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Adolescence is a key phase of development for perturbations in fear extinction, with inability to adequately manage fear a potent factor for developing psychiatric disorders in adulthood. However, while behavioral correlates of adolescent fear regulation are established to a degree, molecular mediators of extinction learning in adolescence remain largely unknown. In this study, we observed fear acquisition and fear extinction (across 4 and 7 days) of adolescent and adult mice of both sexes and investigated how hippocampal levels of different plasticity markers relate to extinction learning. While fear was acquired evenly in males and females of both ages, fear extinction was found to be impaired in adolescent males. We also observed lower levels of GluA1, GLUN2A and GLUN2B subunits in male adolescents following fear acquisition, with an increase in their expression, as well as the activity of Erk-mTOR pathway over subsequent extinction sessions, which was paralleled with improved extinction learning. On the other hand, we detected no changes in plasticity-related proteins after fear acquisition in females, with alterations in GluA1, GluA4 and GLUN2B levels across fear extinction sessions. Additionally, we did not discern any pattern regarding the Erk-mTOR activity in female mice associated with their extinction performance. Overall, our research identifies sex-specific synaptic properties in the hippocampus that underlie developmentally regulated differences in fear extinction learning. We also point out hippocampal NMDA-Erk-mTOR signaling as the driving force behind successful fear extinction in male adolescents, highlighting this pathway as a potential therapeutic target for fear-related disorders in the adolescent population. © 2022 The Authors

Adolescent stress predisposes individuals to increased risk for anxiety and depression in adulthood. The stress response is mediated by the glucocorticoid receptor (GR) via regulation of GR-responsive genes involved in brain reaction to stress. Although dysregulation of GR in depression is well documented, this is the first study investigating the role of GRα isoforms in pathogenesis of depression. We exposed adolescent male and female C57BL/6J mice to chronic unpredictable stress (CUS) for 12 days starting at postnatal day 28 (PND28). Tests evaluating anxiety and depressive-like behaviors were performed at PND70. We analyzed corticosterone concentrations in serum, levels of GRα isoforms (95, 67, 50, 40, and 25 kDa), and mRNA levels of GR-responsive genes (GR, FKBP5, BDNF, and IL-1β) in the hippocampus and the prefrontal cortex (PFC). CUS increased anxiety and depressive-like behavior in adult animals of both sexes, but did not affect corticosterone serum levels, 95 and 67 kDa GR isoforms. However, the levels of shorter GRα isoforms (50, 40, and 25 kDa) were altered in adult mice underwent CUS, in sex- and brain structure–specific way. Changes in gene expression revealed that female depressive-like behavior could be related to increased levels of IL-1β in hippocampus and reduced BDNF levels in both hippocampus and PFC. However, in males, adolescent CUS increased expression of GR in adult hippocampus and BDNF in PFC. These findings suggest that adolescent stress altered levels of GRα isoforms, especially those with lower molecular weight, in sex- and tissue-specific ways, contributing to anxiety and depression in adult mice.

To adapt to the sustained demands of chronic stress, discrete brain circuits undergo structural and functional changes often resulting in anxiety disorders. In some individuals, anxiety disorders precede the development of motor symptoms of Parkinson's disease (PD) caused by degeneration of neurons in the substantia nigra (SN). Here, we present a circuit framework for probing a causal link between chronic stress, anxiety, and PD, which postulates a central role of abnormal neuromodulation of the SN's axon initial segment by brainstem inputs. It is grounded in findings demonstrating that the earliest PD pathologies occur in the stress-responsive, emotion regulation network of the brainstem, which provides the SN with dense aminergic and cholinergic innervation. SN's axon initial segment (AIS) has unique features that support the sustained and bidirectional propagation of activity in response to synaptic inputs. It is therefore, especially sensitive to circuit-mediated stress-induced imbalance of neuromodulation, and thus a plausible initiating site of neurodegeneration. This could explain why, although secondary to pathophysiologies in other brainstem nuclei, SN degeneration is the most extensive. Consequently, the cardinal symptom of PD, severe motor deficits, arise from degeneration of the nigrostriatal pathway rather than other brainstem nuclei. Understanding when and how circuit dysfunctions underlying anxiety can progress to neurodegeneration, raises the prospect of timed interventions for reversing, or at least impeding, the early pathophysiologies that lead to PD and possibly other neurodegenerative disorders. Chapter 32

Fear-related disorders, mainly phobias and post-traumatic stress disorder, are highly prevalent, debilitating disorders that pose a significant public health problem. They are characterized by aberrant processing of aversive experiences and dysregulated fear extinction, leading to excessive expression of fear and diminished quality of life. The gold standard for treating fear-related disorders is extinction-based exposure therapy (ET), shown to be ineffective for up to 35% of subjects. Moreover, ET combined with traditional pharmacological treatments for fear-related disorders, such as selective serotonin reuptake inhibitors, offers no further advantage to patients. This prompted the search for ways to improve ET outcomes, with current research focused on pharmacological agents that can augment ET by strengthening fear extinction learning. Hallucinogenic drugs promote reprocessing of fear-imbued memories and induce positive mood and openness, relieving anxiety and enabling the necessary emotional engagement during psychotherapeutic interventions. Mechanistically, hallucinogens induce dynamic structural and functional neuroplastic changes across the fear extinction circuitry and temper amygdala's hyperreactivity to threat-related stimuli, effectively mitigating one of the hallmarks of fear-related disorders. This paper provides the first comprehensive review of hallucinogens' potential to alleviate symptoms of fear-related disorders by focusing on their effects on fear extinction and the underlying molecular mechanisms. We overview both preclinical and clinical studies and emphasize the advantages of hallucinogenic drugs over current first-line treatments. We highlight 3,4-methylenedioxymethamphetamine and ketamine as the most effective therapeutics for fear-related disorders and discuss the potential molecular mechanisms responsible for their potency with implications for improving hallucinogen-assisted psychotherapy.

Inflammation plays a key role in the pathogenesis of the major depressive disorder. Namely, neuroinflammation can induce the production of neuroactive metabolites that interfere with N-methyl-D-aspartate receptors (NMDAR)-mediated glutamatergic neurotransmission and contribute to depressive-like behaviour. On the other hand, mammalian target of rapamycin (mTOR) activity with synaptogenic effects is the main mediator of antidepressant effects of several potent NMDAR antagonists. In this study, we investigated the specific role of GluN2A subunits of NMDAR on the activity of mTOR signaling and behaviour in lipopolysaccharide (LPS)-induces model of depression. The results showed that mice lacking GluN2A subunit did not display depressive-like behavior after the immune challenge, opposite to LPS-treated wild-type mice. Specifically, in GluN2A knockout mice, we estimated the activity of the mTOR pathway in the hippocampus and prefrontal cortex (PFC) by measuring synaptic levels of upstream regulators (p-Akt, p-ERK, and p-GSK3β) and downstream effectors (p-mTOR, and p-p70S6K) of mTOR activity. In addition, we assessed the changes in the levels of two important synaptic markers, GluA1 and PSD-95. Contrary to downregulated mTOR signaling and decreased synaptic markers in LPS-treated wild-type animals, the resilience of GluN2A KO mice to depressive-like behaviour was paralleled with sustained mTOR signaling activity synaptic stability in hippocampus and PFC. Finally, we disclosed that resistance of GluN2A knockouts to LPS-induced depressive-like behavior was ERK-dependent. These findings demonstrate that GluN2A-ERK-mTOR signaling is a vulnerability factor of inflammation-related depressive behaviour, making this signaling pathway the promising target for developing novel antidepressants.

