The multi-faceted nature of age-associated osteoporosis.

Age-associated osteoporosis (AAOP) poses a significant health burden, characterized by increased fracture risk due to declining bone mass and strength. Effective prevention and early treatment strategies are crucial to mitigate the disease burden and the associated healthcare costs. Current therapeutic approaches effectively target the individual contributing factors to AAOP. Nonetheless, the management of AAOP is complicated by the multitude of variables that affect its development. Main intrinsic and extrinsic factors contributing to AAOP risk are reviewed here, including mechanical unloading, nutrient deficiency, hormonal disbalance, disrupted metabolism, cognitive decline, inflammation and circadian disruption. Furthermore, it is discussed how these can be targeted for prevention and treatment. Although valuable as individual targets for intervention, the interconnectedness of these risk factors result in a unique etiology for every patient. Acknowledgement of the multifaceted nature of AAOP will enable the development of more effective and sustainable management strategies, based on a holistic, patient-centered approach.

An integrated single-cell RNA-seq atlas of the mouse hypothalamic paraventricular nucleus links transcriptomic and functional types.

The hypothalamic paraventricular nucleus (PVN) is a highly complex brain region that is crucial for homeostatic regulation through neuroendocrine signaling, outflow of the autonomic nervous system, and projections to other brain areas. In the past years, single-cell datasets of the hypothalamus have contributed immensely to the current understanding of the diverse hypothalamic cellular composition. While the PVN has been adequately classified functionally, its molecular classification is currently still insufficient. To address this, we created a detailed atlas of PVN transcriptomic cell types by integrating various PVN single-cell datasets into a recently published hypothalamus single-cell transcriptome atlas. Furthermore, we functionally profiled transcriptomic cell types, based on relevant literature, existing retrograde tracing data, and existing single-cell data of a PVN-projection target region. Finally, we validated our findings with immunofluorescent stainings. In our PVN atlas dataset, we identify the well-known different neuropeptide types, each composed of multiple novel subtypes. We identify Avp-Tac1, Avp-Th, Oxt-Foxp1, Crh-Nr3c1, and Trh-Nfib as the most important neuroendocrine subtypes based on markers described in literature. To characterize the preautonomic functional population, we integrated a single-cell retrograde tracing study of spinally projecting preautonomic neurons into our PVN atlas. We identify these (presympathetic) neurons to cocluster with the Adarb2+ clusters in our dataset. Further, we identify the expression of receptors for Crh, Oxt, Penk, Sst, and Trh in the dorsal motor nucleus of the vagus, a key region that the pre-parasympathetic PVN neurons project to. Finally, we identify Trh-Ucn3 and Brs3-Adarb2 as some centrally projecting populations. In conclusion, our study presents a detailed overview of the transcriptomic cell types of the murine PVN and provides a first attempt to resolve functionality for the identified populations.

Glucocorticoid Receptor (GR) antagonism as disease-modifying treatment for MDD with childhood trauma: protocol of the RESET-medication randomized controlled trial.

BACKGROUND: Major depressive disorder (MDD) is a heterogeneous psychiatric disorder. Childhood trauma (CT, emotional/physical/sexual abuse or neglect before the age of 18) is one of the largest and most consistent risk factors for development and poor course of MDD. Overactivity of the HPA-axis and the stress hormone cortisol is thought to play a role in the vulnerability for MDD following exposure to CT. Rodent experiments showed that antagonism of the glucocorticoid receptor (GR) at adult age reversed the effects of early life stress. Similarly, we aim to target MDD in individuals with CT exposure using the GR antagonist mifepristone. METHODS: The RESET-medication study is a placebo-controlled double-blind randomized controlled trial (RCT) which aims to include 158 adults with MDD and CT. Participants will be randomized (1:1) to a 7-day treatment arm of mifepristone (1200 mg/day) or a control arm (placebo). Participants are allowed to receive usual care for MDD including antidepressants. Measurements include three face-to-face meetings at baseline (T0), day 8 (T1), week 6 (T2), and two online follow-up meetings at 12 weeks (T3) and 6 months (T4). A subgroup of participants (N = 80) are included in a fMRI sub-study (T0, T2). The main study outcome will be depressive symptom severity as measured with the Inventory of Depressive Symptomatology-Self Rated (IDS-SR) at T2. Secondary outcomes include, among others, depressive symptom severity at other time points, disability, anxiety, sleep and subjective stress. To address underlying mechanisms mifepristone plasma levels, cortisol, inflammation, epigenetic regulation and fMRI measurements are obtained. DISCUSSION: The RESET-medication study will provide clinical evidence whether GR antagonism is a disease-modifying treatment for MDD in individuals exposed to CT. If effective, this hypothesis-driven approach may extend to other psychiatric disorders where CT plays an important role. TRIAL REGISTRATION: The trial protocol has been registered 01-02-2022 on ClinicalTrials.gov with ID "NCT05217758".

