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

基本信息

批准号：
10670573
负责人：
Julia TCW
金额：
$ 79.29万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
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/10670573
关键词：
Acute Affect Alzheimer&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos s disease patient Alzheimer&apos s disease risk Alzheimer&apos s disease therapeutic Apolipoprotein E Astrocytes Autophagocytosis Autopsy BODIPY Biological Models Brain CRISPR interference CRISPR/Cas technology Cholesterol Clinical Clustered Regularly Interspaced Short Palindromic Repeats Cuprizone Data Defect Dementia Demyelinations Development Disease Drug Targeting Endosomes Excision Exhibits Functional disorder Future Genes Genetic Screening Genetic Transcription Genotype Goals Guide RNA Human Immunodeficient Mouse Impairment In Vitro Intracellular Accumulation of Lipids Knock-out Knowledge Label Late Onset Alzheimer Disease Lipids Longitudinal Studies Lysosomes Measures Mediating Metabolic Microglia Modality Modeling Molecular Molecular Chaperones Myelin NFIA gene Nerve Degeneration Neuroglia Organelles Pathogenesis Pathology Pathway interactions Phagocytes Phagocytosis Phagosomes Phenotype Phosphoric Monoester Hydrolases Phosphotransferases Process RNA library Regulator Genes Reporter Research Project Grants Risk Series Symptoms System Testing Therapeutic Transcriptional Regulation Transfection Treatment Efficacy Variant Western Blotting Xenograft procedure apolipoprotein E-4 astrocyte progenitor autosome base editing brain cell brain endothelial cell causal variant cell type cholesterol biosynthesis chronic demyelination disorder risk droplet sequencing effective therapy endophenotype genetic risk factor genetic variant genome wide association study in vivo induced pluripotent stem cell knock-down lipid transport loss of function neuropathology novel progenitor remyelination risk variant screening single-cell RNA sequencing stem cell model targeted treatment trafficking transcription activator-like effector nucleases transcription factor transcriptome transcriptome sequencing uptake

项目摘要

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氏病（AD）是痴呆症的最常见形式，没有有效的治疗，强调了需要更好地了解AD发病机理。常染色体显性和零星的纵向研究 AD证明病理学在临床症状之前10 - 20年开始，但发育效果与广告相关的遗传变异可能为未来的神经病理学变化提供了底物。许多广告因果GWAS变体与胶质中溶血疗法途径涉及的基因有关。但是，如何这些灾难基因正在影响细胞机制，必须进一步研究以应对该疾病。一重要的问题是这些内溶性途径基因是否在理想情况下融合了一个清晰的可以针对治疗的表型。除其他外，载脂蛋白E（APOE）是最重要的迟到广告的风险 - 风险等位基因APOEɛ4（APOE 44）的Homozygosity将AD风险增加超过15-- 折叠。为了全面评估人APOE4对人脑细胞类型的影响，我们表征了 APOE4四种脑细胞类型中的基因型 - 表型关系：小胶质细胞，星形胶质细胞，脑微血管源自人类诱导多能干细胞（IPSC）的内皮细胞和混合皮质培养物。全球的转录组分析和体外机理研究表明，人APOE 44星形胶质细胞序列溶酶体中的胆固醇，导致胆固醇生物合成目的地升高细胞内胆固醇。我们的数据表明APOE4介导的脂质积累会损害多个细胞内融合溶酶体的贩运途径。因此，我们假设细胞内脂质在APOE 44星形胶质细胞中积聚到溶酶体的堵塞（AIM 1），由上游控制可以通过CRISPRI基因筛查来识别的调节器（AIM 2）。由APOE4领导的内溶性缺陷体外星形胶质细胞可以通过神经变性引起的过度脂质挑战在体内加剧（AIM 3）。为了检验这些假设，在AIM 1中，我们将确定脂质介导的机械缺陷内侧溶性在转录和功能变化中体外人类APOE 44星形胶质细胞。这确定的表型将在验尸后大脑中得到验证。在AIM 2中，使用APOE 44上的CRISPRI屏幕星形胶质细胞，我们将确定靶标，以逆转衰落的吞噬作用和细胞内脂质的积累和通过农作物史确定机制。在AIM 3中，我们将研究机械性内溶性内溶缺陷基线和与脱髓鞘相关的脂质碎片挑战和脱髓鞘相关的质质质细胞的体内异种传输挑战和进一步测试是否针对CRISPRI的星形胶质细胞在体内表现出救出的表型。这个项目的目标是评估脂质运输中APOE4驱动的内溶性和自噬缺陷的分子机制并确定扭转表型的调节目标。此外，该提议的研究项目列出了发现潜在的治疗药物靶标，以应对APOE4驱动的内溶性贩运型内聚型在APOE4载体AD患者中发现。