Characterizing effects of OFC astrocyte plasticity in inflammation response and stress vulnerability with single-cell resolution

重度抑郁症（MDD）源于遗传和环境之间的复杂相互作用。 影响，如压力，这导致额缘基因表达和细胞的持续变化 功能虽然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， 表明神经胶质的失调可能同样重要。特别是，星形胶质细胞是 神经元微环境，并可以通过广泛的分泌和 接触介导的信号传导。慢性应激可导致星形胶质细胞发生广泛的转录组学变化， 这可能导致正常支持功能的丧失，并获得潜在的神经毒性， 同样会破坏周围电路的整体活动[8]。了解这个星形胶质细胞 可塑性及其如何有助于炎症相关精神疾病的病理生理学是一个 这一建议的核心问题。我们的实验室已经实施了FANS偶联ATAC-seq来分析 人类MDD眶额皮质（OFC）中细胞类型特异性调节景观，（Aim 1）是一个大脑区域， 处理基于奖励的决策，并可能介导MDD中的快感缺失症状。使用这种方法， 我确定了一个关键的先驱因子ZBTB 7A，它特异性地调节星形胶质细胞中的染色质结构， 促进由NF-kB驱动的前馈促炎转录循环（ZBTB 7A也直接与NF-kB结合） 互动）。在一系列利用星形胶质细胞特异性病毒操作的研究中，我发现过表达（OE） 这种啮齿类OFC星形胶质细胞中的染色质重塑足以诱导炎性细胞因子的异常表达。 基因，行为缺陷和神经元过度活跃，以应对轻度应激，相比GFP- 表达小鼠。我的研究结果表明，OFC星形胶质细胞中Zbtb 7a的OE启动了星形胶质细胞的变化， 表型对神经元传递有选择性的直接影响。然而，了解具体的 Zbtb 7a OE对星形胶质细胞可塑性的影响以及星形胶质细胞Zbtb 7a OE的非细胞自主作用 需要单细胞分辨率的表达谱分析。因此，在目标2中，我建议执行单核 对来自该相同队列的Zbtb 7a OE动物与Zbtb 7a OE动物的病毒感染的OFC组织进行RNA测序（snRNA-seq）。 GFP，以创建细胞类型特异性变化的综合细胞图谱和转录组学图谱 在眶额皮层的神经和神经胶质细胞群中。我假设Zbtb 7a OE诱导关键基因表达 引起细胞自主适应不良星形胶质细胞表型的变化，逆转了正常的适应性作用， 星形胶质细胞对轻度应激的反应。在目标3中，我将专注于为我的博士后工作确定实验室， 优先考虑多路复用“组学”分析、电路特定方法（包括空间 转录组学）和电生理学，这将建立在我目前的培训，使我能够进一步研究 星形胶质细胞可塑性在精神疾病中的作用目标3还将强调加强必要的 专业发展技能，以促进我的独立研究的进展。

Major Depressive Disorder (MDD) arises from a complex interaction between genetics and environmental influences such as stress, which leads to persistent changes in frontolimbic gene expression and cellular function. Although MDD has been predominantly studied in the context of neuronal function, emerging evidence indicates that dysregulation of glia may be equally important. In particular, astrocytes are key determinants of the neuronal microenvironment and can modulate synaptic efficacy through a wide range of secreted and contact-mediated signaling. Chronic stress can cause astrocytes to undergo broad transcriptomic changes, which can lead to loss of normal supportive functions, and gain of potentially neurotoxic qualities, both of which can be equally disruptive to the overall activity of surrounding circuitry[8]. Understanding this astrocyte plasticity and how it contributes to the pathophysiology of inflammation-related psychiatric disease is a central question in this proposal. Our laboratory has implemented FANS coupled ATAC-seq to profile the cell type-specific regulatory landscape in human MDD orbitofrontal cortex (OFC), (Aim 1) a brain region that processes reward-based decision-making and may mediate anhedonic symptoms in MDD. Using this approach, I identified a key pioneer factor, ZBTB7A, which regulates chromatin structure specifically in astrocytes to facilitate feed-forward pro-inflammatory transcriptional cycles driven by NF-kB (with which ZBTB7A also directly interacts). In a series of studies utilizing astrocyte-specific viral manipulations, I found that overexpressing (OE) this chromatin remodeler in rodent OFC astrocytes was sufficient to induce aberrant expression of inflammatory genes, behavioral deficits, and neuronal hyperactivity in response to a mild stressor, compared to GFP- expressing mice. My findings indicate that OE of Zbtb7a in OFC astrocytes initiates a change in astrocyte phenotype that has selective, direct effects on neuronal transmission. However, understanding the specific impact of Zbtb7a OE on astrocyte plasticity, as well as the non-cell autonomous effects of astrocytic Zbtb7a OE requires expression profiling with single-cell resolution. Therefore, in Aim 2 I propose to perform single-nuclei RNA sequencing (snRNA-seq) on virally-infected OFC tissues from this same cohort of Zbtb7a OE animals vs. GFP in order to create a comprehensive cellular map and transcriptomic profile of cell-type specific changes across neural and glial populations in the OFC. I hypothesize that Zbtb7a OE induces key gene expression changes to elicit cell-autonomous maladaptive astrocyte phenotypes that reverse the normal adaptive role of astrocytes in responding to mild stress. In Aim 3, I will focus on identifying laboratories for my postdoctoral work, prioritizing expertise in multiplexed “omics” profiling, circuit-specific approaches (including spatial transcriptomics), and electrophysiology, which will build on my current training to allow me to further investigate the role of astrocyte plasticity in psychiatric disease. Aim 3 will also emphasize strengthening essential professional development skills in order to facilitate my progress towards independent research.