Article in press - uncorrected proof Horm Mol Biol Clin Invest 2011;7(2):317–326
2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/HMBCI.2011.114

Corticosteroid receptor signalling modes and stress adaptation in the brain

Onno C. Meijer* Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research and Leiden University Medical Center, Leiden, The Netherlands

Abstract Adrenal glucocorticoid hormones modulate neuronal activity to support an adaptive response to stress. They modulate brain circuitry mediating physiological responses, emotion and cognitive processing. Chronically elevated glucocorticoid exposure is however linked to the development of mental disease. Glucocorticoid effects depend on mineralo- and glucocorticoid receptors, which are powerful transcription factors, but also can act via a diversity of non-genomic mechanisms. Here, I review generic factors that determine neuronal glucocorticoid sensitivity, in relation to brain function. First, pre-receptor mechanisms determine ligand availability. Second, there may be considerable variation in the receptor splice- and translation variants. Third, other transcription factors and many transcriptional coregulators interact with steroid receptors, determining nature and magnitude of steroid responses, in part through epigenetic regulation of DNA accessibility. Which factors underlie adaptive and pathogenic effects of stress hormones is largely unknown. Genome-wide identification of the receptor-DNA interactions in specific behavioural and physiological contexts provides a way of assessing the complete genomic range of glucocorticoid modes of action. Novel ligands that induce selective activation of particular receptor signalling modes will aid our understanding of receptor signalling and may allow selective targeting of glucocorticoid effects in emotional or cognitive domains, in research and, hopefully, in clinical settings. Keywords: brain; coregulators; glucocorticoids; selective receptor modulators; stress; transcription.

Abbreviations CORT, adrenal glucocorticoid hormones (corticosterone and cortisol); MR, mineralocorticoid receptor; GR, glucorticoid receptor; CRH, corticotropin releasing hormone; CBG, corticosteroid binding globulin; SRC-1, steroid receptor coactivator-1. *Corresponding author: Onno C. Meijer, Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research and Leiden University Medical Center, PO Box 9503, 2300 RA Leiden, The Netherlands Phone: q31 71 5276070, Fax: q31 71 5274715, E-mail: [email protected] Received June 6, 2011; accepted July 11, 2011

Introduction Brain receptors for adrenal glucocorticoid hormones are crucial for homeostatic balance, in particular in relation to increased glucocorticoid secretion that occurs in response to stress. However, changed glucocorticoid exposure associated with chronic stress can be deleterious and is linked to the onset of psychiatric disease w1x. Potential stressors and the brain regions that support adaptation to these stressors are many. Demands may be made on processes that range from basic physiological regulation (e.g., energy balance, hydration) to processing of memory, fear or reward. As a consequence, the types of cellular adaptations that are brought about by glucocorticoids are highly diverse in different brain regions, and in different stress-contexts. A relatively certain assumption in the midst of the cellular complexity of the brain, is that only two receptor types are responsible for the effects of endogenous glucocorticoids (or: CORT, exclusively corticosterone in rats and mice, and mainly cortisol in humans): mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) w2x. They are members of the nuclear receptor (NR) superfamily, and accordingly can act like transcription factors. MR and GR differ in their affinity for CORT, so that under basal conditions there is a considerable tonic MR-activated gene network, while GR target genes are regulated as a consequence of the circadian CORT peak and – more pronounced – after stress. Besides these classical genomic effects, it is now also well established that both MR and GR can mediate rapid nongenomic effects, likely through membrane-associated forms of the receptor (mMR and mGR), which are however transcribed from the same genes as nuclear MR and GR. These effects are important for modulation of neuronal excitability in the early phases after onset of stressors w3x. For membrane-limited effects the potency of CORT at the MR is 10-fold lower, and in this context, MR should be considered a ‘stress-receptor’, much like GR (while the pharmacological specificity of mMR seems to be conserved in terms of specific antagonists). A certainty that is derived of having only two receptor types, is that of knowing which cells are CORT responsive. The GR is expressed in almost every cell population of the brain (with the notable exception of the suprachiasmatic nucleus, where the biological clock is located w4, 5x), while the MR is more restricted to limbic brain regions and brain stem. Classically mechanistic studies on brain MR and GR have focussed on the hippocampus (important for memory formation), hypothalamus (crucial for endocrine regulation and feedback) and, in recent years, amygdala (in relation to regulation of fear responses and anxiety) and prefrontal

