Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain
Zalachoras, I.; Grootaers, G.; van Weert, L. T.; Aubert, Y.; de Kreij, S. R.; Datson, N. A.; van Roon-Mom, W. M.; Aartsma-Rus, A.; Meijer, O. C.

ABSTRACT: 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.

Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator
Zalachoras, I.; Houtman, R.; Atucha, E.; Devos, R.; Tijssen, A. M.; Hu, P.; Lockey, P. M.; Datson, N. A.; Belanoff, J. K.; Lucassen, P. J.; Joels, M.; de Kloet, E. R.; Roozendaal, B.; Hunt, H.; Meijer, O. C.

Glucocorticoid receptor (GR) antagonism may be of considerable therapeutic value in stress-related psychopathology such as depression. However, blockade of all GR-dependent processes in the brain will lead to unnecessary and even counteractive effects, such as elevated endogenous cortisol levels. Selective GR modulators are ligands that can act both as agonist and as antagonist and may be used to separate beneficial from harmful treatment effects. We have discovered that the high-affinity GR ligand C108297 is a selective modulator in the rat brain. We first demonstrate that C108297 induces a unique interaction profile between GR and its downstream effector molecules, the nuclear receptor coregulators, compared with the full agonist dexamethasone and the antagonist RU486 (mifepristone). C108297 displays partial agonistic activity for the suppression of hypothalamic corticotropin-releasing hormone (CRH) gene expression and potently enhances GR-dependent memory consolidation of training on an inhibitory avoidance task. In contrast, it lacks agonistic effects on the expression of CRH in the central amygdala and antagonizes GR-mediated reduction in hippocampal neurogenesis after chronic corticosterone exposure. Importantly, the compound does not lead to disinhibition of the hypothalamus-pituitary-adrenal axis. Thus, C108297 represents a class of ligands that has the potential to more selectively abrogate pathogenic GR-dependent processes in the brain, while retaining beneficial aspects of GR signaling.

Two Populations of Glucocorticoid Receptor-Binding Sites in the Male Rat Hippocampal Genome
Polman, J. A.; de Kloet, E. R.; Datson, N. A.

In the present study, genomic binding sites of glucocorticoid receptors (GR) were identified in vivo in the rat hippocampus applying chromatin immunoprecipitation followed by next-generation sequencing. We identified 2470 significant GR-binding sites (GBS) and were able to confirm GR binding to a random selection of these GBS covering a wide range of P values. Analysis of the genomic distribution of the significant GBS revealed a high prevalence of intragenic GBS. Gene ontology clusters involved in neuronal plasticity and other essential neuronal processes were overrepresented among the genes harboring a GBS or located in the vicinity of a GBS. Male adrenalectomized rats were challenged with increasing doses of the GR agonist corticosterone (CORT) ranging from 3 to 3000 mug/kg, resulting in clear differences in the GR-binding profile to individual GBS. Two groups of GBS could be distinguished: a low-CORT group that displayed GR binding across the full range of CORT concentrations, and a second high-CORT group that displayed significant GR binding only after administering the highest concentration of CORT. All validated GBS, in both the low-CORT and high-CORT groups, displayed mineralocorticoid receptor binding, which remained relatively constant from 30 mug/kg CORT upward. Motif analysis revealed that almost all GBS contained a glucocorticoid response element resembling the consensus motif in literature. In addition, motifs corresponding with new potential GR-interacting proteins were identified, such as zinc finger and BTB domain containing 3 (Zbtb3) and CUP (CG11181 gene product from transcript CG11181-RB), which may be involved in GR-dependent transactivation and transrepression, respectively. In conclusion, our results highlight the existence of 2 populations of GBS in the rat hippocampal genome.

Understanding stress-effects in the brain via transcriptional signal transduction pathways
Zalachoras, I.; Houtman, R.; Meijer, O. C.

Glucocorticoid hormones exert crucial effects on the brain in relation to physiology, endocrine regulation, mood and cognition. Their two receptor types, glucocorticoid and mineralocorticoid receptors (GR and MR), are members of the nuclear receptor superfamily and act in large measure as transcription factors. The outcome of MR/GR action on the genome depends on interaction with members from different protein families, which are of crucial importance for cross-talk with other neuronal and hormonal signals that impinge on the glucocorticoid sensitive circuitry. Relevant interacting proteins include other transcription factors that may either tether the receptor to the DNA, or that bind in the vicinity of GR and MR to tune the transcriptional response. In addition, transcriptional coregulator proteins constitute the actual signal transduction pathway to the transcription machinery. We review the current evidence for involvement of individual coregulators in GR-dependent effects on stress responses, and learning and memory. We discuss the use of in vitro and in silico tools to predict those coregulators that are of importance for particular brain processes. Finally, we discuss the potential of selective receptor modulators that may only allow a subset of all interactions, thus allowing more selective targeting of glucocorticoid-dependent processes in the brain.

