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

阿尔茨海默病神经炎症的小胶质细胞特异性蛋白质组学机制和生物标志物

基本信息

批准号：
10179808
负责人：
Srikant Rangaraju
金额：
$ 111.7万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2023-10-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10179808
关键词：
APP-PS1 Adult Age Aging Alkynes Alzheimer&apos s Disease Alzheimer&apos s disease model Alzheimer&apos s disease pathology Alzheimer’s disease biomarker Amino Acids Amyloid beta-Protein Animals Anti-Inflammatory Agents Apolipoprotein E Astrocytes Autopsy Azides Bioinformatics Biological Markers Biological Process Biology Brain Cell Separation Cells Cerebrospinal Fluid Cerebrospinal Fluid Proteins Characteristics Chemistry Coupled Dementia Dependence Disease Genetic Genetic Risk Human Human Genetics Immune Immunologic Markers Impaired cognition Inflammasome Inflammation Inflammatory Intervention Knock-in Mouse Knowledge Label Late Onset Alzheimer Disease Lead Lipopolysaccharides LoxP-flanked allele Mass Spectrum Analysis Mediating Messenger RNA Methionine Methionine-tRNA Ligase Methodology Methods Microglia Modeling Molecular Morphologic artifacts Mouse Protein Mus Nerve Degeneration Neurodegenerative Disorders Neuroimmunomodulation Neurons Pathogenesis Pathologic Pathology Phenotype Positioning Attribute Proteins Proteome Proteomics Resolution Risk Role Sampling Sampling Biases Synapses Systems Biology Tamoxifen Testing Transcript Wild Type Mouse abeta accumulation age related analog apolipoprotein E-4 beta amyloid pathology biomarker discovery brain cell cell type disorder control genetic risk factor immune checkpoint immunoregulation in vivo inhibitor/antagonist innovation insight mouse model multidisciplinary mutant neuroinflammation novel novel marker novel strategies novel therapeutic intervention novel therapeutics protein aggregation protein biomarkers proteomic signature selective expression specific biomarkers targeted biomarker transcriptomics transgenic model of alzheimer disease

项目摘要

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.

项目概要阿尔茨海默病（AD）是最常见的神经退行性疾病，其特征是病理性改变大脑中的蛋白质聚集和炎症（神经炎症）是关键的免疫细胞。介导神经炎症的大脑可以被识别出来。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，而不仅仅是在蛋白质水平上我们对 AD 小胶质细胞疾病机制的了解主要是在 mRNA（转录组）水平上形成的。通过转录组学研究，尽管蛋白质是生物过程及其相关性的执行者小胶质细胞蛋白质组学研究的主要障碍是对 mRNA 和蛋白质的依赖性。在分析之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，为了克服这一障碍，我们将使用一种新颖的方法，即产生很少的蛋白质并提供高度偏差的采样。用于体内小胶质细胞特异性蛋白质组标记的策略（ciBONCAT）使我们能够进行新的标记。在小胶质细胞中合成带有叠氮化物标签（叠氮基亮氨酸）的蛋白质。无需细胞分离即可轻松从大脑中分离出来，我们已经证明了使用的可行性。 ciBONCAT 标记成年小鼠的神经和星形胶质细胞蛋白质组，并优化了体内和质量使用大脑、脑脊液 (CSF) 和其他生物液体进行蛋白质组学的光谱测定管道。策略，加上我们在质谱（蛋白质组学）方法方面的丰富专业知识，将使我们能够测试我们的中心假设是β淀粉样蛋白 (Aβ) 积累、APOEε4 和衰老独立且协同影响小胶质细胞的蛋白质组表型，并且这些小胶质细胞介导的机制是通过 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β淀粉样蛋白病理学模型研究 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，我们将使用敲入小鼠模型来确定人类的效果。 APOEε4 和 APOEε3 表达对小胶质细胞蛋白的变化以及 APOEε4 如何影响 Aβ 病理学。在目标 3 中，我们将鉴定 AD 病理小鼠模型脑脊液中小胶质细胞衍生的蛋白质识别反映大脑中小胶质细胞激活或耗竭的新型神经炎症生物标志物 AD小鼠模型。通过这个R01方案的成功完成，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 作为治疗 AD 的有效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）以及定量蛋白质组学、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。