Exploring the choroidal vascular labyrinth and its molecular and structural roles in health and disease.

The choroid is a key player in maintaining ocular homeostasis and plays a role in a variety of chorioretinal diseases, many of which are poorly understood. Recent advances in the field of single-cell RNA sequencing have yielded valuable insights into the properties of choroidal endothelial cells (CECs). Here, we review the role of the choroid in various physiological and pathophysiological mechanisms, focusing on the role of CECs. We also discuss new insights regarding the phenotypic properties of CECs, CEC subpopulations, and the value of measuring transcriptomics in primary CEC cultures derived from post-mortem eyes. In addition, we discuss key phenotypic, structural, and functional differences that distinguish CECs from other endothelial cells such as retinal vascular endothelial cells. Understanding the specific clinical and molecular properties of the choroid will shed new light on the pathogenesis of the broad clinical range of chorioretinal diseases such as age-related macular degeneration, central serous chorioretinopathy and other diseases within the pachychoroid spectrum, uveitis, and diabetic choroidopathy. Although our knowledge is still relatively limited with respect to the clinical features and molecular pathways that underlie these chorioretinal diseases, we summarise new approaches and discuss future directions for gaining new insights into these sight-threatening diseases and highlight new therapeutic strategies such as pluripotent stem cell‒based technologies and gene therapy.

Potential associations between immune signaling genes, deactivated microglia, and oligodendrocytes and cortical gray matter loss in patients with long-term remitted Cushing's disease.

INTRODUCTION: Cushing's disease (CD) is a rare and severe endocrine disease characterized by hypercortisolemia. Previous studies have found structural brain alterations in remitted CD patients compared to healthy controls, specifically in the anterior cingulate cortex (ACC). However, potential mechanisms through which these persistent alterations may have occurred are currently unknown. METHODS: Structural 3T MRI's from 25 remitted CD patients were linked with gene expression data from neurotypical donors, derived from the Allen Human Brain Atlas. Differences in gene expression between the ACC and an unaffected control cortical region were examined, followed by a Gene Ontology (GO) enrichment analysis. A cell type enrichment analysis was conducted on the differentially expressed genes, and a disease association enrichment analysis was conducted to determine possible associations between differentially expressed genes and specific diseases. Subsequently, cortisol sensitivity of these genes in existing datasets was examined. RESULTS: The gene expression analysis identified 300 differentially expressed genes in the ACC compared to the cortical control region. GO analyses found underexpressed genes to represent immune function. The cell type specificity analysis indicated that underexpressed genes were enriched for deactivated microglia and oligodendrocytes. Neither significant associations with diseases, nor evidence of cortisol sensitivity with the differentially expressed genes were found. DISCUSSION: Underexpressed genes in the ACC, the area vulnerable to permanent changes in remitted CD patients, were often associated with immune functioning. The specific lack of deactivated microglia and oligodendrocytes implicates protective effects of these cell types against the long-term effects of cortisol overexposure.

Cortical thickness abnormalities in long-term remitted Cushing's disease.

