A Human iPSC-Based Chimeric Mouse Model of Alzheimers Disease in Down Syndrome

项目摘要 我们研究的目的是为了更好地了解人类小胶质细胞在阿尔茨海默病(AD)中的致病作用。 为治疗唐氏综合征(DS)中的阿尔茨海默病(AD)开辟新的治疗途径 普通人口。我们的研究符合RFA-OD-20-005的目标，因为我们将重点评估 21三体相关的遗传因素及其对人类组织和神经退行性变的影响 一种新的人-鼠嵌合脑模型，我们还将使用基因编辑来移除三重基因。这个 我们研究的基础是，最近的全基因组关联研究表明，许多AD风险基因 由驻留在脑内的巨噬细胞、小胶质细胞高度表达，有时仅由小胶质细胞表达。近期 转录研究也清楚地表明，人和小鼠的小胶质细胞表现出不同的基因。 更重要的是，在正常和疾病条件下，它们的年龄是不同的。这些 研究结果支持利用特定物种的研究工具来研究人类的小胶质细胞功能 大脑老化和退化。我们建议使用一种新的人类诱导多能干细胞(HiPSC)为基础 可概括成人和老年人小胶质细胞特征的嵌合小鼠模型的研究 人小胶质细胞在阿尔茨海默病中的作用而淀粉样β蛋白(Ab)的聚集先于tau、tau 蛋白质病理学在人类中开始的时间比之前认为的要早得多。与被标记的相反 在淀粉样变性转基因小鼠模型中，报告了小胶质细胞在人脑组织中的激活 AD和DS患者，与AD发育特别相关的大脑区域，如海马结构， 表现出低水平和晚期的抗体病理，而过度磷酸化的tau(p-tau)从早期开始积累 疾病的各个阶段。与抗体斑块相比，p-tau的优先聚集可以诱导出完全不同的 小胶质细胞反应。在这里，我们提出了一个新的tau/小胶质细胞衰老假说，人类小胶质细胞 由可溶性p-tau引起的衰老和功能改变可能发生在神经退行性变之前， 与阿尔茨海默病的进展和认知功能下降有因果关系。我们已经创造了控制组和DS小胶质细胞 通过将对照和DS hiPSC来源的小胶质细胞移植到小鼠脑内来构建小鼠嵌合体。我们将描述 人DS和对照HIPSC来源的小胶质细胞对病理性可溶性p-tau的动态反应 小胶质细胞嵌合小鼠大脑，通过使用新发明的机器人四维长期成像 技术我们将通过电生理记录和记录来确定突触功能的变化 DS小胶质嵌合体暴露于病理性可溶性p-tau后的行为表现 控制小胶质细胞嵌合体。此外，单细胞RNA测序分析嵌合小鼠脑和 CRISPR/Cas9介导的三重基因的移除将被用来确定分子机制 小胶质细胞的致病作用。通过了解支撑机制，我们可以开发 防止人类小胶质细胞衰老以减缓DS中AD进展的新治疗策略。

Project Summary The goal of our study is to better understand the pathogenic role of human microglia in Alzheimer’s disease (AD) in Down syndrome (DS) and develop new therapeutic avenues for the treatment of AD in DS as well as AD in general population. Our studies are in line with the goals of RFA-OD-20-005 because we will focus on evaluating the genetic factors associated with trisomy 21 and their impacts on neurodegeneration using human tissue and a novel human-mouse chimeric brain model and we will also use gene editing to remove triplicated genes. The foundation of our studies is that recent genome-wide association studies have shown that many AD risk genes are highly and sometimes exclusively expressed by the brain-resident macrophage, microglia. Recent transcriptomic studies have also clearly demonstrated that human vs. mouse microglia exhibit distinct gene expression profiles, and more importantly, they age differently under both normal and diseased conditions. These findings argue for the utilization of species-specific research tools to investigate microglial functions in human brain aging and degeneration. We propose to use a novel human induced pluripotent stem cell (hiPSC)-based microglial chimeric mouse model that can recapitulate features of adult and aging human microglia to investigate the role of human microglia in AD in DS. While the aggregation of amyloid-beta (Ab) precedes that of tau, tau protein pathology commences in humans much sooner than was previously thought. Contrary to the marked microglial activation reported in amyloidogenic transgenic mouse models, in human brain tissue derived from AD and DS patients, brain regions particularly relevant in AD development, such as the hippocampal formation, exhibit low and late Ab pathology, whereas hyperphosphorylated tau (p-tau) accumulates starting in the early stages of the disease. The preferential accumulation of p-tau over Ab plaques could induce a totally different microglial response. Here we put forward a new tau/microglial senescence hypothesis that human microglial senescence and functional changes, induced by soluble p-tau, likely occur prior to neurodegeneration and is causatively linked to the AD progression and cognitive decline in DS. We have created control and DS microglial mouse chimeras by engrafting control and DS hiPSC-derived microglia into mouse brains. We will characterize the dynamic responses of DS and control hiPSC-derived microglia to pathological soluble p-tau in human microglial chimeric mouse brains, by using newly invented robotic four-dimensional long-term imaging technology. We will determine the changes in synaptic functions by electrophysiological recordings and behavioral performance of DS microglial chimeras after exposure to pathological soluble p-tau, as compared to control microglial chimeras. Moreover, single-cell RNA-sequencing analysis of chimeric mouse brains and CRISPR/Cas9-mediated removal of triplicated genes will be performed to determine the molecular mechanisms underlying the pathogenic role of microglia. By understanding the underpinning mechanisms, we can develop new therapeutic strategies to prevent human microglial senescence to slow the progression of AD in DS.