型之间发生了变化，以及这种变化如何影响APOE功能所涉及的AD发病机理。 将比较脂蛋白，受体和Aβ结合。最后，我们将比较AD的APOE糖胶片 和正常的人脑样品到我们的IPSC数据。 AIM 3将使用普通的APOE等源IPSC来建模衰老 通过两种方法，AD可以确定哪种方法与AD的APOE糖基化更相似。首先 通过破坏Aβ环境，减少在衰老中观察到的特异性硫酰基转移酶的表达 通过引入已知的应用程序突变。这将阐明致病性糖基化的变化如何开始。目标4 将解决异常APOE糖基化的星形胶质细胞是否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的积累。我们的微电极阵列（MEA）分析已显示 星形胶质细胞的APOE基因型会影响神经元网络。我们将使用Aβ的测量和度量 积累以确定这些异常糖基化细胞系对神经元的影响。最终我们会的 已经表征了正常和AD同工型特异性APOE糖基化，并定义了其对APOE的影响 功能，尤其是与AD发病机理有关的功能。我们将确定异常APOE糖基化的异常 Aβ积累，AD发病机理和神经元网络变性的启动子。