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cortical areas (working memory and planning). Yet, many aspects of neuronal MR and GR signalling remain unknown. In contrast, there is a great deal of ever expanding ‘generic’ knowledge on nuclear receptor action in general, much of which is based on the glucocorticoid receptor in different (cellular) model systems. A substantial number of variables that may determine GR/MR responsiveness has thus been identified, including receptor variants, down-stream effector molecules, and epigenetic DNA modifications. However, because of the multitude of cellular contexts present in the brain, and because of the inherent practical limitations of in vivo brain research, it has remained tantalisingly unclear which GR and MR-related mechanisms are at play within the brain, during the diversity of behavioural contexts in which glucocorticoids act. Here, I review a number of mainly transcriptional MR/GR mechanisms, some with proven relevance for the brain, and others that are understudied in a brain context, but may well be of relevance for the efficacy of neuronal glucocorticoid signalling. I conclude that experimental progress will be facilitated by currently available genome wide identification of genomic GR interactins, more efforts to identify new dissociating ligands that activate particular effector pathways of the receptors, and other techniques that allow a more rapid manipulation of NR signalling than in current research practice.

enzyme is an important contributor to overall CORT levels by regeneration of active hormone from the 11-beta dehydro metabolites w10x. In certain brain regions (and in white adipose tissue) type 1 HSD acts as a local CORT sink, increasing available active hormone. In accordance, it has been shown recently that inactivation of 11-beta HSD-1 can protect against age (and hypercortisolism) related memory deficits w11x.

Receptor diversity Already before cloning of GR and MR, their importance as a binary receptor system for CORT in brain was recognised w2x. The notion of high and low affinity nuclear receptors, MR working permissively to set the initial response to a stressor, and GR as a reactive sensor, mediating negative feedback and preparing for subsequent stressors, is still the dominant model in the literature w1x. In recent years this model has become refined by discovery of clear region specific effects in the brain, and the involvement of the membrane-associated receptors. There is as yet no reason to assume that these basic notions need to be fundamentally adjusted. However, the possible presence of multiple MR and GR subtypes adds complexity to the picture. Splice variants

Pre-receptor mechanisms Many factors determine whether and how a particular blood level of CORT will affect GR or MR in a given tissue. At least three pre-receptor mechanisms may cause differential effects, when comparing different individuals, tissues or cells. First, the amount of free plasma CORT that is available for uptake in the target cells can vary as a function of levels of circulating corticosteroid binding globulin (CBG). Also CBG affinity for CORT is not constant, but for example temperature sensitive w6x. Second, many glucocorticoids are substrate for the P-glycoprotein that is expressed at endothelial barriers, such as the blood brain barrier. As an interesting consequence, not only the synthetic steroids dexamethasone w7x and prednisolone, but also cortisol is to some extent hampered in its access to the brain. In contrast, corticosterone seems to enter the brain freely, and therefore in the human brain may actually be present at concentrations that approach that of cortisol. In addition, one interpretation of data on cortisol/corticosterone ratios in human brain, is that overall CORT levels in the brain may be up to six-fold lower than in tissues that are not protected by the P-glycoprotein w8x. A third, powerful prereceptor mechanism is local interconversion of CORT and its low affinity 11-dehydro metabolites, cortisone and 11-dehydrocorticosterone. Inactivation of CORT occurs via the enzyme 11-beta hydroxysteroid dehydrogenase type II (11-beta HSD-2), for example, in kidney, conferring aldosterone selectivity for MRs involved in sodium retention w9x. In the liver, the 11-beta HSD type 1