Long-term remitted Cushing's disease (LTRCD) patients commonly continue to present persistent psychological and cognitive deficits, and alterations in brain function and structure. Although previous studies have conducted gray matter volume analyses, assessing cortical thickness and surface area of LTRCD patients may offer further insight into the neuroanatomical substrates of Cushing's disease. Structural 3T magnetic resonance images were obtained from 25 LTRCD patients, and 25 age-, gender-, and education-matched healthy controls (HCs). T1-weighted images were segmented using FreeSurfer software to extract mean cortical thickness and surface area values of 68 cortical gray matter regions and two whole hemispheres. Paired sample t tests explored differences between the anterior cingulate cortex (ACC; region of interest), and the whole brain. Validated scales assessed psychiatric symptomatology, self-reported cognitive functioning, and disease severity. After correction for multiple comparisons, ROI analyses indicated that LTRCD-patients showed reduced cortical thickness of the left caudal ACC and the right rostral ACC compared to HCs. Whole-brain analyses indicated thinner cortices of the left caudal ACC, left cuneus, left posterior cingulate cortex, right rostral ACC, and bilateral precuneus compared to HCs. No cortical surface area differences were identified. Cortical thickness of the left caudal ACC and left cuneus were inversely associated with anxiety symptoms, depressive symptoms, and disease duration, although certain associations did not persist after correction for multiple testing. In six of 68 regions examined, LTRCD patients had reduced cortical thickness in comparison to HCs. Cortical thickness of the left caudal ACC was inversely associated with disease duration. This suggests that prolonged and excessive exposure to glucocorticoids may be related to cortical thinning of brain structures involved in emotional and cognitive processing.

Importance of the brain corticosteroid receptor balance in metaplasticity, cognitive performance and neuro-inflammation.

Bruce McEwen's discovery of receptors for corticosterone in the rat hippocampus introduced higher brain circuits in the neuroendocrinology of stress. Subsequently, these receptors were identified as mineralocorticoid receptors (MRs) that are involved in appraisal processes, choice of coping style, encoding and retrieval. The MR-mediated actions on cognition are complemented by slower actions via glucocorticoid receptors (GRs) on contextualization, rationalization and memory storage of the experience. These sequential phases in cognitive performance depend on synaptic metaplasticity that is regulated by coordinate MR- and GR activation. The receptor activation includes recruitment of coregulators and transcription factors as determinants of context-dependent specificity in steroid action; they can be modulated by genetic variation and (early) experience. Interestingly, inflammatory responses to damage seem to be governed by a similarly balanced MR:GR-mediated action as the initiating, terminating and priming mechanisms involved in stress-adaptation. We conclude with five questions challenging the MR:GR balance hypothesis.

Isoform switching of steroid receptor co-activator-1 attenuates glucocorticoid-induced anxiogenic amygdala CRH expression.

Maladaptive glucocorticoid effects contribute to stress-related psychopathology. The glucocorticoid receptor (GR) that mediates many of these effects uses multiple signaling pathways. We have tested the hypothesis that manipulation of downstream factors ('coregulators') can abrogate potentially maladaptive GR-mediated effects on fear-motivated behavior that are linked to corticotropin releasing hormone (CRH). For this purpose the expression ratio of two splice variants of steroid receptor coactivator-1 (SRC-1) was altered via antisense-mediated 'exon-skipping' in the central amygdala of the mouse brain. We observed that a change in splicing towards the repressive isoform SRC-1a strongly reduced glucocorticoid-induced responsiveness of Crh mRNA expression and increased methylation of the Crh promoter. The transcriptional GR target gene Fkbp5 remained responsive to glucocorticoids, indicating gene specificity of the effect. The shift of the SRC-1 splice variants altered glucocorticoid-dependent exploratory behavior and attenuated consolidation of contextual fear memory. In conclusion, our findings demonstrate that manipulation of GR signaling pathways related to the Crh gene can selectively diminish potentially maladaptive effects of glucocorticoids.

Understanding stress-effects in the brain via transcriptional signal transduction pathways.

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.

Knockdown of the glucocorticoid receptor alters functional integration of newborn neurons in the adult hippocampus and impairs fear-motivated behavior.

