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.

尽管在神经元功能的背景下，MDD主要是研究的，但新出现的证据表明神经胶质的失调可能同样重要 - 尤其是在慢性神经炎症中的反应中压力，已知有助于MDD的病理生理学。但是，细胞类型特异性驱动这些过程的转录动力学仍然不清楚，因为传统表观遗传染色质可访问性分析方法与细胞类型的特定方法不兼容。我们的实验室最近有实施了新开发的技术风扇（荧光激活的核分选）与ATAC-耦合 SEQ（转座酶可访问的染色质测序测定），以介绍细胞类型特异性调节人类MDD中的景观Orbitrontal Cortex（OFC），这是一个大脑区域，可处理基于奖励的决策 - 制作并可能介导MDD中的鼻鼻症状。有趣的是，我们仅检测到MDD特定的开放神经胶质中的染色质区域（OCR），但没有神经元OFC细胞群。基因集MDD- 特定的OCR显示了调节NF-KB炎症的星形胶质细胞特异性基因的明显酶回复。使用基序发现，我确定了ZBTB7A，这是一种具有识别的染色质重塑蛋白在MDD特异性OCR中，序列明显过多。最近，ZBTB7A已被证明编排染色质的可及性，用于延迟诱导NF-KB靶基因的不同子集，表明 ZBTB7A可能调节慢性NF-KB应力信号从适应性到病理的转导。我的飞行员数据表明，ZBTB7A已在人类MDD和A的OFC中更新临床前慢性社会失败压力（CSD）鼠标模型以及培养的鼠主要星形胶质细胞用LPS处理（一种诱导NF-KB和炎症的化合物）。鉴于这些初步数据，我假设OFC星形胶质细胞中ZBTB7A的上调是促进的致病驱动力通过调节钥匙下游的染色质可及性，MDD中的炎症NF-KB激活靶基因，导致与MDD相关的行为定义。在AIM 1中，我将表征基本机制通过操纵ZBTB7A时在基线和炎症应力下操纵这种染色质改造的方法培养的人类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），表观遗传调节（CHIP-SEQ）和基因表达（RNA-seq）。在AIM 2中，我将使用新颖的星形胶质细胞特异性病毒载体确定ZBTB7A是否需要OFC中的ZBTB7A来影响脆弱性炎症压力诱导的行为在社交失败压力的临床前小鼠模型中定义。一起这些实验将为对疾病病理学的新机械见解提供巨大的潜力在神经炎症和压力中，相对了解的细胞类型，星形胶质细胞的背景。