Childhood trauma (CT) increases the risk for psychopathology through disturbed acquisition and extinction of fear. The effects of CT are mediated by abnormalities of the hypothalamic-pituitary-adrenal axis and glucocorticoid receptor (GR). Since, the alterations in GRα translational isoforms have been documented in psychiatric disorders we sought to: 1) explore whether multiple GRα isoforms in the human peripheral blood mononuclear cells of two independent cohorts (whole cell n = 40; and nuclear extracts n = 43, adult subjects) mediate the effect of CT on negative affectivity (NA) measured by Depression, Anxiety and Stress Scales (DASS), and 2) examine their role/function during fear extinction in the animal model. In multiple regression analysis, CT, nuclear 40-kDa GRα their interactions and FKBP5 explained 22%–35% of variance in DASS scores. Structural equation modeling showed that CT had a significant direct effect on 40-kDa and DASS in both cohorts, and on the nuclear 25-kDa GRα. The association between 40-kDa and total DASS was significantly mediated by nuclear FKBP5, whereas on DASS anxiety, over FKBP5 in both cohorts and nuclear full length GRα. Nuclear 40-kDa GRα and its interaction with CT had a significant direct effect on DASS anxiety. In mice, the successful extinction learning was followed by nuclear translocation of 40-kDa GRα and induction of BDNF exon IV expression. Our data revealed that the association between CT and adult NA in non-clinical subjects is mediated by the GRα translational isoforms, in particular 40-kDa GRα and emphasized its role in fear extinction and neural plasticity. © 2018 Elsevier Inc. Free Author Manuscript available at PMC: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383671/]

The brain is the softest organ in the body, and any change in the mechanical properties of the tissue induces the activation of glial cells, astrocytes and microglia. Amyloid plaques, one of the main pathological features of Alzheimer's disease (AD), are substantially harder than the surrounding brain tissue and can activate astrocytes and microglia resulting in the glial engulfment of plaques. Durotaxis, a migratory preference towards stiffer tissue, is prompting microglia to form a mechanical barrier around plaques reducing amyloid β (Aβ) induced neurotoxicity. Mechanoreceptors are highly expressed in the brain, particularly in microglia. The large increase in the expression of the mechanoreceptor Piezo1 was observed in the brains from AD animal models and AD patients in plaque encompassing glia. Importantly, Piezo1 function is regulated via force-from-lipids through the lipid composition of the membrane and membranous incorporation of polyunsaturated fatty acids (PUFAs) can affect the function of Piezo1 altering mechanosensitive properties of the cell. On the other hand, PUFAs dietary supplementation can alter microglial polarization, the envelopment of amyloid plaques, and immune response and Piezo1 activity was implicated in the similar modulations of microglia behavior. Finally, PUFAs treatment is currently in use in medical trials as the therapy for sickle cell anemia, a disease linked with the mutations in Piezo1. Further studies are needed to elucidate the connection between PUFAs, Piezo1 expression, and microglia behavior in the AD brain. These findings could open new possibilities in harnessing microglia in AD and in developing novel therapeutic strategies.

Hypothalamic–pituitary–adrenal (HPA) axis activity mediates the relationship between childhood trauma (CT) and psychosis. The FKBP5 gene, one of the key regulators of HPA axis activity after stress exposure, has been found associated with psychosis. Allele-specific and CT related FKBP5 demethylation in intron 7 was revealed in different psychiatric disorders. However, no studies have investigated FKBP5 methylation in subjects with different genetic liability for psychosis. A total of 144 participants were included in the study: 48 patients with psychotic disorders, 50 unaffected siblings, and 46 healthy controls. CT was assessed by Childhood Trauma Questionnaire. The FKBP5 rs1360780 was genotyped and FKBP5 methylation analyses were performed using bisulfite conversion followed by Sanger sequencing at three CpG sites in intron 7. Mixed linear model was used to assess group differences depending on rs1360780 T allele and CT. Results showed a significant T allele-dependent decrease of FKBP5 methylation in patients compared to unaffected siblings and controls. Effect of interaction between T allele and CT exposure on FKBP5 demethylation was found in controls. No effect of both risk factors (T allele and CT) on FKBP5 methylation level was found in unaffected siblings. We confirmed previous evidence of the association between the FKBP5 rs1360780 T allele, CT, and decreased FKBP5 methylation in intron 7. Allele-specific FKBP5 demethylation found in patients could shed a light on altered HPA axis activity in a subgroup of patients related to stress-induced psychosis. FKBP5 methylation and potential protective mechanisms in unaffected siblings after trauma exposure require further investigation. © 2021 Elsevier Ltd

The SARS-CoV-2 outbreak caused an unprecedented global public health threat, having a high transmission rate with currently no drugs or vaccines approved. An alternative powerful additional approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 main protease is essential for viral replication and an attractive drug target. In this study, we used the virtual screening (VS) protocol with both long-range and short-range interactions to select candidate SARS-CoV-2 main protease inhibitors. First, the ISM applied for Small Molecules was used for searching the Drugbank database and further followed by molecular docking. After in silico screening of drug space, we identified 57 drugs as potential SARS-CoV-2 main protease inhibitors that we propose for further experimental testing.

Mitochondria play a significant role in pathogenesis of clinical depression and their function can be impaired by inflammation and alterations in hypothalamic–pituitary–adrenal axis. Sexual context is also a relevant factor in the incidence of mood disorders, and could have a strong influence during an immune challenge. Therefore, in this study we investigated whether the effects of seven-day lipopolysaccharide (LPS) treatment on glucocorticoid receptor (GR) could be associated with apoptosis and alterations in energy metabolism in hippocampus of female and male Wistar rats with depressive-like behavior. To that end, we measured the mitochondrial levels of GR and its phosphoisoforms pGR232 and pGR246 in hippocampus of female and male rats, as well as the mRNA levels of two GR-regulated mitochondrial genes, cyclooxygenase -1 and -3 (COX-1 and -3). We also measured alterations in the extrinsic and intrinsic apoptotic pathways in mitochondria and cytosol of hippocampus of these animals, and the levels of cleaved cytosolic poly [ADP-ribose] polymerase-1 (PARP-1) protein. We discovered that even though LPS treatment induced behavioral alterations and affected corticosterone levels and apoptosis in a similar manner in both sexes, it affected mitochondrial GR differently in males and females. Namely, the treatment decreased levels of mitochondrial GR and pGR232/pGR246 ratio only in females, and these alterations were followed by decreased mRNA levels of COX-1 and COX-3 only in this sex. The alterations in COX-1 and COX-3 mRNA levels could indicate impaired oxidative metabolism and diminished mitochondrial function in hippocampus of this sex. © 2020 IBRO

Accumulating evidence strongly suggest that impaired glucocorticoid receptor (GR) signaling is involved in stress-related mood disorders, and nominate GR as a potential target for antidepressants (ADs). It is known that different classes of ADs affects the GR action via modifying its phosphorylation, while the mechanism through which ADs alter GR phosphorylation targeted by GSK3 beta, a kinase modulated via serotonin neurotransmission, are unclear. On this basis, we investigated whether GSK3 beta-GR signaling could be a convergence point of fluoxetine action on brain function and behavior, by examining its effect on GSK3 beta targeted-GR phosphorylation on threonine 171 (pGR171), and expression of GR-regulated genes in the hippocampus of female and male rats exposed to chronic isolation stress. Stress induced sex-specific GSK3 beta-targeted phosphorylation of pGR171 in the nucleus of the hippocampus of stressed animals. Namely, while in females stress triggered coupled action of GSK3 beta-pGR171 signaling, in males changes in pGR171 levels did not correspond to GSK3 beta activity. On the other hand, fluoxetine managed to up regulate this pathway in sex-unbiased manner. Furthermore, fluoxetine reverted stress-induced changes in most of the analyzed genes in males, CRH, 5-HT1a and p11, while in females its effect was limited to CRH. These data further suggest that pGR171 signaling affects cellular localization of GR in response to chronic stress and fluoxetine in both sexes. Collectively, our results describe a novel convergence point between GR signaling and GSK3 beta pathway in rat hippocampus in response to stress and fluoxetine in both sexes and its involvement in fluoxetine-regulated brain function in males.

