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.

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， 对这背后的确切机制的理解是有限的;因此，我们无法减轻这种风险。我们 一直在研究AD中APOE O-糖基化及其变化，并开发了一种详细的定量方法， 用于分析所有糖位点的所有APOE糖型的靶向质谱方法。的理解 由于糖基化，特别是唾液酸，已被证明是重要的，因此糖基-APOE的合成是必不可少的 因为它的脂蛋白结合特性。我们以前已经证明，APOE修饰在细胞中是失调的。 健康人APOE4.4脑与APOE3.3个体相比。我们也知道转移酶， 将单糖单元加到聚糖上的酶，随着年龄的增长而减弱， 减少AD。这使我们推测APOE以亚型依赖的方式糖基化 异常糖基化改变Aβ结合，加重AD发病机制。我们以前的 O-糖蛋白质组学分析表明，APOE糖基化在CSF和血浆之间存在显著差异 APOE，特别是在脂质结合域。在目标1中，我们将使用人类诱导多能干细胞（iPSC）- 来源于星形胶质细胞和肝细胞，APOE的主要来源，并表征APOE的糖谱 产生的脂蛋白及其结合特性，获得了对特定的重要性的理解， 糖基化对APOE功能的影响在目标2和3中，我们将重点关注脑中表达的APOE，比较AD 和健康的iPSC，以及我们创造的遗传变化如何修饰APOE糖基化和Aβ结合。在Aim中 我们将通过以下方法确定APOE基因型如何影响APOE糖基化和APOE的Aβ结合特性： 改变每个细胞系的APOE基因型，以便我们使用具有相同遗传背景的每个基因型 CRISPR/Cas9系统我们初步的糖蛋白组学数据还表明，糖基-APOE在AD中发生了改变， 显示唾液酸化的核心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， 所有APOE基因型的健康和AD iPSC中的唾液酸转移酶表达。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 如果Aim 3中产生的细胞系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基因型影响神经元网络活动，使其成为一种有效的 测试神经胶质对神经元影响的方法。我们将使用MEA和Aβ蓄积的测量来确定 这些异常糖基化的细胞系对共培养的神经元的影响。最终，我们将阐明 每种APOE亚型Aβ结合所需的APOE糖基化以及在AD中如何破坏。我们还将 了解异常APOE糖基化对健康和AD共培养物的影响以及对相关性质的影响 确定APOE糖基化是否主要是AD标志物或参与AD发病机制。