Glucocorticoids (GCs) secreted after stress reduce adult hippocampal neurogenesis, a process that has been implicated in cognitive aspects of psychopathology, amongst others. Yet, the exact role of the GC receptor (GR), a key mediator of GC action, in regulating adult neurogenesis is largely unknown. Here, we show that GR knockdown, selectively in newborn cells of the hippocampal neurogenic niche, accelerates their neuronal differentiation and migration. Strikingly, GR knockdown induced ectopic positioning of a subset of the new granule cells, altered their dendritic complexity and increased their number of mature dendritic spines and mossy fiber boutons. Consistent with the increase in synaptic contacts, cells with GR knockdown exhibit increased basal excitability parallel to impaired contextual freezing during fear conditioning. Together, our data demonstrate a key role for the GR in newborn hippocampal cells in mediating their synaptic connectivity and structural as well as functional integration into mature hippocampal circuits involved in fear memory consolidation.

Specific regulatory motifs predict glucocorticoid responsiveness of hippocampal gene expression.

The glucocorticoid receptor (GR) is an ubiquitously expressed ligand-activated transcription factor that mediates effects of cortisol in relation to adaptation to stress. In the brain, GR affects the hippocampus to modulate memory processes through direct binding to glucocorticoid response elements (GREs) in the DNA. However, its effects are to a high degree cell specific, and its target genes in different cell types as well as the mechanisms conferring this specificity are largely unknown. To gain insight in hippocampal GR signaling, we characterized to which GRE GR binds in the rat hippocampus. Using a position-specific scoring matrix, we identified evolutionary-conserved putative GREs from a microarray based set of hippocampal target genes. Using chromatin immunoprecipitation, we were able to confirm GR binding to 15 out of a selection of 32 predicted sites (47%). The majority of these 15 GREs are previously undescribed and thus represent novel GREs that bind GR and therefore may be functional in the rat hippocampus. GRE nucleotide composition was not predictive for binding of GR to a GRE. A search for conserved flanking sequences that may predict GR-GRE interaction resulted in the identification of GC-box associated motifs, such as Myc-associated zinc finger protein 1, within 2 kb of GREs with GR binding in the hippocampus. This enrichment was not present around nonbinding GRE sequences nor around proven GR-binding sites from a mesenchymal stem-like cell dataset that we analyzed. GC-binding transcription factors therefore may be unique partners for DNA-bound GR and may in part explain cell-specific transcriptional regulation by glucocorticoids in the context of the hippocampus.

Long term sex-dependent psychoneuroendocrine effects of maternal deprivation and juvenile unpredictable stress in rats.

We have analysed the long-term psychoneuroendocrine effects of maternal deprivation (MD) [24 h at postnatal day (PND) 9] and/or exposure to chronic unpredictable stress (CUS) during the periadolescent period (PND 28 to PND 43) in male and female Wistar rats. Animals were tested in the elevated plus maze (EPM, anxiety) at PND 44 and in two memory tests, spontaneous alternation and novel object recognition (NOT) in adulthood. The expression of hippocampal glucocorticoid (GR) and mineralocorticoid (MR) receptors, as well as of synaptophysin, neural cell adhesion molecule and brain-derived neurotrophic factor, was analysed by in situ hybridisation in selected hippocampal regions. Endocrine determinations of leptin, testosterone and oestradiol plasma levels were carried out by radioimmunoassay. Young CUS animals showed decreased anxiety behaviour in the EPM (increased percentage of time and entries in the open arms) irrespective of neonatal treatment. Memory impairments were induced by the two stressful treatments as was revealed by the NOT, with males being most clearly affected. Although each stressful procedure, when considered separately, induced different (always decrements) effects on the three synaptic molecules analysed and affected males and females differently, the combination of MD and CUS induced an unique disruptive effect on the three synaptic plasticity players. MD induced a long-term significant decrease in hippocampal GR only in males, whereas CUS tended to increase MR in males and decrease MR in females. Both neonatal MD and periadolescent CUS induced marked reductions in testosterone and oestradiol in males, whereas MD male animals also showed significantly decreased leptin levels. By contrast, in females, none of the hormones analysed was altered by any of the stressful procedures. Taking our data together in support of the 'two-hit' hypothesis, MD during neonatal life and/or exposure to CUS during the periadolescent period induced a permanent deficit in memory, which was accompanied by a decrement in markers for hippocampal plasticity. The long-term effects on body weight and hormone levels, particularly among males, might reflect sex-dependent lasting metabolic alterations as well as an impaired reproductive function.

