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

阿尔茨海默病患者小胶质细胞代谢和吞噬功能的影像研究 全基因组关联研究表明，阿尔茨海默病的一些最强的遗传风险变异 (AD)涉及仅在小胶质细胞中表达的基因，表明其在AD病理中发挥核心作用。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以及 通过吞噬碎片和分泌细胞因子提供第一道防线。在AD中，以CNS为特征 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AD特有的各种小胶质细胞转录状态，表明保护性和 有害的功能。虽然他们的转录特征很好，但我们缺乏对 驱动保护性/有害小胶质细胞表型形成的分子机制，其功能 特征以及它们如何影响AD疾病的病理。仅通过将转录配置文件连接到 功能细胞状态可以帮助我们确定有希望的新的治疗靶点。具有有害基因的亚种群 功能标志可能代表AD的新治疗靶点。这需要集成新的 技术进入该领域，在那里单个细胞的转录图谱可以通过它们的 功能标志，与小胶质细胞激活剂和病理标志相关。 在这里，我们建议用代谢和显微技术来补充现有的转录数据 阿尔茨海默病患者脑内小胶质细胞的吞噬情况。小胶质细胞转录的异质性 使传统的基于免疫染色的表型难以明确区分 荧光显微镜。相反，我们开发了一线非线性显微镜平台，其中 小胶质细胞的表型可以根据它们的代谢和吞噬特性利用光谱进行区分 相干反斯托克斯拉曼(CARS)和同时双光子激发荧光(TPEF) 显微镜。从显微镜图像中提取的定量数据将汇编成简档； (I)细胞内脂质储存、(Ii)线粒体和(Iii)细胞氧化还原比率的量将整合到 代谢谱，同时(Iv)溶酶体髓鞘/淀粉样蛋白碎片，(V)胞浆髓鞘碎片，和(Vi)积聚 像脂褐素这样不可降解的废物会形成吞噬细胞的轮廓。通过进一步集成映射 使用RNA探针(RNAScope)分发特定的RNA转录本，我们将能够链接 在单细胞水平上的代谢和吞噬功能的转录表达。具体来说， 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 我们已经发现了一组基因，它们通过我们的 全基因组功能CRISPR基因敲除筛选。这将揭示功能失调的脂质的遗传调节。 聚集，有害的小胶质细胞的特征，这可能是新的治疗靶点。我们 可以预见，小胶质细胞的代谢重新编程将成为AD的一条新的治疗途径。

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.