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MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing’s syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing’s disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes.

RNA is abundantly modified by a range of covalent modifications, collectively termed the epitranscriptome. Of these modifications, N6-methyladenosine (m6A) is the most prevalent internal chemical tag in eukaryotic mRNA. Being cotranscriptionally deposited, it regulates almost all aspects of mRNA’s lifetime including maturation into mRNA, stability, distribution and protein translation. While m6A is likely present in all developing and adult mammalian tissues, here we highlight its distribution and reported functions in the mammalian brain. Additionally, we describe its potential to act as an encoding mechanism for activity- and experience-dependent adaptation and memory-formation. Such alterations may be positive when adjusting to outer challenges or negative when involved in maladaptive processes of the brain such as in the development of psychopathologies.

Summary MicroRNAs are important regulators of gene expression and associated with stress-related psychiatric disorders. Here, we report that exposing mice to chronic stress led to a specific increase in microRNA-15a levels in the amygdala-Ago2 complex and a concomitant reduction in the levels of its predicted target, FKBP51, which is implicated in stress-related psychiatric disorders. Reciprocally, mice expressing reduced levels of amygdalar microRNA-15a following exposure to chronic stress exhibited increased anxiety-like behaviors. In humans, pharmacological activation of the glucocorticoid receptor, as well as exposure to childhood trauma, was associated with increased microRNA-15a levels in peripheral blood. Taken together, our results support an important role for microRNA-15a in stress adaptation and the pathogenesis of stress-related psychopathologies., Graphical Abstract, Highlights • miR-15a levels are elevated in the amygdala-Ago2 complex following chronic stress • miR-15a targets FKBP51 and affects behavioral responses to stressful challenges • miR-15a is elevated in peripheral human blood following dexamethasone exposure • miR-15a is elevated in peripheral human blood of patients exposed to childhood trauma, Volk et al. reveal an important role for microRNA-15a in coping with chronic stress, with amygdala-specific manipulation affecting behavioral responses to stressful challenge. Individuals exposed to childhood trauma exhibit increased levels of miR-15a in their peripheral blood, suggesting a target for the treatment of stress-related psychopathologies.

Personality traits offer considerable insight into the biological basis of individual differences. However, existing approaches toward understanding personality across species rely on subjective criteria and limited sets of behavioral readouts, resulting in noisy and often inconsistent outcomes. Here, we introduce a mathematical framework for studying individual differences along dimensions with maximum consistency and discriminative power. We validate this framework in mice, using data from a system for high-throughput longitudinal monitoring of group-housed mice that yields a variety of readouts from all across an individual’s behavioral repertoire. We describe a set of stable traits that capture variability in behavior and gene expression in the brain, allowing for better informed mechanistic investigations into the biology of individual differences.

The selective breeding for extreme behavior on the elevated plus-maze (EPM) resulted in two mouse lines namely high-anxiety behaving (HAB) and low-anxiety behaving (LAB) mice. Using novel behavioral tests we demonstrate that HAB animals additionally exhibit maladaptive escape behavior and defensive vocalizations, whereas LAB mice show profound deficits in escaping from approaching threats which partially results from sensory deficits. We could relate these behavioral distortions to tonic changes in brain activity within the periaqueductal gray (PAG) in HAB mice and the superior colliculus (SC) in LAB mice, using in vivo manganese-enhanced MRI (MEMRI) followed by pharmacological or chemogenetic interventions. Therefore, midbrain-tectal structures govern the expression of both anxiety-like behavior and defensive responses. Our results challenge the uncritical use of the anthropomorphic termsanxietyoranxiety-likefor the description of mouse behavior, as they imply higher cognitive processes, which are not necessarily in place.

