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

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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.