Functional receptor diversity from a single receptor gene basically stems from three levels: splice variants, translation variants, and post-translational modifications. For GR, a Cterminal splice variant exists, GR-beta, which cannot bind CORT, and may act as a dominant negative receptor for the ‘normal’ GR-alpha. While GR-beta was long thought to be exclusive to humans w12x, recent findings identified a similar C-terminal truncation that is generated through an alternative splice event in zebrafish w13x and mouse w14x. Beta forms of GR may not be directly in control of neuronal function, given the paucity of expression w15x, but they can induce GR resistance in other tissues w16x, and may be of relevance in e.g., immune-brain interactions. In fact, in a recent study, a polymorphism in the human GR beta gene was found to be associated with seasonal variations in bipolar disorder w17x. GR-P is another GR splice variant that is C-terminally truncated. In brain it is present at somewhat higher levels than GR-beta, but unless there are local ‘hotspots’ of expression, it seems unlikely that it strongly interferes with neuronal GRalpha w18x. There is considerable variation in the first exon of the GR gene that is spliced into the mRNA w19x. This exon is noncoding, and differential splicing in this case may reflect the differential use of GR promoters. These variants have attracted a lot attention in relation to early life programming of GR sensitivity of particular brain areas w20x. For MR, there are two variant mRNAs with alternative first exons, of which the consequences for protein function are unknown, although relevance of both for the brain has been suggested w21x. Other splice variants have been reported, one variant lacking LBD coding exons w22x, another con-

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taining four additional amino acids in the DBD w23, 24x. Since their identification there has been little follow-up on these findings. It is also unknown to what extent the membrane located steroid receptors depend on alternatively spliced mRNAs (see below). Translation variants

Interestingly, substantial MR and GR diversity may be caused by translation variants, which are derived from one mRNA species through the use of alternative ATG start codons that are embedded in a functional Kozac sequence w25x. Existence of such up to five of such N-terminal variants was proven elegantly for GR containing cell lines w26x, and later also for MR different translation forms were shown w22x. These findings imply that in some cell types there is actually a mixture of different GRs (or MRs) with distinct transcriptional properties. The presence of alternative translation variants is for now only discernable by Western blot analysis, with obvious limitations in terms of spatial resolution. However, work on human brain development has indicated dynamic expression of GR translation variants over development w27x. Immunohistochemical studies with antibodies that are specific for different N-termini would resolve the detailed distribution of translation variants of MR and GR in the brain, but such studies have not been done. Yet, different translation variants may bear relevance for neurobiology, as genetic MR and GR polymorphisms that affect the ration between A- and B-forms of both receptor types have been linked to psychopathology w28, 29x. In conclusion, MR and GR are as yet the two sole receptor types for CORT in any tissue, but the actual distribution of receptor subtypes in brain is not clear. On top of this diversity come post-translational modifications of the receptors, which in part mediate cross-talk with other cellular signalling pathways w25x. Therefore, expression of the receptors is obviously a precondition for sensitivity to hormone, and regulation of expression a prime physiological regulatory mechanism (e.g., w30x), but expression is not sufficient to predict actual steroid responsiveness. Non-genomic effects

Over the last years, it has become increasingly clear that these actions are in fact not restricted to the nucleus, but that in many brain areas non-genomic, membrane associated effects occur at a time scale of minutes after steroid exposure. These can have immediate consequences for the activity of neuronal circuits, as has been elegantly shown in hippocampus and the amygdala w3, 31x. There is good genetic and pharmacological evidence that both mMR and mGR can mediate such rapid effects. Synapses can be modulated by both pre- and postsynaptic mechanisms, and endocannabinoids have been implicated in some of the effects. Importantly, while the relative selectivity of mMR and mGR antagonists is similar to classical nuclear receptors, the potency of corticosterone at these receptors is lower, rendering non-genomic mMR effects relevant for stress-associated levels of CORT. Very recently, a separate non-genomic mode

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of action was proposed in relation to stress-dependent memory consolidation, in which ligand-activated GR acts as a scaffold for nuclear kinases that are subsequently involved in remodelling of chromatin w32x. While the MR/GR genes are necessary for many nongenomic effects, there are many open questions. In relation to membrane signalling these include elucidation of the exact form in which the receptors reside in the membrane, including specific post-translational modification that would be needed to separate them from nuclear receptors. As a consequence, processes that would regulate this type of signalling and modulate mGR/ sensitivity remain to be identified w33x.