Blocking dopamine D2 receptors by haloperidol curtails the beneficial impact of calorie restriction on the metabolic phenotype of high-fat diet induced obese mice.

Calorie restriction is the most effective way of expanding life-span and decreasing morbidity. It improves insulin sensitivity and delays the age-related loss of dopamine receptor D(2) (DRD2) expression in the brain. Conversely, high-fat feeding is associated with obesity, insulin resistance and a reduced number of DRD2 binding sites. We hypothesised that the metabolic benefit of calorie restriction involves the preservation of appropriate DRD2 transmission. The food intake of wild-type C57Bl6 male mice was restricted to 60% of ad lib. intake while they were treated with the DRD2 antagonist haloperidol or vehicle using s.c. implanted pellets. Mice with ad lib. access to food receiving vehicle treatment served as controls. All mice received high-fat food throughout the experiment. After 10 weeks, an i.p. glucose tolerance test was performed and, after 12 weeks, a hyperinsulinaemic euglycaemic clamp. Hypothalamic DRD2 binding was also determined after 12 weeks of treatment. Calorie-restricted (CR) vehicle mice were glucose tolerant and insulin sensitive compared to ad lib. (AL) fed vehicle mice. CR mice treated with haloperidol were slightly heavier than vehicle treated CR mice. Haloperidol completely abolished the beneficial impact of calorie restriction on glucose tolerance and partly reduced the insulin sensitivity observed in CR vehicle mice. The metabolic differences between AL and CR vehicle mice were not accompanied by alterations in hypothalamic DRD2 binding. In conclusion, blocking DRD2 curtails the metabolic effects of calorie restriction. Although this suggests that the dopaminergic system could be involved in the metabolic benefits of calorie restriction, restricting access to high-fat food does not increase (hypothalamic) DRD2 binding capacity, which argues against this inference.

Glucocorticoid ultradian rhythmicity directs cyclical gene pulsing of the clock gene period 1 in rat hippocampus.

In vivo glucocorticoid (GC) secretion exhibits a distinctive ultradian rhythmicity. The lipophilic hormone can rapidly diffuse into cells, although only the pulse peak is of sufficient amplitude to activate the low affinity glucocorticoid receptor (GR). Discrete pulses readily access brain regions such as the hippocampus where GR expression is enriched and known to regulate neuronal function, including memory and learning processes. In the present study, we have tested the hypothesis that GR brain targets are responsive to ultradian GC rhythmicity. We have used adrenalectomised rats replaced with pulses of corticosterone to determine the transcriptional effects of ultradian pulses in the hippocampus. Confocal microscopy confirmed that each GC pulse results in transient GR nuclear localisation in hippocampal CA1 neurones. Concomitant GR activation and DNA binding was demonstrated by synthetic glucocorticoid response element oligonucleotide binding, and verified for the Clock gene Period 1 promoter region by chromatin immunoprecipitation assays. Strikingly each GC pulse induced a 'burst' of transcription of Period 1 measured by heterogeneous nuclear RNA quantitative polymerase chain reaction. The net effect of pulsatile GC exposure on accumulation of the mature transcript was also assessed, revealing a plateau of mRNA levels throughout the time course of pulsatile exposure, indicating the pulse timing works optimally for steady state Per1 expression. The plateau dropped to baseline within 120 min of the final pulse, indicating a relatively short half-life for hippocampal Per1. The significance of this strict temporal control is that any perturbation to the pulse frequency or duration would have rapid quantitative effects on the levels of Per1. This in turn could affect hippocampal function, especially circadian related memory and learning processes.

Recovery from disrupted ultradian glucocorticoid rhythmicity reveals a dissociation between hormonal and behavioural stress responsiveness.

