Development of in vivo quantitative stable isotope probing to quantify microbiome dynamics in Alzheimers Disease

摘要 阿尔茨海默氏病 (AD) 是导致老年人口痴呆的主要原因 影响患者和看护者的生活质量。尽管进行了数十年的研究，但其根本病因学 AD不完全理解。 AD 病理学的普遍假设是淀粉样蛋白级联反应 该假说认为，AD 的致病因素是淀粉样蛋白 - β（Aβ，主要 斑块的组成部分）在大脑中。然而，最近针对 Aβ 的 III 期临床试验尚未取得进展 尽管降低了大脑整体 Aβ 负担，但认知功能仍显着改善。有 人们越来越有兴趣了解 AD 的其他病理特征，这些特征可能会影响 疾病进展。最近对人类和小鼠的研究表明致病性的作用 神经炎症和 AD 中的微生物或改变的微生物群。胃肠道中的微生物群可以影响 通过微生物产生神经递质前体、免疫调节来维持神经健康 代谢物（例如短链脂肪酸），或与迷走神经或肠神经系统的相互作用。 肠道微生物群还可以产生淀粉样蛋白（聚集的不溶性蛋白质，呈现 β 折叠片状） 结构）可能穿过血脑屏障。尽管人们越来越努力地了解肠道 微生物组-脑轴，目前使用 DNA 或 RNA 扩增子测序的技术无法 解决基本的生态问题，例如量化寄主的分类单元特定增长率 相关微生物群。了解微生物生态系统动态对人类具有重大意义 疾病；根据绝对丰度随时间的变化来量化物种的增长或更替 生态系统可以预测种间竞争或微生物演替轨迹 生命早期和晚期，这与宿主健康或疾病风险相关。在本研究中，我们将适应 一种广泛应用于土壤微生物生态学的创新技术，用于研究 AD 中宿主微生物组的动态。 我们将使用定量稳定同位素探测 (qSIP)，这是一种使用同位素富集的技术 底物（例如 18O-水）来测量三重转基因 (3xTg-AD) 和野生动物的肠道微生物组动态 型老鼠。由于 H2O 是通用底物，标记的 18O 将融入微生物和宿主中 生物分子，包括核酸；该功能允许研究人员通过以下方式分离提取的 DNA： 密度来量化特定分类单元的增长率。完成这项研究将导致以下方面的发展 一种新颖的工具，可以在 AD 社区中广泛使用，以更好地了解主机的贡献 微生物群与 AD 进展和神经炎症相关。

ABSTRACT Alzheimer’s Disease (AD) is the leading cause of dementia in the aging population and severely impacts patient’s and caretaker’s quality of life. Despite decades of research, the underlying etiology of AD is incompletely understood. The prevailing hypothesis for AD pathology is the amyloid cascade hypothesis, which posits that the causative agent of AD is the accumulation of amyloid-β (Aβ, the main component of plaques) in the brain. However, recent Phase III clinical trials that target Aβ have not led to significant improvements in cognitive function, despite reducing overall brain Aβ burden. There has been a growing interest in understanding additional pathological features of AD that may impact disease progression. Recent studies in humans and in mice have suggested a role for pathogenic microbes or altered microbiota in neuroinflammation and AD. Microbiota in the GI tract can influence neurological health through microbial production of neurotransmitter precursors, immune-modulatory metabolites (e.g. short chain fatty acids), or interaction with the vagus nerve or enteric nervous system. Gut microbiota can also produce amyloids (aggregated, insoluble proteins exhibiting β-pleated sheet structures) that may cross the blood-brain barrier. Despite growing efforts to understand the gut microbiome-brain axis, current technologies using DNA or RNA amplicon sequencing are unable to address fundamental ecological questions, such as quantifying taxon-specific growth rates of host- associated microbiota. Understanding microbial ecosystem dynamics has large implications for human disease; quantifying growth or turnover of species in terms of absolute abundance change over time in an ecosystem can enable predictions of interspecies competition or trajectories of microbial succession in early and late life, which are associated with host health or disease risk. In this study, we will adapt an innovative technique widely used in soil microbial ecology to study host-microbiome dynamics in AD. We will use quantitative stable isotope probing (qSIP), a technique that uses an isotopically enriched substrate (e.g. 18O-water) to measure gut microbiome dynamics in triple transgenic (3xTg-AD) and wild- type mice. Since H2O is a universal substrate, labeled 18O will incorporate into microbial and host biomolecules, including nucleic acids; this feature allows a researcher to separate extracted DNA by density to quantify taxon-specific growth rates. Completion of this study will lead to the development of a novel tool that can be widely used in the AD community to better understand the contribution of host- associated microbiota to AD progression and neuroinflammation.