Insights into isoform-specific mineralocorticoid receptor action in the hippocampus.

The mineralocorticoid receptor (MR) plays a critical role in the mammalian brain as a mediator of appropriate cellular and behavioural responses under both baseline and stressful conditions. In the hippocampus, the MR has been implicated in several processes, such as neuronal maintenance, adult neurogenesis, inhibitory control of the hypothalamic-pituitary-adrenal axis, and learning and memory. Because of its high affinity for endogenous glucocorticoid hormones, the MR has long been postulated to mediate tonic actions in the brain, but more recent data have expanded on this view, indicating that the MR elicits dynamic responses as well. The complexity of the diverse molecular, cellular, and physiological functions fulfilled by the human, rat and mouse MR could at least partially be explained by the existence of different isoforms of the receptor. The structural and functional characteristics of these isoforms, however, have remained largely unexplored. The present article will review the current knowledge concerning human, rat, and mouse MR isoforms and evaluate seminal studies concerning the roles of the brain MR, with the intent to shed light on the function of its specific isoforms.

Genomic regulation of Krüppel-like-factor family members by corticosteroid receptors in the rat brain.

Hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) mediate glucocorticoid hormone (GC) action in the hippocampus. These receptors bind to glucocorticoid responsive elements (GREs) within target genes, eliciting transcriptional effects in response to stress and circadian variation. Until recently, little was known about the genome-wide targets of hippocampal MRs and GRs under physiological conditions. Following on from our genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus, we investigated the Krüppel-like factors (KLFs) as targets of MRs and GRs throughout the brain under circadian variation and after acute stress. In particular, Klf2, Klf9 and Klf15 are known to be stress and/or GC responsive and play a role in neurobiological processes including synaptic plasticity and neuronal differentiation. We found increased binding of MR and GR to GREs within Klf2, Klf9 and Klf15 in the hippocampus, amygdala, prefrontal cortex, and neocortex after acute stress and resulting from circadian variation, which was accompanied by upregulation of corresponding hnRNA and mRNA levels. Adrenalectomy abolished transcriptional upregulation of specific Klf genes. These results show that MRs and GRs regulate Klf gene expression throughout the brain following exposure to acute stress or in response to circadian variation, likely alongside other transcription factors.

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors.

Glucocorticoid hormones (GCs) - acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) - are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.

Unexpected effects of metyrapone on corticosteroid receptor interaction with the genome and subsequent gene transcription in the hippocampus of male rats.

Glucocorticoid hormones (GCs) play a pivotal role in many stress-related biological processes. In the hippocampus, GCs act through mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) to modify gene transcription. The involvement of GCs in biological processes has been investigated using the corticosterone (CORT)-synthesis blocker metyrapone. How metyrapone affects the action of GC at the genomic level still remains unclear. Therefore, we investigated the effects of this enzyme blocker on plasma CORT levels and hippocampal MR and GR binding to GC responsive elements (GREs) within the GC target genes Fkbp5 (FK506-binding protein 5), Per1 (Period 1) and Sgk1 (Serum- and glucocorticoid-activated kinase 1), as well as the transcriptional responses of these genes under control and acute stress conditions in rats. For comparison, we also investigated these endpoints in rats that had undergone adrenalectomy (ADX). Although metyrapone had no effect on baseline levels of CORT, the drug increased MR and GR to GRE binding within the GC target genes and the transcriptional activity of these genes. As expected, acute forced swim (FS) stress strongly increased plasma CORT levels, hippocampal MR and GR to GRE binding within Fkbp5, Per1 and Sgk1, and the transcriptional activity (mainly hnRNA levels) of these genes. Metyrapone attenuated, but did not abolish, these effects of stress on plasma CORT and MR and GR to GRE binding. The drug effects on FS-induced transcriptional activity were gene-dependent with a reduction seen in Fkbp5 hnRNA (but not Fkbp5 mRNA), an enhancement in Per1 hnRNA (but not Per1 mRNA), and no effect on both Sgk1 hnRNA and mRNA levels. ADX however completely abrogated the effects of FS on plasma CORT, as well as hippocampal MR and GR to GRE binding and transcriptional responses. Thus, in contrast to ADX, metyrapone produced inconsistent effects on GC-sensitive genomic endpoints that question its suitability as a tool in neuroendocrine and other research.

Mineralocorticoid and glucocorticoid receptor-mediated control of genomic responses to stress in the brain.

