Elucidating endolysosomal trafficking dysregulation induced by APOE4 in human astrocytes

阐明人星形胶质细胞中 APOE4 诱导的内溶酶体运输失调

• 批准号: 10835321
• 负责人: Julia TCW
• 金额: $ 4.57万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10835321
• 关键词: Affect; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease patient; Alzheimer' s disease risk; Alzheimer' s disease therapeutic; Apolipoprotein E; Astrocytes; Autopsy; Brain; CRISPR interference; Cholesterol; Clinical; Data; Defect; Dementia; Demyelinations; Development; Disease; Drug Targeting; Exhibits; Future; Genes; Genetic Screening; Genetic Transcription; Genotype; Goals; Human; Impairment; In Vitro; Intracellular Accumulation of Lipids; Knowledge; Late Onset Alzheimer Disease; Lipids; Longitudinal Studies; Lysosomes; Mediating; Microglia; Modality; Molecular; Nerve Degeneration; Neuroglia; Pathogenesis; Pathology; Pathway interactions; Phagocytosis; Phenotype; Research Project Grants; Risk; Symptoms; Testing; Therapeutic; Variant; Xenograft procedure; apolipoprotein E-4; autosome; brain cell; brain endothelial cell; causal variant; cell type; cholesterol biosynthesis; disorder risk; effective therapy; endophenotype; genetic risk factor; genetic variant; genome wide association study; in vivo; induced pluripotent stem cell; lipid transport; neuropathology; novel; risk variant; stem cell model; targeted treatment; trafficking; transcriptome

Project Summary Alzheimer’s disease (AD) is the most common form of dementia without effective treatments, underscoring the need for a better understanding of AD pathogenesis. Longitudinal studies in autosomal dominant and sporadic AD have demonstrated that pathology begins 10-20 years before clinical symptoms, but developmental effects of AD-associated genetic variants likely provide a substrate for future neuropathological changes. Many AD causal GWAS variants are associated with genes involved in endolysosomal pathways in glia. However, how these causal genes are affecting cellular mechanisms has to be further investigated to tackle the disease. One of the important questions is whether these endolysosomal pathway genes converge on ideally one clear phenotype that can be targeted for therapeutics. Among others, Apolipoprotein E (APOE) is the most significant risk for late-onset AD—homozygosity for the risk allele APOE ɛ4 (APOE 44) increases AD risk by more than 15- fold. To comprehensively assess the effect of human APOE4 on human brain cell types, we characterized the APOE4 genotype-phenotype relationship in four brain cell types: microglia, astrocytes, brain microvascular endothelial cells and mixed cortical cultures derived from human induced pluripotent stem cells (iPSCs). Global transcriptome analyses and in vitro mechanism study reveal that human APOE 44 astrocytes sequester cholesterol in lysosomes, leading to upregulated cholesterol biosynthesis despite elevated intracellular cholesterol. Our data suggests that the APOE4-mediated lipid accumulation impairs multiple intracellular trafficking pathways that converge on the lysosome. Therefore, we hypothesize that intracellular lipid accumulation in APOE 44 astrocytes jams trafficking to the lysosome (Aim 1), which are controlled by upstream regulators that can be identified by CRISPRi genetic screening (Aim 2). The APOE4-led endolysosomal defects in vitro astrocytes can be exacerbated in vivo by excessive lipid challenge induced by neurodegeneration (Aim 3). To test these hypotheses, in Aim 1, we will determine the mechanistic defects of lipid-mediated endolysosomal trafficking in vitro human APOE 44 astrocytes in transcriptional and functional changes. The identified phenotypes will be validated in AD post-mortem brain. In Aim 2, using CRISPRi screen on APOE 44 astrocytes, we will identify targets to reverse defected phagocytosis and intracellular lipid accumulation and determine the mechanisms by CROP-seq. In Aim 3, we will investigate mechanistic endolysosomal defects in vivo xenotransplanted astrocytes at baseline and during demyelination-associated lipid debris challenge and further test if CRISPRi-targeted astrocytes exhibit rescued phenotypes in vivo. The goal of this project is to assess the molecular mechanisms of APOE4-driven endolysosomal and autophagic defects in lipid trafficking and identify regulatory targets that reverse the phenotype. Further, this proposed research project sets out to uncover potential therapeutic drug targets to tackle APOE4-driven endolysosomal trafficking endophenotypes found in APOE4 carrier AD patients.

项目摘要 阿尔茨海默病(AD)是最常见的痴呆症形式，没有有效的治疗方法，强调了 需要更好地了解AD的发病机制。常染色体显性和散发性的纵向研究 AD已经证明，病理学开始于临床症状之前10-20年，但对发育的影响 与AD相关的遗传变异可能为未来的神经病理变化提供了底物。许多AD GWA型突变体与胶质细胞内溶酶体途径相关的基因有关。然而，如何 这些致病基因正在影响细胞机制，必须进一步研究才能应对这种疾病。一 其中一个重要的问题是，这些内溶酶体途径基因是否会聚在理想的一个透明的 可作为治疗靶点的表型。其中，载脂蛋白E(ApoE)是最重要的 晚发性AD的风险-风险等位基因apoEɛ4(ApoE 44)的纯合子使AD风险增加超过15%- 收牌。为了全面评估人类载脂蛋白4对人脑细胞类型的影响，我们对 四种脑细胞类型：小胶质细胞、星形胶质细胞、脑微血管细胞ApoE4基因-表型关系的研究 人诱导多能干细胞(IPSCs)来源的内皮细胞和混合皮质培养物。全球 转录组分析和体外机制研究表明，人载脂蛋白44星形胶质细胞隔离 溶酶体中的胆固醇，导致细胞内胆固醇生物合成上调 胆固醇。我们的数据表明，APOE4介导的脂质堆积损害了细胞内的多个 聚集在溶酶体上的运输途径。因此，我们假设细胞内的脂质 APOE 44星形胶质细胞中的积聚阻碍了运输到溶酶体(Aim 1)，溶酶体受上游控制 可通过CRISPRi基因筛查确定的调节子(目标2)。载脂蛋白4所致的内溶酶体缺陷 体外培养的星形胶质细胞可因神经变性引起的过多脂质刺激而在体内恶化(目的 3)。为了验证这些假设，在目标1中，我们将确定脂质介导的机制缺陷 体外培养人载脂蛋白44星形胶质细胞内溶酶体转运在转录和功能上的变化。这个 已确定的表型将在AD死后脑中得到验证。在AIM 2中，在APOE 44上使用CRISPRi屏幕 星形胶质细胞，我们将确定靶点，逆转有缺陷的吞噬和细胞内脂质堆积 根据作物生长顺序确定机理。在目标3中，我们将研究机械性内溶酶体缺陷 体内异种移植星形胶质细胞在基线和脱髓鞘相关脂碎屑激发期间 进一步测试CRISPRi靶向星形胶质细胞是否在体内表现出被拯救的表型。这个项目的目标是 评估APOE4驱动的内溶酶体和自噬缺陷在脂质运输中的分子机制 并确定逆转表型的调控目标。此外，这项拟议的研究项目旨在 发现潜在的治疗药物靶点以应对APOE4驱动的内溶酶体运输内表型 在APOE4携带者AD患者中发现。