Mitochondrial dysfunction can result from the interplay between elevated inflammatory markers and alterations in hypothalamic–pituitary–adrenal (HPA) axis, and can contribute to pathogenesis of major depression. Therefore, in this study we investigated whether the effects of lipopolysaccharide (LPS) on glucocorticoid receptor (GR) could be associated with alterations in mitochondrial apoptotic signaling in the prefrontal cortex of male and female Wistar rats with depressive-like behavior. To that end, we measured LPS-induced alterations in the extrinsic and intrinsic apoptotic pathways in mitochondria and cytosol of PFC of female and male rats, as well as the levels of cleaved cytosolic PARP-1. We also measured the mitochondrial levels of GR and its phosphoisoforms pGR232 and pGR246, as well as the mRNA levels of two GR-regulated mitochondrial genes, COX-1 and COX-3. We discovered that although seven-day LPS treatment evoked depressive-like behavior and induced apoptosis in the PFC of both sexes, it affected apoptotic cascades in both sexes differently. In females the treatment initiated both intrinsic and extrinsic apoptotic cascade, while in males only intrinsic cascade was engaged. Alterations in intrinsic apoptotic pathway were more associated with GR alterations in males, where LPS treatment decreased levels of mitochondrial GR and increased pGR232/pGR246 ratio. Alterations in mitochondrial GR could be associated with changes in expression of genes involved in oxidative metabolism in the PFC of this sex, and could, in combination with elevated levels of BCL-2 and decreased levels of BAX detected in this cell fraction, mitigate the detrimental effect of LPS on mitochondria in male PFC.

In 2012, alcohol liver disease resulted in 3.3 million—5.9% of global deaths. This study introduced whey protection capacity against chronic alcohol-induced liver injury. Rats were orally administered to 12% ethanol solution in water (ad libitum, average 8.14 g of ethanol/kg body weight (b.w.)/day) alone or combined with whey (per os, 2 g/kg b.w./day). After 6-week treatment, chronic ethanol consumption induced significant histopathological liver changes: congestion, central vein dilation, hepatic portal vein branch dilation, Kupffer cells hyperplasia, fatty liver changes, and hepatocytes focal necrosis. Ethanol significantly increased liver catalase activity and glutathione reductase protein expression without significant effects on antioxidative enzymes: glutathione peroxidase (GPx), copper–zinc-containing superoxide dismutase (CuZnSOD) and manganese-containing superoxide dismutase (MnSOD). Co-treatment with whey significantly attenuated pathohistological changes induced by ethanol ingestion and increased GSH-Px and nuclear factor kappa B (NF-κB) protein expression. Our results showed positive effects of whey on liver chronically exposed to ethanol, which seem to be associated with NF-κB-GPx signaling. © The Author(s) 2019.

Recent reports have demonstrated that lipopolysaccharide (LPS)-induced depressive-like behaviour is mediated via NMDA receptor. In this study, we further investigated the role of GluN2 A subunit of NMDA receptor in synaptic processes in the prefrontal cortex (PFC) and hippocampus of GluN2 A knockout (KO) mice in LPS-induced depressive-like behavior. Our data suggest that LPS-treated mice, lacking GluN2 A subunit, did not exhibit depressive-like behaviour. This was accompanied by unaltered levels of IL-6 and significant changes in neuroplasticity markers and glutamate receptor subunits composition in PFC and hippocampus. In particular, an immune challenge in GluN2 A KO mice resulted in unchanged PSA-NCAM levels and proBDNF increase in both brain structures as well as in increase in BDNF levels in hippocampus. Furthermore, the absence of GluN2 A resulted in increased levels of all NCAM isoforms in PFC upon LPS which was followed with a decrease in GluN1 and GluN2B subunits. The levels of AMPA receptor subunits (GluA1, GluA3, and GluA4) in the hippocampus of GluN2 A mice were unaltered upon the treatment and abundantly present in the PFC of KO mice. These results indicate that the GluN2 A subunit is critical in neuroinflammation-related depression, that its absence abolishes LPS-induced depressive phenotype, sustains PSA-NCAM levels, increases proBDNF signalling in the PFC and hippocampus and potentiates synaptic stabilization through NCAM in the PFC upon an immune challenge. © 2018 Elsevier B.V.

Preclinical Research Mitochondria are cell organelles crucial to the production of cellular energy. Several lines of evidence have indicated that mitochondrial dysfunction could be related to the pathophysiology of CNS diseases including bipolar disorder, major depressive disorder, and schizophrenia. These changes include impaired energy metabolism in the brain, co-morbidity with mitochondrial diseases, the effects of psychotropics on mitochondrial function, increased mitochondrial DNA (mtDNA) deletion in the brain, and association with mtDNA polymorphisms. Additionally, psychotropic drug treatments can alter energy metabolism and may affect mitochondrial processes. This review focuses on recent findings regarding the effects of antidepressants on mitochondrial processes in psychiatric disorders. Drug Dev Res 77 : 400-406, 2016. © 2016 Wiley Periodicals, Inc.

Objectives. To develop a structural equation model of negative affectivity (NA) that involves interaction of glucocorticoid receptor (GR) signaling, personality dimensions and recent stressful life events. Methods. Seventy participants -35 diagnosed with major depression and 35 healthy controls, were enrolled in the study. Morning plasma cortisol levels were determined by chemiluminescent immunometric assays. Molecular parameters (total nuclear and cytoplasmatic GR, nuclear GR phosphorylated at serine 211 (pGR-211) and at serine 226 (pGR-226) and cytoplasmic FKBP51) were analysed from peripheral blood lymphocytes by Western blot. NA, personality dimensions and stressful life events were assessed by self-report instruments. Results. GR signalling parameters had direct independent effect on measures of NA, with pGR-226 levels showing the strongest correlation, followed by FKBP51 and pGR-211 levels. Neuroticism and extraversion also demonstrated strong independent effect on NA, while recent stressful events did not predict NA directly, but demonstrated a significant effect on personality dimensions. Cortisol, total nuclear GR and total cytoplasmatic GR levels were excluded from the model due to non-significant correlations with NA. Conclusions. Negative affectivity is a transdiagnostic factor in vulnerability to affective disorders and possible therapeutic target. Molecular signature of negative affectivity should incorporate GR phosphorylation with other known biological underpinnings.