Brain target genes: cell specificity In any target cell, there are hundreds of ‘primary’ target genes for a given nuclear receptor, which all depend on binding to the DNA, either directly via hormone response elements (GRE in case of GR/MR) or indirectly via interactions with non-receptor transcription factors w34, 35x. In terms of generic steroid hormone mechanisms, the total number of genes that show some kind of transcriptional response to a single hormone stimulus may in some cells actually be in the order of )20% of the genome, as was recently suggested by a genome wide ‘run-on’ assay for the estrogen receptor w36x. On the other hand, the typical overlap between cell lines in actual binding sites, and gene regulation with detectable consequences at the mRNA levels is likely as low as 5% w37x. Certainly in the brain, very cell-specific responses to MR and GR activation are at hand, as a consequence of the huge variety in demands on different brain structures in the context of adaptation to stressors. The cellular basis of CORT effects must differ for negative feedback regulation in the hypothalamus w38x, modulation of emotional reactivity w39x and reward sensitivity w40x in the limbic brain, or changes in cognitive processing w41, 42x. In fact, cell populations in the hippocampus that are both relevant for the effects of CORT on learning and memory have very divergent responses w43x. As a consequence of all this cellular diversity, the current situation is that much of the substantial (and ever expanding) ‘generic’ knowledge on MR and GR mechanisms of action, still needs to be translated to the brain. The best exception is perhaps regulation of hypothalamic peptide hormones, which form the base for direct output of the brain, i.e., CRH and vasopressin w38, 44x. However, even if expression of pituitary secretagogue mRNA is relatively well understood, this does obviously not equal actual control over HPA activity. In general, the link between the coordinated effects of CORT on gene regulation (programs) and its functional neurobiological outcomes remains to be determined. A complication is that the magnitude of acute transcriptional responses to MR/GR activation in brain tends to be modest (notwithstanding its functional significance). This may be due to experimental ‘dilution’ effects as a consequence of cellular heterogeneity, which may be solved by

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transcriptome analysis after laser microdissection w45x, but it also seems to be an intrinsic characteristic of differentiated neurons. An aspect of target gene identification that receives little attention is variation in potency of agonist-activated GR on different genes. The EC50 of different genes in a single cell type may differ by more than an order of magnitude w46x. This makes sense, as GR-sensitive components of the circadian clock will have to respond to the modest variations in circadian rhythms, whereas high hormone levels or sustained stressors will call upon different sets of target genes, that may actually have a lower sensitivity for GR w47x. In fact, the point has been made that the two classical pharmacological characteristics of dose-response relationships, EC50 and maximal gene induction can be regulated separately w48x, which would have consequences for the extent to which genes are actually stress-responsive.

Postreceptor diversity in nuclear signalling: protein-protein and protein-DNA interactions Once activated by hormone, and present in the nucleus, GR and MR can engage in many interactions that lead to changes in gene expression. There is a fundamental dichotomy between effects that are dependent on direct binding of the receptors to response elements in the DNA, and those that depend on protein-protein interactions with other (non-receptor) transcription factors. These two type of effects depend on different combinations of receptor domains, and can, for example, be mechanistically separated to a large extent (but not completely) by mutations in the receptor dimerisation domain w49, 50x. Work with GRdim/dim mice, which carry such a GR that is impaired in direct GRE binding, has shown that the transcriptional response is essential for a number of prominent cellular and behavioural responses to CORT w51, 52x. These DNA binding dependent effects are generally thought of as stimulatory of gene expression, but recently a ‘negative (n)GRE’ element has been characterised that is used in a widespread manner in the genome w53x. It is unknown what the behaviour of dimer mutants on these repressive elements is, but given the different orientation of the GR monomers on this element, it may well be unaffected by the mutation in the dimer interface. MR activity was as yet not tested on these motifs, which bear little resemblance to ‘spurious’ nGREs that had been identified before on CRH and POMC genes w54, 55x. Better-known transrepression mechanisms are those involving binding of GR to AP-1, NF-kB and others w56x. Even if this transrepression mode is well established in the immune system, and even if both AP-1 and NF-kB are known to be of crucial importance for neuronal function, there are tantalisingly few data on functional cross talk of this kind in the brain (with interesting exceptions w57x). Irrespective of what cis-acting elements are involved in transcriptional MR/GR effects, a first prerequisite is that these DNA elements have to be accessible for binding. A major determinant of initial responsiveness is of course

whether or non-receptor binding sites are available as a consequence of chromatin structure w37x, and epigenetic changes of the DNA itself. In this respect there is not only (defining) cell type specificity, but also considerable variation at many places in the genome that is a consequence of individual history (e.g., w20, 58x).

