Microglia-specific proteomic mechanisms and biomarkers of neuroinflammation in Alzheimer’s disease

项目总结 阿尔茨海默病(AD)是最常见的神经退行性疾病，以病理性为特征 大脑中的蛋白质聚集和炎症(神经炎症)。小胶质细胞是重要的免疫细胞。 负责调节神经炎症的大脑。新的治疗策略和AD生物标志物可以在以下情况下识别 我们可以在蛋白质水平上定义阿尔茨海默病小胶质细胞中发生的分子变化，而不仅仅是在 MRNA(转录)水平。我们对阿尔茨海默病小胶质细胞疾病机制的认识大多是形成的 通过转录学研究，尽管蛋白质是生物过程的参与者和相互关系 信使核糖核酸和蛋白质之间的关系很差。小胶质细胞蛋白质组学研究的主要障碍是依赖于 分析前纯化小胶质细胞的分离策略。从大脑中分离小胶质细胞会产生伪影， 产生的蛋白质非常少，并提供高度偏向的采样。为了克服这一障碍，我们将用一部小说 小胶质细胞特异性蛋白质组体内标记策略(CiBONCAT)。CiBONCAT允许我们新标记 在小胶质细胞中合成的带有叠氮标签的蛋白质(叠氮酮亮氨酸)。这些叠氮标记的蛋白质可以 很容易与大脑隔离，不需要细胞隔离。我们已经论证了使用 CiBONCAT标记成年小鼠神经元和星形胶质细胞蛋白质组，并对体内和质量进行了优化 使用脑、脑脊液(CSF)和其他生物流体的蛋白质组学光谱分析管道。这部小说 战略，再加上我们在质谱学(蛋白质组学)方法方面的广泛专业知识，将使我们能够测试 我们的中心假设是淀粉样β蛋白(Aβ)堆积，载脂蛋白ε4，与衰老独立和 协同影响小胶质细胞的蛋白质组表型，这些小胶质细胞介导的机制是 通过免疫生物标记物反映在脑脊液中的AD病理。在目标1中，我们将使用ciBONCAT来定义 两种进行性淀粉样β蛋白病理模型在AD中发生的小胶质细胞特异性蛋白改变 (APP-PS1和5xFAD)。在目标2中，我们将使用敲入小鼠模型来确定人类 载脂蛋白ε4和载脂蛋白ε3在小胶质细胞蛋白上的表达变化以及载脂蛋白ε4对Aβ病理的影响。 在目标3中，我们将在AD病理模型的小鼠脑脊液中鉴定小胶质细胞衍生蛋白 寻找反映脑内小胶质细胞激活或耗竭的新的神经炎症生物标志物 广告鼠标模型。通过R01提案的顺利完成，我们将获得新的分子 对小胶质细胞介导的AD机制的洞察，并将建立ciBONCAT作为一种强有力的方法 研究神经变性的特定细胞类型的机制。我们在小胶质细胞方面的多学科专业知识 AD病理学的生物学和转基因模型(Rangaraju博士)，以及定量蛋白质组学、系统生物学 和脑脊液生物标记物的发现(塞弗里德博士)，使我们处于独特的地位，可以执行这项创新的R01提案。

PROJECT SUMMARY Alzheimer's disease (AD) is the most common neurodegenerative disease that is characterized by pathological protein aggregation and inflammation in the brain (neuroinflammation). Microglia are the key immune cells of the brain that mediate neuroinflammation. Novel therapeutic strategies and AD biomarkers can be identified if we can define the molecular changes occurring in microglia in AD at the protein level, and not just at the mRNA (transcriptomic) level. Our knowledge about microglial disease mechanisms in AD are mostly shaped by transcriptomic studies although proteins are the enactors of biological processes and the correlation between mRNA and protein is poor. The major barrier to microglial proteomic studies is the dependence on isolation strategies to purify microglia before analyses. Isolation of microglia from brain induces artefacts, yields very little protein and provides highly biased sampling. To overcome this barrier, we will use a novel strategy (ciBONCAT) for microglia-specific proteomic labeling in-vivo. CiBONCAT allows us to label newly synthesized proteins in microglia with an azide tag (azidonorleucine). These azide-tagged proteins can be easily isolated from the brain without need for cell isolation. We have demonstrated the feasibility of using ciBONCAT to label neuronal and astrocytic proteomes in adult mice, and have optimized the in-vivo and mass spectrometry pipelines for proteomics using brain, cerebrospinal fluid (CSF) and other biofluids. This novel strategy, coupled with our extensive expertise in mass spectrometry (proteomics) methods, will allow us to test our central hypothesis that amyloid beta (Aβ) accumulation, APOEε4, and aging independently and synergistically impact proteomic phenotypes of microglia, and that these microglia-mediated mechanisms are reflected in the CSF via immune biomarkers of AD pathology. In Aim 1, we will use ciBONCAT to define microglia-specific protein alterations occurring in AD using two models of progressive amyloid beta pathology (APP-PS1 and 5xFAD). In Aim 2, we will use knock-in mouse models to determine the effect of human APOEε4 and APOEε3 expression on microglial proteins changes and how APOEε4 impacts with Aβ pathology. In Aim 3, we will identify microglia-derived proteins in the cerebrospinal fluid in mouse models of AD pathology to identify novel biomarkers of neuroinflammation that reflect activation or depletion of microglia in the brain in AD mouse models. Through the successful completion of this R01 proposal, we will obtain novel molecular insights into microglia-mediated AD mechanisms and will establish ciBONCAT as a powerful approach to investigate cell type-specific mechanisms of neurodegeneration. Our multidisciplinary expertise in microglial biology and transgenic models of AD pathology (Dr Rangaraju), and quantitative proteomics, systems biology and CSF biomarker discovery (Dr Seyfried), uniquely position us to execute this innovative R01 proposal.