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Dynamics of Tau protein, Amyloid beta oligomer, and APOE isoforms at the neurovascular unit

神经血管单元 Tau 蛋白、β 淀粉样蛋白寡聚体和 APOE 亚型的动态

基本信息

批准号：
10710705
负责人：
Britta Engelhardt
金额：
$ 37.4万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-03 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10710705
关键词：
Abeta clearance Adopted Alleles Alzheimer&apos s Disease Alzheimer&apos s disease patient Alzheimer&apos s disease risk Amyloid Amyloid beta-Protein Apolipoprotein E Astrocytes Automobile Driving Award Basement membrane Binding Biological Assay Blood Blood - brain barrier anatomy Blood Vessels Blood brain barrier dysfunction Brain Brain Injuries Cells Cerebrum Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Complex Data Development Endothelial Cells Enzyme-Linked Immunosorbent Assay Failure Generations Genotype Glass Health Human Image In Vitro Inflammatory Injury Knock-out LDL-Receptor Related Protein 1 Lipoprotein Receptor Lipoproteins Measures Mediating Membrane Microglia Modeling Molecular Molecular Conformation Monitor Morphology Mus Parents Pathologic Pathology Pathway interactions Pattern Pericytes Permeability Phase Pilot Projects Positron-Emission Tomography Production Protein Isoforms Proteins Recombinants Reporter Resolution Resources Risk Factors Role Sepsis Side Silicon Testing Tissue Microarray Tissues Toxin Variant Vascular Diseases abeta oligomer apolipoprotein E-3 apolipoprotein E-4 blood-brain barrier disruption blood-brain barrier function blood-brain barrier permeabilization brain endothelial cell cell type cytokine digital experimental study glial activation induced pluripotent stem cell live cell microscopy microphysiology system monomer mouse model mutant neuroinflammation neurotoxicity neurovascular injury neurovascular unit parent grant pre-formed fibril response small molecule synergism tau Proteins tau interaction tau mutation tau-1 tool translational study

项目摘要

This supplement will establish the utility of the µSiM-hNVU platform for mechanistic and translational studies on the mutant proteins tau, amyloid beta oligomer (Aβ), and apoE4 and their roles in blood-brain barrier (BBB) dysfunction and neuroinflammation at the neurovascular unit (NVU) in Alzheimer’s Disease (AD). The µSiM- hNVU (microphysiological system featuring a silicon membrane) is an in vitro platform we developed under the parent grant (R61/R33 HL154249) as a tool to study neurovascular injury mechanisms during sepsis (McCloskey et al., Adv Health Mater. 2022, e2200804). The µSiM is uniquely capable of high resolution and live cell microscopy and provides superior exchange of soluble factors between ‘blood’ and ‘brain’ compartments compared to other tissue chip platforms. While less is known about the role of tau proteins in the genesis of AD vascular pathology than Aβ and apoE4, there is intriguing evidence that interactions between all three factors drive neuroinflammation in the NVU microenvironment. These interactions likely relate to the common ability of these mutant proteins to bind the low-density lipoprotein receptor–related protein 1 (LRP1). The studies here will utilize the µSiM-hNVU to examine both direct effects, and competition, between Aβ, tau and apoE4, to alter BBB function, Aβ clearance, and microglial activation (as a marker of neuroinflammation). The supplement will leverage the full suite of µSiM-compatible assays and cellular resources developed in the parent award including iPSC derived quad cultures featuring endothelial cells, pericytes, astrocytes and microglia isogenic for APOE ε alleles 3/3 or 4/4. Aim 1 will examine the impact of Aβ1-42, tau and NVU APOE ε genotypes on BBB function. Studies will add Aβ1- 42 oligomers and/or tau proteins at a range of concentration to the brain side of the µSiM-hNVU while monitoring small molecule permeability, cytokine production and cellular activation. Molecular mechanisms associated with the strongest combined d microglial and BBB responses will be identified through single cell/nucelli sequencing of all 4 cell types isolated from the µSiM-hNVU. Aim 2 will assess the effect of tau and apoE3 & 4 on Aβ1-42 transport across the BBB. Tau mutants, in monomeric, pre-formed fibrils and hyper- phosphorylated forms and recombinant apoE3 & 4 proteins at a range of concentrations will be added to the brain side of the µSiM-hNVU along with a fixed concentration of Aβ1-42 oligomer to study efflux of the Aβ oligomer to the blood side and influx to the brain side. To examine the role of LRP1, we will generate an LRP1 knock out in hiPSCs with CRISPR and generate both endothelial cells and pericytes. We expect this supplement to establish the µSiM-hNVU as a new tool for mechanistic and translational studies in AD and provide preliminary data on the complex interactions of key protein risk factors driving AD-associated neuroinflammation.

本补充文件将确立µSiM-hNVU平台在机制和转化研究中的实用性突变蛋白tau、淀粉样β寡聚体（Aβ）和apoE 4及其在血脑屏障（BBB）中的作用阿尔茨海默病（AD）中神经血管单位（NVU）的功能障碍和神经炎症。微SiM- hNVU（具有硅膜的微生理系统）是我们在以下条件下开发的体外平台：父母补助金（R61/R33 HL 154249）作为研究神经血管损伤机制的工具，败血症（McCloskey等人，高级健康材料。2022，e2200804）。µSiM具有独特的高分辨率和活细胞显微镜检查，并提供“血液”和“大脑”之间可溶性因子的上级交换与其他组织芯片平台相比，虽然对tau蛋白在肿瘤中的作用知之甚少， AD血管病理的发生要比Aβ和apoE 4多，有有趣的证据表明，在NVU微环境中，所有三个因素之间的相互作用驱动神经炎症。这些互动可能与这些突变蛋白结合低密度脂蛋白受体相关的共同能力有关。蛋白1（LRP 1）。这里的研究将利用µSiM-hNVU来检查直接影响和竞争，在Aβ、tau和apoE 4之间，改变BBB功能、Aβ清除和小胶质细胞活化（作为神经炎症）。该补充将利用全套µ SiM兼容的分析和蜂窝在父母奖中开发的资源，包括iPSC衍生的具有内皮细胞的四元培养物，周细胞、星形胶质细胞和小胶质细胞与APOE ε等位基因3/3或4/4等基因。目的1将研究Aβ1-42、tau和NVU APOE ε基因型对BBB功能的影响。研究将添加Aβ1- 42种寡聚体和/或tau蛋白以一定浓度范围转移到µSiM-hNVU的脑侧，监测小分子渗透性、细胞因子产生和细胞活化。分子机制与最强的组合d小胶质细胞和BBB反应相关的将通过单个从µSiM-hNVU分离的所有4种细胞类型的细胞/nucelli测序。目标2将评估tau的影响， apoE 3和4对Aβ1-42跨血脑屏障转运的影响。Tau突变体，在单体，预形成的原纤维和超- 磷酸化形式和重组apoE 3和4蛋白在一系列浓度下将被添加到沿着使用固定浓度的Aβ1-42寡聚体的µSiM-hNVU脑侧，以研究Aβ的外排低聚物进入血液侧并流入脑侧。为了检查LRP 1的作用，我们将生成一个LRP 1 用CRISPR敲除hiPSC，并产生内皮细胞和周细胞。我们预计这一将µSiM-hNVU作为AD机制和翻译研究的新工具，提供了关于驱动AD相关疾病的关键蛋白质风险因素之间复杂相互作用的初步数据。神经炎症