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.
Excess glucocorticoid exposure affects emotional and cognitive brain functions. The extreme form, Cushing's syndrome, is adequately modelled in the AdKO2.0 mouse, consequential to adrenocortical ...hypertrophy and hypercorticosteronemia. We previously reported that the AdKO2.0 mouse brain undergoes volumetric changes that resemble closely those of Cushing's syndrome human patients, as well as changes in expression of glial related marker proteins. In the present work, the expression of genes related to glial and neuronal cell populations and functions was assessed in regions of the anterior brain, hippocampus, amygdala and hypothalamus. Glucocorticoid target genes were consistently regulated, including CRH mRNA suppression in the hypothalamus and induction in amygdala and hippocampus, even if glucocorticoid receptor protein was downregulated. Expression of glial genes was also affected in the AdKO2.0 mouse brain, indicating a different activation status in glial cells. Generic markers for neuronal cell populations, and cellular integrity were only slightly affected. Our findings highlight the vulnerability of glial cell populations to chronic high levels of circulating glucocorticoids.
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.
Glucocorticoid stress hormones are produced in response to hypothalamic–pituitary–adrenal (HPA) axis activation. Glucocorticoids are essential for physiology and exert numerous actions via binding to ...the glucocorticoid receptor (GR). Relacorilant is a highly selective GR antagonist currently undergoing a phase 3 clinical evaluation for the treatment of endogenous Cushing’s syndrome. It was found that increases in serum adrenocorticotropic hormone (ACTH) and cortisol concentrations after relacorilant treatment were substantially less than the increases typically observed with mifepristone, but it is unclear what underlies these differences. In this study, we set out to further preclinically characterize relacorilant in comparison to the classical but non-selective GR antagonist mifepristone. In human HEK-293 cells, relacorilant potently antagonized dexamethasone- and cortisol-induced GR signaling, and in human peripheral blood mononuclear cells, relacorilant largely prevented the anti-inflammatory effects of dexamethasone. In mice, relacorilant treatment prevented hyperinsulinemia and immunosuppression caused by increased corticosterone exposure. Relacorilant treatment reduced the expression of classical GR target genes in peripheral tissues but not in the brain. In mice, relacorilant induced a modest disinhibition of the HPA axis as compared to mifepristone. In line with this, in mouse pituitary cells, relacorilant was generally less potent than mifepristone in regulating Pomc mRNA and ACTH release. This contrast between relacorilant and mifepristone is possibly due to the distinct transcriptional coregulator recruitment by the GR. In conclusion, relacorilant is thus an efficacious peripheral GR antagonist in mice with only modest disinhibition of the HPA axis, and the distinct properties of relacorilant endorse the potential of selective GR antagonist treatment for endogenous Cushing’s syndrome.
Angelman Syndrome (AS) is a severe neurodevelopmental disorder, caused by the neuronal absence of the ubiquitin protein ligase E3A (UBE3A). UBE3A promotes ubiquitin-mediated protein degradation and ...functions as a transcriptional coregulator of nuclear hormone receptors, including the glucocorticoid receptor (GR). Previous studies showed anxiety-like behavior and hippocampal-dependent memory disturbances in AS mouse models. Hippocampal GR is an important regulator of the stress response and memory formation, and we therefore investigated whether the absence of UBE3A in AS mice disrupted GR signaling in the hippocampus. We first established a strong cortisol-dependent interaction between the GR ligand binding domain and a UBE3A nuclear receptor box in a high-throughput interaction screen. In vivo, we found that UBE3A-deficient AS mice displayed significantly more variation in circulating corticosterone levels throughout the day compared to wildtypes (WT), with low to undetectable levels of corticosterone at the trough of the circadian cycle. Additionally, we observed an enhanced transcriptomic response in the AS hippocampus following acute corticosterone treatment. Surprisingly, chronic corticosterone treatment showed less contrast between AS and WT mice in the hippocampus and liver transcriptomic responses. This suggests that UBE3A limits the acute stimulation of GR signaling, likely as a member of the GR transcriptional complex. Altogether, these data indicate that AS mice are more sensitive to acute glucocorticoid exposure in the brain compared to WT mice. This suggests that stress responsiveness is altered in AS which could lead to anxiety symptoms.
Glucocorticoids are key executors of the physiological response to stress. Previous studies in mice showed that the androgen receptor (AR) influenced the transcriptional outcome of glucocorticoid ...treatment in white and brown adipocytes and in the liver. In the brain, we observed that chronic hypercorticism induced changes in gene expression that tended to be more pronounced in male mice. In the present study, we investigated if glucocorticoid signaling in the brain could be modulated by androgen. After chronic treatment with corticosterone, dihydrotestosterone, a combination of both, and corticosterone in combination with the AR antagonist enzalutamide, we compared the expression of glucocorticoid receptor (NR3C1, also abbreviated GR) target genes in brain regions where AR and GR are co-expressed, namely: prefrontal cortex, hypothalamus, hippocampus, ventral tegmental area and substantia nigra. We observed that androgen affected glucocorticoid signaling only in the prefrontal cortex and the substantia nigra. Dihydrotestosterone and corticosterone independently and inversely regulated expression of
and
in prefrontal cortex. AR antagonism with enzalutamide attenuated corticosterone-induced expression of
in the prefrontal cortex and of
and
in the substantia nigra. Additionally, in the substantia nigra, AR antagonism increased expression of
and
, two genes coding for key components of the dopaminergic system. Our data indicate that androgen influence over glucocorticoid stimulation in the brain is not a dominant phenomenon in the context of high corticosterone levels, but can occur in the prefrontal cortex and substantia nigra.
