The role of histone demethylase KDM5B in ethanol-induced microglial activation

组蛋白去甲基化酶 KDM5B 在乙醇诱导的小胶质细胞激活中的作用

基本信息

批准号：
10247833
负责人：
Stanley M Stevens
金额：
$ 34.11万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-10 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10247833
关键词：
Acute Affect Alcohol abuse Alcoholism Alcohols Amino Acids Bioinformatics Biological Brain Brain Injuries Carbon Cell Culture Techniques Cells ChIP-seq Chromatin Chromatin Structure Chronic Classification Coculture Techniques Code Data Development Dose Drug Targeting Epigenetic Process Ethanol Excision Exhibits Functional disorder Future Genetic Genetic Transcription Histone H3 Histones Immune Immunoprecipitation In Vitro Inflammatory Inflammatory Response KDM5B gene Mass Spectrum Analysis Mediating Metabolism Methylation Methyltransferase Microglia Modification Mus Neuraxis Neurons Outcome Pathway interactions Pharmacology Phenotype Process Proteins Proteome Proteomics Publishing Role Site Small Interfering RNA Stable Isotope Labeling Structure Testing Time Validation activity-based protein profiling alcohol exposure base brain cell chromatin immunoprecipitation epigenetic therapy glial activation histone demethylase histone methylation histone methyltransferase improved in vitro activity in vivo innovation knock-down molecular dynamics multidisciplinary neuroinflammation next generation sequencing novel overexpression response small molecule inhibitor treatment strategy validation studies

项目摘要

Ethanol has profound effects on the central nervous system (CNS) including pathophysiological sequelae resulting from glial cell activation. Microglia, as the resident immune cells of the brain, have been implicated in neuroinflammatory processes that occur from chronic ethanol exposure. Emerging evidence now suggests, however, that microglia can exhibit activation phenotypes other than a pro-inflammatory state depending on dose and time of ethanol exposure. Based on recent proteomic analyses of ethanol-treated microglia, we demonstrate that a significant portion of the ethanol-induced proteome response in microglia can be attributed to changes in the activity of KDM5B, a histone demethylase that catalyzes the removal of tri-methylation on Lys 4 of histone H3 (H3K4me3). Moreover, we have strong preliminary data that show ethanol induces histone methylation changes both in vitro and in vivo and that experimental modulation of KDM5B activity affects histone methylation status and subsequent pro-inflammatory response of microglia. Therefore, we hypothesize that (a) methylation of H3K4me3 and its potential impact on the overall histone methylation code is an important epigenetic mechanism that influences ethanol-induced activation of microglia and (b) changes in KDM5B-mediated histone methylation promotes the exposure time-dependent transition of microglia to a pro- inflammatory phenotype. In order to test our hypothesis, we will 1) determine ethanol-induced changes in the histone methylation code and related impact on microglial activation phenotype upon genetic and pharmacological modulation of KDM5B activity in vitro and 2) characterize the ethanol dose- and time- dependent role of KDM5B on chromatin structural and functional changes related to microglial activation phenotype in vivo. In Aim 1, unbiased, mass spectrometry-based approaches will be utilized in order to determine the impact of KDM5B activity on the histone methylation code and on the activity of epigenetic writers such as methyltransferases. Additionally, we will accurately define activation phenotype driven by histone methylation through a novel bioinformatics approach developed by our lab. In Aim 2, we will employ mass spectrometry, ChIP-Seq and computational approaches to determine chromatin functional and structural consequences related to ethanol-induced histone methylation changes in vivo. This project will be the first of its kind to accurately classify activation phenotype of microglia through novel proteomics and bioinformatics- based approaches in order to better understand microglial functional changes that occur during chronic ethanol exposure. Moreover, this project will clarify the role of the histone demethylase, KDM5B, in histone methylation changes that regulate ethanol-induced microglial phenotype, potentially allowing for the development of novel epigenetic therapies for the treatment of CNS dysfunction resulting from alcohol abuse.

乙醇对中枢神经系统 (CNS) 具有深远影响，包括病理生理学后遗症胶质细胞激活的结果。小胶质细胞作为大脑的常驻免疫细胞，与慢性乙醇暴露引起的神经炎症过程。现在新出现的证据表明，然而，小胶质细胞可以表现出促炎状态以外的激活表型，具体取决于乙醇暴露的剂量和时间。根据最近对乙醇处理的小胶质细胞的蛋白质组学分析，我们证明小胶质细胞中乙醇诱导的蛋白质组反应的很大一部分可归因于 KDM5B 的活性发生变化，KDM5B 是一种组蛋白去甲基化酶，可催化去除三甲基化组蛋白 H3 (H3K4me3) 的赖氨酸 4。此外，我们有强有力的初步数据表明乙醇诱导组蛋白甲基化在体外和体内都会发生变化，KDM5B 活性的实验调节会影响组蛋白甲基化状态和随后的小胶质细胞促炎症反应。因此，我们假设 (a) H3K4me3 的甲基化及其对整个组蛋白甲基化代码的潜在影响是影响乙醇诱导的小胶质细胞激活的重要表观遗传机制以及（b） KDM5B 介导的组蛋白甲基化促进小胶质细胞暴露时间依赖性转变为亲炎症表型。为了检验我们的假设，我们将 1）确定乙醇引起的变化组蛋白甲基化密码及对小胶质细胞活化表型的相关影响对遗传和 KDM5B 活性的体外药理学调节以及 2) 表征乙醇剂量和时间 KDM5B 对小胶质细胞激活相关染色质结构和功能变化的依赖作用体内表型。在目标 1 中，将利用公正的、基于质谱的方法来确定 KDM5B 活性对组蛋白甲基化密码和表观遗传活性的影响作家，如甲基转移酶。此外，我们将准确定义由以下驱动的激活表型通过我们实验室开发的新型生物信息学方法进行组蛋白甲基化。在目标 2 中，我们将采用质谱法、ChIP-Seq 和计算方法来确定染色质功能和结构与乙醇诱导的体内组蛋白甲基化变化相关的后果。该项目将是其第一个通过新颖的蛋白质组学和生物信息学对小胶质细胞的激活表型进行准确分类- 基于方法，以便更好地了解慢性乙醇过程中发生的小胶质细胞功能变化接触。此外，该项目将阐明组蛋白去甲基化酶 KDM5B 在组蛋白甲基化中的作用调节乙醇诱导的小胶质细胞表型的变化，可能允许开发新的表观遗传疗法用于治疗因酗酒引起的中枢神经系统功能障碍。