The early postnatal environment is critical for its capacity to influence adult behavior, and is associated with traits of altered physiological and neurobiological function and long-term predisposition to depression. Here we describe the delayed effects of maternal deprivation (MD) in male and female Wistar pups on their physical development and behavior in adulthood in tasks designed to explore depressive-like (forced swimming test, FST), and anxiety-like behaviors (elevated plus maze, EPM). We observed that MD led to reduced body weight in adulthood, anxiety-like traits in the EPM test and increased activity in the phases of the FST. Particularly, a consistent sexual dimorphism was observed in the responses to MD. A lower increase in body weight during maturation of MD rats was more pronounced in males than in females. MD anxiogenic effects were more pronounced in females, while in FST only MD males showed a marked increase in swimming activity followed by decreased immobility. [Projekat Ministarstva nauke Republike Srbije, br. III41029]

Considerable evidence shows an association of depression with the immune system and emphasizes the importance of gender in the etiology of the disease and the response to inflammatory stimuli. We examined the influence of immune-challenged systems on depressive-like behavior in female rats in the context of gonadal hormones. We used a neuroinflammatory model of depression elicited by lipopolysaccharide (LPS) administration on naive and ovariectomized (OVX) female rats, and examined the effects of estradiol (E2) and/or progesterone (P4) replacement therapy on animal behavior, as assessed by the forced swimming test (FST). We found that LPS and OVX increase immobility in the FST, while LPS also decreased body weight in naive female rats. Further, even though P4 application alone showed beneficial effects on the behavioral profile (it reduced immobility and increased climbing), supplementation of both hormones (E2 and P4) together to OVX rats failed to do so. When OVX rats were exposed to LPS-induced immune challenge, neither hormone individually nor their combination had any effect on immobility, however, their joint supplementation increased climbing behavior. In conclusion, our study confirmed that both LPS and OVX induced depressive-like behavior in female rats. Furthermore, our results potentiate P4 supplementation in relieving the depressive-like symptomatology in OVX rats, most likely through fine-tuning of different neurotransmitter systems. In the context of an activated immune system, the application of E2 and/or P4 does not provide any advantageous effects on depressive-like behavior.

Background: Epigenetic changes of the FKBP5 locus in adults were reported to be allele specific and trauma related in different patient populations. However, DNA methylation status of FKBP5 and its relation to the circulating protein levels are still unknown in schizophrenia spectrum disorders.

Depression is a disease of an abnormal brain energy metabolism also marked with increased apoptosis in specific brain regions. Mounting evidence indicates that the mitochondrial oxidative phosphorylation and apoptosis are novel targets for the actions of estrogen receptors (ERs). In this study, we examined the effects of antidepressant (AD) fluoxetine (FLU) treatment on the mitochondria] ER alpha (ER alpha), ER beta (total and phospho-pER(beta) and their association with cytochrome c (cyt oxidase activity and apoptotic Bcl2/Bax-molecules in the hippocampal mitochondria of chronically isolated (CPSI) female and male rats depicting depression. Impaired behaviour induced by CPSI was followed by decreased corticosterone (CORT) in both sexes and downregulation of cyt c oxidase in males. CPSI did not affect the ERa in either of sexes, but it decreased mitochondrial ER beta and increased pER beta in both sexes. Stress-reduced ER beta is associated with a decrease in mitochondrial energetic processes in males and with apoptotic mechanisms in females. FLU normalized behaviour in both sexes and increased cyt c oxidase in females. FLU elevated ERa in males, increased ER beta and decreased pERB in both sexes. The AD-induced alterations of ER beta paralleled with bioenergetics and pro-survival pathways in females. In conclusion, sex-unspecific regulation of ER beta by the stress and by AD and its differential convergence with bioenergetics and apoptotic pathways in females and males implies its role as a vulnerability factor in the stress response and emphasizes mitochondrial ER beta-dependent pathways as an important gateway of ADs action, at least in females. (C) 2017 Elsevier B.V. All rights reserved.

Inflammation plays a critical role in pathogenesis of depression and can affect the hypothalamic-pituitary-adrenal axis activity. Accordingly, in this study we investigated the role of hippocampal glucocorticoid receptor in mediating the effects of inflammation on behaviour of female and male Wistar rats. We studied the effects of lipopolysaccharide on the levels of glucocorticoid receptors and its co-chaperones FK506 binding protein 52 and FK506 binding protein 51, the levels of glucocorticoid receptor phospho-isoforms, pGR-232 and pGR-246, and glucocorticoid receptor up-stream kinases. In order to assess transcriptional activity of glucocorticoid receptor, we measured mRNA levels of several glucocorticoid receptor-regulated genes. We demonstrated that lipopolysaccharide induced depressive-like behaviour and elevated serum corticosterone in both sexes. However, it affected glucocorticoid receptor signalling in the nucleus of females and males differently - in females it elevated levels of glucocorticoid receptors, pGR-246 and FK506 binding protein 52, while in males it decreased levels of glucocorticoid receptor, both co-chaperons and pGR-246. Alterations in pGR-246 were associated with alterations of c-Jun N-terminal kinases. Altered nuclear levels of total glucocorticoid receptors and pGR-246 were accompanied by sex-specific reduction in brain-derived neurotrophic factor and cyclooxygenase-2 mRNA and sex-unspecific reduction in the expression of p11 and glucocorticoid receptor genes. These alterations may ultimately affect different glucocorticoid receptor -associated processes involved in depressive-like behaviour in males and females.

The impaired glucocorticoid receptor (GR) signaling has long been considered one of the cornerstones in understanding the pathophysiology of depression. Since the phosphorylation of GR is very important for GR function, in this study we investigated whether GR phosphorylation at serine 211 (pGR-S211) and serine 226 (pGR-S226) is altered in patients with current episode of major depressive disorder (MDD). Particularly, in 30 MDD patients and 35 controls we assessed the levels of nuclear total GR (tGR), pGR-S211 and pGR-S226 in peripheral blood mononuclear cells (PBMC) using Western blot technique, along with plasma cortisol concentrations from the same blood samples. Our results demonstrated increased phosphorylation of GR at S226 (p LT 0.001) and, to a less extent, at S211 (p LT 0.05) in MDD patients compared to controls. Consequently, the pGR-S211/pGR-S226 ratio was decreased (p LT 0.05) implying reduced transcriptional activity of GR in MDD patients. MDD subjects had higher cortisol levels than controls and cortisol concentrations were positively correlated with PBMC pGR-S226 levels from the same blood samples. There was no difference in the levels of tGR between MDD and control subjects. The study showed that altered phosphorylation of GR could contribute to impaired GR function related to the pathophysiology of depression. (c) 2012 Elsevier Inc. All rights reserved.

Background Mounting evidence demonstrates enhanced systemic levels of inflammatory mediators in depression, indicating that inflammation may play a role in the etiology and course of mood disorders. Indeed, proinflammatory cytokines induce a behavioral state of conservation- withdrawal resembling human depression, characterized by negative mood, fatigue, anhedonia, psychomotor retardation, loss of appetite, and cognitive deficits. Neuroinflammation also contributes to non-responsiveness to current antidepressant (AD) therapies. Namely, response to conventional AD medications is associated with a decrease in inflammatory biomarkers, whereas resistance to treatment is accompanied by increased inflammation. Methods In this review, we will discuss the utility and shortcomings of pharmacologic AD treatment strategies focused on inflammatory pathways, applied alone or as an adjuvant component to current AD therapies. Results Mechanisms of cytokine actions on behavior involve activation of inflammatory pathways in the brain, resulting in changes of neurotransmitter metabolism, neuroendocrine function, and neuronal plasticity. Selective serotonin reuptake inhibitors exhibit the most beneficial effects in restraining the inflammation markers in depression. Different anti-inflammatory agents exhibit AD effects via modulating neurotransmitter systems, neuroplasticity markers and glucocorticoid receptor signaling. Anti-inflammatory add-on therapy in depression highlights such treatment as a candidate for enhancement strategy in patients with moderate-to-severe depression. Conclusion The interactions between the immune system and CNS are not only involved in shaping behavior, but also in responding to therapeutics. Even though, substantial evidence from animal and human research support a beneficial effect of anti-inflammatory add-on therapy in depression, further research with special attention on safety, particularly during prolonged periods of antiinflammatory co-treatments, is required.

