Glucocorticoid stress hormones are powerful modulators of brain function and can affect mood and cognitive processes. The hippocampus is a prominent glucocorticoid target and expresses both the ...glucocorticoid receptor (GR: Nr3c1) and the mineralocorticoid receptor (MR: Nr3c2). These nuclear steroid receptors act as ligand‐dependent transcription factors. Transcriptional effects of glucocorticoids have often been deduced from bulk mRNA measurements or spatially informed individual gene expression. However, only sparse data exists allowing insights on glucocorticoid‐driven gene transcription at the cell type level. Here, we used publicly available single‐cell RNA sequencing data to assess the cell‐type specificity of GR and MR signaling in the adult mouse hippocampus. The data confirmed that Nr3c1 and Nr3c2 expression differs across neuronal and non‐neuronal cell populations. We analyzed co‐expression with sex hormones receptors, transcriptional coregulators, and receptors for neurotransmitters and neuropeptides. Our results provide insights in the cellular basis of previous bulk mRNA results and allow the formulation of more defined hypotheses on the effects of glucocorticoids on hippocampal function.
Abstract
In the limbic brain, mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) both function as receptors for the naturally occurring glucocorticoids (corticosterone/cortisol) but ...mediate distinct effects on cellular physiology via transcriptional mechanisms. The transcriptional basis for specificity of these MR- vs GR-mediated effects is unknown. To address this conundrum, we have identified the extent of MR/GR DNA-binding selectivity in the rat hippocampus using chromatin immunoprecipitation followed by sequencing. We found 918 and 1450 nonoverlapping binding sites for MR and GR, respectively. Furthermore, 475 loci were co-occupied by MR and GR. De novo motif analysis resulted in a similar binding motif for both receptors at 100% of the target loci, which matched the known glucocorticoid response element (GRE). In addition, the Atoh/NeuroD consensus sequence was found in co-occurrence with all MR-specific binding sites but was absent for GR-specific or MR-GR overlapping sites. Basic helix-loop-helix family members Neurod1, Neurod2, and Neurod6 showed hippocampal expression and were hypothesized to bind the Atoh motif. Neurod2 was detected at rat hippocampal MR binding sites but not at GR-exclusive sites. All three NeuroD transcription factors acted as DNA-binding–dependent coactivators for both MR and GR in reporter assays in heterologous HEK293 cells, likely via indirect interactions with the receptors. In conclusion, a NeuroD family member binding to an additional motif near the GRE seems to drive specificity for MR over GR binding at hippocampal binding sites.
Genome-wide identification of corticosteroid receptor binding loci on the DNA suggests that at MR-exclusive binding sites, NeuroD factors cooperate with MR binding and/or functionality.
Glucocorticoids exert their pleiotropic effects by activating the glucocorticoid receptor (GR), which is expressed throughout the body. GR‐mediated transcription is regulated by a multitude of ...tissue‐ and cell type‐specific mechanisms, including interactions with other transcription factors such as the androgen receptor (AR). We previously showed that the transcription of canonical glucocorticoid‐responsive genes is dependent on active androgen signaling, but the extent of this glucocorticoid‐androgen crosstalk warrants further investigation. In this study, we investigated the overall glucocorticoid‐androgen crosstalk in the hepatic transcriptome. Male mice were exposed to GR agonist corticosterone and AR antagonist enzalutamide in order to determine the extent of androgen‐dependency after acute and chronic exposure. We found that a substantial proportion of the hepatic transcriptome is androgen‐dependent after chronic exposure, while after acute exposure the transcriptomic effects of glucocorticoids are largely androgen‐independent. We propose that prolonged glucocorticoid exposure triggers a gradual upregulation of AR expression, instating a situation of androgen dependence which is likely not driven by direct AR‐GR interactions. This indirect mode of glucocorticoid‐androgen interaction is in accordance with the absence of enriched AR DNA‐binding near AR‐dependent corticosterone‐regulated genes after chronic exposure. In conclusion, we demonstrate that glucocorticoid effects and their interaction with androgen signaling are dependent on the duration of exposure and believe that our findings contribute to a better understanding of hepatic glucocorticoid biology in health and disease.