Prior history of chronic stress changes the transcriptional response to glucocorticoid challenge in the dentate gyrus region of the male rat hippocampus
Datson, N. A.; van den Oever, J. M.; Korobko, O. B.; Magarinos, A. M.; de Kloet, E. R.; McEwen, B.

Chronic stress is a risk factor for several neuropsychiatric diseases such as depression and psychosis. In response to stress glucocorticoids (GCs) are secreted that bind to the glucocorticoid receptor (GR), a ligand-activated transcription factor that regulates the transcription of gene networks in the brain necessary for coping with stress, recovery and adaptation. Chronic stress particularly affects the dentate gyrus (DG) subregion of the hippocampus, causing several functional and morphological changes with consequences for learning and memory, that are likely adaptive, but at the same time make DG neurons more vulnerable to subsequent challenges.The aim of this study was to investigate the transcriptional response of DG neurons to a GC-challenge in male rats previously exposed to chronic restraint stress (CRS). An intriguing finding of the current study was that having a history of CRS had profound consequences for the subsequent response to acute GC-challenge, differentially affecting the expression of several hundreds of genes in the DG compared to challenged non-stressed control animals. This enduring effect of prior stress exposure suggests that epigenetic processes may be involved. In line with this, CRS indeed affected the expression of several genes involved in chromatin structure and epigenetic processes, including Asf1, Ash1l, Hist1h3f and Tp63. The data presented here indicate that CRS alters the transcriptional response to a subsequent GC-injection. We propose that this altered transcriptional potential forms part of the molecular mechanism underlying the enhanced vulnerability for stress-related disorders like depression caused by chronic stress.

Functional profile of the binary brain corticosteroid receptor system: Mediating, multitasking, coordinating, integrating
de Kloet, E. R.

This contribution to Willem Hendrik Gispen's 'Festschrift' is focused on the action of the naturally occurring corticosteroids, cortisol and corticosterone, which are secreted from the adrenals in hourly pulses and after stress with the goal to maintain resilience and health. To achieve this goal the action of the corticosteroids displays an impressive diversity, because it is cell-specific and context-dependent in coordinating the individual's response to changing environments. These diverse actions of corticosterone are mediated by mineralocorticoid- and glucocorticoid-receptors that operate as a binary system in concert with neurotransmitter and neuropeptide signals to activate and inhibit stress reactions, respectively. Classically MR and GR are gene transcription factors, but recently these receptors appear to mediate also rapid non-genomic actions on excitatory neurotransmission suggesting that they integrate functions over time. Hence the balance of receptor-mediated actions is crucial for homeostasis. This balanced function of mineralo- and glucocorticoid-receptors can be altered epigenetically by a history of traumatic (early) life events and the experience of repeated stressors as well as by predisposing genetic variants in signaling pathways of these receptors. One of these variants, mineralocorticoid receptor haplotype 2, is associated with dispositional optimism in appraisal of environmental challenges. Imbalance in receptor-mediated corticosterone actions was found to leave a genomic signature highlighting the role of master switches such as cAMP response element-binding protein and mammalian target of rapamycin to compromise health, and to promote vulnerability to disease. Diabetic encephalopathy is a pathology of imbalanced corticosterone action, which can be corrected in its pre-stage by a brief treatment with the antiglucocorticoid mifepristone.

Lifetime achievement from a brain-adrenal perspective: On the CRF-urocortin-glucocorticoid balance
de Kloet, E. R.

This contribution dedicated to Wylie Vale is focused on the action of the glucocorticoid hormone aimed to counterbalance the stress response orchestrated by the corticotrophin releasing factor (CRF) and urocortin (Ucn) family of peptides. It appears that the release and action of these stress hormones themselves are subjected to intrinsic self-regulatory feedback loops that operate as checks and balances in stress adaptation. One of these feedback loops is operated by the mineralocorticoid (MR) and glucocorticoid receptors (GR) that mediate in complementary fashion the action of endogenous cortisol/corticosterone in brain circuits underlying the onset and termination of the stress response. By affecting appraisal processes MR has an important role in coordinating emotional expression and cognitive flexibility with the onset of the stress response, while GR's role is prominent in the management of behavioral and physiological adaptations during the recovery phase. Genetic variation in interaction with environmental input and experience-related factors can modulate this balance between susceptibility and recovery governed by a balanced MR:GR signaling. Thanks to the Wylie Vale School of scientists a parallel balanced regulation between the CRF/CRF-1 and Ucn/CRF-2 receptor systems is being uncovered, leading inexorably to the question: how do the CRF/Ucn and glucocorticoid systems interact in multiple brain sites to maintain homeostasis and health?