Glucocorticoids mediate numerous essential processes in the human body via binding to the glucocorticoid receptor (GR). Excessive GR signaling can cause disease, and GR antagonists can be used to ...treat many symptoms of glucocorticoid-induced pathology. The purpose of this study was to characterize the tissue-specific properties of the selective GR antagonist CORT125281. We evaluated the antagonistic effects of CORT125281 upon acute and subchronic corticosterone exposure in mice. In the acute corticosterone setting, hypothalamus-pituitary-adrenal-axis activity was investigated by measurement of basal- and stress-induced corticosterone levels, adrenocorticotropic hormone levels and pituitary proopiomelanocortin expression. GR signaling was evaluated by RT-PCR analysis of GR-responsive transcripts in liver, muscle, brown adipose tissue (BAT), white adipose tissue (WAT) and hippocampus. Pretreatment with a high dose of CORT125281 antagonized GR activity in a tissue-dependent manner. We observed complete inhibition of GR-induced target gene expression in the liver, partial blockade in muscle and BAT and no antagonism in WAT and hippocampus. Tissue distribution only partially explained the lack of effective antagonism. CORT125281 treatment did not disinhibit the hypothalamus-pituitary-adrenal neuroendocrine axis. In the subchronic corticosterone setting, CORT125281 partially prevented corticosterone-induced hyperinsulinemia, but not hyperlipidemia and immune suppression. In conclusion, CORT125281 antagonizes GR transcriptional activity in a tissue-dependent manner and improves corticosterone-induced hyperinsulinemia. Tailored dosing of CORT125281 may allow tissue-specific inhibition of GR transcriptional activity.
Glucocorticoids are powerful modulators of brain function. They act via mineralocorticoid and glucocorticoid receptors (MR and GR). These are best understood as transcription factors. Although many ...glucocorticoid effects depend on the modulation of gene transcription, it is a major challenge to link gene expression to function given the large‐scale, apparently pleiotropic genomic responses. The extensive sets of MR and GR target genes are highly specific per cell type, and the brain contains many different (neuronal and non‐neuronal) cell types. Next to the set “trait” of cellular context, the “state” of other active signaling pathways will affect MR and GR transcriptional activity. Here, we discuss receptor specificity and contextual factors that determine the transcriptional outcome of MR/GR signaling, experimental possibilities offered by single‐cell transcriptomics approaches, and reflect on how to make sense of lists of target genes in relation to understanding the functional effects of steroid receptor activation.
Glucocorticoid hormones affect the brain in the context of stress, medical treatment and disease. They have massive effects on gene expression in many different neuronal and non‐neuronal cell types in the brain. We discuss challenges and possible solutions in identifying those target genes that are responsible for particular adaptive, pathogenic of therapeutic effects.
Glucocorticoids regulate numerous processes in human physiology, but deregulated or excessive glucocorticoid receptor (GR) signaling contributes to the development of various pathologies including ...metabolic syndrome. For this reason, GR antagonists have considerable therapeutic value. Yet, the only GR antagonist that is clinically approved to date - mifepristone - exhibits cross-reactivity with other nuclear steroid receptors like the progesterone receptor. In this study, we set out to identify novel selective GR antagonists by combining rational chemical design with an unbiased in vitro and in vivo screening approach. Using this pipeline, we were able to identify CORT125329 as the compound with the best overall profile from our octahydro series of novel GR antagonists, and demonstrated that CORT125329 does not exhibit cross-reactivity with the progesterone receptor. Further in vivo testing showed beneficial activities of CORT125329 in models for excessive corticosterone exposure and short- and long-term high-fat diet-induced metabolic complications. Upon CORT125329 treatment, most metabolic parameters that deteriorated upon high-fat diet feeding were similarly improved in male and female mice, confirming activity in both sexes. However, some sexually dimorphic effects were observed including male-specific antagonism of GR activity in brown adipose tissue and female-specific lipid lowering activities after short-term CORT125329 treatment. Remarkably, CORT125329 exhibits beneficial metabolic effects despite its lack of GR antagonism in white adipose tissue. Rather, we propose that CORT125329 treatment restores metabolic activity in brown adipose tissue by stimulating lipolysis, mitochondrial activity and thermogenic capacity. In summary, we have identified CORT125329 as a selective GR antagonist with strong beneficial activities in metabolic disease models, paving the way for further clinical investigation.
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•Pharmacochemical optimization of GR antagonist without progesterone receptor binding.•CORT125329 exhibits beneficial metabolic activities in male and female mice.•CORT125329 displays tissue-specific GR antagonism in mice.•CORT125329 treatment activates metabolic pathways in brown adipose tissue.
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