Imaging the metabolic and phagocytic landscape of microglia in Alzheimer’s disease

对阿尔茨海默病中小胶质细胞的代谢和吞噬景观进行成像

• 批准号: 10190479
• 负责人: Sarah C Heilshorn
• 金额: $ 15.75万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10190479
• 关键词: Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease diagnosis; Alzheimer' s disease pathology; Alzheimer' s disease risk; Amyloid; Amyloid beta-Protein; Brain; Cells; Characteristics; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Data; Deposition; Disease; Disease Progression; Environment; Exposure to; Fluorescence Microscopy; Gene Expression Profile; Genes; Genetic; Genetic Risk; Genetic Transcription; Health; Heterogeneity; Human; Human Cell Line; Human Genome; Image; Immune; In Situ Hybridization; Individual; Inflammatory; Information Distribution; Intervention; Knock-out; Link; Lipids; Lipofuscin; Maps; Messenger RNA; Metabolic; Microglia; Microscope; Microscopy; Mitochondria; Molecular; Myelin; Nerve Degeneration; Neurites; Oxidation-Reduction; Pathologic; Pathology; Phagocytes; Phenotype; Play; Proteins; RNA; RNA Probes; Role; Route; Tissues; Transcript; base; brain cell; brain parenchyma; brain tissue; cytokine; disorder prevention; fluorophore; gene discovery; genome wide association study; genome-wide; imaging probe; lipid metabolism; metabolic imaging; metabolic phenotype; metabolic profile; microscopic imaging; new technology; new therapeutic target; novel therapeutics; protein aggregation; reduce symptoms; response; risk variant; single cell analysis; single-cell RNA sequencing; symptom treatment; therapeutic target; trait; two-photon; wasting

Imaging the Metabolic and Phagocytic Landscape of Microglia in Alzheimer’s Disease Genome-wide association studies show that some of the strongest genetic risk variants for Alzheimer’s disease (AD) involve genes exclusively expressed in microglia, indicating its central role in AD pathology. Microglia are the resident immune cells of the brain, essential for maintaining the health and function of the brain, as well as providing a first line of defense by phagocytizing debris and secreting cytokines. In AD, characterized by a CNS environment with chronic exposure to cellular debris and protein aggregation, recent single-cell RNA sequencing has uncovered a variety of microglial transcriptional states specific to AD, indicating both protective and detrimental functions. While their transcriptional profiles are well-characterized, we lack an understanding of the molecular mechanisms that drive the formation of protective/detrimental microglial phenotypes, their functional characteristics and how they inform AD disease pathology. Only by connecting transcriptional profiles to functional cell-states can we identify promising, new therapeutic targets. Subpopulations with detrimental functional signatures may represent novel therapeutic targets for AD. This requires the integration of new technologies into the field, where the transcriptional profile of individual cells can be complemented by their functional signatures and correlated with microglia-activating agents and pathological hallmarks. Here, we propose to complement available transcriptional data with microscopy of the metabolic and phagocytic landscape of microglia in the human AD brain. The heterogeneity of microglial transcription makes it difficult to unambiguously distinguish phenotypes based on immunostaining in conventional fluorescence microscopy. Instead, we have developed a front-line nonlinear microscopy platform, where microglial phenotypes can be distinguished based on their metabolic and phagocytic profiles using spectral coherent anti-Stokes Raman (CARS) and simultaneous two-photon excited fluorescence (TPEF) microscopy. The profiles will be compiled from quantitative data extracted from the microscopy images; amounts of (i) intracellular lipid stores, (ii) mitochondria, and (iii) the cellular redox ratio will be integrated into the metabolic profile, while (iv) lysosomal myelin/amyloid debris, (v) cytosolic myelin debris, and (vi) accumulating undegradable waste as lipofuscin will form the phagocytic profile. By further integrating a capability to map the distribution of specific RNA transcripts using RNA probes (RNAScope), we will be able to link transcriptional expression to the metabolic and phagocytic profiles at the single cell level. Specifically, we will investigate the metabolic and phagocytic signatures of microglia in human AD brain tissues that express a set of genes, which we have discovered to modulate lipid accumulation in human immune cells through our functional genome-wide CRISPR knock-out screens. This will reveal genetic regulators of dysfunctional lipid accumulation, characteristic for detrimental microglia, which may represent novel therapeutic targets. We envision that metabolic reprogramming of microglia will become a new therapeutic route for AD.

阿尔茨海默病中小胶质细胞代谢和吞噬景观的成像 全基因组关联研究表明，阿尔茨海默病的一些最强遗传风险变异 (AD)涉及仅在小胶质细胞中表达的基因，表明其在AD病理学中的中心作用。小胶质细胞是 大脑的常驻免疫细胞，对维持大脑的健康和功能至关重要，以及 通过吞噬碎片和分泌细胞因子提供第一道防线。在以CNS为特征的AD中 长期暴露于细胞碎片和蛋白质聚集的环境，最近的单细胞RNA测序 已经发现了多种AD特异性的小胶质细胞转录状态，这表明AD既具有保护性， 有害功能。虽然他们的转录谱是很好的特点，我们缺乏了解， 驱动保护性/有害性小胶质细胞表型形成的分子机制，其功能 特征以及它们如何告知AD疾病病理学。只有将转录谱与 功能性细胞状态，我们可以确定有前途的，新的治疗靶点。有害亚群 功能特征可能代表AD的新治疗靶点。这就需要整合新的 技术进入该领域，其中单个细胞的转录谱可以通过它们的 功能特征并与小胶质细胞激活剂和病理标志相关。 在这里，我们建议补充可用的转录数据与显微镜的代谢和 人AD脑中小胶质细胞的吞噬景观。小胶质细胞转录的异质性 这使得难以在常规免疫组织化学中基于免疫染色明确区分表型， 荧光显微镜相反，我们开发了一个前线非线性显微镜平台， 小胶质细胞表型可以基于它们的代谢和吞噬概况使用光谱分析来区分。 相干反斯托克斯拉曼（汽车）和同时双光子激发荧光（TPEF） 显微镜将根据从显微镜图像中提取的定量数据编制概况; （i）细胞内脂质储存，（ii）线粒体，和（iii）细胞氧化还原比的量将整合到细胞内。 代谢谱，而（iv）溶酶体髓鞘/淀粉样蛋白碎片，（v）胞质髓鞘碎片，和（vi）积累 不可降解的废物如脂褐素将形成吞噬作用。通过进一步集成映射 使用RNA探针（RNAScope）的特定RNA转录本的分布，我们将能够连接 在单细胞水平上，转录表达与代谢和吞噬特征相关。具体地说， 我们将研究人类AD脑组织中小胶质细胞的代谢和吞噬特征， 一组基因，我们已经发现，通过我们的研究， 功能性全基因组CRISPR敲除筛选。这将揭示功能失调的脂质的遗传调节因子 积累，有害的小胶质细胞的特征，这可能代表新的治疗靶点。我们 我们可以预见，小胶质细胞的代谢重编程将成为AD的一种新的治疗途径。