Ultradian release of glucocorticoids is thought to be essential for homeostasis and health. Furthermore, deviation from this pulsatile release pattern is considered to compromise resilience to stress-related disease, even after hormone levels have normalised. In the present study, we investigate how constant exposure to different concentrations of corticosterone affects diurnal and ultradian pulsatility. The rate of recovery in pulsatile hypothalamic-pituitary-adrenal (HPA) activity after withdrawal of exogenous corticosterone is also examined. Finally, the behavioural and neuroendocrine responsiveness to an audiogenic stressor is studied. Adrenally intact male rats were subcutaneously implanted with vehicle, 40% or 100% corticosterone pellets for 7 days. The continuous release of corticosterone from these implants abolished diurnal and ultradian corticosterone variation, as measured with high-frequency automated blood sampling. Pellet removal on post-surgery day 8 allowed rapid recovery of endogenous rhythms in animals previously exposed to daily average concentrations (40%) but not after exposure to high concentrations (100%) of corticosterone. Behavioural and neuroendocrine responsiveness to stress was distinctly different between the treatment groups. Audiogenic stimulation 1 day after pellet removal resulted in a similar corticosterone response in animals previously exposed to 40% corticosterone or vehicle. The 40% pellet group, however, showed less and shorter behavioural activity (i.e. locomotion, risk assessment) to noise stress compared to 100% corticosterone and vehicle-treated animals. In conclusion, unlike the animals impanted with 100% corticosterone, we find that basal HPA axis activity in the 40% group, which had mean daily levels of circulating corticosterone in the physiological range, rapidly reverts to the characteristic pulsatile pattern of corticosterone secretion. Upon reinstatement of the ultradian rhythm, and despite the fact that these animals did not differ from controls in their response to noise stress, they did show substantial changes in their behavioural response to stress.

Glucocorticoid signaling and stress-related limbic susceptibility pathway: about receptors, transcription machinery and microRNA.

BACKGROUND: Stress is essential for health, but if coping with stress fails, the action of the stress hormones cortisol and corticosterone (CORT) becomes dysregulated, precipitating a condition favorable for increased susceptibility to psychopathology. We focus on the question how the action of CORT can change from protective to harmful. APPROACH: CORT targets the limbic brain, where it affects cognitive processes and emotional arousal. The magnitude and duration of the CORT feedback signal depends on bio-availability of the hormone, the activity of the CORT receptor machinery and the stress-induced drive. If CORT action becomes dysregulated, we postulate that this is linked to compromised receptor regulation in the limbic brain's susceptibility pathway. RESULTS: CORT action on gene transcription is mediated by high affinity mineralocorticoid (MR) and 10 fold lower affinity glucocorticoid (GR) receptors that also can mediate fast non-genomic actions. MR and GR operate a feedback loop that involves access and binding to the receptors, activation and shuttling of the CORT receptor complexes, which require interaction with coregulators and transcription factors for transcriptional outcome. CORT modulates the expression of gene transcripts encoding specific chaperones, motor proteins and transcription factors as well as its own receptors. The emerging evidence of microRNAs operating translational control points to further fine-tuning in receptor signaling. CONCLUSION: Imbalance in MR:GR-mediated actions caused by receptor variants and epigenetic modulations have been proposed as risk factor in stress-related disease. We here provide key regulatory steps in the activation, transport and regulation of CORT receptors that may sensitize susceptibility pathways underlying psychopathology.

Nuclear receptor coregulators differentially modulate induction and glucocorticoid receptor-mediated repression of the corticotropin-releasing hormone gene.

