An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants.

The zebrafish as a model system for glucocorticoid receptor research.

Glucocorticoids regulate a plethora of physiological processes, and are widely used clinically as anti-inflammatory drugs. Their effects are mediated by the glucocorticoid receptor (GR), a ligand-activated transcription factor. Currently, zebrafish embryos are being developed into a model system for GR research, since they are easy to manipulate genetically and their phenotype can easily be visualized because of their transparent bodies. In addition, the zebrafish GR gene shows a relatively high level of similarity with its human equivalent. First, both the zebrafish and the human genome contain only a single gene encoding the GR. In all other fish species studied thus far, two GR genes have been found. Second, the zebrafish contains a C-terminal GR splice variant with high similarity to the human GRbeta, which has been shown to be a dominant-negative inhibitor of the canonical GRalpha and may be involved in glucocorticoid resistance. Thus, zebrafish embryos are potentially a useful model system for glucocorticoid receptor research, but currently only a limited number of tools is available. In this review, we discuss which tools are available and which need to be developed, in order to exploit the full potential of the zebrafish as a model system for GR research.

AUUUA motifs in the 3'UTR of human glucocorticoid receptor alpha and beta mRNA destabilize mRNA and decrease receptor protein expression.

An association between a gene polymorphism of the human glucocorticoid receptor (hGR) gene and rheumatoid arthritis has recently been suggested. This polymorphism contains an A to G mutation in the 3'UTR of exon 9beta, which encodes the 3'UTR of the mRNA of the hGRbeta isoform. The hGRbeta isoform can act as a dominant negative inhibitor of hGRalpha, and therefore may contribute to glucocorticoid resistance. The A to G mutation is located in an AUUUA motif, which is known to destabilize mRNA. In the present study, the importance of the mutation in this AUUUA motif was further characterized and mutations in other AUUUA motifs in the 3'UTR of hGRbeta and hGRalpha mRNA were studied. hGRbeta and hGRalpha expression vectors, carrying mutations in one AUUUA motif or all AUUUA motifs were transiently transfected into COS-1 cells. Each transfected vector was analyzed for the mRNA expression level, the mRNA turnover rate and the protein expression level. The naturally occurring mutation in the 3'UTR of hGRbeta mRNA increased mRNA stability and protein expression. Mutation of two other AUUUA motifs in the 3'UTR of hGRbeta, or mutation of all four AUUUA motifs resulted in a similar effect. Mutation of the most 5' AUUUA motif did not alter hGRbeta mRNA expression or mRNA stability. Mutation of all 10 AUUUA motifs in the 3'UTR of hGRalpha mRNA increased hGRalpha mRNA expression and mRNA stability as well as expression of the receptor protein level. Thus, the naturally occurring mutation in an AUUUA motif in the 3'UTR of hGRbeta mRNA results not only in increased mRNA stability, but also in increased receptor protein expression, which may contribute to glucocorticoid resistance. A similar role is suggested for two other AUUUA motifs in the 3'UTR of hGRbeta mRNA and for the 10 AUUUA motifs that are present in the 3'UTR of hGRalpha.

A human glucocorticoid receptor gene variant that increases the stability of the glucocorticoid receptor beta-isoform mRNA is associated with rheumatoid arthritis.

OBJECTIVE: To study the occurrence and function of polymorphism in the human glucocorticoid receptor (hGR) gene in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). METHODS: We used single stranded conformation polymorphism (SSCP) and direct sequencing to study the hGR gene in 30 patients with RA, 40 with SLE, and 24 controls. A newly identified polymorphism was transfected in COS-1 cells and the stability of the mRNA containing the polymorphism was tested using real-time PCR. RESULTS: A polymorphism in the hGR gene in exon9beta, in an "ATTTA" motif, was found to be significantly associated with RA. Introduction of this polymorphism in the hGRb mRNA was found to significantly increase stability in vitro compared to the wild-type sequence. CONCLUSION: Our findings show an association between RA and a previously unreported polymorphism in the hGR gene. This polymorphism increased stability of hGRbeta mRNA, which could contribute to an altered glucocorticoid sensitivity since the hGRbeta is thought to function as an inhibitor of hGRalpha activity.

Correlation between hippocampal BDNF mRNA expression and memory performance in senescent rats.