Coregulator diversity A key to discover the way in which GR and MR affect different aspects of brain function may lie in a class of proteins known as ‘coregulators’. These proteins constitute the actual signal transduction machinery of nuclear receptors, once bound to the DNA. They bind to ligand-activated receptors on the DNA to recruit histone modifying proteins, as well as the complexes that form a bridge from the receptor to the transcription initiation complex w59, 60x. There are in the order of 165 of such proteins that determine nature (coactivation or corepression) and magnitude of the response after nuclear receptor activation. Coregulators in general interact with many of the 48 types of nuclear receptors, but there is a degree of specificity, so that many but not all known coregulators will be directly relevant for MR/GR actions. Importantly, coregulators can have pronounced cell-specific expression w61x, as well as differential interactions with particular nuclear receptors w62x. At the same time, there is clear gene specificity for coregulator modulation of a particular receptor. This gene specificity may be caused by interdependence of the nuclear receptor with other transcription factors that are regulating the gene, as the coregulators have been put forward as integrators of the diverse transcription factors that simultaneously affect a particular gene w63x. Interestingly, the gene specificity may also be intrinsic to particular GREs, because of allosteric modulation of the receptor by the DNA element it binds to, inducing a particular conformation of the receptor, and hence a specificity of receptor-interacting proteins w64x. Finally, because the exact conformation of nuclear receptors is important for subsequent coregulator recruitment, ligands are major determinants of the subset of coregulators that is involved in gene regulation. GR and MR have highly homologous DNA binding domains, and in vitro no distinction can be made between DNA binding of the two. In some systems, GR can actually take on the role of MR, through regulation of MR target genes, for example, cultured kidney cells that have lost MR expression, but that are responsive to glucocorticoids w65x. However, in hippocampal CA1 cells, MR and GR have opposed intrinsic effects on neuronal excitability, calling for diverse (sets of) target genes, which have indeed been found in whole hippocampus w66x. The differences in MR and GR transcriptional activity may be due differences in terms of MR and GR in acting as repressor of other transcription factors (as determined in heterologous systems) w67x. On the other hand there is also evidence for MR specific coregulators w68x – after all, there is only very limited homology between MR and GR in N-terminal part of the molecule.

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Indeed, the tuning of coregulator recruitment by ligands is apparent from reports on coactivators that are specific for aldosterone, rather than cortisol-occupied MR w69x. Coregulators in brain

Coregulators are of major importance for brain function in general, given the many nuclear receptors effects on the brain. A number of coregulator kockout mice are embryonically lethal, and others are associated with severe neurodegenerative phenotype upon ageing, as a consequence of involvement in cell metabolism and oxygen radical scavenging w70x. Other coregulators have proven importance in mediating the effects of steroid hormones on the brain in relation to adaptation and behaviour. Most of this work has been done in the realm of the sex steroids, and there is convincing evidence showing that both programming and activational effects of sex steroids depend on particular coactivator pathways w71x. MR/GR coregulators

MR and GR are no exception in terms of generic signalling pathways, and like other nuclear receptors depend on coregulator pathways. A case in point is regulation of the CRH gene, a prominent transcriptional GR target in the brain. Its downregulation by chronically elevated CORT is considered an essential part of negative feedback regulation of the HPA axis. Yet, in the central amygdala and other extra-hypothalamic sites, GR transactivates the CRH gene w72x. Opposite effects of a single transcription factor at one and the same promoter obviously call for additional factors, which determine the outcome of GR (or MR) signalling on a particular gene. We addressed this question, initially focussing on the members of the first family of coregulators that was discovered, the p160 Steroid Receptor Coactivators w73x, and observed a striking cell specific expression of two SRC-1 splice variants, termed SRC-1a and SRC-1e, the former being very abundant in hypothalamic nuclei and anterior pituitary, the latter more evenly distributed over the brain w74x. These variants had earlier been shown to differentially affect ER-mediated signalling w75x. In particular, SRC-1a was shown to harbour a repression domain, which could impede classical coactivation on positively regulated ER target genes, and which rendered it incapable of strong coactivation at some GR/MR driven reporters w61x. Based on this repression domain, and on its localisation in the core of the HPA axis, where negative feedback action of CORT occurs, we tested modulation of repression of the CRH gene by overexpressed SRC-1a and 1e. Indeed, SRC-1a is capable of increasing the potency of GR in suppressing CRH-reporter gene activity, whereas SRC-1e is not w76x. Thus, SRC-1a seems to play a specific role in negative feedback actions of CORT. Studies in knockout mice partially supported a SRC-1a – negative feedback hypothesis: total absence of SRC lead to strong GR resistance that is specific for anterior pituitary POMC mRNA suppression w77x and CRH mRNA repression

