Investigating the cause of APOE4-associated microglial activation and its resulting neurotoxicity of tauopathy-afflicted neurons

项目摘要 载脂蛋白E（APOE）的等位基因是晚发性阿尔茨海默病的主要危险因素 (AD)尽管直接原因仍然是一个争论的来源。最近的数据表明，APOE基因是 在AD和其他神经退行性疾病中发现的一种新的小胶质细胞活化状态中上调， 疾病相关小胶质细胞（DAM）。此外，APOE 4表达已显示诱导神经毒性。 在存在各种刺激的情况下激活小胶质细胞，包括tau蛋白病折磨的神经元，尽管 这种激活的机制尚未阐明。为了揭示APOE 4- 相关的小胶质细胞激活及其导致的tau蛋白病折磨神经元的神经毒性，我已经设计了一个 一系列创新实验，利用尖端技术和新资源。在目标1中，单一- 细胞RNA测序将在从一种新的小鼠系纯化的小胶质细胞上进行，其中人APOE 3 和APOE 4小鼠与EC-Tau小鼠杂交，EC-Tau小鼠主要表达促聚集突变tau蛋白 内嗅皮层（EC）。在目标2中，将在以下研究中研究APOE 4对小胶质细胞活化的影响： 设置人类小胶质细胞，使用新创建的等基因APOE人类干细胞系和强大的 生物信息学分析。在目标3中，将使用稳健的体外共培养试验，以揭示 负责APOE 4相关小胶质细胞活化和由此产生的神经毒性的机制， 确定抑制这些事件的新的治疗策略。由于APOE 4和小胶质细胞活化 显著影响AD，发现这两个重要参与者之间的机制联系， 调节APOE 4相关的小胶质细胞活化的策略将代表在 AD领域，并将大大推进我们预防或减缓AD进展的目标，特别是在 APOE 4携带者。

Project Summary Possession of the ɛ4 allele of apolipoprotein E (APOE) is a major risk factor for late onset Alzheimer's disease (AD), although the direct cause remains a source of debate. Recent data has shown that the APOE gene is upregulated in a novel microglial activation state found in AD and other neurodegenerative diseases termed disease associated microglia (DAM). Furthermore, APOE4 expression has been shown to induce a neurotoxic activation of microglia in the presence of various stimuli, including tauopathy-afflicted neurons, although the mechanism of this activation has yet to be elucidated. In order to discover the role and mechanism of APOE4- associated microglial activation and its resulting neurotoxicity of tauopathy-afflicted neurons, I have devised an innovative set of experiments that utilize both cutting-edge technology and novel resources. In Aim 1, single- cell RNA-sequencing will be performed on microglia purified from a novel mouse line in which human APOE3 and APOE4 mice are crossed with EC-Tau mice, which express a pro-aggregating mutant tau protein primarily in the entorhinal cortex (EC). In Aim 2, APOE4's effects on microglial activation will be investigated in the setting of human microglia, using both a newly created isogenic APOE human stem cell line and powerful bioinformatics analyses. And in Aim 3, a robust in vitro co-culture assay will be utilized in order to both uncover the mechanism responsible for APOE4-associated microglial activation and the resulting neurotoxicity and to identify novel therapeutic strategies for inhibiting these events. As both APOE4 and microglial activation significantly impact AD, discovery of the mechanistic link between these two important players and therapeutic strategies for modulating APOE4-associated microglial activation would represent a major breakthrough in the AD field and would greatly advance our goal of preventing or slowing the progression of AD, especially among APOE4 carriers.