APOE and microglia-mediated progression in tau pathology in AD

我们以前报道过小胶质细胞和细胞外小泡在tau中起着关键作用。 传播。我们的发现在许多研究中都得到了重现，表明小胶质细胞和 EVS在动物模型和人类生物标本中的tau病理发展。在当前的 资助期(1RF1 AG054199)，我们发现：1)胶质细胞和疾病相关蛋白在 阿尔茨海默病脑源性EVS(AD EVS)与对照EVS的比较，2)高度传染性 AD EVS表现为神经元摄取和tau聚集能力增加，3)tau的强劲传播 通过接种只含有300pg tau的AD EV在老年C57BL/6小鼠的整个海马区 老鼠的大脑。我们尚未阐明AD EV获得致病功能的潜在机制。 我们假设，抗体诱导的炎症条件导致AD的小胶质细胞激活，从而改变 小胶质细胞EV的分子组成，导致它们向神经元的传递增加。载脂蛋白E4 通过增加抗体沉积和MGnD诱导来增强这一过程。这个假说已经得到了发展。 根据以往的研究和我们的初步数据显示：(1)Clec7a+神经退行性小胶质细胞 在APPNL-G-F/NL-G-F敲入中，围绕抗体斑块的(MGnD)在加速tau增殖中起关键作用 (2)MGnD上调EV标志物；(3)AD EV和APP小鼠脑源性EV显示 与对照组相比，MGnD和细胞黏附分子增加；(4)AD EV显示tau种植增加 与对照组相比，APOE4的活动和神经元摄取比非APOE4的AD更严重 APOE4敲入APP/PS1小鼠加速抗体沉积、小胶质细胞激活和 APOE3基因敲除APP/PS1小鼠MGnD标志物表达的比较建议的理由是 研究是鉴定在AD和APOE4中唯一表达的EV蛋白和脂质成分 背景知识将成为了解EV介导的疾病进展的分子基础。我们的研究 将填补MGnD诱导和通过小胶质细胞tau种子EV产生tau繁殖之间缺失的环节。 在目标1中，我们将描述AD EVS和tau-EV中易于聚集的tau的化学和分子结构。 相互作用的分子。我们还将表征来自APOE4/4和APOE4/4的EV的蛋白质和脂肪组成。 APOE3/3EVS，以表征细胞起源的差异，炎性和毒性分子的参与， 以及体外和体内的tau播种活性。在目标2中，我们将击倒参与小胶质细胞激活的载脂蛋白E 和Smpd3，参与EV的合成，以确定MGnD诱导和胶质EV产生对tau的作用 传播。在目标3中，我们将区分APOE3/3和APOE4/4背景下的同源人类IPSCs 小胶质细胞样细胞(IMGL)，并在MGnD条件下鉴定iMGL EV的致病性。我们会 对iMGL EVS进行蛋白质组和脂肪组学分析，以验证我们在AIM 1中对人类AD EVS的发现。 这项工作有望为肠胃病的EV生物学提供基本的分子基础。

We have previously reported that microglia and extracellular vesicles (EVs) play pivotal roles in tau propagation. Our findings have been reproduced in numerous studies showing involvement of microglia and EVs in the development of tau pathology in animal models and human biospecimens. During the current funded period (1RF1 AG054199), we identified: 1) Enrichment of glial and disease-associated proteins in Alzheimer’s disease brain-derived EVs (AD EVs) compared to control EVs, 2) Highly transmissible nature of AD EVs demonstrated by increased neuronal uptake and tau aggregation potency, 3) Robust tau propagation throughout the entire hippocampus by inoculating AD EVs containing just 300 pg of tau in aged C57BL/6 mouse brains. We have yet to elucidate the underlying mechanism of AD EVs gaining pathogenic functions. We hypothesize that Ab-induced inflammatory conditions in AD leads to microglial activation, which alters molecular compositions of microglial EVs, leading to their increased transmission to neurons. APOE4 enhances this process by augmented Ab deposition and MGnD induction. The hypothesis has been developed based on the previous studies and our preliminary data showing that (1) Clec7a+ neurodegenerative microglia (MGnD) surrounding Ab plaques play a key role in accelerating tau propagation in APPNL-G-F/NL-G-F knock-in (APP KI) mice; (2) MGnD upregulates EV markers; (3) AD EVs and APP mouse brain-derived EVs show elevated MGnD and cell adhesion molecules compared to controls; (4) AD EVs show increased tau seeding activity and neuronal uptake compared to controls, which is exacerbated in APOE4 over non-APOE4 AD cohorts; and (5) APOE4 knock-in APP/PS1 mice accelerates Ab deposition, microglial activation, and expression of MGnD markers compared to APOE3 knock-in APP/PS1 mice. The rationale of the proposed research is that identification of EV protein and lipid composition uniquely expressed in AD and APOE4 background will serve as the basis of molecular understanding of EV-mediated disease progression. Our study will fill the missing link between MGnD induction and tau propagation by microglial tau-seeding EV production. In Aim 1, we will delineate the chemical and molecular structure of aggregation-prone tau in AD EVs and tau- interacting molecules. We will also characterize the protein and lipid compositions of EVs from APOE4/4 and APOE3/3 EVs to characterize the difference in cellular origin, involvement of inflammatory and toxic molecules, and tau seeding activity in vitro and in vivo. In Aim 2, we will knock down Apoe, involved in microglial activation and Smpd3, involved in EV synthesis, to determine the role of MGnD induction and glial EV production on tau propagation. In Aim 3, we will differentiate isogenic human iPSCs in APOE3/3 and APOE4/4 background to microglia-like cells (iMGL), and characterize pathogenicity of iMGL EVs under MGnD condition. We will perform proteomic and lipidomic profiling of iMGL EVs to validate our findings from human AD EVs in Aim 1. The proposed work is expected to obtain the fundamental molecular bases for EV biology in tauopathy.