Hippocampal glucocorticoid target genes associated with enhancement of memory consolidation.

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.

Corticosteroid Action in the Brain: The Potential of Selective Receptor Modulation.

Glucocorticoid hormones have important effects on brain function in the context of acute and chronic stress. Many of these are mediated by the glucocorticoid receptor (GR). GR has transcriptional activity which is highly context-specific and differs between tissues and even between cell types. The outcome of GR-mediated transcription depends on the interactome of associated coregulators. Selective GR modulators (SGRMs) are a class of GR ligands that can be used to activate only a subset of GR-coregulator interactions, thereby giving the possibility to induce a unique combination of agonistic and antagonistic GR properties. We describe SGRM action in animal models of brain function and pathology, and argue for their utility as molecular filters, to characterize context-specific GR interactome and transcriptional activity that are responsible for particular glucocorticoid-driven effects in cognitive processes such as memory consolidation. The ultimate objective of this approach is to identify molecular processes that are responsible for adaptive and maladaptive effects of glucocorticoids in the brain.

Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions.

Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones.

Mechanistic Insights in NeuroD Potentiation of Mineralocorticoid Receptor Signaling.

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.

Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain.

BACKGROUND: Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon. METHODS: For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants. RESULTS: We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression. CONCLUSIONS: We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function.

Transcriptional glucocorticoid effects in the brain: Finding the relevant target genes.

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 and Mineralocorticoid Receptors in the Brain: A Transcriptional Perspective.

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.

Sex-Dependent Modulation of Acute Stress Reactivity After Early Life Stress in Mice: Relevance of Mineralocorticoid Receptor Expression.

Early life stress (ELS) is considered a major risk factor for developing psychopathology. Increasing evidence points towards sex-dependent dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis as a contributing mechanism. Additionally, clinical studies suggest that the mineralocorticoid receptor (MR) may further confer genetic vulnerability/resilience on a background of ELS. The link between ELS, sex and the HPA axis and how this interacts with MR genotype is understudied, yet important to understand vulnerability/resilience to stress. We used the early life-limited nesting and bedding model to test the effect of ELS on HPA properties in adult female and male mice carrying a forebrain-specific heterozygous knockout for MR. Basal HPA axis activity was measured by circadian peak and nadir corticosterone levels, in addition to body weight and weight of stress-sensitive tissues. HPA axis reactivity was assessed by mapping corticosterone levels after 10 min immobilization. Additionally, we measured the effects of ELS on steroid receptor [MR and glucocorticoid receptor (GR)] levels in the dorsal hippocampus and medial prefrontal cortex (mPFC) with western blot. Finally, behavioral reactivity towards a novel environment was measured as a proxy for anxiety-like behavior. Results show that HPA axis activity under rest conditions was not affected by ELS. HPA axis reactivity after immobilization was decreased by ELS in females and increased, at trend-level in males. This effect in females was further exacerbated by low expression of the MR. We also observed a sex*ELS interaction regarding MR and GR expression in the dorsal hippocampus, with a significant upregulation of MR in males only. The sex-dependent interaction with ELS was not reflected in the behavioral response to novel environment and time spent in a sheltered compartment. We did find increased locomotor activity in all groups after a history of ELS, which attenuated after 4 h in males but not females regardless of condition. Our findings support that ELS alters HPA axis functioning sex-dependently. Genetic predisposition to low MR function may render females more susceptible to the harmful effect of ELS whereas in males low MR function promotes resilience. We propose that this model may be a useful tool to investigate the underlying mechanisms of sex-dependent and genetic vulnerability/resilience to stress-related psychopathology.

Identification of mineralocorticoid receptor target genes in the mouse hippocampus.

Brain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) respond to the same glucocorticoid hormones but can have differential effects on cellular function. Several lines of evidence suggest that MR-specific target genes must exist and might underlie the distinct effects of the receptors. The present study aimed to identify MR-specific target genes in the hippocampus, a brain region where MR and GR are co-localised and play a role in the stress response. Using genome-wide binding of both receptor types, we previously identified MR-specific, MR-GR overlapping and GR-specific putative target genes. We now report altered gene expression levels of such genes in the hippocampus of forebrain MR knockout (fbMRKO) mice, killed at the time of their endogenous corticosterone peak. Of those genes associated with MR-specific binding, the most robust effect was a 50% reduction in Jun dimerization protein 2 (Jdp2) mRNA levels in fbMRKO mice. Down-regulation was also observed for the MR-specific Nitric oxide synthase 1 adaptor protein (Nos1ap) and Suv3 like RNA helicase (Supv3 l1). Interestingly, the classical glucocorticoid target gene FK506 binding protein 5 (Fkbp5), which is associated with MR and GR chromatin binding, was expressed at substantially lower levels in fbMRKO mice. Subsequently, hippocampal Jdp2 was confirmed to be up-regulated in a restraint stress model, posing Jdp2 as a bona fide MR target that is also responsive in an acute stress condition. Thus, we show that MR-selective DNA binding can reveal functional regulation of genes and further identify distinct MR-specific effector pathways.

NeuroD Factors Discriminate Mineralocorticoid From Glucocorticoid Receptor DNA Binding in the Male Rat Brain.

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.

A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain.

Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a "selective modulator" type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.