Successful coping with stressful events involves adaptive and cognitive processes in the brain that make the individual more resilient to similar stressors in the future. Stressful events result in the secretion of glucocorticoids (GCs) from the adrenal glands into the blood stream. Early work proved instrumental for developing the concept that these hormones act in the brain to coordinate physiological and behavioral responses to stress through binding to two different GC-binding receptors. Once activated these receptors translocate to the nucleus where they act on target genes to facilitate (or sometimes inhibit) transcription. There are two types of receptors in the brain, the mineralocorticoid receptor (MR), and glucocorticoid receptor (GR). This review summarizes recent work which provides new insights regarding the genomic action of these receptors, both under baseline conditions and following exposure to acute stress. This work is discussed alongside the extensive studies undertaken in this field previously and new, and exciting "big data" studies which have generated a wealth of relevant data. The consequence of these new insights will challenge existing assumptions about the role of MRs and GRs and pave the way for the implementation of novel and improved methodologies to identify the role these corticosteroid receptors have in stress-related behavioral adaptation.

Rapid Down-Regulation of Glucocorticoid Receptor Gene Expression in the Dentate Gyrus after Acute Stress in vivo: Role of DNA Methylation and MicroRNA Activity.

BACKGROUND: Although glucocorticoid receptors (GRs) in the hippocampus play a vital role in the regulation of physiological and behavioural responses to stress, the regulation of receptor expression remains unclear. This work investigates the molecular mechanisms underpinning stress-induced changes in hippocampal GR mRNA levels in vivo. METHODS: Male Wistar rats were killed either under baseline conditions or after forced swim stress (FSS; 15 min in 25°C water). Rat hippocampi were micro-dissected (for mRNA, microRNA, and DNA methylation analysis) or frozen whole (for chromatin immunoprecipitation). In an additional experiment, rats were pre-treated with RU486 (a GR antagonist) or vehicle. RESULTS: FSS evoked a dentate gyrus-specific reduction in GR mRNA levels. This was related to an increased DNMT3a protein association with a discreet region of the Nr3c1 (GR gene) promoter, shown here to undergo increased DNA methylation after FSS. FSS also caused a time-dependent increase in the expression of miR-124a, a microRNA known to reduce GR mRNA expression, which was inversely correlated with a reduction in GR mRNA levels 30 min after FSS. FSS did not affect GR binding to a putative negative glucocorticoid response element within the Nr3c1 gene. CONCLUSIONS: Acute stress results in decreased GR mRNA expression specifically in the dentate gyrus. Our results indicate that a complex interplay of multiple molecular mechanisms - including increased DNA methylation of discrete CpG residues within the Nr3c1 gene, most likely facilitated by DNMT3a, and increased expression of miR-124a - could be responsible for these changes.

Molecular and Epigenetic Mechanisms Underlying Cognitive and Adaptive Responses to Stress.

Consolidation of contextual memories after a stressful encounter is essential for the survival of an organism and in allowing a more appropriate response to be elicited should the perceived threat reoccur. Recent evidence has explored the complex role that epigenetic mechanisms play in the formation of such memories, and the underlying signaling pathways are becoming more apparent. The glucocorticoid receptor (GR) has been shown to play a key role in these events having both genomic and non-genomic actions in the brain. GR has been shown to interact with the extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) signaling pathway which, in concert, drives epigenetic modifications and chromatin remodeling, resulting in gene induction and memory consolidation. Evidence indicates that stressful events can have an effect on the offspring in utero, and that epigenetic marks altered early in life may persist into adulthood. A new and controversial area of research, however, suggests that epigenetic modifications could be inherited through the germline, a concept known as transgenerational epigenetics. This review explores the role that epigenetic processes play in the central nervous system, specifically in the consolidation of stress-induced memories, the concept of transgenerational epigenetic inheritance, and the potential role of epigenetics in revolutionizing the treatment of stress-related disorders through the emerging field of pharmacoepigenetics and personalized medical treatment.

Acute Stress Enhances Epigenetic Modifications But Does Not Affect the Constitutive Binding of pCREB to Immediate-Early Gene Promoters in the Rat Hippocampus.

