iPSC Macrophages/microglia and genome-wide CRISPR/Cas9 screening to investigate lipid accumulation in the pathophysiology of Alzheimer's Disease

iPSC 巨噬细胞/小胶质细胞和全基因组 CRISPR/Cas9 筛选研究阿尔茨海默病病理生理学中的脂质积累

• 批准号: 2886802
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2886802
• 关键词: iPSC; Macrophages; microglia; genome; wide

BackgroundLipid accumulation in the cell cytoplasm of Alzheimer's brains was observed in the earliest descriptions of the disease. However, the vast majority of biomedical research in Alzheimer's disease (AD) has focused on the two gross histopathological targets of amyloid plaques and neurofibrillary tangles. The result of this research has been over 200 clinical trials focusing on these two targets that failed to generate an effective therapy. There are several reasons for this failure; among these are the poor understanding of the fundamental pathophysiology of Alzheimer's disease and the poor choice of disease models to more faithfully mimic the human cells most associated with AD pathology.Recently, GWAS studies have identified lipid metabolism as an important pathway in Alzheimer's disease (Jansen et al., 2019), with GWAS hits, such as Trem2 and ApoE, involved in lipid sensing and processing, in addition to regulation of inflammatory processes. A greater understanding of lipid regulation, and in particular of neuroinflammation, would be fundamental to understanding the pathophysiology of AD. Macrophages and microglia containing accumulations of lipids upon inflammation have been described (Nadjar, 2018). Lipid droplets, consisting of neutral lipids such as triacylglycerols and diacylglycerols, have been associated with inflammation and cytokine storage, but also with homeostatic functions, such as storage of fatty acids that could support phagocytosis and proper mitochondria functioning.All these studies strongly evidence that fatty acids can modulate microglial phagocytic activity. More studies are needed however to understand the mechanisms by which microglia interact with fatty acids and how they can shift microglia from one phenotype/function to another. Moreover, we still need to address the net outcome of these functional shifts for brain homeostasis, since depending on the context, increasing microglial phagocytosis might be beneficial (e.g. clearance of A plaques) or detrimental (e.g. phagocytosis of functional synapses).Research DesignThis project will investigate whether altering microglia lipid accumulation can improve microglial functions in human cell models. By modelling the disease effectively using patient-derived iPSC differentiated into microglia, targets affecting lipid accumulation will be identified through genome-wide CRISPR/Cas9 screening. Firstly, a robust assay for lipid droplet formation in human iPSC-derived microglia will be developed and comparisons between disease and isogenic lines explored. A genome-wide CRISPR/Cas9 screen will then be performed to identify important genes involved in lipid accumulation and potential targets for therapeutic validation. Further assessments will then be made of the consequences of lipid droplet formation and modulation by compounds on phagocytosis, ROS and cytokine production. Finally, the effect of lipid droplet modulation on complex co-culture models including neurons and astrocytes will be investigated.ImplicationUsing human iPSC-derived microglia will enable more faithful modelling of the cells associated with the disease, and by investigating this unexplored role of microglial lipid accumulation in the pathophysiology of AD, we aim to identify new targets that can be explored as potential new strategies for therapeutic intervention in Alzheimer's disease.ReferencesJansen, I. E. et al. 2019. Genome-wide meta-analysis identifies new loci and functional pathways influencing Alzheimer's disease risk. Nature Genetics 51(3), pp. 404-413. doi: 10.1038/s41588-018-0311-9Nadjar, A. 2018. Role of metabolic programming in the modulation of microglia phagocytosis by lipids. Prostaglandins Leukot Essent Fatty Acids 135, pp. 63-73. doi: 10.1016/j.plefa.2018.07.006

背景 在对该疾病的最早描述中就观察到了阿尔茨海默氏症大脑细胞质中的脂质积累。然而，阿尔茨海默病（AD）的绝大多数生物医学研究都集中在淀粉样蛋白斑和神经原纤维缠结这两个总体组织病理学目标上。这项研究的结果是针对这两个目标进行了 200 多项临床试验，但未能产生有效的治疗方法。造成这种失败的原因有很多；其中包括对阿尔茨海默病的基本病理生理学了解甚少，以及疾病模型选择不当，无法更忠实地模拟与 AD 病理学最相关的人类细胞。最近，GWAS 研究发现脂质代谢是阿尔茨海默病的重要途径（Jansen et al., 2019），GWAS 命中的 Trem2 和 ApoE 除了参与炎症过程的调节外，还参与脂质传感和处理。更好地了解脂质调节，特别是神经炎症，对于了解 AD 的病理生理学至关重要。已经描述了炎症时含有脂质积累的巨噬细胞和小胶质细胞（Nadjar，2018）。脂滴由三酰甘油和二酰甘油等中性脂质组成，与炎症和细胞因子储存有关，但也与稳态功能有关，例如可以支持吞噬作用和正常线粒体功能的脂肪酸储存。所有这些研究有力地证明，脂肪酸可以调节小胶质细胞的吞噬活性。然而，需要更多的研究来了解小胶质细胞与脂肪酸相互作用的机制以及它们如何将小胶质细胞从一种表型/功能转变为另一种表型/功能。此外，我们仍然需要解决这些功能转变对大脑稳态的最终结果，因为根据具体情况，增加小胶质细胞吞噬作用可能是有益的（例如A斑块的清除）或有害的（例如功能性突触的吞噬作用）。将研究改变小胶质细胞脂质积累是否可以改善人类细胞模型中的小胶质细胞功能。通过使用分化为小胶质细胞的患者来源的 iPSC 有效地对疾病进行建模，将通过全基因组 CRISPR/Cas9 筛选来确定影响脂质积累的靶标。首先，将开发一种针对人类 iPSC 衍生的小胶质细胞中脂滴形成的强大测定方法，并探索疾病和同基因系之间的比较。然后将进行全基因组 CRISPR/Cas9 筛选，以确定参与脂质积累的重要基因和治疗验证的潜在靶点。然后将进一步评估脂滴形成和化合物对吞噬作用、ROS 和细胞因子产生的调节的影响。最后，将研究脂滴调节对包括神经元和星形胶质细胞在内的复杂共培养模型的影响。 意义：使用人类 iPSC 衍生的小胶质细胞将能够更忠实地对与疾病相关的细胞进行建模，并通过研究小胶质细胞脂质的这种尚未探索的作用随着 AD 病理生理学的积累，我们的目标是确定新的靶点，这些靶点可以作为阿尔茨海默氏病治疗干预的潜在新策略进行探索。 2019。全基因组荟萃分析确定了影响阿尔茨海默病风险的新位点和功能途径。 《自然遗传学》51(3)，第 404-413 页。 doi: 10.1038/s41588-018-0311-9Nadjar, A. 2018。代谢编程在脂质调节小胶质细胞吞噬作用中的作用。前列腺素白细胞精华脂肪酸 135，第 63-73 页。 doi：10.1016/j.plefa.2018.07.006