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: 9789808
• 负责人: Sarah Ann Flowers
• 金额: $ 38.88万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789808
• 关键词: Address; Affect; Age; Aging; Alzheimer' s Disease; Alzheimer' s disease risk; Amyloid; Amyloid beta-Protein; Apolipoprotein E; Astrocytes; Binding; Brain; CRISPR/Cas technology; Cell Line; Cells; Coculture Techniques; Data; Disease; Disease Marker; Enzymes; Expression Profiling; Genetic; Genotype; Glycoproteins; Health; Hepatocyte; High Density Lipoproteins; Human; Individual; Inflammation; Late Onset Alzheimer Disease; Lipid Binding; Lipoprotein Binding; Location; Measures; Microelectrodes; Modeling; Modification; Monitor; Monosaccharides; Mutation; Nervous system structure; Neuroglia; Neurons; Pathogenesis; Pathology; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Plasma; Polysaccharides; Process; Property; Protein Isoforms; Proteins; Research Personnel; Risk; Role; Sampling; Sialic Acids; Sialyltransferases; Skin; Source; Stem cells; System; Techniques; Testing; Transferase; Very low density lipoprotein; Work; abeta accumulation; base; genetic risk factor; glycoproteomics; glycosylation; glycosyltransferase; human model; improved; induced pluripotent stem cell; novel; preference; response; stem cell differentiation; sugar; tool

Abstract Despite the critical importance of the O-glycoprotein apolipoprotein E (APOE) on Alzheimer’s disease (AD) risk, understanding of the precise mechanism behind this is limited; hence we are unable to mitigate this risk. We have been investigating APOE O-glycosylation and its changes in AD and have developed a detailed quantitative targeted mass spectrometric approach for the analysis of all APOE glycoforms at all glycosites. An understanding of glyco-APOE is essential given that glycosylation and in particular, sialic acid, has been shown to be important for its lipoprotein binding properties. We have previously shown that APOE modification is dysregulated in the healthy human APOE4.4 brain, compared to APOE3.3 individuals. We also know that transferases, the enzymes that add the monosaccharide units to glycans, wane with age and sialyltransferases have been shown to be reduced in AD. This leads us to hypothesize that APOE is glycosylated in an isoform dependent manner and that aberrant glycosylation alters Aβ binding, exacerbating AD pathogenesis. Our previous O-glycoproteomic analyses has shown that APOE glycosylation differs dramatically between CSF and plasma APOE, particularly in the lipid binding domain. In Aim 1 we will use human induced pluripotent stem cell (iPSC)- derived astrocytes and hepatocytes, the major sources of APOE, and characterize the glycoprofile of the APOE produced and its lipoprotein binding properties, gaining an understanding of the importance of specific glycosylation to APOE function. In Aims 2 and 3 we will focus on APOE expressed in the brain, comparing AD and healthy iPSCs and how the genetic changes we create modify APOE glycosylation and Aβ binding. In Aim 2 we will determine how APOE genotype affects APOE glycosylation and the Aβ binding properties of APOE by altering the APOE genotype of each cell line so we have each genotype with the same genetic background using the CRISPR/Cas9 system. Our preliminary glycoproteomic data also suggests that glyco-APOE is altered in AD, showing a reduction in sialylated core 1 glycans. In Aim 3, we will utilize the cell lines created in Aim 2 and modify sialyltransferase expression in healthy and AD iPSCs of all APOE genotypes. We will determine the glycosylation changes that impact Aβ binding and, therefore, may be involved in AD pathogenesis. In Aim 4, we will address if the cell lines produced in Aim 3 have an effect on neuronal network activity as well as amyloid accumulation by co-culturing the astrocyte cell lines with iPSC-derived neurons. Our micro-electrode array (MEA) analyses have shown that the APOE genotype of added astrocytes affects neuronal network activity making it an effective approach to test the glia impact on neurons. We will use MEAs and measures of Aβ accumulation to determine the effect of these aberrantly glycosylated cell lines on co-cultured neurons. Ultimately, we will elucidate the APOE glycosylation required for Aβ binding for each APOE isoform and how this is disrupted in AD. We will also understand the effect of aberrant APOE glycosylation on healthy and AD co-cultures and on properties relating to AD pathogenesis, determining if APOE glycosylation is primarily an AD marker or involved in AD pathogenesis.

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， 对这背后的精确机制的了解是有限的；因此我们无法降低这种风险。我们 一直在研究 APOE O-糖基化及其在 AD 中的变化，并制定了详细的定量方法 用于分析所有糖位点的所有 APOE 糖型的靶向质谱方法。一种理解 鉴于糖基化，特别是唾液酸，已被证明很重要，糖基化 APOE 的作用至关重要 因其脂蛋白结合特性。我们之前已经表明 APOE 修饰在 健康人类 APOE4.4 大脑与 APOE3.3 个体相比。我们还知道转移酶 将单糖单元添加到聚糖上的酶会随着年龄的增长而减弱，唾液酸转移酶已被证明 AD减少。这使我们推测 APOE 以异构体依赖性方式糖基化 异常的糖基化会改变 Aβ 结合，加剧 AD 发病机制。我们之前的 O-糖蛋白组学分析表明，CSF 和血浆中的 APOE 糖基化存在显着差异 APOE，特别是在脂质结合域中。在目标 1 中，我们将使用人类诱导多能干细胞 (iPSC) - 衍生的星形胶质细胞和肝细胞（APOE 的主要来源），并表征 APOE 的糖谱 产生及其脂蛋白结合特性，了解特定的重要性 APOE 功能的糖基化。在目标 2 和 3 中，我们将重点关注大脑中表达的 APOE，比较 AD 和健康的 iPSC，以及我们产生的基因变化如何改变 APOE 糖基化和 Aβ 结合。瞄准 2 我们将确定 APOE 基因型如何影响 APOE 糖基化以及 APOE 的 Aβ 结合特性 改变每个细胞系的 APOE 基因型，使每个基因型具有相同的遗传背景，使用 CRISPR/Cas9系统。我们的初步糖蛋白组学数据还表明糖-APOE 在 AD 中发生改变， 显示唾液酸化核心 1 聚糖减少。在目标 3 中，我们将利用目标 2 中创建的细胞系并修改 所有 APOE 基因型的健康和 AD iPSC 中唾液酸转移酶的表达。我们将确定糖基化 影响 Aβ 结合的变化，因此可能参与 AD 发病机制。在目标 4 中，我们将解决 Aim 3 中产生的细胞系是否对神经元网络活动以及淀粉样蛋白积累有影响 通过将星形胶质细胞系与 iPSC 衍生的神经元共培养。我们的微电极阵列 (MEA) 分析 已经表明，添加星形胶质细胞的 APOE 基因型会影响神经元网络活动，使其成为有效的 测试神经胶质细胞对神经元影响的方法。我们将使用 MEA 和 Aβ 积累的测量来确定 这些异常糖基化细胞系对共培养神经元的影响。最终，我们将阐明 每种 APOE 亚型的 Aβ 结合所需的 APOE 糖基化以及 AD 中这种糖基化是如何被破坏的。我们还将 了解异常 APOE 糖基化对健康和 AD 共培养物以及相关特性的影响 AD 发病机制，确定 APOE 糖基化是否主要是 AD 标记或参与 AD 发病机制。