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The brain is the central coordinator of the human stress reaction. At the same time, peripheral endocrine and neural stress signals act on the brain modulating brain function. Here, three experimental studies are presented demonstrating this dual role of the brain in stress. Study I shows that centrally acting insulin, an important regulator of energy homeostasis, attenuates the stress related cortisol secretion. Studies II and III show that specific components of the stress reaction modulate learning and memory retrieval, two important aspects of higher-order brain function.
Aggression is one of the most researched topics in psychology. This is understandable, since aggression behavior does a lot of harm to individuals and groups. A lot is known already about the biology of aggression, but one system that seems to be of vital importance in animals has largely been overlooked: the hypothalamic-pituitary-adrenal (HPA) axis. Menno Kruk and Jószef Haller and their research teams developed rodent models of adaptive, normal, and abnormal aggressive behavior. They found the acute HPA axis (re)activity, but also chronic basal levels to be causally relevant in the elicitation and escalation of aggressive behavior. As a mediating variable, changes in the processing of relevant social information is proposed, although this could not be tested in animals. In humans, not a lot of research has been done, but there is evidence for both the association between acute and basal cortisol levels in (abnormal) aggression. However, not many of these studies have been experimental of nature. rnrnOur aim was to add to the understanding of both basal chronic levels of HPA axis activity, as well as acute levels in the formation of aggressive behavior. Therefore, we did two experiments, both with healthy student samples. In both studies we induced aggression with a well validated paradigm from social psychology: the Taylor Aggression Paradigm. Half of the subjects, however, only went through a non-provoking control condition. We measured trait basal levels of HPA axis activity on three days prior. We took several cortisol samples before, during, and after the task. After the induction of aggression, we measured the behavioral and electrophysiological brain response to relevant social stimuli, i.e., emotional facial expressions embedded in an emotional Stroop task. In the second study, we pharmacologically manipulated cortisol levels 60min before the beginning of the experiment. To do that, half of the subjects were administered 20mg of hydrocortisone, which elevates circulating cortisol levels (cortisol group), the other half was administered a placebo (placebo group). Results showed that acute HPA axis activity is indeed relevant for aggressive behavior. We found in Study 1 a difference in cortisol levels after the aggression induction in the provoked group compared to the non-provoked group (i.e., a heightened reactivity of the HPA axis). However, this could not be replicated in Study 2. Furthermore, the pharmacological elevation of cortisol levels led to an increase in aggressive behavior in women compared to the placebo group. There were no effects in men, so that while men were significantly more aggressive than women in the placebo group, they were equally aggressive in the cortisol group. Furthermore, there was an interaction of cortisol treatment with block of the Taylor Aggression Paradigm, in that the cortisol group was significantly more aggressive in the third block of the task. Concerning basal HPA axis activity, we found an effect on aggressive behavior in both studies, albeit more consistently in women and in the provoked and non-provoked groups. However, the effect was not apparent in the cortisol group. After the aggressive encounter, information processing patterns were changed in the provoked compared to the non-provoked group for all facial expressions, especially anger. These results indicate that the HPA axis plays an important role in the formation of aggressive behavior in humans, as well. Importantly, different changes within the system, be it basal or acute, are associated with the same outcome in this task. More studies are needed, however, to better understand the role that each plays in different kinds of aggressive behavior, and the role information processing plays as a possible mediating variable. This extensive knowledge is necessary for better behavioral interventions.
The glucocorticoid (GC) cortisol, main mediator of the hypothalamic-pituitary-adrenal axis, has many implications in metabolism, stress response and the immune system. GC function is mediated mainly via the glucocorticoid receptor (GR) which binds as a transcription factor to glucocorticoid response elements (GREs). GCs are strong immunosuppressants and used to treat inflammatory and autoimmune diseases. Long-term usage can lead to several irreversible side effects which make improved understanding indispensable and warrant the adaptation of current drugs. Several large scale gene expression studies have been performed to gain insight into GC signalling. Nevertheless, studies at the proteomic level have not yet been made. The effects of cortisol on monocytes and macrophages were studied in the THP-1 cell line using 2D fluorescence difference gel electrophoresis (2D DIGE) combined with MALDI-TOF mass spectrometry. More than 50 cortisol-modulated proteins were identified which belonged to five functional groups: cytoskeleton, chaperones, immune response, metabolism, and transcription/translation. Multiple GREs were found in the promoters of their corresponding genes (+10 kb/-0.2 kb promoter regions including all alternative promoters available within the Database for Transcription Start Sites (DBTSS)). High quality GREs were observed mainly in cortisol modulated genes, corroborating the proteomics results. Differential regulation of selected immune response related proteins were confirmed by qPCR and immuno-blotting. All immune response related proteins (MX1, IFIT3, SYWC, STAT3, PMSE2, PRS7) which were induced by LPS were suppressed by cortisol and belong mainly to classical interferon target genes. Mx1 has been selected for detailed expression analysis since new isoforms have been identified by proteomics. FKBP51, known to be induced by cortisol, was identified as the strongest differentially expressed protein and contained the highest number of strict GREs. Genomic analysis of five alternative FKBP5 promoter regions suggested GC inducibility of all transcripts. 