Investigating the Effect of APOE Alleles on Neuro-Immunity of Human Brain Borders in Normal Aging and Alzheimer's Disease Using Single-Cell Multi-Omics and In Vitro Organoids

项目摘要 神经炎症和先天免疫反应已经成为阿尔茨海默病的最前沿病理学 疾病（AD）。越来越多的证据表明，载脂蛋白E4（APOE 4），最重要的AD 遗传风险因素，赋予正常衰老和AD大脑促炎状态。然而，大多数研究 这些研究集中在APOE4在破坏星形胶质细胞和小胶质细胞的稳态功能中的作用， 脑实质硬脑膜淋巴系统和免疫系统库的最新发现 脑边缘细胞的免疫学研究突出了边缘免疫在脑老化和AD中的关键作用。APOE高度 在边缘相关巨噬细胞（BAM）、硬脑膜淋巴管和脉络丛中表达。因此，在本发明中， 这些研究提出了一个关键问题，即APOE4是否调节正常人的边界相关免疫， 老化和AD。尽管最近对小鼠BAM进行了单细胞转录组学研究18，但关于 人脑边缘细胞的状态与正常衰老和AD有关，APOE 4对边缘细胞的影响与正常衰老和AD无关， 国家、豁免权和职能。本申请的总体目标是研究APOE对 等位基因对正常衰老和AD中人脑边界细胞状态和功能的影响。我们的中央 假设AD和APOE4改变了脑边缘区域的细胞状态，APOE4加重了 在对照和AD同基因边缘细胞模型中的分子和细胞表型。我们制定了这个 基于上述文献假设和我们的转录组学分析显示不同的细胞 转录组签名和强APOE表达的BAM在人脑边界。我们将测试这个 通过结合离体人边界区域和体外边界的单细胞多组学分析的假设 区域建模具体来说，我们将首先分析单细胞转录组和染色质可及性， 来自对照和AD的APOE4和非APOE4携带者的死后脑膜和脉络丛的相同细胞 个体这一目标将使我们能够揭示细胞类型，细胞状态，区域特异性和细胞间 在人脑边界的通信，并确定AD和APOE4特异性细胞的上游主调节因子 目标1）。为了研究APOE 4在人脑边缘的功能，我们将培养多细胞脉络膜， 通过将iPSC衍生的免疫细胞引入脉络丛类器官来建立脉络丛3D模型。然后我们将生成 携带APOE3或APOE4等位基因的对照和AD等基因脉络丛模型，以检查 APOE4在细胞自主和非细胞自主方式中的细胞、分子和功能作用 (Aim 2）。我们的研究将揭示人类边界相关免疫的重要见解，并揭示一种新的 AD病理生理学的潜在机制。AD和APOE4特异性细胞状态的上游调节因子 本研究中确定的免疫抑制剂将作为调节脑边缘免疫的理想治疗靶点。我们 脉络丛模型提供了一个易处理的系统来测试其他遗传靶点、外在因素和候选基因。 药物，开辟了一个新的途径，针对脑边界的AD治疗干预。

PROJECT SUMMARY Neuroinflammation and innate immune response have emerged as the forefront pathology of Alzheimer’s disease (AD). Accumulated evidence has shown that Apolipoprotein E4 (APOE4), the most significant AD genetic risk factor, confers a pro-inflammatory state in the normal aging and AD brains. However, most studies have centered around the roles of APOE4 in disrupting the homeostatic functions of astrocytes and microglia in brain parenchyma. Recent discoveries of a lymphatic system in the dura meninges and a repertoire of immune cells at brain borders have highlighted the critical role of border immunity in brain aging and AD. APOE is highly expressed in the border-associated macrophages (BAMs), dura lymphatic vessels, and choroid plexus. Thus, those studies raise a critical question on whether APOE4 regulates the border-associated immunity in normal aging and AD. Despite the recent single-cell transcriptomic study on mouse BAMs18, little information exists on cell states of human brain borders related to normal aging and AD, neither the APOE4’s effects on border cell states, immunity, and functions. The overall goal of this application is to investigate the effect of APOE alleles on cell states and functions of human brain borders in normal aging and AD. Our central hypothesis is that AD and APOE4 alter cell states in brain border regions, and APOE4 exacerbates molecular and cellular phenotypes in control and AD isogenic border cell models. We formulated this hypothesis based on literature as mentioned above and our transcriptomic analysis showing distinct cell transcriptomic signature and strong APOE expression in BAMs at human brain borders. We will test this hypothesis by combining single-cell multi-omics profiling of ex vivo human border regions and in vitro border region modeling. Specifically, we will first profile the single-cell transcriptome and chromatin accessibility from the same cells of postmortem meninges and choroid plexus of APOE4 and non-APOE4 carriers from control and AD individuals. This Aim will enable us to reveal cell types, cell states, regional specificity, and intercellular communications at human brain borders and identify upstream master regulators of AD- and APOE4-specific cell states (Aim 1). To study the APOE4’s functions in human brain borders, we will develop multicellular choroid plexus 3D models by introducing iPSC-derived immune cells to choroid plexus organoids. We will then generate control and AD isogenic choroid plexus models carrying either APOE3 or APOE4 alleles to examine the APOE4’s cellular, molecular, and functional effects in both cell-autonomous and non-cell-autonomous manners (Aim 2). Our study will shed significant insights into human border-associated immunity and reveal a novel mechanism underlying AD pathophysiology. The upstream regulators of AD- and APOE4-specific cell states identified in this study will serve as ideal therapeutic targets for modulating the brain border immunity. Our choroid plexus model provides a tractable system to test other genetic targets, extrinsic factors, and candidate drugs, opening a new avenue of targeting the brain borders for AD therapeutic interventions.