Elucidating endolysosomal trafficking dysregulation induced by APOE4 in human astrocytes

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，强调了 需要更好地了解AD的发病机制。常染色体显性遗传和散发性的纵向研究 AD已经证明，病理学在临床症状出现之前10-20年就开始了，但发育影响 AD相关的遗传变异可能为未来的神经病理学变化提供了基础。许多AD 致病性GWAS变异体与参与神经胶质细胞内溶酶体途径的基因相关。但如何 这些致病基因正在影响细胞机制，必须进一步研究以解决这种疾病。一 一个重要的问题是，这些内溶酶体途径基因是否在理想情况下会聚在一个明确的 表型，可以靶向治疗。其中，载脂蛋白E（APOE）是最重要的 风险等位基因APOE 44（APOE 44）的晚发型AD风险纯合性增加AD风险超过15- 折为了全面评估人APOE 4对人脑细胞类型的影响，我们表征了 APOE 4基因型-表型在四种脑细胞类型中的关系：小胶质细胞、星形胶质细胞、脑微血管 内皮细胞和源自人诱导多能干细胞（iPSC）的混合皮质培养物。全球 转录组分析和体外机制研究揭示人APOE 44星形胶质细胞隔离 溶酶体中的胆固醇，导致胆固醇生物合成上调，尽管细胞内胆固醇水平升高， 胆固醇我们的数据表明，APOE 4介导的脂质积累损害了多个细胞内， 汇聚在溶酶体上的运输途径。因此，我们假设细胞内脂质 APOE 44在星形胶质细胞中的积累阻碍了向溶酶体的运输（Aim 1），这是由上游调控的。 可以通过CRISPRi基因筛选鉴定的调节剂（Aim 2）。APOE 4导致的内溶酶体缺陷 体外星形胶质细胞在体内可因神经变性诱导的过度脂质挑战而恶化（Aim 3）。为了验证这些假设，在目标1中，我们将确定脂质介导的 体外人APOE 44星形胶质细胞内溶酶体运输的转录和功能变化。的 将在AD死后脑中验证鉴定的表型。在Aim 2中，使用CRISPRi筛选APOE 44 星形胶质细胞，我们将确定目标，以扭转缺陷吞噬和细胞内脂质积累， 通过CROP-seq确定机制。在目标3中，我们将研究在哺乳动物中的机制性内溶酶体缺陷。 基线和脱髓鞘相关脂质碎片激发期间的体内异种移植星形胶质细胞， 进一步测试CRISPRi靶向的星形胶质细胞是否在体内表现出拯救的表型。该项目的目标是 评估脂质运输中APOE 4驱动的内溶酶体和自噬缺陷的分子机制 并确定逆转表型的调节靶点。此外，这项拟议的研究项目旨在 发现潜在的治疗药物靶点，以解决APOE 4驱动的内溶酶体运输内表型 在APOE 4携带者AD患者中发现。

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.