Nuclear receptor coregulators are proteins that modulate the transcriptional activity of steroid receptors and may explain cell-specific effects of glucocorticoid receptor action. Based on the uneven distribution of a number of coregulators in CRH-expressing cells in the hypothalamus of the rat brain, we tested the hypothesis that these proteins are involved as mediators in the glucocorticoid-induced repression of the CRH promoter. Therefore, we assessed the role of coregulator proteins on both induction and repression of CRH in the AtT-20 cell line, a model system for CRH repression by glucocorticoids. The steroid receptor coactivator 1a (SRC1a), SRC-1e, nuclear corepressor (N-CoR), and silencing mediator of the retinoid and thyroid hormone receptor (SMRT) were studied in this system. We show that the concentration of glucocorticoid receptor and the type of ligand, i.e. corticosterone or dexamethasone, determines the repression. Furthermore, overexpression of SRC1a, but not SRC1e, increased both efficacy and potency of the glucocorticoid receptor-mediated repression of the forskolin-induced CRH promoter. Unexpectedly, cotransfection of the corepressors N-CoR and SMRT did not affect the corticosterone-dependent repression but resulted in a marked decrease of the forskolin stimulation of the CRH gene. Altogether, our data demonstrate that 1) the concentration of the receptor, 2) the type of ligand, and 3) the coregulator recruited all determine the expression and the repression of the CRH gene. We conclude that modulation of coregulator activity may play a role in the control of the hypothalamus-pituitary-adrenal axis.

The dynamic pattern of glucocorticoid receptor-mediated transcriptional responses in neuronal PC12 cells.

The aim of the current study was (i) to examine the overlap in the pattern of glucocorticoid receptor (GR)-mediated transcriptional responses between different neuronal substrates and (ii) to assess the nature of these responses by differentiating between primary and downstream GR-responsive genes. For this purpose, nerve growth factor-differentiated catecholaminergic PC12 cells were used in which endogenous GRs were activated briefly with a high dose of corticosterone followed by gene expression profiling 1 and 3 h afterwards using Affymetrix GeneChips. The results revealed a strikingly similar temporal pattern to that which was reported previously in hippocampus, with only down-regulated genes 1 h after GR activation and the majority of genes up-regulated 3 h after GR activation. Real-time quantatitive PCR of transcripts in cycloheximide-treated cells showed that all five GR-responsive genes selected from the 1-h time point were primary responsive, whereas all four GR-responsive genes selected from the 3-h time point were downstream responsive. At the level of individual genes, the overlap with the previously generated hippocampal data sets was small, illustrating the cell-type specifity of GR-mediated genomic responses. Finally, we identified a number of interesting genes, such as SWI/SNF, synaptosomal-associated protein 25 and certain Rab proteins which may play a role in the effects of glucocorticoids on catecholaminergic neuronal functioning.

Understanding stress through the genome.

Stress-induced glucocorticoid hormones support coping with and adaptation to different stressors. They act to modulate gene expression in a tissue and stressor-specific manner through activation of corticosteroid receptors, which act as transcription factors. Here, a number of recent insights in gene regulation under the influence of glucocorticoids are discussed. Emphasis is put on distinct classes of target genes that may be defined, based on categorization of (combinations of) transcription factor binding sites in responsive genes. These categories depend on insights into different mechanisms of transcriptional regulation, such as transactivation vs transrepression, and high affinity vs low affinity hormone receptor response elements. It is argued that such classes, based on mechanistic understanding of transcription regulation, in combination with the availability of complete genomic sequences and expression data from different organs, may enhance our understanding of the way in which organisms deal with different forms of stress.

Steroid receptor coregulator diversity: what can it mean for the stressed brain?

Glucocorticoid hormones modulate brain function and as such are crucial for responding and adjusting to physical and psychological stressors. Their effects are mediated via mineralo- and glucocorticoid receptors, which in large measure act as transcription factors to modulate transcription of target genes, in a receptor-, cell-, and state-specific manner. The nature and magnitude of these transcriptional effects depend on the presence and activity of downstream proteins, such as steroid receptor coactivators and corepressors (together: coregulators), many of which are expressed in the brain. We address the role of coregulators for mineralo- and glucocorticoid receptor-mediated modulation of gene transcription. We first address evidence from cell lines for the importance of coregulator stoichiometry for steroid signaling. The in vivo importance of coregulators-when possible specifically for glucocorticoid signaling in the brain-is discussed based on knockout mice, transient knockdown of steroid receptor coactivators, and distribution and regulation of coactivator expression in the brain. We conclude that for a better understanding of modulation of brain function by glucocorticoids, it is necessary to take into account the role of coregulators, and to assess their importance relative to changes in hormone levels and receptor expression.