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

项目摘要 阿尔茨海默病（Alzheimer's disease，AD）是最常见的神经退行性疾病， 蛋白质聚集和大脑中的炎症（神经炎症）。小胶质细胞是免疫系统的关键免疫细胞， 调节神经炎症的大脑新的治疗策略和AD生物标志物可以确定， 我们可以在蛋白质水平上定义AD小胶质细胞中发生的分子变化，而不仅仅是在细胞水平上。 mRNA（转录组）水平。我们对AD中小胶质细胞疾病机制的认识主要是 虽然蛋白质是生物过程的参与者， mRNA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 分离策略以在分析前纯化小胶质细胞。从脑中分离小胶质细胞诱导伪影， 产生非常少的蛋白质并且提供高度偏差的采样。为了克服这一障碍，我们将使用一本小说 小胶质细胞特异性蛋白质组学标记的ciBONCAT策略。CiBONCAT让我们可以在新的标签 在小胶质细胞中用叠氮标签（叠氮正亮氨酸）合成蛋白质。这些叠氮化物标记的蛋白质可以是 容易从脑中分离而不需要细胞分离。我们已经证明了使用 ciBONCAT用于标记成年小鼠的神经元和星形胶质细胞蛋白质组，并优化了体内和质量 用于使用脑、脑脊液（CSF）和其他生物流体的蛋白质组学的光谱分析管道。这本小说 战略，加上我们在质谱（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β淀粉样蛋白（A β）积累、APOE ε 4和衰老独立， 协同影响小胶质细胞蛋白质组表型，这些小胶质细胞介导的机制 通过AD病理学的免疫生物标志物反映在CSF中。在目标1中，我们将使用ciBONCAT来定义 使用两种进行性淀粉样蛋白β病理模型研究AD中发生的小胶质细胞特异性蛋白质改变 （APP-PS1和5xFAD）。在目标2中，我们将使用基因敲入小鼠模型来确定人 APOE ε 4和APOE ε 3在小胶质细胞蛋白上的表达变化以及APOE ε 4如何影响A β病理 在目标3中，我们将在AD病理学小鼠模型的脑脊液中鉴定小胶质细胞衍生的蛋白质 为了鉴定神经炎症的新生物标志物，其反映了脑中小胶质细胞的激活或耗竭， AD小鼠模型。通过R01提案的成功完成，我们将获得新的分子 深入了解小胶质细胞介导的AD机制，并将建立ciBONCAT作为一种强大的方法， 研究神经变性的细胞类型特异性机制。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），定量蛋白质组学，系统生物学 和CSF生物标志物发现（Seyfried博士），使我们能够执行这一创新的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.