The transcriptional response to chronic stress and glucocorticoid receptor blockade in the hippocampal dentate gyrus
Datson, N. A.; Speksnijder, N.; Mayer, J. L.; Steenbergen, P. J.; Korobko, O.; Goeman, J.; de Kloet, E. R.; Joels, M.; Lucassen, P. J.

The dentate gyrus (DG) of the hippocampus plays a crucial role in learning and memory. This subregion is unique in its ability to generate new neurons throughout life and integrate these new neurons into the hippocampal circuitry. Neurogenesis has further been implicated in hippocampal plasticity and depression. Exposure to chronic stress affects DG function and morphology and suppresses neurogenesis and long-term potentiation (LTP) with consequences for cognition. Previous studies demonstrated that glucocorticoid receptor (GR) blockade by a brief treatment with the GR antagonist mifepristone (RU486) rapidly reverses the stress and glucocorticoid effects on neurogenesis. The molecular pathways underlying both the stress-induced effects and the RU486 effects on the DG are, however, largely unknown. The aim of this study was therefore (1) to investigate by microarray analysis which genes and pathways in the DG are sensitive to chronic stress and (2) to investigate to what extent blockade of GR can normalize these stress-induced effects on DG gene expression. Chronic stress exposure affected the expression of 90 genes in the DG (P < 0.01), with an overrepresentation of genes involved in brain development and morphogenesis and synaptic transmission. RU486 treatment of stressed animals affected expression of 107 genes; however, mostly different genes than those responding to stress. Interestingly, we found CREBBP to be normalized by RU486 treatment to levels observed in control animals, suggesting that CREB-signaling may play a central role in mediating the chronic stress effects on neurogenesis, LTP and calcium currents. The identified genetic pathways provide insight into the stress-induced adaptive plasticity of the hippocampal DG that is so central in learning and memory and will direct future studies on the functional outcome and modulation of these stress effects.(c) 2010 Wiley Periodicals, Inc.

Mineralocorticoid and glucocorticoid receptors at the neuronal membrane, regulators of nongenomic corticosteroid signalling
Groeneweg, F. L.; Karst, H.; de Kloet, E. R.; Joels, M.

The balance between corticosteroid actions induced via activation of the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) determines the brain's response to stress. While both receptors are best known for their delayed genomic role, it has become increasingly evident that they can also associate with the plasma membrane and act as mediators of rapid, nongenomic signalling. Nongenomic corticosteroid actions in the brain are required for the coordination of a rapid adaptive response to stress; membrane-associated MRs and GRs play a major role herein. However, many questions regarding the underlying mechanism are still unresolved. How do MR and GR translocate to the membrane and what are their downstream signalling partners? In this review we discuss these issues based on insights obtained from related receptors, most notably the estrogen receptor alpha.

Glucocorticoids Modulate the mTOR Pathway in the Hippocampus: Differential Effects Depending on Stress History
Polman, J. A.; Hunter, R. G.; Speksnijder, N.; van den Oever, J. M.; Korobko, O. B.; McEwen, B. S.; de Kloet, E. R.; Datson, N. A.

Glucocorticoid (GC) hormones, released by the adrenals in response to stress, are key regulators of neuronal plasticity. In the brain, the hippocampus is a major target of GC, with abundant expression of the GC receptor. GC differentially affect the hippocampal transcriptome and consequently neuronal plasticity in a subregion-specific manner, with consequences for hippocampal information flow and memory formation. Here, we show that GC directly affect the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational control and has long-lasting effects on the plasticity of specific brain circuits. We demonstrate that regulators of the mTOR pathway, DNA damage-induced transcript (DDIT)4 and FK506-binding protein 51 are transcriptionally up-regulated by an acute GC challenge in the dentate gyrus (DG) subregion of the rat hippocampus, most likely via a GC-response element-driven mechanism. Furthermore, two other mTOR pathway members, the mTOR regulator DDIT4-like and the mTOR target DDIT3, are down-regulated by GC in the rat DG. Interestingly, the GC responsiveness of DDIT4 and DDIT3 was lost in animals with a recent history of chronic stress. Basal hippocampal mTOR protein levels were higher in animals exposed to chronic stress than in controls. Moreover, an acute GC challenge significantly reduced mTOR protein levels in the hippocampus of animals with a chronic stress history but not in unstressed controls. Based on these findings, we propose that direct regulation of the mTOR pathway by GC represents an important mechanism regulating neuronal plasticity in the rat DG, which changes after exposure to chronic stress.