2D DIGE combined with 2D immunoblotting revealed the existence of several previously unknown FKBP51 isoforms, possibly resulting from these transcripts. Additionally multiple post-translational modifications were found, which could lead to different subcellular localization in monocytes and macrophages as seen by confocal microscopy. Similar results were obtained for the different cellular subsets of human peripheral blood mononuclear cells (PBMCs). FKBP51 was found to be constitutively phosphorylated with up to 8 phosphosites in CD19+ B lymphocytes. Differential Co-immunoprecipitation for cytoplasm and nucleus allowed us to identify new potential interaction partners. Nuclear FKBP51 was found to interact with myosin 9, whereas cytosolic FKBP51 with TRIM21 (synonym: Ro52, Sjögren`s syndrome antigen). The GR has been found to interact with THOC4 and YB1, two proteins implicated in mRNA processing and transcriptional regulation. We also applied proteomics to study rapid non-genomic effects of acute stress in a rat model. The nuclear proteome of the thymus was investigated after 15 min restraint stress and compared to the non-stressed control. Most of the identified proteins were transcriptional regulators found to be enriched in the nucleus probably to assist gene expression in an appropriate manner. The proteomic approach allowed us to further understand the cortisol mediated response in monocytes/macrophages. We identified several new target proteins, but we also found new protein variants and post-translational modifications which need further investigation. Detailed study of FKBP51 and GR indicated a complex regulation network which opened a new field of research. We identified new variants of the anti-viral response protein MX1, displaying differential expression and phosphorylation in the cellular compartments. Further, proteomics allowed us to follow the very early effects of acute stress, which happen prior to gene expression. The nuclear thymocyte proteome of restraint stressed rats revealed an active preparation for subsequent gene expression. Proteomics was successfully applied to study differential protein expression, to identify new protein variants and phosphorylation events as well as to follow translocation. New aspects for future research in the field of cortisol-mediated immune modulation have been added.
Stress represents a significant problem for Western societies inducing costs as high as 3-4 % of the European gross national products, a burden that is continually increasing (WHO Briefing, EUR/04/5047810/B6). The classical stress response system is the hypothalamic-pituitary-adrenal (HPA) axis which acts to restore homeostasis after disturbances. Two major components within the HPA axis system are the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Cortisol, released from the adrenal glands at the end of the HPA axis, binds to MRs and with a 10 fold lower affinity to GRs. Both, impairment of the HPA axis and an imbalance in the MR/GR ratio enhances the risk for infection, inflammation and stress related psychiatric disorders. Major depressive disorder (MDD) is characterised by a variety of symptoms, however, one of the most consistent findings is the hyperactivity of the HPA axis. This may be the result of lower numbers or reduced activity of GRs and MRs. The GR gene consists of multiple alternative first exons resulting in different GR mRNA transcripts whereas for the MR only two first exons are known to date. Both, the human GR promoter 1F and the homologue rat Gr promoter 1.7 seem to be susceptible to methylation during stressful early life events resulting in lower 1F/1.7 transcript levels. It was proposed that this is due to methylation of a NGFI-A binding site in both, the rat promoter 1.7 and the human promoter 1F. The research presented in this thesis was undertaken to determine the differential expression and methylation patterns of GR and MR variants in multiple areas of the limbic brain system in the healthy and depressed human brain. Furthermore, the transcriptional control of the GR transcript 1F was investigated as expression changes of this transcript were associated with MDD, childhood abuse and early life stress. The role of NGFI-A and several other transcription factors on 1F regulation was studied in vitro and the effect of Ngfi-a overexpression on the rat Gr promoter 1.7 in vivo. The susceptibility to epigenetic programming of several GR promoters was investigated in MDD. In addition, changes in methylation levels have been determined in response to a single acute stressor in rodents. Our results showed that GR and MR first exon transcripts are differentially expressed in the human brain, but this is not due to epigenetic programming. We showed that NGFI-A has no effect on endogenous 1F/1.7 expression in vitro and in vivo. We provide evidence that the transcription factor E2F1 is a major element in the transcriptional complex necessary to drive the expression of GR 1F transcripts. In rats, highly individual methylation patterns in the paraventricular nucleus of the hypothalamus (PVN) suggest that this is not related to the stressor but can rather be interpreted as pre-existing differences. In contrast, the hippocampus showed a much more uniform epigenetic status, but still is susceptible to epigenetic modification even after a single acute stress suggesting a differential "state‟ versus "trait‟ regulation of the GR gene in different brain regions. The results of this thesis have given further insight in the complex transcriptional regulation of GR and MR first exons in health and disease. Epigenetic programming of GR promoters seems to be involved in early life stress and acute stress in adult rats; however, the susceptibility to methylation in response to stress seems to vary between brain regions.