Corticotropin-releasing factor (CRF) and its type 1 receptor (CRFR(1)) play an important role in the responses to stressful challenges. Despite the well established expression of CRFR(1) in granular cells (GrCs), its role in procedural motor performance and memory formation remains elusive. To investigate the role of CRFR(1) expression in cerebellar GrCs, we used a mouse model depleted of CRFR(1) in these cells. We detected changes in the cellular learning mechanisms in GrCs depleted of CRFR(1) in that they showed changes in intrinsic excitability and long-term synaptic plasticity. Analysis of cerebella transcriptome obtained from KO and control mice detected prominent alterations in the expression of calcium signaling pathways components. Moreover, male mice depleted of CRFR(1) specifically in GrCs showed accelerated Pavlovian associative eye-blink conditioning, but no differences in baseline motor performance, locomotion, or fear and anxiety-related behaviors. Our findings shed light on the interplay between stress-related central mechanisms and cerebellar motor conditioning, highlighting the role of the CRF system in regulating particular forms of cerebellar learning. SIGNIFICANCE STATEMENT Although it is known that the corticotropin-releasing factor type 1 receptor (CRFR(1)) is highly expressed in the cerebellum, little attention has been given to its role in cerebellar functions in the behaving animal. Moreover, most of the attention was directed at the effect of CRF on Purkinje cells at the cellular level and, to this date, almost no data exist on the role of this stress-related receptor in other cerebellar structures. Here, we explored the behavioral and cellular effect of granular cell-specific ablation of CRFR(1). We found a profound effect on learning both at the cellular and behavioral levels without an effect on baseline motor skills.

SummaryN6-Methyladenosine (m6A) is an abundant internal RNA modification that regulates transcript processing and translation. The regulation of brain m6A by stressful stimuli in vivo and its role in the stress response are currently unknown.Here, we provide a detailed analysis of the stress-epitranscriptome using m6A-Seq, global and gene-specific m6A measurements. We show that stress exposure and glucocorticoids alter m6A and its regulatory network in a region- and time-specific manner. We demonstrate that depletion of the methyltransferase Mettl3 and the demethylase Fto in adult neurons increases fear memory, and alters the transcriptome response to fear as well as synaptic plasticity. Finally, we report that regulation of m6A is impaired in major depressive disorder patients following glucocorticoid receptor activation.Our findings indicate that brain m6A represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the m6A-response may contribute to the pathophysiology of stress-related psychiatric disorders.Highlightsm6A RNA methylation in adult mouse brain is regulated by stressBrain m6A levels are temporally and spatially regulated by stressMettl3 and Fto-KO alter fear memory, transcriptome response and synaptic plasticityThe m6A-glucocorticoid-response is impaired in major depressive disorder patientseTOC blurbEngel et al. demonstrate a brain-area-specific and time-dependent role for the mRNA modification, m6A, in stress-response regulation. Manipulating m6A-enzymes alters fear-memory, transcriptome-response and synaptic-plasticity. Altered m6A dynamics in depressed patients suggest an involvement of m6A-modifications in stress-related psychiatric disorders.

Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of “master sex-determining genes” at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.

Covalent RNA modifications were recently rediscovered as abundant RNA chemical tags. Similarly to DNA epigenetic modifications, they have been proposed as essential regulators of gene expression. Here we focus on 3 of the most abundant adenosine methylations: N6-methyladenosine (m6 A), N6,2'-O-dimethyladenosine (m6 Am) and N1-methyladenosine (m1 A). We review the potential role of these modifications on mature mRNA in regulating gene expression within the adult brain, nervous system function and normal and pathological behavior. Dynamic mRNA modifications, summarized as the epitranscriptome, regulate transcript maturation, translation and decay, and thus crucially determine gene expression beyond primary transcription regulation. However, the extent of this regulation in the healthy and maladapted adult brain is poorly understood. Analyzing this novel layer of gene expression control in addition to epigenetics and posttranslational regulation of proteins will be highly relevant for understanding the molecular underpinnings of behavior and psychiatric disorders.

Binge eating (BE) is a common aberrant form of eating behavior, characterized by overconsumption of food in a brief period of time. Recurrent episodes of BE constitute the BE disorder, which mostly affects females and is associated with early-life adversities. Here, we show that corticotropin releasing factor (CRF)-induced prenatal stress (PNS) in late gestation predisposes female offspring to BE-like behavior that coincides with hypomethylation of hypothalamic miR-1a and downstream dysregulation of the melanocortin system through Pax7/Pax3. Moreover, exposing the offspring to a methyl-balanced diet during adolescence prevents the dysregulation and predisposition from being triggered. We demonstrate that gestational programming, per se, will not lead to BE-like behavior, but pre-existing alterations due to prenatal programming are revealed only when challenged during adolescence. We provide experimental evidence for long-term epigenetic abnormalities stemming from PNS in predisposing female offspring to BE disorder as well as a potential non-invasive prevention strategy.