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.

项目摘要阿尔茨海默氏病（AD）是最常见的神经退行性疾病，其特征是病理学大脑中蛋白质聚集和炎症（神经炎症）。小胶质细胞是关键的免疫细胞介导神经炎症的大脑。如果可以确定新颖的热策略和AD生物标志物我们可以定义蛋白质水平的AD中小胶质细胞中发生的分子变化，而不仅仅是在 mRNA（转录组）水平。我们对AD中小胶质疾病机制的了解主要是形状的通过转录组学研究，尽管蛋白质是生物过程的启示器和相关性 mRNA和蛋白质之间很差。小胶质细胞研究的主要障碍是对分析之前净化小胶质细胞的隔离策略。从大脑中分离小胶质细胞会诱导伪影，产生的蛋白质很少，提供了高度偏见的采样。为了克服这个障碍，我们将使用小说小胶质细胞特异性蛋白质组学标记的策略（Ciboncat）。 ciboncat允许我们新标记带有叠氮化标签（氮杂氨瓜）的小胶质细胞中合成的蛋白质。这些叠氮化物标记的蛋白可以是易于与大脑隔离，而无需细胞隔离。我们已经证明了使用的可行性在成年小鼠中标记神经元和星形胶质细胞蛋白质组的ciboncat，并优化了体内和质量使用脑，脑脊液（CSF）和其他生物流体的蛋白质组学的光谱管道。这本小说策略，再加上我们广泛的质谱法专业知识（蛋白质组学）方法，将使我们能够测试我们的核心假设是淀粉样β（Aβ）积累，APOEε4和衰老独立和衰老小胶质细胞的协同影响蛋白质组学表型，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在CSF中反映。在AIM 1中，我们将使用Ciboncat定义使用两种进行性淀粉样β病理学模型在AD中发生的小胶质细胞特异性蛋白质改变（App-PS1和5XFAD）。在AIM 2中，我们将使用敲入鼠标模型来确定人类的效果小胶质细胞蛋白上的APOEε4和APOEε3表达会改变以及APOEε4如何用Aβ病理影响。在AIM 3中，我们将在AD病理的小鼠模型中鉴定脑脊液中的小胶质细胞衍生的蛋白确定反映大脑中小胶质细胞激活或部署的神经炎症的新型生物标志物 AD鼠标模型。通过成功完成此R01提案，我们将获得新的分子对小胶质细胞介导的AD机制的见解，并将建立Ciboncat作为一种有力的方法研究神经变性的细胞类型特异性机制。我们的小胶质学上的多学科专业知识 AD病理学的生物学和转基因模型（Rangaraju博士）和定量蛋白质组学，系统生物学以及CSF Biomarker Discovery（Seyfried博士），独特地定位了我们执行这一创新的R01提案。