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

本附录将建立用于机械和翻译研究的µSIM-hNVU平台的实用程序 突变蛋白tau、A-β和apoE4在血脑屏障中的作用 阿尔茨海默病(AD)患者神经血管单位(NVU)的功能障碍和神经炎症。？SIM卡- Hnvu是我们在中国农业大学博士学位论文《硅膜微生理系统》 作为研究神经血管损伤机制的工具的父母资助(R61/R33 HL154249) 败血症(McCloskey等人，Adv Health Mater.2022、e2200804)。µSIM是唯一能够实现高分辨率的设备 和活细胞显微镜，在“血”和“脑”之间提供更好的可溶性因子交换。 与其他组织芯片平台相比。虽然人们对tau蛋白在体内的作用知之甚少 AD血管病理的发生比Aβ和apoE4，有耐人寻味的证据相互作用 所有这三个因素之间的关系会导致NVU微环境中的神经炎症。这些相互作用很可能 与这些突变蛋白与低密度脂蛋白受体结合的共同能力有关 蛋白1(LRP1)。这里的研究将利用µSIM-hNVU来检查直接影响和竞争， 在Aβ、tau和apoE4之间，改变血脑屏障功能、Aβ清除和小胶质细胞激活(作为 神经炎症)。该补充将利用与µSIM兼容的全套分析和蜂窝 在父奖项中开发的资源包括IPSC衍生的以内皮细胞为特征的四倍体培养， APOEε等位基因3/3或4/4与周细胞、星形胶质细胞和小胶质细胞同源。 目的1研究Aβ1-42、tau和NVU apoEε基因对血脑屏障功能的影响。研究将增加一个β1- 42个低聚物和/或tau蛋白在一定浓度范围内进入µSIM-hNVU的脑侧，而 监测小分子通透性、细胞因子产生和细胞活化。分子机制 与最强的联合小胶质细胞和血脑屏障反应相关的将通过单一 从µSIM-hNVU分离的所有4种细胞类型的细胞/核测序。目标2将评估tau和 β1-42上的APOE3和4跨边界边界的传输。Tau突变体，在单体，预形成的纤维和超- 磷酸化形式和重组apoE3&4蛋白将在一定浓度范围内添加到 用固定浓度的β1-42寡聚体研究A-β的外流 低聚物进入血液侧并流入脑侧。为了检查LRP1的角色，我们将生成一个LRP1 用CRISPR敲除HiPSCs，同时产生内皮细胞和周细胞。我们预料到了这一点 补充建立µSIM-hNVU作为AD和HNVU中机械和翻译研究的新工具 提供有关推动AD相关的关键蛋白质风险因素复杂相互作用的初步数据 神经炎。

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.