Investigating chromatin-based mechanisms of astrocyte pathology during inflammation response and stress-induced behaviors

研究炎症反应和应激诱导行为期间基于染色质的星形胶质细胞病理学机制

基本信息

批准号：
10246177
负责人：
Sasha Fulton
金额：
$ 1万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-14 至 2021-08-23
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10246177
关键词：
ATAC-seq Affect Affective Affinity Chromatography Anhedonia Animals Anxiety Astrocytes Automobile Driving Autopsy Bacteria Behavior Behavioral Binding Binding Sites Biological Assay Brain region Cell Nucleus Cell physiology Cells ChIP-seq Chromatin Chromatin Remodeling Factor Chronic Clinical Complex Control Animal Coupled Cultured Cells DNA-Binding Proteins Data Data Set Decision Making Disease Epigenetic Process Fellowship Female Fluorescence Functional disorder Gene Expression Gene Targeting Genes Genetic Genetic Transcription Genomic Segment Goals Human Inflammation Inflammatory Inflammatory Response Investigation Laboratories Laboratory Research Lentivirus Vector Major Depressive Disorder Measures Mediating Mental disorders Mentorship Messenger RNA Methods Molecular Morphology Mus NF-kappa B Neuroglia Neurons Nuclear Pathogenicity Pathologic Pathology Pathway interactions Phenotype Population Predisposition Process Proteins Regulation Regulatory Element Research Personnel Rewards Ribosomes Rodent Model Role Saline Signal Transduction Sorting - Cell Movement Stress Sucrose Symptoms System Techniques Testing Therapeutic Therapeutic Agents Tissues Training Transcriptional Regulation Translating Transposase Up-Regulation Viral Vector base behavioral phenotyping career cell cortex cell type cellular transduction chromatin remodeling epigenetic regulation experimental study genetic regulatory protein genome-wide insight knock-down male molecular phenotype mouse model neuroinflammation neurotransmission novel overexpression pre-clinical preference response social social defeat social stress targeted treatment transcription factor transcriptome sequencing vector

项目摘要

Although MDD has been predominantly studied in the context of neuronal function, emerging evidence indicates that dysregulation of glia may be equally important – particularly during chronic neuroinflammation in response to stress, which is known to contribute to the pathophysiology of MDD. However, the cell-type specific transcriptional dynamics driving these processes remain unclear because traditional epigenetic chromatin accessibility profiling methods are not compatible with cell-type specific approaches. Our laboratory has recently implemented the newly developed technique FANS (Fluorescence-Activated Nuclear Sorting)-coupled ATAC- seq (Assay for Transposase-Accessible Chromatin-Sequencing) to profile the cell type-specific regulatory landscape in human MDD in orbitofrontal cortex (OFC), a brain region that processes reward-based decision- making and may mediate anhedonic symptoms in MDD. Interestingly, we only detected MDD-specific open chromatin regions (OCRs) in the glial, but not the neuronal OFC cell population. Gene set analyses of MDD- specific OCRs showed significant enrichment of astrocyte-specific genes regulating NF-KB inflammation response. Using motif discovery, I identified ZBTB7A, a chromatin remodeling protein with recognition sequences significantly overrepresented in MDD-specific OCRs. Recently, ZBTB7A has been shown to orchestrate chromatin accessibility for a distinct subset of delayed induction NF-Kb target genes, suggesting that ZBTB7A may regulate the transduction of chronic NF-Kb stress signals from adaptive to pathological. My pilot data has shown that ZBTB7A is upregulated in the OFC of both human MDD and in OFC astrocytes of a preclinical chronic social defeat stress (CSDS) mouse model, as well as in cultured murine primary astrocytes treated with LPS (a compound which induces NF-KB and inflammation). Given these preliminary data, I hypothesize that upregulation of ZBTB7A in OFC astrocytes acts as a pathogenic driver of pro- inflammatory NF-Kb activation in MDD through modulation of chromatin accessibility at key downstream target genes, leading to MDD-related behavioral deficits. In Aim 1, I will characterize the basic mechanisms of manipulating this chromatin remodeler at baseline and during inflammation stress by manipulating ZBTB7A levels in a cultured human primary astrocyte system, then treating with LPS or saline followed by assessment of astrocyte reactivity (IHC), chromatin accessibility (via ATAC-seq), epigenetic regulation (ChIP-seq) and gene expression (RNA-seq). In Aim 2, I will explore the therapeutic potential of targeting Zbtb7a by using novel astrocyte-specific viral vectors to determine if Zbtb7a is necessary and sufficient in the OFC to affect vulnerability to inflammation stress-induced behavioral deficits in a preclinical mouse model of social defeat stress. Together these experiments will offer enormous potential for new mechanistic insights into disease pathology in the context of a relatively understudied cell-type, astrocytes, in neuroinflammation and stress.

尽管 MDD 主要是在神经功能的背景下研究的，但新出现的证据表明神经胶质细胞的失调可能同样重要——特别是在慢性神经炎症反应期间众所周知，压力会导致 MDD 的病理生理学，但其细胞类型具有特异性。驱动这些过程的转录动力学仍不清楚，因为传统的表观遗传染色质可及性分析方法与细胞类型特定方法不兼容。实施了新开发的技术FANS（荧光激活核分选）耦合ATAC- seq（转座酶可及染色质测序分析）来分析细胞类型特异性调控人类MDD中眶额皮层（OFC）的景观，这是一个处理基于奖励的决策的大脑区域可能会介导 MDD 害羞的快感缺失症状，但我们只检测到 MDD 特有的开放性。神经胶质细胞中的染色质区域 (OCR)，但不是 MDD 的神经元 OFC 细胞群的基因集分析。特异性 OCR 显示调节 NF-KB 炎症的星形胶质细胞特异性基因显着富集通过基序发现，我发现了 ZBTB7A，一种具有识别功能的染色质重塑蛋白。最近，ZBTB7A 已被证明可以在 MDD 特异性 OCR 中显着过度表达。协调延迟诱导 NF-Kb 靶基因的一个独特子集的染色质可及性，表明 ZBTB7A 可能调节慢性 NF-Kb 应激信号从适应性到病理性的转导。数据显示，ZBTB7A 在人类 MDD 和 OFC 星形胶质细胞的 OFC 中表达上调。临床前慢性社交失败应激 (CSDS) 小鼠模型以及培养的小鼠原代星形胶质细胞考虑到这些初步数据，我认为 LPS（一种诱导 NF-KB 并治疗炎症的化合物）。 OFC 星形胶质细胞中 ZBTB7A 的上调是亲- MDD 中通过调节关键下游染色质可及性激活炎症 NF-Kb 在目标 1 中，我将描述其基本机制。通过操纵 ZBTB7A 在基线和炎症应激期间操纵这种染色质重塑剂培养的人原代星形胶质细胞系统中的水平，然后用 LPS 或盐水处理，然后评估星形胶质细胞反应性 (IHC)、染色质可及性（通过 ATAC-seq）、表观遗传调控 (ChIP-seq) 和基因在目标 2 中，我将探索使用新型药物靶向 Zbtb7a 的治疗潜力。星形胶质细胞特异性病毒载体，以确定 Zbtb7a 是否对 OFC 脆弱性有必要和充分的影响在社交失败压力的临床前小鼠模型中，炎症压力诱导的行为缺陷。这些实验将为疾病病理学的新机制见解提供巨大的潜力。星形胶质细胞在神经炎症和应激中的研究相对较少。