Stressful experiences evoke, among others, a rapid increase in brain (nor)epinephrine (NE) levels and a slower increase in glucocorticoid hormones (GCs) in the brain. Microglia are key regulators of ...neuronal function and contain receptors for NE and GCs. These brain cells may therefore potentially be involved in modulating stress effects on neuronal function and learning and memory. In this review, we discuss that stress induces (1) an increase in microglial numbers as well as (2) a shift toward a pro‐inflammatory profile. These microglia have (3) impaired crosstalk with neurons and (4) disrupted glutamate signaling. Moreover, microglial immune responses after stress (5) alter the kynurenine pathway through metabolites that impair glutamatergic transmission. All these effects could be involved in the impairments in memory and in synaptic plasticity caused by (prolonged) stress, implicating microglia as a potential novel target in stress‐related memory impairments.
Abstract
Adrenal glucocorticoid hormones are crucial for maintenance of homeostasis and adaptation to stress. They act via the mineralocorticoid receptors (MRs) and glucocorticoid receptors ...(GRs)—members of the family of nuclear receptors. MRs and GRs can mediate distinct, sometimes opposite, effects of glucocorticoids. Both receptor types can mediate nongenomic steroid effects, but they are best understood as ligand-activated transcription factors. MR and GR protein structure is similar; the receptors can form heterodimers on the DNA at glucocorticoid response elements (GREs), and they share a number of target genes. The transcriptional basis for opposite effects on cellular physiology remains largely unknown, in particular with respect to MR-selective gene transcription. In this review, we discuss proven and potential mechanisms of transcriptional specificity for MRs and GRs. These include unique GR binding to “negative GREs,” direct binding to other transcription factors, and binding to specific DNA sequences in conjunction with other transcription factors, as is the case for MRs and NeuroD proteins in the brain. MR- and GR-specific effects may also depend on specific interactions with transcriptional coregulators, downstream mediators of transcriptional receptor activity. Current data suggest that the relative importance of these mechanisms depends on the tissue and physiological context. Insight into these processes may not only allow a better understanding of homeostatic regulation but also the development of drugs that target specific aspects of disease.
Glucocorticoids regulate memory consolidation, facilitating long-term storage of relevant information to adequately respond to future stressors in similar conditions. This effect of glucocorticoids ...is well-established and is observed in multiple types of behaviour that depend on various brain regions. By and large, higher glucocorticoid levels strengthen event-related memory, while inhibition of glucocorticoid signalling impairs consolidation. The mechanism underlying this glucocorticoid effect remains unclear, but it likely involves the transcriptional effects of the glucocorticoid receptor (GR). We here used a powerful paradigm to investigate the transcriptional effects of GR in the dorsal hippocampus of mice after training in an auditory fear conditioning task, aiming to identify a shortlist of GR target genes associated to memory consolidation. Therefore, we utilized in an explorative study the properties of selective GR modulators (CORT108297 and CORT118335), alongside the endogenous agonist corticosterone and the classical GR antagonist RU486, to pinpoint GR-dependent transcriptional changes. First, we confirmed that glucocorticoids can modulate memory strength via GR activation. Subsequently, by assessing the specific effects of the available GR-ligands on memory strength, we established a pharmacological filter which we imposed on the hippocampal transcriptome data. This identified a manageable shortlist of eight genes by which glucocorticoids may modulate memory consolidation, warranting in-depth follow-up. Overall, we showcase the strength of the concept of pharmacological transcriptome filtering, which can be readily applied to other research topics with an established role of glucocorticoids.
•Glucocorticoid receptors modulate consolidation of auditory fear conditioning.•Selective glucocorticoid receptor modulators used to study glucocorticoid signalling.•Identification of a shortlist of genes associated to memory consolidation.•Glucocorticoids affect memory consolidation via both neuronal and non-neuronal cells.•Introducing the concept of a pharmacological transcriptome filter to study transcriptional effects.
