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

项目总结 神经炎症和先天免疫反应已成为阿尔茨海默氏症的最重要的病理因素 疾病(AD)。越来越多的证据表明，载脂蛋白E4(APOE4)，最重要的AD 遗传风险因素，在正常衰老和阿尔茨海默病的大脑中赋予促炎状态。然而，大多数研究 已经集中在APOE4在破坏星形胶质细胞和小胶质细胞的动态平衡功能中的作用 脑实质。硬脑膜淋巴系统和免疫谱系的最新发现 大脑边界的细胞强调了边界免疫在大脑老化和阿尔茨海默病中的关键作用。APOE高度 表达于边界相关巨噬细胞(BAM)、硬脑膜淋巴管和脉络丛。因此， 这些研究提出了一个关键问题，即APOE4是否调节正常的边界相关免疫 衰老和AD。尽管最近对小鼠BAMs18进行了单细胞转录研究，但关于BAMs18的信息很少 人脑边缘细胞状态与正常衰老和阿尔茨海默病的关系，载脂蛋白4‘S对边缘细胞的影响 州、豁免权和职能。本应用程序的总体目标是调查APOE的效果 正常衰老和阿尔茨海默病患者脑细胞状态和功能的等位基因。我们的中央 假说是AD和APOE4改变了大脑边缘区域的细胞状态，而APOE4加剧了 对照和AD等基因边缘细胞模型中的分子和细胞表型。我们制定了这个方案 基于上述文献的假设和我们的转录分析显示不同的细胞 人脑边界BAMS的转录转录特征和强APOE表达。我们将对此进行测试 结合体外人类交界区和体外交界区的单细胞多组学分析的假说 区域建模。具体地说，我们将首先描述单细胞转录组和染色质的可及性 对照组和AD患者APOE4携带者和非APOE4携带者死后脑膜和脉络丛的相同细胞 个人。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 人类大脑边界的通讯，并识别AD和APOE4特异性细胞的上游主调节因子 国家(目标1)。为了研究载脂蛋白4‘S在人脑边界的功能，我们将建立多细胞脉络膜 通过将IPSC来源的免疫细胞引入脉络丛器官建立神经丛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 载脂蛋白E4‘S在细胞自主和非细胞自主方式中的细胞、分子和功能效应 (目标2)。我们的研究将为人类边界相关免疫提供重要的见解，并揭示一种新的 阿尔茨海默病的病理生理学机制。AD和APOE4特异性细胞状态的上游调节因子 本研究确定的将作为调节大脑边界免疫的理想治疗靶点。我们的 脉络丛模型提供了一个易于处理的系统来测试其他遗传目标、外部因素和候选基因 药物，为AD治疗干预开辟了一条针对大脑边界的新途径。

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.