Brain-derived neurotrophic factor (BDNF) has been suggested to be involved in memory processes. In the present study, the association between memory impairment at senescence and BDNF expression in the hippocampus was studied in 30-32-month-old Brown Norway rats, which had been maternally deprived early in life. These animals display a bimodal distribution in their spatial learning ability: rats are either non-impaired or impaired. BDNF mRNA expression in the hippocampus was compared between non-impaired and impaired rats. We measured BDNF mRNA expression in the hippocampus 3 h after training in the Morris water maze ('post-training') and at 1 month after training ('basal'). Non-impaired performers displayed a higher post-training BDNF mRNA level in the CA1 region than impaired rats. In addition, only in the non-impaired performers post-training BDNF mRNA levels in CA1 and dentate gyrus were increased as compared to basal levels. Thus, we have demonstrated that in senescent rats, hippocampal BDNF expression in response to water maze training is associated with memory performance.

Comparison of serum S-100 protein levels following stroke and traumatic brain injury.

Temporal changes in serum S-100 protein levels were compared between patients with ischemic stroke, transient ischemic attack (TIA) and traumatic brain injury (TBI). In addition, S-100 levels were correlated with clinical severity and outcome. Measurements were done with a LIA-mat((R)) Sangtec((R)) 100 using an automated immunoluminometric assay. Serum S-100 was measured in 21 stroke patients, 18 TIA patients and ten TBI patients on days 1 (0-24 h), 2, 3, 4, 5 or 6 and 8 or 9. In a control group of 28 healthy volunteers one measurement was done. For the stroke and TIA patients, National Institutes of Health Stroke Scale (NIHSS) scores were obtained on admission and on day 10. For the TBI patients, Glasgow Coma Scale (GCS) scores were obtained on admission and Glasgow Outcome Scale (GOS) scores were obtained after 6 months. Changes in serum S-100 levels over the first 3 days were significantly different between stroke and TBI patients (P=0.014) and between stroke and TIA patients (P=0.006). Peak concentrations of S-100 were most often observed on day 3 or 4 after stroke and on day 1 or 2 after TBI. In the stroke patients individual S-100 peak levels correlated well with the NIHSS score on admission (r=0.58 P=0.014) and the change in NIHSS score between day 10 and day 1 (r=0.65, P=0. 005). In the TBI patients a good correlation between individual peak levels of S-100 and the GCS score on admission (r=-0.81, P=0.010) and the GOS score 6 months after the trauma was found (r=-0.87, P=0. 004). We conclude that there is a significant difference in temporal changes of S-100 levels between ischemic stroke and TBI patients. This suggests different pathophysiological mechanisms. The results of this study further confirm that peak levels of serum S-100 correlate with neurological deficit resulting from either stroke or TBI.

Corticosterone effects on BDNF expression in the hippocampus. Implications for memory formation.

The adrenal steroid corticosterone has profound effect on the structure and function of the hippocampus. Probably as a result of that, it modulates memory formation. In this review, the question is addressed if the corticosterone effects on memory processes are mediated by alterations in the expression of the neurotrophin Brain-Derived Neurotrophic Factor (BDNF) in the hippocampus. First, studies are described investigating the effect of corticosterone on BDNF expression in the rat hippocampus. It appears that corticosterone suppresses the BDNF expression at the mRNA and protein level in a subfield-specific way. Second, a model for the mechanism of action is proposed. In this model, activated mineralocorticoid and glucocorticoid receptors repress transcriptional activity of the BDNF promoter site-specifically via interaction with other transcription factors. Third, the implications for learning and memory are discussed. Studies show that during water maze training, corticosterone levels rise significantly, but the BDNF expression is not suppressed in any hippocampal subfield. Furthermore, high BDNF expression levels in specific subfields correlate with a good memory performance. Therefore, we suggest that the resistance of the hippocampal BDNF expression to suppression by corticosterone, as seen after water maze training, may contribute to an optimal memory performance.

Circadian variation in BDNF mRNA expression in the rat hippocampus.

BDNF mRNA levels in the hippocampus were studied during the circadian cycle by in situ hybridization. These levels display a circadian pattern, which may be due to regulation by corticosterone. This may have consequences for hippocampal functioning at different time points of the circadian cycle.

Corticosterone effects on BDNF mRNA expression in the rat hippocampus during morris water maze training.

Corticosterone and Brain-Derived Neurotrophic Factor (BDNF) have both been shown to be involved in spatial memory formation in rats. In the present study we have investigated the effect of corticosterone on hippocampal BDNF mRNA expression after training in the Morris water maze in young adult Wistar rats. Therefore, we first studied BDNF mRNA levels in the hippocampus in relation to corticosterone levels at several time points after 4 training trials in the Morris water maze. Corticosterone levels were significantly increased after this procedure, and hippocampal BDNF mRNA levels only displayed a minor change: an increase in CA1 at 1 hr after training. However, in a previous study we observed dramatically decreased hippocampal BDNF mRNA levels in dentate gyrus and CA1 at 3 hr after injection of corticosterone. In order to analyze this discrepancy, we subsequently investigated if hippocampal BDNF mRNA expression is affected by corticosterone at 3 hr after water maze training. Therefore, we incorporated ADX animals and ADX animals which were injected with corticosterone in our study. ADX animals which were subjected to water maze training displayed similar hippocampal BDNF mRNA levels 3 hr after training compared to control ADX animals. Furthermore, ADX animals which were injected with corticosterone showed decreased BDNF mRNA levels in all hippocampal regions compared to control ADX animals. Water maze training did not alter this effect. Thus, the increased corticosterone levels during water maze training do not affect hippocampal BDNF mRNA expression, although exogenous corticosterone is effective under these conditions. Hence, our results suggest that in this situation BDNF is resistant to regulation by endogenous corticosterone, which may be important for learning and memory processes.