Preclinical Research Mitochondria are cell organelles crucial to the production of cellular energy. Several lines of evidence have indicated that mitochondrial dysfunction could be related to the pathophysiology of CNS diseases including bipolar disorder, major depressive disorder, and schizophrenia. These changes include impaired energy metabolism in the brain, co-morbidity with mitochondrial diseases, the effects of psychotropics on mitochondrial function, increased mitochondrial DNA (mtDNA) deletion in the brain, and association with mtDNA polymorphisms. Additionally, psychotropic drug treatments can alter energy metabolism and may affect mitochondrial processes. This review focuses on recent findings regarding the effects of antidepressants on mitochondrial processes in psychiatric disorders. Drug Dev Res 77 : 400-406, 2016. © 2016 Wiley Periodicals, Inc.

Chronic stress is a contributing risk factor in the development of psychiatric illnesses, including depressive disorders. The mechanisms of their psychopathology are multifaceted and include, besides others, alterations in the brain plasticity. Previously, we investigated the effects of chronic social stress in the limbic brain structures of Wistar rats (hippocampus, HIPPO, and prefrontal cortex, PFC) and found multiple characteristics that resembled alterations described in some clinical studies of depression. We extended our investigations and followed the behavior of stressed animals by the open field test (OFT) and forced swimming test (FST), and the expression and polysialylation of synaptic plasticity markers, neural cell adhesion molecule (NCAM) and L1, in the HIPPO and PFC. We also determined the adrenal gland mass and plasma corticosterone (CORT) as a terminal part of the hypothalamic-pituitary-adrenal axis activity. Our data indicated that stressed animals avoided the central zone in the OFT and displayed decreased swimming, but prolonged immobility in the FST. The animals exhibited marked hypertrophy of the adrenal gland cortex, in spite of decreased serum CORT. Simultaneously, the stressed animals exhibited an increase in NCAM mRNA expression in the HIPPO, but not in the PFC. The synaptosomal NCAM of the HIPPO was markedly polysialylated, while cortical PSA-NCAM was significantly decreased. The results showed that chronic social isolation of Wistar rats causes both anxiety-like and depression-like behavior. These alterations are parallel with molecular changes in the limbic brain, including diminished NCAM sialylation in the PFC. Together with our previous results, the current observations suggest that a chronic social isolation model may potentially be used to study molecular mechanisms that underlie depressive symptomatology. Copyright (c) 2012 S. Karger AG, Basel

30th Congress of the European-College-of-Neuropsychopharmacology (ECNP), Sep 02-05, 2017, Paris, France

Background Hypothalamic–pituitary–adrenal (HPA) axis dysregulation is a potential neurobiological mechanism proposed by vulnerability-stress model for schizophrenia. Recent studies highlighted the role of functional genetic variants of FKBP5 gene, which affect the activity of HPA axis following stress exposure, and supported the hypothesis of increased stress sensitivity in schizophrenia. Additionally, FKBP5 demethylation in Intron 7 was observed in stress-related conditions thus the purpose of this pilot study was to investigate FKBP5 methylation levels at Intron 7 in schizophrenia. Methods Ethnically homogeneous Serbian sample of 24 schizophrenia spectrum patients and 24 controls matched by age and gender with patients’ group, were analyzed regarding DNA methylation levels at three CpG sites and average methylation level in Intron 7. Accordingly, we covered the area of GRE that has been reported to be associated with altered stress responsiveness. Epigenetic changes in FKBP5 Intron 7 were measured by Sanger sequencing and compared between the groups using t test as appropriate. Results Analyses revealed decreased FKBP5 methylation at the three targeted CpG sites (CpG1, CpG2, and CpG3) in patients compared to controls (p=0.026, p=0.017, and p=0.027, respectively). Similarly, when we averaged methylation scores of the observed region in Intron 7, significant demethylation was detected in patients (p=0.003). Discussion To the best of our knowledge this is the first study which explored FKBP5 epigenetic changes in schizophrenia. Conclusively to previous stress-related conditions, our preliminary results revealed significant decrease of FKBP5 methylation levels in Intron 7 in patients with schizophrenia. FKBP5 demethylation presents further insight into the reported FKBP5 genetic influence in psychosis and could be promising terapeutic target for the prevention of the onset and the course of schizophrenia.

Chronic neuroendocrine stress usually leads to the elevation of the stress hormones and increased metabolic rate, which is frequently accompanied by oxidative damage to the CNS. In the present study we hypothesized that chronic psychosocial isolation (CPSI) of male Wistar rats, characterized by decreased serum corticosterone (CORT), unaltered catecholamines (CTs), and low blood glucose (GLU), may also promote oxidative imbalance in the CNS, by targeting antioxidant defense system. To test it, we have examined the relation between these input signals and protein expression/activity of antioxidant enzymes (AOEs): superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GLR) in the hippocampus (HIPPO) of CPSI animals. We found that CPSI did not affect SODs or CAT, but decreased activity of GPx and compromised GLR, an enzyme highly dependent on blood GLU for its substrate precursor. Further, we have tested whether the CPSI experience altered AOEs response to a novelty stress, and found that it attenuated peroxide-metabolizing enzymes, CAT and GPx, and decreased GLR activity, even though blood GLU was restored. The altered ratios of hippocampal AOEs in CPSI animals, which were worsened under the combined stress conditions, may lead to the accumulation of peroxide products and oxidative imbalance. The mechanism by which CPSI generate oxidative imbalance in the HIPPO is most likely based on poor systemic energy conditions set by this stress. Such conditions may cause functional decline of CNS structures, such as HIPPO, and are likely to promote state linked to onset of many mood disorders.

Mitochondrial dysfunction is increasingly recognized as a key component in compromised neuroendocrine stress response and. among other etiological causes. it may also involve action of glucocorticoid hormones. In the current study we followed glucocorticoid receptor and Identified its mitochondrial phosphoisophorms in hippocampus and prefrontal brain cortex of Wistar male rats subjected to acute. chronic and combined neuroendocrine stresses. In both brain structures chronic social isolation caused m,irked increase in mitochondrial glucocorticoid receptor that was preferentially phosphorylated at serine 232 compared to serine 246 or serine 171. This increase corresponded with the decreased expression of mitochondrially encoded cytochrome oxidase subunits 1 and 3 in hippocampus, and with their increased expression in prefrontal brain cortex. Prefrontal brain cortex appeared to be more sensitive to chronic stress, since it exibited higher levels of mitochondrial Bax and cytoplasmic Bcl2 compared to hippocampus. Chronic stress also altered the response of both brain structures to subsequent acute stress according to the studied parameters. Therefore, prolonged social isolation may cause susceptibility to mitochondria triggered proapototic signalling. which at least in part may be mediated by the glucocorticoid receptor dependent mechanism. (C) 2009 Elsevier Ltd All rights reserved.

