Developing Fluorescence Lifetime Imaging Microscopy (FLIM) as a novel method to measure microglial metabolism in situ

开发荧光寿命成像显微镜（FLIM）作为原位测量小胶质细胞代谢的新方法

• 批准号: 10040954
• 负责人: Kimberley D Bruce
• 金额: $ 15.55万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2022-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040954
• 关键词: Adult; Alzheimer' s Disease; Brain; Brain Diseases; Brain Injuries; Cell physiology; Cells; Cues; Data; Demyelinations; Development; Disease; Environment; Experimental Autoimmune Encephalomyelitis; Explosion; Fatty Acids; Fluorescence; Future; Gene Expression; Genetic Transcription; Glycolysis; Heterogeneity; Immune; In Situ; In Vitro; Intervention; Life; Life Cycle Stages; Lipids; Lipoproteins; Measurement; Measures; Metabolic; Metabolism; Methodology; Methods; Microglia; Mitochondria; Mus; NADH; Neurodegenerative Disorders; Oxidative Phosphorylation; Pathogenesis; Peripheral; Phenotype; Play; Process; Production; Resolution; Signaling Molecule; Techniques; Transcript; Treatment Efficacy; Up-Regulation; aged; aging brain; base; brain cell; brain tissue; cell type; design; fatty acid oxidation; fluorescence lifetime imaging; in vivo; injured; innovation; insight; macrophage; metabolic abnormality assessment; metabolic profile; novel; novel strategies; prevent; single-cell RNA sequencing; tool

ABSTRACT It is now becoming increasingly clear that microglia play a key role in the development of neurodegenerative diseases (ND) such as Alzheimer’s disease (AD). Microglia are highly plastic and respond to environmental cues by switching to a plethora of activation states, some of which are adaptive, while others are maladaptive. Understanding the heterogeneity of microglial activation in the context of AD may help design effective therapeutics that dampen the detrimental—but augment the beneficial—effects of activated microglia. In recent years, single-cell RNA seq (scRNAseq) analyses of microglia ex vivo have helped to define microglial heterogeneity in unprecedented detail. A striking feature of these studies is the identification of microglial clusters with common transcriptional profiles that arise during development and disease, characterized by (in part) altered expression of genes involved in lipid and lipoprotein metabolism. This is consistent with our current understanding of macrophage metabolism, where homeostatic macrophages maintain high rates of mitochondrial oxidative phosphorylation and fatty acid oxidation, compared to LPS-stimulated macrophages that metabolically shift towards glycolysis and fatty acid synthesis. In support, our preliminary data suggest that microglia show a similar metabolic polarization in vitro. Since microglial metabolism is one of the most prominent and consistent changes that follow activation/polarization, we hypothesize that microglial metabolism is a key component of the phenotypic switching involved in the development of AD. Moreover, we hypothesize that modulation of microglial metabolism may be a novel strategy to prevent, delay or reverse the onset of AD. However, our understanding of microglial metabolism is largely extrapolated from peripheral macrophages. In addition, scRNAseq studies may not be representative since the process of microglial isolation can profoundly alter cellular metabolism. In fact, no measurements of endogenous microglial metabolism are currently available. Fluorescence lifetime imaging microscopy (FLIM), is a state-of-the-art technique that measures the endogenous fluorescence of metabolic co-factors (e.g NADH and FAD), which have altered lifetimes depending on the metabolic status of the cell. In the proposed study, we will use FLIM as an innovative methodology to make precise measurements of microglial metabolism in situ. We will use FLIM to measure endogenous microglial metabolism in the early and aged brain to determine the metabolism of microglia in their native environment, and throughout life (AIM I). We will also perform FLIM in 5XFAD mice to define microglial metabolism in situ during AD pathogenesis (AIM II). This study will not only help develop a novel tool with which to measure microglial metabolism, but will also be the first the determine microglial metabolism in situ, guiding future studies of metabolic interventions to treat AD and beyond.

摘要 现在越来越清楚的是，小胶质细胞在神经退行性疾病（ND）如阿尔茨海默病（AD）的发展中起着关键作用。小胶质细胞具有高度可塑性，并通过切换到过多的激活状态来响应环境提示，其中一些是适应性的，而另一些是适应不良的。 了解AD背景下小胶质细胞激活的异质性可能有助于设计有效的治疗方法，抑制激活的小胶质细胞的神经毒性作用，但增加神经毒性作用。 近年来，离体小胶质细胞的单细胞RNA测序（scRNAseq）分析有助于以前所未有的细节定义小胶质细胞的异质性。 这些研究的一个显著特征是鉴定具有在发育和疾病期间出现的共同转录谱的小胶质细胞簇，其特征在于（部分）改变了参与脂质和脂蛋白代谢的基因的表达。这与我们目前对巨噬细胞代谢的理解是一致的，其中稳态巨噬细胞与代谢转向糖酵解和脂肪酸合成的LPS刺激的巨噬细胞相比，维持高速率的线粒体氧化磷酸化和脂肪酸氧化。作为支持，我们的初步数据表明，小胶质细胞在体外显示出类似的代谢极化。 由于小胶质细胞代谢是激活/极化后最突出和最一致的变化之一，因此我们假设小胶质细胞代谢是AD发展中所涉及的表型转换的关键组成部分。 此外，我们推测调节小胶质细胞代谢可能是预防、延迟或逆转AD发病的一种新策略。 然而，我们对小胶质细胞代谢的理解很大程度上是从外周巨噬细胞推断的。 此外，scRNAseq研究可能不具有代表性，因为小胶质细胞分离的过程可以深刻地改变细胞代谢。 事实上，目前还没有内源性小胶质细胞代谢的测量方法。 荧光寿命成像显微镜（FLIM）是一种最先进的技术，用于测量代谢辅因子（如NADH和FAD）的内源性荧光，这些辅因子的寿命取决于细胞的代谢状态。 在拟议的研究中，我们将使用FLIM作为一种创新的方法来精确测量原位小胶质细胞代谢。 我们将使用FLIM来测量早期和老年大脑中的内源性小胶质细胞代谢，以确定小胶质细胞在其天然环境中和整个生命中的代谢（AIM I）。 我们还将在5XFAD小鼠中进行FLIM，以确定AD发病机制（AIM II）期间的原位小胶质细胞代谢。 这项研究不仅有助于开发一种测量小胶质细胞代谢的新工具，而且也将是第一个原位确定小胶质细胞代谢的方法，指导未来治疗AD及其他疾病的代谢干预研究。