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

项目摘要 我们的研究目的是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 在唐氏综合征（DS），并开发新的治疗途径，治疗AD的DS以及AD的 一般人口。我们的研究符合RFA-OD-20-005的目标，因为我们将重点评估 与21三体相关的遗传因素及其对使用人体组织的神经变性的影响， 一种新的人-小鼠嵌合脑模型，我们还将使用基因编辑来删除三重基因。的 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 是由脑内巨噬细胞，小胶质细胞高度表达的，有时是专门表达的。最近 转录组学研究也清楚地表明，人和小鼠小胶质细胞表现出不同的基因， 表达谱，更重要的是，它们在正常和患病条件下的年龄不同。这些 研究结果表明，利用物种特异性研究工具来研究人类小胶质细胞的功能， 大脑老化和退化。我们建议使用一种新的基于人诱导多能干细胞（hiPSC）的 小胶质细胞嵌合小鼠模型，可以重现成年和衰老的人类小胶质细胞的特征，以研究 人类小胶质细胞在DS中AD中的作用。虽然淀粉样蛋白-β（Ab）的聚集先于tau的聚集，但tau 蛋白质病理学在人类中的开始比以前认为的要早得多。与标记相反 在淀粉样蛋白生成转基因小鼠模型中报告的小胶质细胞活化，在源自 AD和DS患者，与AD发展特别相关的脑区域，如海马结构， 表现出低和晚期Ab病理学，而过度磷酸化tau（p-tau）在早期开始积累， 疾病的阶段。p-tau相对于Ab噬斑的优先积累可以诱导完全不同的 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，即人类小胶质细胞 由可溶性p-tau诱导的衰老和功能变化可能发生在神经变性之前， 与AD进展和DS中的认知下降有因果关系。我们创造了对照组和DS小胶质细胞 通过将对照和DS hiPSC衍生的小胶质细胞移植到小鼠脑中来制备小鼠嵌合体。我们将描述 DS和对照hiPSC衍生的小胶质细胞对人类病理性可溶性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来确定突触功能的变化， DS小胶质细胞嵌合体在暴露于病理性可溶性p-tau后的行为表现，与 对照小胶质细胞嵌合体。此外，嵌合小鼠脑的单细胞RNA测序分析和 将进行CRISPR/Cas9介导的三重基因去除以确定分子机制。 小胶质细胞的致病作用。通过了解基础机制，我们可以开发 新的治疗策略，以防止人类小胶质细胞衰老，以减缓DS中AD的进展。

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.