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

虽然MDD主要是在神经元功能的背景下进行研究，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中， 压力，这是众所周知的，有助于MDD的病理生理学。然而，细胞类型特异性 驱动这些过程的转录动力学仍然不清楚， 可访问性分析方法与特定于小区类型的方法不兼容。我们的实验室最近 实施了新开发的技术FANS（荧光激活核分选）偶联ATAC- seq（转座酶可降解染色质测序试验），以分析细胞类型特异性调控蛋白 人类MDD中眶额皮层（OFC）的景观，OFC是一个处理基于奖励的决策的大脑区域， 导致并可能介导抑郁症的享乐缺失症状。有趣的是，我们只检测到MDD特异性开放 染色质区域（OCR）的神经胶质细胞，但不是神经元OFC细胞群。MDD的基因集分析- 特异性OCR显示调节NF-κ B炎症的星形胶质细胞特异性基因显著富集 反应使用基序发现，我鉴定了ZBTB 7A，一种染色质重塑蛋白， 在MDD特异性OCR中显著过度表达的序列。最近，ZBTB 7A被证明可以 协调染色质对延迟诱导NF-Kb靶基因的不同亚群的可及性，表明 ZBTB 7A可能调节慢性NF-κ B应激信号从适应性向病理性的传导。我的飞行员 数据显示，ZBTB 7A在人MDD的OFC和人MDD的OFC星形胶质细胞中上调， 临床前慢性社会失败应激（CSDS）小鼠模型以及培养的鼠原代星形胶质细胞中 用LPS（一种诱导NF-κ B和炎症的化合物）处理。根据这些初步数据，我 假设ZBTB 7A在OFC星形胶质细胞中的上调作为促凋亡的致病驱动因素， MDD中炎症性NF-Kb活化通过调节关键下游染色质可及性 靶基因，导致MDD相关的行为缺陷。在目标1中，我将描述基本机制 在基线和炎症应激期间，通过操纵ZBTB 7A 在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，随后评估 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 表达（RNA-seq）。在目的2中，我将探索通过使用新的靶向Zbtb 7a的治疗潜力。 星形胶质细胞特异性病毒载体，以确定Zbtb 7a在OFC中是否是必要的和足够的，以影响脆弱性 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。一起 这些实验将提供巨大的潜力，为新的机制洞察疾病病理学， 在神经炎症和应激中，星形胶质细胞是一种研究相对不足的细胞类型。

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.