Jürgen Zschocke

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.

Antidepressants inhibit DNA methyltransferase 1 through reducing G9a levels

The discovery of epigenetic processes as possible pivotal regulatory mechanisms in psychiatric diseases raised the question of how psychoactive drugs may impact the epigenetic machinery. In the present study we set out to explore the specificity and the mode of action of the reported inhibitory effect of the TCA (tricyclic antidepressant) amitriptyline on DNMT (DNA methyltransferase) activity in primary astrocytes from the rat cortex. We found that the impact on DNMT was shared by another TCA, imipramine, and by paroxetine, but not by venlafaxine or the mood stabilizers carbamazepine and valproic acid. DNMT activity in subventricular neural stem cells was refractory to the action of ADs (antidepressants). Among the established DNMTs, ADs primarily targeted DNMT1. The reduction of enzymatic DNMT1 activity was neither due to reduced DNMT1 expression nor due to direct drug interference. We tested putative DNMT1-inhibitory mechanisms and discovered that a known stimulator of DNMT1, the histone methyltransferase G9a, exhibited decreased protein levels and interactions with DNMT1 upon AD exposure. Adding recombinant G9a completely reversed the AD repressive effect on DNMT1 function. In conclusion, the present study presents a model where distinct ADs affect DNMT1 activity via G9a with important repercussions for possible novel treatment regimes.

DNA methylation dependent silencing of the human glutamate transporter EAAT2 gene in glial cells

Glutamate is the major excitatory neurotransmitter in the CNS that is cleared from the extracellular space by a family of high‐affinity glutamate transporters. The astroglial glutamate transporter EAAT2 is thought to carry out the uptake of the vast quantity of glutamate, and dysregulation of EAAT2 expression is involved in the pathogenesis of neurological disorders with marked excitotoxic components. Here, we present a novel epigenetic mechanism by which the human EAAT2 gene is kept in a silent state. Sequence inspection identified a classical CpG island at the EAAT2 promoter. Bisulfite analysis of the DNA methylation profile revealed that lack of EAAT2 expression in human glioma cell lines was associated with a densely methylated EAAT2 promoter. In contrast, EAAT2 positive normal human brain tissue used as reference displayed hypomethylation of the same promoter regions. In vitro methylation of EAAT2 promoter sequences indeed altered the binding properties of nuclear factors to the respective DNA sites as illustrated by electrophoretic mobility shift assay. Moreover, we observed a reduced activity of a methylated EAAT2 promoter construct as compared to the unmethylated control, both in a human glioma cell line and rodent primary astrocytes. Further supporting a role of DNA methylation for EAAT2 silencing, inhibition of DNA methyltransferases robustly enhanced EAAT2 mRNA transcription in several cell lines tested. In conclusion, the idea is put forward of an epigenetic mode of EAAT2 regulation based on the differential methylation of the gene promoter.

XAP2 inhibits glucocorticoid receptor activity in mammalian cells.

XAP2 is member of a protein family sharing the TPR protein interaction motif. It displays close homology to the immunophilins FKBP51 and FKBP52 that act via the Hsp90 folding machinery to regulate the glucocorticoid receptor (GR). We show that XAP2 inhibits GR by reducing its responsiveness to hormone in transcriptional activation. The effect of XAP2 on GR requires its interaction with Hsp90 through the TPR motif. The PPIase‐like region turned out to be enzymatically inactive. Thus, PPIase activity is not essential for the action of XAP2 on GR, similarly to FKBP51 and FKBP52.

Chaperoning epigenetics: FKBP51 decreases the activity of DNMT1 and mediates epigenetic effects of the antidepressant paroxetine

Chaperone switching at the kinase CDK5 mediates epigenetic effects of antidepressants. Antidepressants chaperone DNA methylation Epigenetic changes are associated with depression. Some depressed patients have increased DNA methylation and decreased expression of the gene encoding BDNF, a secreted factor important for synaptic plasticity. Rein et al. found that some antidepressants inhibit epigenetic changes by causing a switch in chaperone binding to the DNA methyltransferase DNMT1. The chaperones FKBP51 and FKBP52 competed for binding to CDK5, a kinase that activates DNMT1. The authors found that cells from depressed patients or cultured mouse astrocytes exposed to the antidepressant paroxetine favored the FKBP51-CDK5 interaction, resulting in reduced activity of DNMT1 and DNA methylation, and increased the expression of BDNF. These effects of paroxetine on patient blood cells isolated before therapy correlated with a positive clinical response to antidepressants, suggesting that a simple blood test may aid in personalizing treatment for depression. Epigenetic processes, such as DNA methylation, and molecular chaperones, including FK506-binding protein 51 (FKBP51), are independently implicated in stress-related mental disorders and antidepressant drug action. FKBP51 associates with cyclin-dependent kinase 5 (CDK5), which is one of several kinases that phosphorylates and activates DNA methyltransferase 1 (DNMT1). We searched for a functional link between FKBP51 (encoded by FKBP5) and DNMT1 in cells from mice and humans, including those from depressed patients, and found that FKBP51 competed with its close homolog FKBP52 for association with CDK5. In human embryonic kidney (HEK) 293 cells, expression of FKBP51 displaced FKBP52 from CDK5, decreased the interaction of CDK5 with DNMT1, reduced the phosphorylation and enzymatic activity of DNMT1, and diminished global DNA methylation. In mouse embryonic fibroblasts and primary mouse astrocytes, FKBP51 mediated several effects of paroxetine, namely, decreased the protein-protein interactions of DNMT1 with CDK5 and FKBP52, reduced phosphorylation of DNMT1, and decreased the methylation and increased the expression of the gene encoding brain-derived neurotrophic factor (Bdnf). In human peripheral blood cells, FKBP5 expression inversely correlated with both global and BDNF methylation. Peripheral blood cells isolated from depressed patients that were then treated ex vivo with paroxetine revealed that the abundance of BDNF positively correlated and phosphorylated DNMT1 inversely correlated with that of FKBP51 in cells and with clinical treatment success in patients, supporting the relevance of this FKBP51-directed pathway that prevents epigenetic suppression of gene expression.

