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

摘要 现在越来越清楚的是，小胶质细胞在阿尔茨海默病(AD)等神经退行性疾病(ND)的发展中起着关键作用。小胶质细胞是高度可塑性的，通过切换到过多的激活状态来对环境提示做出反应，其中一些是适应性的，而另一些是不适应的。了解阿尔茨海默病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，减弱激活的小胶质细胞的不利影响，但增强其有益影响。近年来，对体外培养的小胶质细胞的单细胞RNA序列(ScRNAseq)分析已经帮助以前所未有的详细程度定义了小胶质细胞的异质性。这些研究的一个显著特点是识别具有共同转录特征的小胶质细胞簇，这些小胶质细胞簇在发育和疾病期间出现，其特征是(部分)参与脂类和脂蛋白代谢的基因表达发生变化。这与我们目前对巨噬细胞代谢的理解是一致的，与内毒素刺激的巨噬细胞代谢转向糖酵解和脂肪酸合成相比，动态平衡的巨噬细胞保持较高的线粒体氧化磷酸化和脂肪酸氧化速率。作为支持，我们的初步数据表明，小胶质细胞在体外表现出类似的代谢极化。由于小胶质细胞代谢是激活/极化后最显著和持续的变化之一，我们假设小胶质细胞代谢是参与AD发生发展的表型转换的关键组成部分。此外，我们假设小胶质细胞代谢的调节可能是预防、延迟或逆转AD发病的新策略。然而，我们对小胶质细胞代谢的了解在很大程度上是从外周巨噬细胞推断出来的。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，目前还没有测量内源性小胶质细胞代谢的方法。荧光寿命成像显微镜(FLiM)是一种最先进的技术，它测量代谢辅助因子(如NADH和FAD)的内源荧光，这些辅助因子根据细胞的代谢状态改变寿命。在这项拟议的研究中，我们将使用FOLIM作为一种创新的方法，在原位精确测量小胶质细胞的新陈代谢。我们将使用FOLIM来测量早期和老年脑中的内源性小胶质细胞代谢，以确定小胶质细胞在其自然环境中和整个生命过程中的代谢(AIM I)。我们还将在5XFAD小鼠身上进行FLIM，以确定AD发病过程中小胶质细胞的原位代谢(AIM II)。这项研究不仅将有助于开发一种新的工具来测量小胶质细胞的代谢，而且还将是第一个原位测定小胶质细胞代谢的工具，指导未来治疗AD和其他疾病的代谢干预研究。

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.