Theo Rein

Polymorphisms in FKBP5 are associated with increased recurrence of depressive episodes and rapid response to antidepressant treatment

The stress hormone–regulating hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the causality as well as the treatment of depression. To investigate a possible association between genes regulating the HPA axis and response to antidepressants and susceptibility for depression, we genotyped single-nucleotide polymorphisms in eight of these genes in depressed individuals and matched controls. We found significant associations of response to antidepressants and the recurrence of depressive episodes with single-nucleotide polymorphisms in FKBP5, a glucocorticoid receptor–regulating cochaperone of hsp-90, in two independent samples. These single-nucleotide polymorphisms were also associated with increased intracellular FKBP5 protein expression, which triggers adaptive changes in glucocorticoid receptor and, thereby, HPA-axis regulation. Individuals carrying the associated genotypes had less HPA-axis hyperactivity during the depressive episode. We propose that the FKBP5 variant–dependent alterations in HPA-axis regulation could be related to the faster response to antidepressant drug treatment and the increased recurrence of depressive episodes observed in this subgroup of depressed individuals. These findings support a central role of genes regulating the HPA axis in the causality of depression and the mechanism of action of antidepressant drugs.

Allele-specific FKBP5 DNA demethylation mediates gene–childhood trauma interactions

Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma–dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders.

Gene Expression Patterns Associated with Posttraumatic Stress Disorder Following Exposure to the World Trade Center Attacks

BACKGROUND Although genetic risk factors for posttraumatic stress disorder (PTSD) in similarly traumatized cohorts can be confounded with risk for type of exposure, the primary risk for exposure to the 9/11 attack on New York City was proximity, allowing study of PTSD risk in a sample that is not confounded by exposure-related risk. METHODS Thirty-five Caucasians (15 with PTSD, stratified for exposure, age, and gender) were selected from a population-representative sample of persons exposed to the attack from which longitudinal data had been collected in four previous waves. Whole blood gene expression and cortisol levels were obtained. RESULTS Seventeen probe sets were differentially expressed in PTSD. Identified genes were generally involved in hypothalamic-pituitary-adrenal (HPA) axis, signal transduction, or brain and immune cell function. FKBP5, a modulator of glucocorticoid receptor (GR) sensitivity, showed reduced expression in PTSD, consistent with enhanced GR responsiveness. FKBP5 expression was predicted by cortisol when entered with PTSD severity in regression analysis. Quantitative polymerase chain reaction confirmed significant reductions in FKBP5. Also less expressed in PTSD were STAT5B, a direct inhibitor of GR, and major histocompatibility complex (MHC) Class II. CONCLUSIONS Consistent with observations of HPA axis dysfunction in PTSD, several genes involved in glucocorticoid signaling are differentially expressed among those with current PTSD.

FK506 binding protein 5 shapes stress responsiveness: modulation of neuroendocrine reactivity and coping behavior.

BACKGROUND The Hsp90 cochaperone FK506 binding protein 5 (FKBP5) is an established regulator of the glucocorticoid receptor (GR), and numerous genetic studies have linked it to stress-related diseases such as major depression or posttraumatic stress disorder. However, translational studies including genetic animal models are lacking. METHODS Mice deficient of FKBP5 were generated and analyzed in comparison with wildtype littermates. They were subjected to several test paradigms characterizing their emotionality, stress reactivity, and coping behavior as well as hypothalamus-pituitary-adrenal axis function and regulation. Moreover, protein expression of GR and FKBP5 was determined in different brain structures 8 days after stress exposure. The combined dexamethasone/corticotropin-releasing hormone test was performed both in mice and healthy human subjects of different FKBP5 genotypes. The GR function was evaluated by reporter gene assays. RESULTS Under basal conditions, deletion of FKBP5 did not change exploratory drive, locomotor activity, anxiety-related behavior, stress-coping, or depression-like behavior. After exposure to different acute stressors of sufficient intensity, however, it led to a more active coping behavior. Moreover, loss of FKBP5 decreased hypothalamus-pituitary-adrenal axis reactivity and GR expression changes in response to stressors. In mice and humans, the FKBP5 genotype also determined the outcome of the dexamethasone/corticotropin-releasing hormone test. CONCLUSIONS This study in mice and humans presents FKBP5 as a decisive factor for the physiological stress response, shaping neuroendocrine reactivity as well as coping behavior. This lends strong support to the concept emerging from human studies of FKBP5 as important factor governing gene-environment interactions relevant for the etiology of affective disorders.

