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.

项目概要阿尔茨海默病（AD）是最常见的痴呆症，目前尚无有效的治疗方法，这强调了需要更好地了解常染色体显性和散发性 AD 发病机制。 AD 已证明病理学开始于临床症状前 10-20 年，但对发育的影响 AD 相关的遗传变异可能为未来的神经病理学变化提供基础。因果 GWAS 变异与神经胶质细胞内溶酶体途径相关的基因相关。这些致病基因正在影响细胞机制，必须进一步研究以解决该疾病。重要的问题之一是这些内溶酶体途径基因是否在理想情况下汇聚到一个明确的其中，载脂蛋白 E (APOE) 是最重要的治疗靶点。迟发性 AD 的风险 — 风险等位基因 APOE ɛ4 (APOE 44) 的纯合性使 AD 风险增加超过 15- 为了全面评估人 APOE4 对人脑细胞类型的影响，我们对人脑细胞类型进行了表征。四种脑细胞类型中的 APOE4 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。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，导致尽管细胞内胆固醇水平升高，但胆固醇生物合成上调我们的数据表明 APOE4 介导的脂质积累会损害多种细胞内的脂质。因此，我们捕获了细胞内脂质。 APOE 44 星形胶质细胞中的积累会阻碍向溶酶体的运输（目标 1），而溶酶体由上游控制可以通过 CRISPRi 基因筛选来识别的调节因子（目标 2）。体外星形胶质细胞在体内可能因神经变性引起的过度脂质挑战而加剧（目的 3). 为了检验这些假设，在目标 1 中，我们将确定脂质介导的机制缺陷。内溶酶体运输体外人 APOE 44 星形胶质细胞的转录和功能变化。在目标 2 中，将使用 APOE 44 上的 CRISPRi 筛选在 AD 死后大脑中验证所识别的表型。星形胶质细胞，我们将确定逆转缺陷吞噬作用和细胞内脂质积累的靶标通过 CROP-seq 确定机制在目标 3 中，我们将研究机械内溶酶体缺陷。在基线和脱髓鞘相关脂质碎片挑战期间体内异种移植的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挽救的表型。评估脂质运输中 APOE4 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监管目标。此外，该拟议的研究项目旨在发现潜在的治疗药物靶点来解决 APOE4 驱动的内溶酶体运输内表型发现于 APOE4 携带者 AD 患者。