Glucocorticoids enhance memory consolidation of emotionally arousing events via largely unknown molecular mechanisms. This glucocorticoid effect on the consolidation process also requires central ...noradrenergic neurotransmission. The intracellular pathways of these two stress mediators converge on two transcription factors: the glucocorticoid receptor (GR) and phosphorylated cAMP response element‐binding protein (pCREB). We therefore investigated, in male rats, whether glucocorticoid effects on memory are associated with genomic interactions between the GR and pCREB in the hippocampus. In a two‐by‐two design, object exploration training or no training was combined with post‐training administration of a memory‐enhancing dose of corticosterone or vehicle. Genomic effects were studied by chromatin immunoprecipitation followed by sequencing (ChIP‐seq) of GR and pCREB 45 min after training and transcriptome analysis after 3 hr. Corticosterone administration induced differential GR DNA‐binding and regulation of target genes within the hippocampus, largely independent of training. Training alone did not result in long‐term memory nor did it affect GR or pCREB DNA‐binding and gene expression. No strong evidence was found for an interaction between GR and pCREB. Combination of the GR DNA‐binding and transcriptome data identified a set of novel, likely direct, GR target genes that are candidate mediators of corticosterone effects on memory consolidation. Cell‐specific expression of the identified target genes using single‐cell expression data suggests that the effects of corticosterone reflect in part non‐neuronal cells. Together, our data identified new GR targets associated with memory consolidation that reflect effects in both neuronal and non‐neuronal cells.
Administration of corticosterone after object location training enhanced long‐term hippocampal memory. Glucocorticoid receptor and phosphorylated cAMP response element‐binding protein were co‐bound at the hippocampal genome and exclusively GR DNA‐binding levels were altered after corticosterone, yet binding of these two transcription factors was not affected by training nor was any interaction detected. Corticosterone altered the transcriptome in a context‐independent manner—partially via direct GR‐regulation—and reflects involvement of both neuronal and non‐neuronal cells in the male rat hippocampus.
Mineralocorticoid receptor (MR)-mediated signaling in the brain has been suggested as a protective factor in the development of psychopathology, in particular mood disorders. We recently identified ...genomic loci at which either MR or the closely related glucocorticoid receptor (GR) binds selectively, and found members of the NeuroD transcription factor family to be specifically associated with MR-bound DNA in the rat hippocampus. We show here using forebrain-specific MR knockout mice that GR binding to MR/GR joint target loci is not affected in any major way in the absence of MR. Neurod2 binding was also independent of MR binding. Moreover, functional comparison with MyoD family members indicates that it is the chromatin remodeling aspect of NeuroD, rather than its direct stimulation of transcription, that is responsible for potentiation of MR-mediated transcription. These findings suggest that NeuroD acts in a permissive way to enhance MR-mediated transcription, and they argue against competition for DNA binding as a mechanism of MR- over GR-specific binding.
Glucocorticoid signaling is context dependent, and in certain scenarios, glucocorticoid receptors (GRs) are able to engage with other members of the nuclear receptor subfamily. Glucocorticoid ...signaling can exert sexually dimorphic effects, suggesting a possible interaction with androgen sex hormones. We therefore set out to determine the crosstalk between glucocorticoids and androgens in metabolic tissues including white adipose tissue, liver and brown adipose tissue. Thereto we exposed male C57BL/6J mice to elevated levels of corticosterone in combination with an androgen receptor (AR) agonist or an AR antagonist. Systemic and local glucocorticoid levels were determined by mass spectrometry, and tissue expression of glucocorticoid-responsive genes and protein was measured by RT-qPCR and Western blot, respectively. To evaluate crosstalk in vitro, cultured white and brown adipocytes were exposed to a combination of corticosterone and an AR agonist. We found that AR agonism potentiated transcriptional response to GR in vitro in white and brown adipocytes and in vivo in white and brown adipose tissues. Conversely, AR antagonism substantially attenuated glucocorticoid signaling in white adipose tissue and liver. In white adipose tissue, this effect could partially be attributed to decreased 11B-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid regeneration upon AR antagonism. In liver, attenuated GR activity was independent of active glucocorticoid ligand levels. We conclude that androgen signaling modulates GR transcriptional output in a tissue-specific manner.
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