Successful adaptation to stress involves actions of glucocorticoid receptor (GR), a steroid-dependent transcription factor, abundant in hippocampus. Another transcription factor, nuclear factor kappa B (NF kappa B) is considered as an important stress sensor implicated in adaptive synaptic plasticity. Numerous stress-related genes are regulated by both hippocampal GR and NF kappa B, including neural cell adhesion molecules (NCAM and L1), involved in plasticity, and genes that encode apoptotic proteins (bax and bcl-2). We presumed that the ratio of nuclear NF kappa B to nuclear GR may determine the degree of proplastic or proapoptotic signaling under stress. To test this presumption we have investigated effects of acute, chronic and combined stress on compartmental levels and ratios of NF kappa B and GR proteins, and in parallel, changes in their mRNA expression. In addition, the expression of plasticity (NCAM, L1) and apoptotic (bax, bcl-2) genes, as well as, Bax and Bcl-2 proteins redistribution between mitochondrial and cytoplasmic compartments, were followed. When glucocorticoid levels were low, as found in chronic stress, GR was not efficiently translocated to the nucleus. This resulted in its lower nuclear level relative to the nuclear NF kappa B. Such conditions did not affect proplastic induction of NCAM mRNA, but were related to the onset of proapoptotic signaling illustrated by relocation of mitochondrial Bcl-2 protein to its soluble cytoplasmic form. Because these Bcl-2 rearrangements were not reversed by subsequent acute stress, representing more stable alterations, it is concluded that chronic social isolation of Wistar rats led to the initiation of proapoptotic signaling that may be etiologically related to compromised adaptive response of central nervous system. Link to erratum: [https://vinar.vin.bg.ac.rs/handle/123456789/10581]

Chronic stress and impaired glucocorticoid receptor (GR) feedback are important factors for the compromised hypothalamic-pituitary-adrenal (HPA) axis activity. We investigated the effects of chronic 2 1 day isolation of Wistar rats on the extrinsic negative feedback part of I-IPA axis: hippocampus (HIPPO) and prefrontal cortex (PFC). In addition to serum corticosterone (CORT), we followed GR subcellular localization, GR phosphorylation at serine 232 and serine 246, expression of GR, regulated genes: GR, CRF and brain-derived neurotropic factor (BDNF), and activity of c-Jun N-terminal kinase (JNK) and Cdk5 kinases that phosphorylate GR. These parameters were also determined in animals subjected to acute 30 min immobilization, which was taken as normal adaptive response to stress. In isolated animals, we found decreased CORT, whereas, in animals exposed to acute immobilization, CORT was markedly increased. Even though the GR was predominantly localized in the nucleus of HIPPO and PFC in acute, but not in chronic stress, the expression of GR, CRF, and BDNF genes was similarly regulated under both acute and chronic stresses. Thus, the transcriptional activity of GR under chronic isolation did not seem to be exclusively dependent on high serum CORT levels nor on the subcellular location of the GR protein. Rather, it resulted front the increased Cdk5 activation and phosphorylation of the nuclear GR at serine 232 and the decreased JNK activity reflected in decreased phosphorylation of the nuclear GR at serine 246. Our study suggests that this nuclear isoform of hippocampal and cortical GR may be related to hypocorticism i.e. HPA axis hypoactivity under chronic isolation stress. journal of Endocrinology (2009) 202, 87-97

The glucocorticoid receptor (GR) protein is a cytosolic ligand-dependent transcription factor with numerous functions regulated by post-translational modifications, including phosphorylation/dephosphorylation. Among the functions most extensively affected by GR phosphorylation are the modulation of its transcriptional activity, alterations in its interaction pattern with cofactors, nuclear translocation and selective gene transactivation. Intensive analysis of the intracellular distribution of GR phosphoisoforms and their interaction with proteins of other cellular signalling networks required the use of [?-32P]ATP as a phosphate donor, and special laboratory protection measures to avoid external irradiation and contamination. In the present study, simple and easy-to-use non-radioactive protein mobility shift assays (NMS assays) were developed using one- and/or two-dimensional gel electrophoresis based on differences in the pI and molecular mass of GR phosphoisoforms. The GR isoforms were immunodetected with specific monoclonal or polyclonal anti-GR antibodies by Western blot in three diverse systems, namely yeast BJ2168 cells expressing wild-type rat GR, rat hepatoma GRH2 cells grown in culture and brain tissue from Wistar rat experimental animals. The results obtained using the NMS assay were similar to previous results obtained with the [?-32P] ATP standard assay.

Glucocorticoids are key regulators of the neuroendocrine stress response in the hippocampus. Their action is partly mediated through the subfamily of MAPKs termed c-jun-N-terminal kinases (JNKs), whose activation correlates with neurodegeneration. The stress response also involves activation of cell protective mechanisms through various heat shock proteins (HSPs) that mediate neuroprotection. We followed both JNKs and Hsp70 signals in the cytoplasmic and nuclear compartments of the hippocampus of Wistar male rats exposed to acute, chronic, and combined stress. The activity of JNK1 was decreased in both compartments by all three types of stress, while the activity of cytoplasmic JNK2/3 was elevated in acute and unaltered or lowered in chronic and combined stress. Under all stress conditions, Hsp70 translocation to the nucleus was markedly increased. The results suggest that neurodegenerative signaling of JNKs may be counteracted by increase of nuclear Hsp70, especially under chronic stress. Link to erratum: [https://vinar.vin.bg.ac.rs/handle/123456789/3785]

ims: Periodic leg movements in sleep (PLMS) are a frequent finding in polysomnography. Most patients with restless legs syndrome (RLS) display PLMS. However, since PLMS are also often recorded in healthy elderly subjects, the clinical significance of PLMS is still discussed controversially. Leg movements are seen concurrently with arousals in obstructive sleep apnoea (OSA) may also appear periodically. Quantitative assessment of the periodicity of LM/PLM as measured by inter movement intervals (IMI) is difficult. This is mainly due to influencing factors like sleep architecture and sleep stage, medication, inter and intra patient variability, the arbitrary amplitude and sequence criteria which tend to broaden the IMI distributions or make them even multi-modal. Methods: Here a statistical method is presented that enables eliminating such effects from the raw data before analysing the statistics of IMI. Rather than studying the absolute size of IMI (measured in seconds) we focus on the shape of their distribution (suitably normalized IMI). To this end we employ methods developed in Random Matrix Theory (RMT). Patients: The periodicity of leg movements (LM) of four patient groups (10 to 15 each) showing LM without PLMS (group 1), OSA without PLMS (group 2), PLMS and OSA (group 3) as well as PLMS without OSA (group 4) are compared. Results: The IMI of patients without PLMS (groups 1 and 2) and with PLMS (groups 3 and 4) are statistically different. In patients without PLMS the distribution of normalized IMI resembles closely the one of random events. In contrary IMI of PLMS patients show features of periodic systems (e.g. a pendulum) when studied in normalized manner. Conclusions: For quantifying PLMS periodicity proper normalization of the IMI is crucial. Without this procedure important features are hidden when grouping LM/PLM over whole nights or across patients. The clinical significance of PLMS might be eluded when properly separating random LM from LM that show features of periodic systems.