The immediate early genes (IEGs) c-Fos and Egr-1 are rapidly and transiently induced in sparse neurons within the hippocampus after exposure to an acute stressor. The induction of these genes is a critical part of the molecular mechanisms underlying successful behavioral adaptation to stress. Our previous work has shown that transcriptional activation of c-Fos and Egr-1 in the hippocampus requires formation of a dual histone mark within their promoter regions, the phosphorylation of serine 10 and acetylation of lysine 9/14 of histone H3. In the present study, using chromatin immuno-precipitation (ChIP), we found that an increase in the formation of H3K9ac-S10p occurs within the c-Fos and Egr-1 promoters after FS stress in vivo and that these histone modifications were located to promoter regions containing cAMP Responsive Elements (CREs), but not in neighboring regions containing only Serum Responsive Elements (SREs). Surprisingly, however, subsequent ChIP analyses showed no changes in the binding of pCREB or CREB-binding protein (CBP) to the CREs after FS. In fact, pCREB binding to the c-Fos and Egr-1 promoters was already highly enriched under baseline conditions and did not increase further after stress. We suggest that constitutive pCREB binding may keep c-Fos and Egr-1 in a poised state for activation. Possibly, the formation of H3K9ac-S10p in the vicinity of CRE sites may participate in unblocking transcriptional elongation through recruitment of additional epigenetic factors.

Acute stress enhances heterodimerization and binding of corticosteroid receptors at glucocorticoid target genes in the hippocampus.

A stressful event results in secretion of glucocorticoid hormones, which bind to mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the hippocampus to regulate cognitive and affective responses to the challenge. MRs are already highly occupied by low glucocorticoid levels under baseline conditions, whereas GRs only become substantially occupied by stress- or circadian-driven glucocorticoid levels. Currently, however, the binding of MRs and GRs to glucocorticoid-responsive elements (GREs) within hippocampal glucocorticoid target genes under such physiological conditions in vivo is unknown. We found that forced swim (FS) stress evoked increased hippocampal RNA expression levels of the glucocorticoid-responsive genes FK506-binding protein 5 (Fkbp5), Period 1 (Per1), and serum- and glucocorticoid-inducible kinase 1 (Sgk1). Chromatin immunoprecipitation (ChIP) analysis showed that this stressor caused substantial gene-dependent increases in GR binding and surprisingly, also MR binding to GREs within these genes. Different acute challenges, including novelty, restraint, and FS stress, produced distinct glucocorticoid responses but resulted in largely similar MR and GR binding to GREs. Sequential and tandem ChIP analyses showed that, after FS stress, MRs and GRs bind concomitantly to the same GRE sites within Fkbp5 and Per1 but not Sgk1 Thus, after stress, MRs and GRs seem to bind to GREs as homo- and/or heterodimers in a gene-dependent manner. MR binding to GREs at baseline seems to be restricted, whereas after stress, GR binding may facilitate cobinding of MR. This study reveals that the interaction of MRs and GRs with GREs within the genome constitutes an additional level of complexity in hippocampal glucocorticoid action beyond expectancies based on ligand-receptor interactions.

Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus.

Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5'-cytosine-phosphate-guanine-3') sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental stimuli in terms of gene expression and behavior.

Distinct epigenetic and gene expression changes in rat hippocampal neurons after Morris water maze training.

Gene transcription and translation in the hippocampus is of critical importance in hippocampus-dependent memory formation, including during Morris water maze (MWM) learning. Previous work using gene deletion models has shown that the immediate-early genes (IEGs) c-Fos, Egr-1, and Arc are crucial for such learning. Recently, we reported that induction of IEGs in sparse dentate gyrus neurons requires ERK MAPK signaling and downstream formation of a distinct epigenetic histone mark (i.e., phospho-acetylated histone H3). Until now, this signaling, epigenetic and gene transcriptional pathway has not been comprehensively studied in the MWM model. Therefore, we conducted a detailed study of the phosphorylation of ERK1/2 and serine10 in histone H3 (H3S10p) and induction of IEGs in the hippocampus of MWM trained rats and matched controls. MWM training evoked consecutive waves of ERK1/2 phosphorylation and H3S10 phosphorylation, as well as c-Fos, Egr-1, and Arc induction in sparse hippocampal neurons. The observed effects were most pronounced in the dentate gyrus. A positive correlation was found between the average latency to find the platform and the number of H3S10p-positive dentate gyrus neurons. Furthermore, chromatin immuno-precipitation (ChIP) revealed a significantly increased association of phospho-acetylated histone H3 (H3K9ac-S10p) with the gene promoters of c-Fos and Egr-1, but not Arc, after MWM exposure compared with controls. Surprisingly, however, we found very little difference between IEG responses (regarding both protein and mRNA) in MWM-trained rats compared with matched swim controls. We conclude that exposure to the water maze evokes ERK MAPK activation, distinct epigenetic changes and IEG induction predominantly in sparse dentate gyrus neurons. It appears, however, that a specific role for IEGs in the learning aspect of MWM training may become apparent in downstream AP-1- and Egr-1-regulated (second wave) genes and Arc-dependent effector mechanisms.