FKBP51 and FKBP52 in signaling and disease

FKBP51 and FKBP52 are diverse regulators of steroid hormone receptor signaling, including receptor maturation, hormone binding and nuclear translocation. Although structurally similar, they are functionally divergent, which is largely attributed to differences in the FK1 domain and the proline-rich loop. FKBP51 and FKBP52 have emerged as likely contributors to a variety of hormone-dependent diseases, including stress-related diseases, immune function, reproductive functions and a variety of cancers. In addition, recent studies have implicated FKBP51 and FKBP52 in Alzheimers disease and other protein aggregation disorders. This review summarizes our current understanding of FKBP51 and FKBP52 interactions within the receptor-chaperone complex, their contributions to health and disease, and their potential as therapeutic targets for the treatment of these diseases.

Valproate and Amitriptyline Exert Common and Divergent Influences on Global and Gene Promoter-Specific Chromatin Modifications in Rat Primary Astrocytes

Aberrant biochemical processes in the brain frequently go along with subtle shifts of the cellular epigenetic profile that might support the pathogenic progression of psychiatric disorders. Although recent reports have implied the ability of certain antidepressants and mood stabilizers to modulate epigenetic parameters, studies comparing the actions of these compounds under the same conditions are lacking. In this study, we screened amitriptyline (AMI), venlafaxine, citalopram, as well as valproic acid (VPA), carbamazepine, and lamotrigine for their potential actions on global and local epigenetic modifications in rat primary astrocytes. Among all drugs, VPA exposure evoked the strongest global chromatin modifications, including histone H3/H4 hyperacetylation, 2MeH3K9 hypomethylation, and DNA demethylation, as determined by western blot and luminometric methylation analysis, respectively. CpG demethylation occurred independently of DNA methyltransferase (DNMT) suppression. Strikingly, AMI also induced slight cytosine demethylation, paralleled by the reduction in DNMT enzymatic activity, without affecting the global histone acetylation status. Locally, VPA-induced chromatin modifications were reflected at the glutamate transporter (GLT-1) promoter as shown by bisulfite sequencing and acetylated histone H4 chromatin immunoprecipitation analysis. Distinct CpG sites in the distal part of the GLT-1 promoter were demethylated and enriched in acetylated histone H4 in response to VPA. For the first time, we could show that these changes were associated with an enhanced transcription of this astrocyte-specific gene. In contrast, AMI failed to stimulate GLT-1 transcription and to alter promoter methylation levels. In conclusion, VPA and AMI globally exerted chromatin-modulating activities using different mechanisms that divergently precipitated at an astroglial gene locus.

Cross-talk of vitamin D and glucocorticoids in hippocampal cells

There is growing evidence for a role of vitamin D3 signalling in the brain. In this study, we investigated the influence of vitamin D3, in combination with glucocorticoids, on differentiation of the hippocampal progenitor line HIB5, as well as survival of rat primary hippocampal cells. In HIB5, pre‐treatment with dexamethasone (Dex) alone inhibited neurite outgrowth and abolished activation of the mitogen‐activated protein kinase (MAPK) pathway during platelet‐derived growth factor (PDGF)‐induced differentiation, consistent with previous findings. Interestingly, pre‐treating HIB5 with vitamin D3 significantly reduced these effects of Dex and, in addition, lowered the transactivational function of the glucocorticoid receptor (GR) in transient reporter gene assays. A further impact of vitamin D3 on glucocorticoid effects was observed in a rat primary hippocampal culture known to be particularly sensitive to prolonged GR activation. In this model, Dex induced considerable cell death after 72 h of exposure in vitro. However, 24 h of pre‐treatment with low doses of vitamin D3 substantially reduced the degree of Dex‐induced apoptosis in primary hippocampal cells. Taken together, our experiments demonstrate a cross‐talk between vitamin D3 and glucocorticoids in two hippocampal models, a feature that may have important implications in disorders with dysregulated glucocorticoid signalling, including major depression.