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in the paraventricular nucleus of the hypothalamus (PVN) w78x. However, in these constitutive knockout mice, there is not a very robust phenotype on actual activation of the HPAaxis in response to acute or chronic stress, perhaps due to compensatory changes during development w79x. Interestingly, also the upregulation of CRH mRNA in the central nucleus of the amygdala was compromised in total absence of SRC-1. Other CNS GR target genes, such as GILZ w80x were unaffected by the absence of SRC-1. Thus, SRC-1 splice variants may act as corepressors as well as coactivators of GR on the CRH promoter w78x.

Selective receptor modulators There are at least two separate reasons why the post-receptor MR/GR mechanisms are of interest. First, in view of the very substantial cell- and context specific effects that must occur in different cells and brain regions, it may be expected that particular signalling modes underlie coordinated gene programs. Such coordinated programs are clearly apparent in instances of cell fate determination, for example, in relation brown fat development w81x. Likewise, the SRC-1 dependence of both POMC and CRH downregulation via GR w77, 78x seems to allow coordinated regulation of the HPA axis by CORT. Thus, particular signalling pathways likely shed light on the cellular processes that underlie adaptive changes in response to stress. Second, there is the important possibility that differential receptor signalling modes can be selectively activated by ligands. These so-called ‘dissociating ligands’, or ‘selective receptor modulators’ (in case of GR ligands: SEGRMs) induce a conformation of the receptor that allows only part of the molecular interactions that are available after binding of a full agonist. Much research has been devoted to GR ligands capable of separating the clinically highly relevant anti-inflammatory effects from side effects, such as metabolic changes and osteoporosis w56x. It was originally assumed that this separation could be made based on separating DNA-binding dependent GR effects from transrepression of pro-inflammatory factors like NF-kB, and some ligands in fact seem to be able to selectively activate such transrepression pathways w82x. However, a number of dissociating ligands also induce selective interactions with coactivators w83x. In this respect, array-based techniques for the detection of receptor-coregulator interactions will aid the characterisation of new ligands with dissociating characteristics w84x. Thus, effects of steroids that are linked to specific receptor signalling modes may be targeted in a specific manner by future dissociating ligands, either for research purposes, or in fact for clinical applications w85x. In Figure 1, this notion is illustrated, based on the involvement of the two SRC-1 splice variants that seem to mediate feedback regulation on the CRH gene in the core of the HPA-axis (which would be wanted during chronic stress), and feedforward stimulatory effects on anxiogenic CRH gene expression in the amygdala (which would be unwanted in most chronic stress situations).

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Figure 1 The principle of selective activation of corticosteroid receptor signalling modes. Whereas full agonists allow interaction of GR with many different transcription factors and coregulators. SEGRMs or dissociating ligands induce a different conformation that is able to only activate particular GR effector pathways, and may antagonise the others. This notion is well developed in the field of anti-inflammatory drugs (separating immune suppression from metabolic side effects), but also applicable to different brain circuits and processes.

Dynamic regulation On top of the molecular diversity of receptors and receptorinteracting proteins, it becomes more and more clear that the actual pattern of CORT exposure is an important modifier of signalling efficacy. Stress-induced CORT secretion is superimposed on the circadian variation, which actually exists of hourly ultradian peaks w86x. Basal GR transcriptional activation follows these ultradian hormone pulses w87x. Disturbances of this basal pulsatility, as may occur as a consequence of chronic stress, affect the transcriptional response to superimposed acute CORT challenges w88x. In fact, controlled infusion of corticosterone in adrenalectomised rats suggests that the neuronal and behavioural response to stress is affected by the timing of the stressor relative to the onset of an ultradian pulse w89x. Which signalling modes are involved in transducing amplitude and phase of ultradian peaks into functional responses is as yet unknown.