Peripheral inflammation induced by lipopolysaccharide (LPS) causes behavioural changes indicative for depression. The possible mechanisms involve the interference with neuroinflammatory, neuroendocrine, and neurotrophic processes. Apart from heterogeneity in the molecular background, sexual context may be another factor relevant to the manifestation of mood disturbances upon an immune challenge. We investigated sex-dependent effects of a 7-day LPS treatment of adult Wistar rats on depressive-like behaviour and their relation with hypothalamic neuroendocrine factor, corticotrophin-releasing hormone (CRH), proplastic brain-derived neurotropic factor (BDNF), pro-inflammatory cyclooxygenase-2 (COX-2) and nuclear factor kappa beta (NFkB). Also, their regulators, the glucocorticoid receptor (GR) and CCAAT enhancer-binding protein (C/EBP) beta were followed. LPS induced depressive-like behaviour in females was associated with the increased hypothalamic CRH and decreased BDNF, but not with COX-2. These changes were paralleled by an increase in nuclear GR, NFkB and 20 kDa C/EBP beta. LPS also altered behaviour in males and increased CRH expression, but in contrast to females, this was accompanied with the elevated COX-2, accumulation of cytosolic GR and elevated nuclear 38 kDa C/EBP beta and NFkB. In conclusion, depressive-like phenotype induced by LPS in both sexes emerges from similar HPA axis activation and sex-specific alterations of hypothalamic molecular signalling: in males it is related to compromised control of neuroinflamation connected with cytoplasmic GR retention, while in females it is related to diminished proplastic capacity of BDNF. Sex-dependent mechanisms by which inflammation alters hypothalamic processes and cause pathological behaviour in animals, could be operative in the treatment of depression-related brain inflammation. (C) 2015 Elsevier B.V. All rights reserved.

Glucocorticoid resistance is a common finding in major depressive disorder. Increased glucocorticoid receptor (GR) phosphorylation at serine 226 is associated with increased glucocorticoid resistance. Previously we have demonstrated that depressed patients exhibit higher levels of GR phosphorylated at serine 226 compared to healthy controls. The enzyme that is involved in this specific GR phosphorylation is c-Jun N-terminal kinase (JNK). We propose that modulation of glucocorticoid phosphorylation at serine 226, by targeting JNK signaling pathway, could be a potential strategy for antidepressant treatment. We base this assumption on the results of previous research that examined GR phosphorylation and JNK signaling in animal models and human studies. We also discuss the potential challenges in targeting JNK signaling pathway in depression. (C) 2015 Elsevier Ltd. All rights reserved.

Gamma-irradiation (gamma-IR) is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl)-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD), specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. Gamma-IR treatment had a significant (P < 0.001) antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control) to 49% (IR cells), with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

Background: Hypothalamic-pituitary-adrenal (HPA) axis dysregulation is a proposed etiological mechanism of psychosis. Recent studies highlighted impact of the FKBP5 gene and its functional variant rs1360780, which risk (T) allele affects the activity of HPA axis following stress exposure, on psychotic patients exposed to early trauma (1). Additionally, risk allele and trauma dependent FKBP5 demethylation in intron 7 was observed in traumatized individuals (2). Thus, the purpose of this pilot study was to investigate influence of the risk allele and trauma on FKBP5 DNA methylation levels at intron 7 in psychotic patients and to compare it with healthy individuals. Methods: The sample consisted of 24 psychosis spectrum patients and 24 controls matched by age and gender. All participants were genotyped for rs1360780 and divided into 2 groups depending on the presence of the risk allele (risk and nonrisk group). DNA methylation levels at 3 CpG sites (CpG1, CpG2, and CpG3) in intron 7 were analyzed by Sanger sequencing. Early-life adversities were measured by Childhood Trauma Questionnaire. Pearson correlation and t test were performed as appropriate. Results: Analyses revealed decreased FKBP5 methylation at targeted CpG sites and averaged methylation level (AML) at intron 7 in patients compared to controls (P = .026, P = .017, P = .027, and P = .003, respectively). Decreased AML and methylation at CpG3 were observed comparing risk and nonrisk patients’ groups (P = .018 and P = .016, respectively). Additionally, decreased methylation was found in risk patients’ group compared to risk controls’ group. No differences were found comparing nonrisk groups. Furthermore, strong negative associations between trauma and methylation at CpG3 and AML were observed only in risk controls’ group (r = −0.707, P = .007; r = −0.741, P = .004, respectively). Conclusion: Our preliminary results revealed allele-specific epigenetic changes of the FKBP5 gene in psychotic patients, which is in line with previous reports in traumatized individuals. Trauma-related demethylation in risk controls’ group supports the hypothesis that psychotic and stress-related conditions could share common neurobiological underlying mechanism, such as HPA axis dysregulation, particularly in individuals with genetic predisposition for altered stress response.References1.Daskalakis NP, Binder EB. Schizophrenia in the spectrum of gene-stress interactions: the FKBP5 example. Schizophr Bull. 2015;41:323–329.2.Klengel T, Mehta D, Anacker C et al. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions. Nat Neurosci. 2013;16:33–41.

Knowledge of the antioxidant defense in the stress-responding structures of the CNS is of crucial importance, since oxidative damage is a phenomenon accompanying many stress-related disorders. Regulation of antioxidative and anti-inflammatory defense through Nrf2 (nuclear factor 2 eritroid related factor 2) pathway has emerged as a promising approach for neuroprotection. In this study, we used chronic social isolation of male Wistar rats to induce depressive-like behavior. We hypothesized that Nrf2 Keap1 pathway is compromised in the limbic brain after prolonged stress. Since subcellular trafficking of Nrf2 and its inhibitor Keap1 (Kelch ECH associating protein 1) is essential for the activation of Nrf2, we determined their protein level in cytosolic and nuclear comp& linents of hippocampus and prefrontal cortex (PEG). We also determined mRNA levels of Nr12-regulated genes involved in the production and utilization of glutathione, glutamate cysteine ligase (Gclm), glutathione S-transferase (Gsta3) and glutathione reductase (Gsr). Our results showed that chronic isolation induced anxiety and depressive-like behavior, decreased Nrf2 and in parallel increased Keap1 and nuclear factor kappa B (NF kappa B) in the hippocampus, which were not accompanied by expression profiles of Nrf2-regulated genes. Chronically stressed rats challenged with acute stress failed to induce any response of examined genes in either of brain structures, even though Nrf2/Keap1 was altered, while in naive animals Nrf2 activity corresponded witli an expression of Nrf2-regulated genes. Our results reveal maladaptive character of chronic stress at Nrf2/Keap1 level followed by pro-inflammatory conditions, and suggest a possible role of these alterations in pathogenesis of depressive/anxiety disorders. (C) 2015 Elsevier B.V. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia [III41029]

A growing body of evidence indicates that mitogen-activated protein kinase (MAPK) participates in various stress-induced responses and is considered to be one of the pathophysiological mechanisms in depression. Surprisingly, the effect of antidepressants on MAPKs is almost unexplored, particularly from the perspective of sexes. The present study investigates the cytoplasm-nuclear distribution of MAPK family, c-Jun N-terminal kinases (JNKs) 1, 2 and 3; extracellular signal-regulated kinases (ERKs) 1 and 2; and p38 kinases, as well as their phosphoisoforms in the hippocampus of chronically stressed female and male rats and upon chronic fluoxetine treatment. Additionally, we analysed crosstalk between MAPK signalling and depressive-like behaviour which correlated with brain-derived neurotrophic factor (BDNF) expression. Our results emphasize a gender-specific and compartment-dependent response of MAPKs to stress and fluoxetine. In females, stress decreased pp38 and pJNK and induced cytosolic retention of pERKs which reduced all nuclear pMAPKs. These changes correlated with altered BDNF expression and behaviour. Similarly, in males, stress decreased pp38 but promoted nuclear translocation of pJNKs and pERKs. These stress alterations of pMAPKs in males were not associated with BDNF expression and depressive-like behaviour. Fluoxetine treatment in stressed females upregulated whole pMAPK signalling particularly those in nucleus which was followed with BDNF expression and normalization of behaviour. In stressed males, fluoxetine affected only cytosolic pJNKs, while nuclear pMAPK signalling and BDNF expression were unaffected even though fluoxetine normalized behaviour. Overall, our results suggest existence of gender-specific mechanism of fluoxetine on nuclear pMAPK/BDNF signalling and depressive-like behaviour and reinforce the antidepressant dogma that females and males respond differently to certain antidepressants.