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co‐chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co‐chaperone Tpr2, which contains two chaperone‐binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co‐chaperones, Tpr2 induces ATP‐independent dissociation of Hsp90 but not of Hsp70 from chaperone–substrate complexes. Excess Tpr2 inhibits the Hsp90‐dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.

Genetic markers for PTSD risk and resilience among survivors of the World Trade Center attacks

We have previously reported the differential expression of 17 probe sets in survivors of the 9/11 attacks with current posttraumatic stress disorder (PTSD) compared to similarly exposed survivors with no lifetime PTSD. The current study presents an expanded analysis of these subjects, including genotype at FKBP5, a modulator of glucocorticoid receptor (GR) sensitivity. It includes data from additional subjects who developed PTSD following 9/11 but then recovered, distinguishing expression profiles associated with risk for developing PTSD, resilience, and symptom recovery. 40 Caucasians (20 with and 20 without PTSD, matched for exposure, age, and gender) were selected from a population-representative sample of persons exposed to the 9/11 attacks from which longitudinal data had been collected in four previous waves. Whole blood gene expression and cortisol levels were obtained and genome-wide gene expression was analyzed. 25 probe sets were differentially expressed in PTSD. Identified genes were generally involved in hypothalamic-pituitary-adrenal axis, signal transduction, or in brain and immune cell function. STAT5B, a direct inhibitor of GR, and nuclear factor I/A, both showed reduced expression in PTSD. Comparison of lifetime versus current PTSD identified overlapping genes with altered expression suggesting enduring markers, while some markers present only in current PTSD may reflect state measures. As a follow-up, direct comparisons of expression in current PTSD, lifetime-only PTSD, and control groups identified FKBP5 and MHC Class II as state markers, and also identified several trait markers. An analysis of indirect effects revealed that homozygosity for any of 4 PTSD risk-related polymorphisms at FKBP5 predicted FKBP5 expression, which mediated indirect effects of genotype on plasma cortisol and PTSD severity.

The activated glucocorticoid receptor inhibits the transcription factor T-bet by direct protein-protein interaction

Glucocorticoids (GCs) immunosuppression acts via regulation of several transcription factors (TF), including activating protein (AP)‐1, NF‐κB, and NFAT. GCs inhibit Th1 cytokines and promote a shift toward Th2 differentiation. Th1 phenotype depends on TF T‐bet. In this study, we examined GC regulation of T‐bet. We found that GCs inhibit T‐bet transcriptional activity. We show that glucocorticoid receptor (GR) physically interacts with T‐bet both in transfected cell lines and in primary splenocyte cultures with endogenous GR and T‐bet. This interaction also blocks GR‐dependent transcription. We show both in vitro and in vivo at endogenous binding sites that the mechanism underlying T‐bet inhibition further involves reduction of T‐bet binding to DNA. Using specific mutations of GR, we demonstrate that the first zinc finger region of GR is required for T‐bet inhibition. GCs additionally inhibit T‐bet both at mRNA and protein expression levels, revealing another layer of GR action on T‐bet. Finally, we examined the functional consequences of GR/T‐bet interaction on IFN‐gamma, showing that GCs inhibit transcriptional activity of T‐bet on its promoter. In view of the crucial role of T‐bet in T cell differentiation and inflammation, we propose that GR inhibitory interaction with T‐bet may be an important mechanism underlying the immunosuppressive properties of GCs.—Liberman, A. C., Refojo, D., Druker, J., Toscano, M., Rein, T., Holsboer, F., Arzt, E. The activated glucocorticoid receptor inhibits the transcription factor T‐bet by direct protein‐protein interaction. FASEB J. 21, 1177–1188 (2007)