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Abstract

Major depressive disorder (MDD) is a complex mental disease with a high medical, social, and economic burden. Despite this harm, still little is known about the pathophysiology of MDD, while available pharmacotherapy is only partially effective. Therefore, understanding depression by investigating the neurobiology of the disease is an essential step to help patients. Numerous studies have discovered alterations in functional, morphological, cellular, and transcriptional properties of brain circuits controlling mood. Common findings reveal abnormal activity, changes in cellular composition, and molecular profile in the prefrontal cortex, a brain center of stress response. Nevertheless, investigation of the underlying pathology at cellular resolution has been hampered by the lack of tools enabling studies at required cellular and molecular specificity. Previous studies demonstrated that astrocytes mediate transcriptional effects of main stress hormones, glucocorticoids, on the brain. We, therefore, hypothesized that astrocytes’ dysfunctions mediate biological symptoms of depression and that it would be possible to identify disease relevant cell-type-specific transcriptional changes. In this project, we conducted a systematic approach to investigate changes in astrocytes’ transcriptome in human depression and a rodent model of chronic stress. We established methods for efficient and specific RNA isolation from astrocytes and subsequently applied them to test our research hypothesis. Out of several available methods, we selected a strategy based on fluorescent-based cell sorting of human nuclei labelled with cell type-specific antibodies. When our project started, such methods were available only for two brain cell types: neurons and oligodendrocytes. Thus, we provide the first positive selection approach for isolating astrocytic nuclei from frozen human postmortem brain samples. Furthermore, we adopted standard strategies for isolation of astrocytes from adult mouse brain, and we optimized those techniques to be now applied for small tissue volume, like prefrontal cortex or hypothalamus. Next, we employed these novel methods for gene expression studies of astrocytes’ nuclei from frozen tissue samples from healthy controls and depressed suicides and in astrocytes isolated from the adult mice brain samples collected from animals exposed to chronic stress. As a result, we report novel genes linking astrocyte-specific molecular processes with altered neurobiological functions in depression. In summary, we developed protocols to facilitate gene expression profiling of astrocytes in humans and mice. Our findings suggest that changes in astrocytes’ molecular profile may underlie the neurotransmitter imbalance in depression. This study points to astrocyte-specific pathways as potential therapeutic targets for reversing psychiatric phenotypes.