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

研究背景早在阿尔茨海默病的早期描述中，就观察到阿尔茨海默病患者脑细胞质中的脂质堆积。然而，绝大多数阿尔茨海默病(AD)的生物医学研究都集中在淀粉样斑块和神经原纤维缠结这两个大体组织病理学指标上。这项研究的结果是200多项临床试验，重点放在这两个未能产生有效治疗的目标上。造成这一失败的原因有几个；其中包括对阿尔茨海默病的基本病理生理学缺乏了解，以及没有选择更真实地模拟与AD病理最相关的人类细胞的疾病模型。最近，GWAS研究已确定脂代谢是阿尔茨海默病的一条重要途径(Jansen等人，2019年)，GWAS HITS，如TREM2和ApoE，除了调节炎症过程外，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，将是了解AD的病理生理学的基础。巨噬细胞和小胶质细胞含有炎症时积累的脂质已被描述(Nadjar，2018)。脂滴由三酰甘油和二酰甘油等中性脂类组成，与炎症和细胞因子的储存有关，但也与体内平衡功能有关，如支持吞噬功能的脂肪酸的储存和适当的线粒体功能。所有这些研究都有力地证明了脂肪酸可以调节小胶质细胞的吞噬活性。然而，还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净结果，因为根据上下文的不同，增加小胶质细胞的吞噬可能是有益的(例如，清除A斑块)，也可能是有害的(例如，吞噬功能突触)。研究设计本项目将调查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。通过使用患者来源的分化为小胶质细胞的IPSC有效地对疾病进行建模，将通过全基因组CRISPR/Cas9筛选确定影响脂质积累的靶点。首先，将开发一种可靠的方法来检测人IPSC来源的小胶质细胞中脂滴的形成，并探索疾病和等基因系之间的比较。然后将进行全基因组CRISPR/Cas9筛查，以确定与脂质积累有关的重要基因和治疗验证的潜在靶点。然后将对脂滴的形成和化合物对吞噬、ROS和细胞因子产生的调节的后果进行进一步的评估。最后，我们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。含义使用人类IPSC来源的小胶质细胞将使与疾病相关的细胞能够更真实地建模，通过研究这种未知的小胶质细胞脂质积累在AD病理生理学中的作用，我们旨在确定新的靶点，这些靶点可以被探索为潜在的治疗阿尔茨海默病的新策略。2019年。全基因组荟萃分析确定了影响阿尔茨海默病风险的新的基因座和功能途径。《自然遗传学》51(3)，第404-413页。DOI：10.1038/s41588-0180311-9Nadjar，A.代谢程序在脂质调节小胶质细胞吞噬功能中的作用。前列腺素Leukot Essen脂肪酸135，第63-73页。DOI：10.1016/j.plefa.2018.07.006

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