Glucocorticoids, epigenetic control and stress resilience.

Glucocorticoid hormones play a pivotal role in the response to stressful challenges. The surge in glucocorticoid hormone secretion after stress needs to be tightly controlled with characteristics like peak height, curvature and duration depending on the nature and severity of the challenge. This is important as chronic hyper- or hypo-responses are detrimental to health due to increasing the risk for developing a stress-related mental disorder. Proper glucocorticoid responses to stress are critical for adaptation. Therefore, the tight control of baseline and stress-evoked glucocorticoid secretion are important constituents of an organism's resilience. Here, we address a number of mechanisms that illustrate the multitude and complexity of measures safeguarding the control of glucocorticoid function. These mechanisms include the control of mineralocorticoid (MR) and glucocorticoid receptor (GR) occupancy and concentration, the dynamic control of free glucocorticoid hormone availability by corticosteroid-binding globulin (CBG), and the control exerted by glucocorticoids at the signaling, epigenetic and genomic level on gene transcriptional responses to stress. We review the beneficial effects of regular exercise on HPA axis and sleep physiology, and cognitive and anxiety-related behavior. Furthermore, we describe that, possibly through changes in the GABAergic system, exercise reduces the impact of stress on a signaling pathway specifically in the dentate gyrus that is strongly implicated in the behavioral response to that stressor. These observations underline the impact of life style on stress resilience. Finally, we address how single nucleotide polymorphisms (SNPs) affecting glucocorticoid action can compromise stress resilience, which becomes most apparent under conditions of childhood abuse.

Stress, epigenetic control of gene expression and memory formation.

Making memories of a stressful life event is essential for an organism's survival as it allows it to adapt and respond in a more appropriate manner should the situation occur again. However, it may be envisaged that extremely stressful events can lead to formation of traumatic memories that are detrimental to the organism and lead to psychiatric disorders such as post-traumatic stress disorder (PTSD). The neurotransmitter glutamate and the ERK MAPK signaling pathway play a principal role in learning and memory. Glucocorticoid hormones acting via the glucocorticoid receptor have been shown to strengthen the consolidation of memories of stressful events. The ERK MAPK signaling pathway and glucocorticoid receptor-mediated actions have recently been shown to drive epigenetic modifications and conformational changes in the chromatin, stimulating the expression of neuroplasticity-related genes involved in stress-related learning and memory processes. The main epigenetic regulatory mechanisms are histone modifications and DNA (de-)methylation. Recently, studies have demonstrated that these processes are acting together in concert to regulate gene expression required for memory consolidation. This review explores the role of stress in learning and memory paradigms and the participating signaling pathways and epigenetic mechanisms and the enzymes that control these modifications during the consolidation process of memory formation.

Epigenetic mechanisms in stress and adaptation.

Epigenetic mechanisms are processes at the level of the chromatin that control the expression of genes but their role in neuro-immuno-endocrine communication is poorly understood. This review focuses on epigenetic modifications induced by a range of stressors, both physical and psychological, and examines how these variations can affect the biological activity of cells. It is clear that epigenetic modifications are critical in explaining how environmental factors, which have no effect on the DNA sequence, can have such profound, long-lasting influences on both physiology and behavior. A signaling pathway involving activation of MEK-ERK1/2, MSK1, and Elk-1 signaling molecules has been identified in the hippocampus which results in the phospho-acetylation of histone H3 and modification of gene expression including up-regulation of immediate early genes such as c-Fos. This pathway can be induced by a range of challenging experiences including forced swimming, Morris water maze learning, fear conditioning and exposure to the radial maze. Glucocorticoid (GC) hormones, released as part of the stress response and acting via glucocorticoid receptors (GRs), enhance signaling through the ERK1/2/MSK1-Elk-1 pathway and thereby increase the impact on epigenetic and gene expression mechanisms. The role of synergetic interactions between these pathways in adaptive responses to stress and learning and memory paradigms is discussed, in addition we speculate on their potential role in immune function.