Astrocyte-specific in vivo molecular signatures of APOE genetic risk in Alzheimer's disease

项目摘要 阿尔茨海默病（Alzheimer's disease，AD）是一种常见的神经退行性疾病。载脂蛋白E 载脂蛋白E（APOE）是发展AD的最普遍的风险因素。APOE基因型可能导致疾病 发病机制和风险通过影响星形胶质细胞分子谱和随后的生物学功能。数据从 我的实验室在人类死后AD和对照脑蛋白质组中发现了一个共表达的星形胶质细胞蛋白质模块， APOE ε4个体中参与糖代谢和炎症的小胶质细胞蛋白， 与MAPK/ERK通路密切相关。因为星形胶质细胞在整个大脑中很丰富， 产生高水平的apoE蛋白，并在大脑内稳态中发挥关键作用，它们更有可能有助于 在APOE风险的背景下，AD的疾病易感性和发病机制。为了解决APOE基因型 影响星形胶质细胞功能，我的实验室最近开发了细胞类型特异性的蛋白质体内生物素化 （CIBOP）作为一种新的方法来定量星形胶质细胞在其天然状态下的总蛋白质组和磷酸化蛋白质组 而不需要细胞分离。在这种方法中，生物素连接酶TurboID在细胞中选择性表达， 感兴趣的类型使用条件Cre/lox遗传策略。在我们的初步研究中使用星形细胞-CIBOP， 我们已经成功地从小鼠脑中获得了星形胶质细胞的天然状态蛋白质组，并定量了星形胶质细胞- 衍生的细胞因子和MAPK/ERK信号磷酸化蛋白。利用这种高度创新的方法， 我目前研究的目标是首次确定APOE ε4基因型的星形胶质细胞样免疫应答 并确定MAPK/ERK通路是否是这种反应的机制。我的主要假设是， ε4基因型通过增加细胞因子和补体增强星形胶质细胞的促炎性特征 以ERK信号依赖的方式产生。我将使用星形胶质细胞-CIBOP小鼠， 人APOE 4/4和APOE 3/3基因敲入遗传背景，以确定APOE的不同影响 遗传风险对体内星形胶质细胞分子特征的影响。在目标1中，我将检验APOE 4/4 与APOE 3/3相比，星形胶质细胞将表现出升高的促炎细胞因子水平， 补体蛋白产生、MAPK/ERK信号传导放大和脂质代谢异常变化。在 目的2，我将检验APOE 4星形胶质细胞的过度促炎反应，启动了 全身性炎症激发（LPS）引起的炎症反应是通过ERK信号转导介导的。使用生物信息学方法 蛋白质组学和转录组学数据以及验证小鼠和人脑组织中的IHC方法，我将 鉴定体内APOE 4/4和3/3星形胶质细胞反应性状态的分子变化，并鉴定ERK- APOE依赖和ERK非依赖机制。我的工作将为进一步的研究打下基础 星形胶质细胞介导的AD发病机制，在未来的研究中采用CIBOP来探索细胞类型- 除了AD和星形胶质细胞之外，神经变性的特定机制。

PROJECT ABSTRACT Alzheimer’s disease (AD) is a common and burdensome neurodegenerative disease. Apolipoprotein E (APOE) is the most prevalent risk factor for developing AD. APOE genotype may contribute to disease pathogenesis and risk by impacting astrocyte molecular profiles and subsequent biological functions. Data from my lab in human post-mortem AD and control brain proteomes identified a module of co-expressed astrocytic and microglial proteins in APOE ε4 individuals that were involved in sugar metabolism and inflammation and strongly correlated with the MAPK/ERK pathway. Because astrocytes are abundant throughout the brain, produce high levels of apoE protein, and play a critical role in brain homeostasis, they are more likely to contribute to disease vulnerability and pathogenesis of AD in the context of APOE risk. To address how APOE genotype impacts astrocyte function, my laboratory recently developed cell type-specific in vivo biotinylation of proteins (CIBOP) as a novel approach to quantify the total and phospho-proteomes of astrocytes in their native state without the need for cell isolation. In this approach, the biotin ligase, TurboID, is selectively expressed in the cell type of interest using a conditional Cre/lox genetic strategy. Using astrocyte-CIBOP in our preliminary studies, we have successfully obtained native-state proteomes of astrocytes from mouse brain, and quantified astrocyte- derived cytokines and MAPK/ERK signaling phospho-proteins. Leveraging this highly innovative methodology, my goal in the current study is to for the first time define the astrocyte-like immune response to APOE ε4 genotype and determine if the MAPK/ERK pathway is a mechanism of this response. My central hypothesis is that APOE ε4 genotype augments pro-inflammatory profiles of astrocytes via increased cytokine and complement production in an ERK signaling-dependent manner. I will use the astrocyte-CIBOP mice derived on homozygous human APOE 4/4 and APOE 3/3 knock-in genetic backgrounds to determine the differential impacts of APOE genetic risk on molecular signatures of astrocytes in vivo. In Aim 1, I will test the hypothesis that APOE 4/4 astrocytes, compared to APOE 3/3, will exhibit elevated levels of pro-inflammatory cytokines, increased complement protein production, amplified MAPK/ERK signaling, and aberrant changes in lipid metabolism. In Aim 2, I will test the hypothesis that the exaggerated pro-inflammatory response of APOE4 astrocytes, initiated by systemic inflammatory challenge (LPS), is mediated via ERK signaling. Using bioinformatic approaches on proteomic and transcriptomic data and validation IHC approaches in mouse and human brain tissues, I will identify molecular changes in reactivity states of APOE 4/4 and 3/3 astrocytes in vivo and identify ERK- dependent and ERK-independent mechanisms of APOE. My work will lay the foundation to further investigate astrocyte-mediated mechanisms of AD pathogenesis to employ CIBOP in future studies to explore cell type- specific mechanisms of neurodegeneration, beyond AD and astrocytes.