Downregulation of BDNF mRNA and protein in the rat hippocampus by corticosterone.

Previously, we showed that corticosterone regulates BDNF mRNA levels in the hippocampus. In the present study, we have investigated the time course and dose-dependency of this effect at both the mRNA and the protein level. Corticosterone was administered in doses of 30 and 1000 microgram/kg b.w. subcutaneously to adrenalectomized animals. At 3, 6, 12 and 24 h after administration BDNF and trkB mRNA levels in hippocampal subfields were measured by in situ hybridization. Our results show a dose-dependent decrease in BDNF mRNA in dentate gyrus and CA1 at 3 h. After the high dose, this decrease was 70% and 40% respectively. In addition, ELISA was performed to study if this downregulation is also detectable at the protein level. Hippocampal tissue was used from adrenalectomized animals which had received 1000 microgram/kg b.w. corticosterone 4 and 6 h before decapitation. At both time points, a decrease in BDNF protein was observed; 17% at 4 h and 14% at 6 h after corticosterone, as compared to the vehicle injected controls. TrkB mRNA levels were not affected by corticosterone. However, between 6 and 24 h after treatment, increases in trkB mRNA were observed. In conclusion, we have found a transient, dose-dependent decrease in BDNF mRNA and protein in the hippocampus, which may underly changes in neuronal plasticity in the hippocampus after short-term changes in corticosterone concentrations.

Corticosterone regulates expression of BDNF and trkB but not NT-3 and trkC mRNA in the rat hippocampus.

Corticosterone has profound effects on growth, differentiation, and synaptic transmission of hippocampal neurons by activation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). In the present study we tested if neurotrophins can be implicated in these effects. For this purpose we injected 30, 300, and 1,000 microg corticosterone s.c. (per kg body weight) in adrenalectomized rats and measured the mRNA levels of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase (trk)B, neurotrophin (NT)-3, and trkC in hippocampal cell fields at 6 hr after steroid administration by in situ hybridization. NT-3 and trkC mRNA did not show significant changes in any hippocampal region after the various doses of corticosterone. BDNF mRNA decreased after corticosterone administration dose dependently, resulting in a maximal suppression of 35, 20, and 50% in dentate gyrus, CA3, and CA1, respectively. Interestingly, trkB responded to corticosterone in an inverted U-shaped fashion in CA3 and dentate gyrus: the low dose of corticosterone increased trkB mRNA expression in both regions by approximately 30%, while the effect of the two higher doses was not different from the vehicle injected controls. In conclusion, we found differential effects of low and high doses of corticosterone on BDNF and trkB expression in hippocampus, which suggests involvement of a coordinated MR- and GR-mediated action.

Molecular dissection of corticosteroid action in the rat hippocampus. Application of the differential display techniques.

Both adrenal steroids and glutamate are crucial for hippocampal cell viability. In order to identify adrenal steroid- and glutamate-responsive genes controlling hippocampal cell viability, we have used the PCR-based differential display method. We have described the characteristics of this technique and how it can be automated. Using differential display, we have identified a number of rat hippocampal genes of which the expression is affected by a combination of the glutamate analog kainic acid and adrenalectomy. Administration of kainic acid or removal of the adrenals alone gave a limited number of differentially displayed genes. Therefore, our results indicate that the main mode of corticosteroid receptor-controlled gene expression in the hippocampus is interaction with other transcription factors (e.g., CREB, AP-1) and not by binding to hormone-responsive elements of corticosterone-specific genes. Characterization by multiplex PCR experiments of a differentially displayed fragment of which the expression is increased by the combination of kainic acid and adrenalectomy confirmed our differential display results. Further characterization by DNA sequence analysis of the corresponding full-length cDNA clone revealed a gene product with 91.4% sequence identity with the mouse transcription factor KROX-20, suggesting that we have cloned the rat homolog. This finding suggests a role of KROX-20 in corticosteroid- and kainic acid-controlled hippocampal plasticity.