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) 对阿尔茨海默病 (AD) 风险至关重要， 与 APOE3 和 APOE2 相比，APOE4 亚型增加了风险，而 APOE2 降低了风险，精确的机制 这背后的真相仍然难以捉摸。我们使用定量糖蛋白组学表征了 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， 特别是在脂质结合域中。我们还开发了一系列 APOE 结合测定法来确定 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了 APOE 结合特性。我们还知道转移酶，即 将单糖添加到聚糖中，随着年龄的增长而减弱，AD 中唾液酸转移酶减少，并且有 APOE3.3 和 APOE4.4 人脑之间 APOE 修饰的差异。在一起，这使得 对于充分理解 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，异常糖基化会改变 APOE 结合特性，加剧 AD 发病。我们将使用一系列糖生物学和 iPSC 技术来实现四个目标。目标 1 将使用 正常 APOE 同基因 iPSC 来源的星形胶质细胞和肝细胞是 APOE 的主要产生者。我们将 表征其 APOE 糖谱和相关的结合特性，以了解组织和 亚型特异性 APOE 糖基化差异及其功能影响。转移酶表达也将 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 源自健康和 AD APOE 同基因 iPSC，并确定 AD 如何改变 APOE 糖基化；这怎么 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 的 APOE 糖谱 和正常人脑样本到我们的 iPSC 数据。 Aim 3 将使用正常的 APOE 同基因 iPSC 来模拟衰老 和 AD 通过两种方法来确定哪种更接近 AD 的 APOE 糖基化。首先由 减少衰老过程中出现的特定唾液酸转移酶的表达，其次是通过破坏 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是如何开始的。目标 4 将解决具有异常 APOE 糖基化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 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 基因型影响神经元网络。我们将使用 MEA 和 Aβ 的测量 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 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 发病机制相关的功能。我们将确定 APOE 糖基化的异常程度 Aβ 积累、AD 发病机制和神经元网络变性的促进者。