Investigating the effects of APOE and APOE-related AD risk genes on human microglia activity and lipid metabolism in aging and disease

摘要 了解阿尔茨海默病(AD)与衰老相关的分子和细胞机制 降低风险和延长寿命的基因变异是基因转换的关键瓶颈 将发现转化为有效的疗法。我们的提案旨在通过关注生物过程来填补这一空白 (胆固醇/脂类清除)、细胞类型(小胶质细胞)和与人类遗传学有关的基因。大脑是 体内大多数富含胆固醇/脂肪的器官。因此，老年人/患病的大脑中的组织损伤会产生大量 大量富含胆固醇/脂质的细胞碎片。巨噬细胞(MΦ，就像大脑中的小胶质细胞一样)是一种细胞类型 专门研究维持和恢复组织的动态平衡，主要是通过它们吞噬细胞和 清除凋亡细胞和其他细胞碎片(泡沫化)和2)协调先天免疫反应。 当受到富含胆固醇/脂质的细胞碎片的挑战时，MΦ上调了几个基因的表达 参与胆固醇/脂类代谢、胞吐和AD。这一基因表达谱通常被称为 AS DAM/LAM(用于疾病相关的小胶质细胞/脂质相关的巨噬细胞)。最激动人心的 DAM/LAM基因是载脂蛋白E(ApopoProtein E，APOE)，是胆固醇代谢和AD的主要基因，也是非常重要的基因之一。 很少有基因与长寿有关。 我们和其他人表明，在全基因组关联中发现了常见的非编码AD风险等位基因 研究(GWA)富含MΦ特异性增强子，与这些细胞类型和MΦ特异性密切相关 基因表达调控(以及转录因子如SPI1/PU.1)在AD发病机制中的作用基因的通径分析 这些等位基因还与胆固醇代谢、吞噬作用和先天免疫有关。类似地，罕见的编码 AD风险等位基因影响在MΦ中高表达或特异表达的基因的结构/功能 在胞吐中的作用(例如，TREM2、ABCA7、ABI3)。我们的主要假设是AD/衰老相关的遗传 变异体通过影响基因的表达或结构从而影响参与基因的活性来调节风险/寿命 吞噬清除富含胆固醇/脂肪的细胞碎片，从而损害小胶质细胞维持大脑的能力 衰老和疾病期间的组织动态平衡。我们的方法是使用同基因的人iPSC来源的小胶质细胞 携带AD/衰老相关基因和基因修饰的(IMGL)评估其对小胶质细胞的影响 在基线和与衰老/疾病相关的情况下，脂代谢、基因表达和功能作用， 在体外(2D和3D培养)和体内(小鼠脑异种移植43)。特别是，我们将重点关注APOE 基因分型(Aim 1)、BHLHE40/41和NR1H2/3转录因子(Aim 2)及其治疗相互作用 (目标3)。

SUMMARY Understanding the molecular and cellular mechanisms linking Alzheimer’s disease (AD) and aging-associated genetic variants to reduced risk and increased longevity is a critical bottleneck for the translation of genetic findings into effective therapeutics. Our proposal aims to fill this gap by focusing on biological processes (cholesterol/lipid clearance), cell types (microglia), and genes implicated by human genetics. The brain is the most cholesterol/lipid-rich organ in the body. Hence, tissue damage in the aged/diseased brain produces large amounts of cholesterol/lipid-rich cellular debris. Macrophages (MΦ, like microglia in the brain) are cell types specialized in maintenance and restoration of tissue homeostasis, mainly via their ability to 1) phagocytose and clear apoptotic cells and other cellular debris (efferocytosis) and 2) orchestrate an innate immune response. When challenged with cholesterol/lipid-rich cellular debris, MΦ upregulate the expression of several genes involved in cholesterol/lipid metabolism, efferocytosis and AD. This gene expression profile is often referred to as DAM/LAM (for disease-associated microglia/ lipid-associated macrophages). The most upregulated DAM/LAM gene is Apolipoprotein E (APOE), a major gene for cholesterol metabolism and AD, and one of very few genes associated with longevity. We and others showed that common non-coding AD risk alleles identified in genome-wide association studies (GWAS) are enriched in MΦ-specific enhancers, strongly implicating these cell types and MΦ-specific gene expression regulation (and transcription factors like SPI1/PU.1) in the etiology of AD. Pathway analysis of these alleles also implicated cholesterol metabolism, phagocytosis, and innate immunity. Similarly, rare coding AD risk alleles impact the structure/function of genes highly or specifically expressed in MΦ and with critical roles in efferocytosis (e.g., TREM2, ABCA7, ABI3). Our main hypothesis is that AD/aging-associated genetic variants modulate risk/longevity by affecting expression or structure and thus activity of genes involved in phagocytic clearance of cholesterol/lipid-rich cellular debris, thereby impairing microglia ability to maintain brain tissue homeostasis during aging and disease. Our approach is to use isogenic human iPSC-derived microglia (iMGL) carrying AD/aging-associated genotypes and gene modifications to assess their impact on microglial lipid metabolism, gene expression, and functional roles, at baseline and in aging/disease-relevant contexts, both in vitro (2D and 3D cultures) and in vivo (mouse brain xenografts43). In particular, we will focus on APOE genotypes (Aim 1), BHLHE40/41 and NR1H2/3 transcription factors (Aim 2), and their therapeutic interactions (Aim 3).