Expert opinion There are many factors that determine the outcome of a particular transient of prolonged activation of corticosteroid receptors. The cellular diversity of the brain, and the different cellular contexts that are the consequence of different stressors call for a proper understanding of the brain circuitry involved in particular adaptations, and analysis of MR/GR signalling in those particular circuits and contexts. With respect to the genomic signalling mode, the advances of chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) allows for a truly complete picture of primary GR or MR genomic targets, and in combination with deepsequencing based measurement of expression will also advance functional analysis of transcriptional changes w90x. The application of ChIP-seq to the brain will certainly be a superior new starting point for the unbiased analysis of targets and – via analysis of the DNA sequences associated with binding – signalling modes of MR and GR in different conditions. Combining GR/MR binding with that of crosstalk partners or specific coregulators will allow mapping to

common loci on the DNA, and yield testable hypothesis on the nature of molecular interactions in transcriptional control. A caveat may, however, be the amount of material that is needed for such analysis, which will allow only analysis of relative large populations of cells. The analysis of diffusely distributed networks of cells w91x may escape such an approach, although progressively lower amounts of input material that will be needed for novel techniques may resolve this issue, like it has done for mRNA analysis which can now be done on laser-microdissected neuronal populations w45x. Data on dynamic epigenetic changes of the DNA are already accumulating in relation to stress-responsiveness. Such changes are known to affect the expression levels of factors like GR and CRH w20, 58x. Long-term epigenetic changes that affect binding of MR/GR itself, or of their accessory proteins, will likely be discovered in the near future. More tools for the manipulation of particular GR/MR signalling pathways are also becoming available. The GRdim/dim mouse has been a strong example for the power of such techniques. Having ligands available that allow comparable selective activation of diverse signalling modes, will allow similar analysis without the need for transgenesis. Also other molecular tools may be used, such as virus-mediated shRNA knockdown of particular pathways. A – for neuroscience – rather recent but promising option is to use modified antisense oligonucleotide (AON)-mediated exon-skipping w92x. Such AONs are taken up very effectively by neurons once past the blood-brain barrier, without the need for viral delivery, and may be used to bias expression of genes to particular splice variants, or to generate internal deletions of protein domains that are crucial for particular modes of signalling w93x.

Outlook In spite of the major technological advancements described above, it is hard to envisage anything like high-throughput understanding of stress signalling. Pathways will be selective

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for particular circuits under particular circumstances, although some common interactions should exist, for example, related to CREB-GR signalling in relation to modulation of cognitive processes by arousal in multiple brain areas w94x. However, it will become easier to translate the advancement in ‘generic’ nuclear receptor biology to the brain, given the more comprehensive nature of present day genomics techniques, and progress in manipulation of site- and cell-specific protein activity. A major challenge will be to relate the way non-genomic signalling interacts with subsequent transcriptional responses w3x. Corticosteroid receptor ligands may become very relevant for actual use in psychopathology. GR antagonists can abrogate psychotic-depressive symptoms associated with Cushing’s disease w95x and may well be of use in psychotic major depression w96x. GR agonists are now used experimentally for their capacity to facilitate extinction learning in diseases linked to conditioned fear, such as PTSD and phobias w97x. MR agonists have been shown to facilitate the clinical response to classical antidepressant treatment w98x. Development of new compounds that target the selective signalling pathways underlying such effects with greater specificity or efficacy will be the best possible outcome of the search for selective MR/GR mechanisms.

Highlights • Apart from HPA axis activation there are numerous factors that determine whether MR and GR will be occupied, which include CBG, the 11-beta-hydroxysteroid dehydrogenases, and barrier proteins like P-glycoprotein. • Corticosteroid receptor diversity goes beyond merely ‘MR and GR’, given splice and in particular translation variants, and the many post-translationally modified receptor flavours that can exist. • There is strong evidence for specific GR/MR output mechanisms being involved in CORT effects on the brain, including transcription factors and coregulators. For most effects however, the actual signalling mechanism is unknown. • The mechanisms of non-genomic signalling, and the way in which non-genomic signalling interacts with classical transcriptional regulation is a major challenge for the near future. • The presently available genomic tools, including ChIP-seq will be of great help to elucidate the distinct genomic MR/ GR signalling modes in particular brain processes. • In parallel, the identification of new dissociating ligands will help experimental analysis of pathways, and bears promise for putting such knowledge to clinical use in stress related disorders.

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