The CpG island shore of the GLT-1 gene acts as a methylation-sensitive enhancer.

Astrocytic lineage commitment and brain region‐dependent specialization of glia are partly ascribed to epigenetic processes. Clearance of glutamate is an essential task, which astrocytes assume in a temporal‐spatial fashion by distinct glutamate transporter expression. Glutamate transporter subtype 1 (GLT‐1) is predominant in cortex (CTX), while it plays an inferior role in cerebellum (CER). Here, we set out to identify regulatory elements that could account for the differences in brain region‐specific activity as well as response to dexamethasone (DEX) or epigenetic factors. We found a distal promoter element at the shore of the CpG island exhibiting enhancer function in response to DEX in reporter gene assays. This shore region showed slight enrichment in repressive trimethyl‐histone H3 (Lys27) and under‐representation of acetyl‐histone H4 (H4ac) marks in DEX nonresponsive CER astrocytes as determined by chromatin immunoprecipitation. In addition, CpG sites of the shore region displayed higher methylation in CER than in CTX cells. Targeted in vitro methylation of CpG sites within the shore abrogated the stimulatory effects of DEX. Interestingly, the shore was characterized by a pronounced epigenetic plasticity in CTX cells since DEX exposure elicited an increase of H4ac in CTX in comparison to DEX nonresponsive CER. The transcriptional activity of this region was also affected by histone deacetylase inhibitors in a methylation‐ and brain region‐dependent manner. Together, our study highlights the impact of an epigenetically adaptive DNA element of the GLT‐1 promoter being decisive for brain region‐specific activity and reactivity.

FKBP51 inhibits GSK3β and augments the effects of distinct psychotropic medications

Psychotropic medications target glycogen synthase kinase 3β (GSK3β), but the functional integration with other factors relevant for drug efficacy is poorly understood. We discovered that the suggested psychiatric risk factor FK506 binding protein 51 (FKBP51) increases phosphorylation of GSK3β at serine 9 (pGSK3βS9). FKBP51 associates with GSK3β mainly through its FK1 domain; furthermore, it also changes GSK3βs heterocomplex assembly by associating with the phosphatase PP2A and the kinase cyclin-dependent kinase 5. FKBP51 acts through GSK3β on the downstream targets Tau, β-catenin and T-cell factor/lymphoid enhancing factor (TCF/LEF). Lithium and the antidepressant (AD) paroxetine (PAR) functionally synergize with FKBP51, as revealed by reporter gene and protein association analyses. Deletion of FKBP51 blunted the PAR- or lithium-induced increase in pGSK3βS9 in cells and mice and attenuated the behavioral effects of lithium treatment. Clinical improvement in depressive patients was predicted by baseline GSK3β pathway activity and by pGSK3βS9 reactivity to ex vivo treatment of peripheral blood mononuclear lymphocytes with lithium or PAR. In sum, FKBP51-directed GSK3β activity contributes to the action of psychotropic medications. Components of the FKBP51–GSK3β pathway may be useful as biomarkers predicting AD response and as targets for the development of novel ADs.

Hsp70 Cochaperones HspBP1 and BAG-1M Differentially Regulate Steroid Hormone Receptor Function

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

Antidepressants encounter autophagy in neural cells

The prevailing view of antidepressants’ (ADs) mode of action primarily focuses on their impact on neurotransmitter circuits, since the corresponding transporters and receptors are common targets of ADs. However, mounting evidence points to additional target structures, which may either support the beneficial effects or account for undesired side effects of ADs. Recently, we analyzed the influence of three ADs of different classes on autophagy-related processes in primary astrocytes and neurons. While amitriptyline (AMI) and citalopram (CIT) upregulate the expression of autophagic markers such as LC3B-II or Beclin 1, venlafaxine fails to exert these effects. Autophagy triggered by AMI and CIT is functional in terms of autophagic flux, and is partially mediated by class III PtdIns 3-kinase- and ROS dependent-pathways. Together, our study’s results highlight a novel mode of action of ADs beyond monoaminergic neurotransmission.

Functional interaction of estrogen receptor α and caveolin isoforms in neuronal SK-N-MC cells

Estrogen receptors (ERs) are expressed in neuronal cells and exhibit a wide variety of activities in the central nervous system. The actions of ERs are regulated in a hormone-dependent manner as well as by a number of co-activators and -repressors. A recently identified co-activator of ERalpha is caveolin-1 which has been shown to mediate the ligand-independent activation of this steroid receptor. In the present study we have demonstrated that neuronal SK-N-MC cells lacking functional ERalpha show high levels of caveolin-1/-2 specific transcripts and proteins. Ectopic expression of ERalpha in SK-N-MC cells leads to the transcriptional suppression of caveolin-1 and -2 genes. This silencing event is accompanied by changes in the methylation pattern of the caveolin-1 promoter. Certain CpG dinucleotides were methylated in the caveolin-1 promoter region of the SK-ERalpha cells whereas the same sites were non-methylated in control SK-N-MC cells, implicating a gene silencing mechanism including hypermethylation of DNA. In addition, inhibitors of methyltransferases or histone deacetylases, enzymes involved in the establishment and maintenance of silenced chromatin status, partially restored caveolin transcription in SK-ERalpha cells. In conclusion, our observations provide a possible mechanism of negative feedback regulation of ERalpha co-activator caveolin by the steroid receptor itself in this cellular model.