Sex and Estrous Cycle Effects on Anxiety- and Depression-Related Phenotypes in a Two-Hit Developmental Stress Model

Stress during sensitive developmental periods can adversely affect physical and psychological development and contribute to later-life mental disorders. In particular, adverse experiences during childhood dramatically increase the risk for the development of depression and anxiety disorders. Although women of reproductive age are twice as likely to develop anxiety and depression than men of the corresponding age, little is known about sex-specific factors that promote or protect against the development of psychopathology. To examine potential developmental mechanisms driving sex disparity in risk for anxiety and depression, we established a two-hit developmental stress model including maternal separation in early life followed by social isolation in adolescence. Our study shows complex interactions between early-life and adolescent stress, between stress and sex, and between stress and female estrogen status in shaping behavioral phenotypes of adult animals. In general, increased locomotor activity and body weight reduction were the only two phenotypes where two stressors showed synergistic activity. In terms of anxiety- and depression-related phenotypes, single exposure to early-life stress had the most significant impact and was female-specific. We show that early-life stress disrupts the protective role of estrogen in females, and promotes female vulnerability to anxiety- and depression-related phenotypes associated with the low-estrogenic state. We found plausible transcriptional and epigenetic alterations in psychiatric risk genes, Nr3c1 and Cacna1c, that likely contributed to the stress-induced behavioral effects. In addition, two general transcriptional regulators, Egr1 and Dnmt1, were found to be dysregulated in maternally-separated females and in animals exposed to both stressors, respectively, providing insights into possible transcriptional mechanisms that underlie behavioral phenotypes. Our findings provide a novel insight into developmental risk factors and biological mechanisms driving sex differences in depression and anxiety disorders, facilitating the search for more effective, sex-specific treatments for these disorders

The Role of Egr1 in Driving Sex-Specific Transcriptional Programs and Anxiety-Related Behavior

Women have a two-fold increased risk for anxiety and depression disorders compared to men, though the molecular mechanisms underlying this phenomenon are understudied. Clinical and epidemiological data indicate ovarian hormone fluctuations are a critical female-specific risk factor for these disorders. Accordingly, we previously demonstrated that anxiety-like behavior in female mice varies across the estrous cycle. We also showed estrous cycle-dependent changes in neuronal chromatin accessibility and gene expression in the ventral hippocampus, a region critical for emotion regulation in rodents. Analysis of these data identified Egr1, a transcription factor and estrogen-responsive immediate early gene product, as a candidate regulator of chromatin regulation and gene expression across the estrous cycle. To test this, in the current study, I first profiled behavior and Egr1 expression across the four estrous cycle phases, revealing that high-estrogenic proestrus females have lower anxiety indices. However, following the withdrawal of estrogen levels that characterizes the other phases, anxiety-related behavior increased. In parallel, I observed cycling Egr1 mRNA expression across the estrous cycle, following estrogen levels. To mechanistically link Egr1 to the estrous cycle-dependent behavioral and molecular phenotype, I performed AAV-mediated, neuronal-specific overexpression of Egr1 in the ventral hippocampus of intact-male and ovariectomized-female mice. I then performed anxiety- and depression-related behavioral tests, as well as cell type-specific genomics assays profiling neuronal chromatin (ATAC-seq) and gene expression (RNA-seq). Results from behavioral tests implicate Egr1 in driving cyclical changes in anxiety- and depression-related behaviors in females, with no effect of Egr1 overexpression in males, indicating the effect is sex-specific. Further, Egr1 overexpression induced sex-specific changes in gene expression and chromatin organization, including Egr1-directed chromatin opening, in ventral hippocampal neurons that give crucial insights into this behavioral phenotype, including female-specific regulation of genes related to serotonin and anxiety. Overlapping these data with the previously generated estrous cycle data demonstrated that Egr1-induced chromatin regulation facilitates the downregulation of anxiety-related genes and the upregulation of synaptic plasticity-related genes during the high-estrogenic proestrus phase. Taken together, these results causally link Egr1 to estrous cycle-dependent gene regulation and behavioral plasticity and establish a foundation for developing sex-specific treatments for anxiety and depression

Cell type-specific chromatin accessibility analysis in the mouse and human brain

The Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) is becoming popular in the neuroscience field where chromatin regulation is thought to be involved in neurodevelopment, activity-dependent gene regulation, hormonal and environmental responses, and pathophysiology of neuropsychiatric disorders. The advantages of using ATAC-seq include a small amount of material needed, fast protocol, and the ability to capture a range of gene regulatory elements with a single assay. With increasing interest in chromatin research, it is an imperative to have feasible, reliable assays that are compatible with a range of neuroscience study designs. Here we tested three protocols for neuronal chromatin accessibility analysis, including a varying brain tissue freezing method followed by fluorescence-activated nuclei sorting (FANS) and ATAC-seq. Our study shows that the cryopreservation method impacts the number of open chromatin regions identified from frozen brain tissue using ATAC-seq. However, we show that all protocols generate consistent and robust data and enable the identification of functional regulatory elements in neuronal cells. Our study implies that the broad biological interpretation of chromatin accessibility data is not significantly affected by the freezing condition. We also reveal additional challenges of doing chromatin analysis on post-mortem human brain tissue. Overall, ATAC-seq coupled with FANS is a powerful method to capture cell-type-specific chromatin accessibility information in mouse and human brain. Our study provides alternative brain preservation methods that generate high-quality ATAC-seq data while fitting in different study designs, and further encourages the use of this method to uncover the role of epigenetic (dys)regulation in the brain