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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：
10227481
负责人：
Stanley M Stevens
金额：
$ 35.98万
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 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10227481
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.

酒精对中枢神经系统(CNS)有深远的影响，包括病理生理后遗症由神经胶质细胞激活所致。小胶质细胞作为大脑的常驻免疫细胞，已被卷入慢性酒精暴露引起的神经炎性反应。新出现的证据表明，然而，小胶质细胞可以表现出不同于促炎状态激活表型，这取决于酒精暴露剂量和时间。基于最近对乙醇处理的小胶质细胞的蛋白质组学分析，我们证明乙醇诱导的小胶质细胞蛋白质组反应的很大一部分可以归因于对KDM5B活性的变化，KDM5B是一种组蛋白去甲基酶，催化去除三甲基化组蛋白H3的赖氨酸4(H3K4me3)。此外，我们有强有力的初步数据表明乙醇可以诱导组蛋白体内和体外甲基化改变以及KDM5B活性的实验调节影响组蛋白甲基化状态和随后的小胶质细胞的促炎反应。因此，我们假设 (A)H3K4me3的甲基化及其对整个组蛋白甲基化密码的潜在影响是一种影响乙醇诱导的小胶质细胞活化的重要表观遗传学机制和(B) KDM5B介导组蛋白甲基化促进小胶质细胞向亲组蛋白的转化炎症表型。为了检验我们的假设，我们将1)确定乙醇诱导的组蛋白甲基化密码及其对小胶质细胞激活表型的影响 KDM5B体外活性的药理调节；2)乙醇剂量和时间的表征 KDM5B在小胶质细胞激活相关染色质结构和功能改变中的依赖作用活体表型。在目标1中，将利用基于质谱学的无偏见方法，以便确定KDM5B活性对组蛋白甲基化密码和表观遗传学活性的影响作家，如甲基转移酶。此外，我们将准确定义由以下因素驱动的激活表型通过我们实验室开发的一种新的生物信息学方法实现组蛋白甲基化。在目标2中，我们将使用确定染色质功能和结构的质谱学、芯片序列和计算方法与体内乙醇诱导组蛋白甲基化改变有关的后果。这个项目将是它的第一个通过新的蛋白质组学和生物信息学准确分类小胶质细胞的激活表型- 为更好地了解慢性酒精过程中发生的小胶质细胞功能变化曝光。此外，该项目将阐明组蛋白去甲基酶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，潜在地允许新的酒精滥用所致中枢神经系统功能障碍的表观遗传学治疗。