Background: Oxidative stress is reliably observed in major depressive disorder (MDD). However, molecular data on the principal cellular redox-sensitive transcriptional factors and the levels of their downstream-regulated antioxidant enzymes in MDD are scarce. Methods: In the peripheral blood mononuclear cells (PBMC) of subjects with a current episode of MDD (n = 30) and healthy controls (n = 35), we investigated alterations in the levels of redox-sensing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein, its inhibitor Keap1, and nuclear factor-κB (NF-κB), along with their cognate downstream effectors, the antioxidant enzymes (AOEs): manganese and copper zinc superoxide dismutase (MnSOD and CuZnSOD, respectively), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GLR). Results: MDD subjects exhibited higher levels of Nrf2 and its regulator Keap1, as well as NF-κB in the cytoplasm of PBMC compared to controls. This state was further reflected by increased levels of MnSOD, CuZnSOD and CAT proteins and by the lack of correlation between MnSOD and CAT, which could indicate impaired oxidative detoxification capacity in MDD patients. Moreover, increased levels of MnSOD, CuZnSOD and CAT in MDD patients positively correlated with levels of Nrf2, while increased levels of SODs were also positively related to NF-κB. There were no differences regarding the levels of GPx and GLR proteins, but the ratio of GLR/GPx was reduced, suggesting diminished capacity of GPx in antioxidative defence in PBMC of MDD subjects. Conclusion: These data provide evidence that MDD is characterized by up-regulation of redox-sensitive transcriptional factors (Nrf2 and NF-κB) and AOEs (MnSOD, CuZnSOD and CAT), indicating pro-oxidative state in the PBMC of MDD patients.

Alterations in the antioxidative defense parameters upon chronic stress are considered critical for pathophysiology of stress related psychiatric disorders, and their status in blood serves as biomarker for effects of pharmacological treatments. The present study was designed to investigate the modulation of erythrocyte antioxidant enzymes (AOEs): CuZn superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GLR) activities and their protein expression in Wistar male rats subjected to chronic psychosocial isolation and/or to pharmacological treatment with fluoxetine. Chronically isolated animals exhibited decreased levels of serum corticosterone, as opposed to other chronic stress paradigms. In addition to that, SOD, CAT and GPx status was not altered either by chronic psychosocial isolation or by fluoxetine treatment. In contrast, GLR activity and its protein level were both markedly reduced by fluoxetine. Since, GLR is crucial for overall oxido-reductive balance through maintaining optimal ratio of reduced/oxidized glutathione level (GSH/GSSG) in erythrocytes, these results could indicate that in spite of numerous beneficial effects of fluoxetine, it may compromise both haemoglobin function and oxygen transport.

One of the most consistent biological findings in major depression (MDD) is the altered activity of the hypothalamic-pituitary-adrenal (HPA) axis. It is not surprising that glucocorticoid receptor (GR), the common mechanism for stress-related changes in brain function, is a potential target of antidepressant drugs and therapies. All effective antidepressant treatments should trigger and maintain GR-related cellular processes necessary for recovery from MDD. Classic antidepressants act indirectly, by affecting the dynamic interplay between serotonin neurotransmission and HPA. On the other hand, certain compounds acting at supra-hypothalamic, HPA axis, glucocorticoid receptors, and post-receptor levels are being considered as new therapeutic options with the potential to modulate the aforementioned system in affective disorders directly. Different classes of drugs pharmacologically modify the HPA axis. This article summarizes the efficacy of classic antidepressants, as well as drugs classified as "antiglucocorticoids" (GR agonists, GR antagonists, dehydroepiandrosterone- DHEA, steroid synthesis inhibitors drugs, etc) in their capacity to heal glucocorticoid-mediated damage in depression. New avenues investigating the potential therapeutic benefits of antiglucocorticoids in affective disorders are at the proof-of-concept stage and future developments in this area deserve the full attention of psychiatrists and neuroscientists, as the current pharmacological treatment of MDD is far from perfect.

Chronic psychosocial isolation stress (CPSI) modulates glucocorticoid receptor (GR) functioning in Wistar male rat hippocampus (HIPPO) through alteration of nuclear GR phosphorylation and its upstream kinases signaling, which parallels animal depressive-like behavior. The current study investigated potential gender specificities regarding the effect of chronic therapy by an antidepressant fluoxetine (FLU) on GR signaling in HIPPO and depressive-like behavior in CPSI animals. FLU was administrated to female and male naive or CPSI rats for 21 days and GR protein, its phosphorylation status and upstream kinases, as well as GR and BDNF mRNA were followed in HIPPO together with animal serum corticosterone (CORT) and depressive-like behavior. The results showed that CPSI increased immobility in males versus hyperactivity in females and disrupted nuclear pGR232-Cdk5 pathway and JNK signaling in a gender-specific way. In contrast, in both genders CPSI increased the nuclear levels of GR and pGR246 but decreased CORT and mRNA levels of GR and BDNF. Concomitant FLU normalized the depressive-like behavior and altered the nuclear pGR232-Cdk5 signaling in a gender-specific manner. In both females and males, FLU reversed the nuclear levels of GR and pGR246 without affecting CORT and GR mRNA levels. In contrast, FLU exhibited gender-specific effect on BDNF mRNA in CPSI animals, by increasing it in females, but not in males. In spite of normalization the total nuclear GR level upon FLU treatment in both gender, down-regulation of GR mRNA is possibly maintained through prevalence of pGR232 isoform only in males. The gender-specific alterations of pGR232-Cdk5 signaling and BDNF gene expression in HIPPO and normalization of depressive-like behavior upon FLU treatment distinguishes this signaling pathway as potential future antidepressant target for gender-specific therapy of stress related mood disorders. (C) 2013 Elsevier Ltd. All rights reserved.

The mechanism of maladaptive chronic stress response involves altered phosphorylation of the glucocorticoid receptor (GR). In this study, we investigated if important depressogenic vulnerability factors, such as neuroticism and self-reports of negative affective states, may be associated with alterations in levels of the GR and GR phosphoisoforms in peripheral blood mononuclear cells (PBMC) of healthy adults. In 21 women and 16 men we evaluated PMBC levels of total GR (tGR), GR phosphorylated at serine 211 (pGR-S211) and serine 226 (pGR-S226) and correlated these data with personality traits and current reports of stress, anxiety and depression. Also, we assessed plasma cortisol levels in all tested subjects. Our results showed that in women nuclear pGR-S226 was positively correlated with neuroticism and current reports of depression, anxiety and stress, while the ratio of nuclear pGR-S211/pGR-S226 was negatively correlated with reports of depression. None of the aforementioned correlations were significant in men. No significant relations between cortisol levels and any of GR parameters were observed. These preliminary findings highlight the value of GR phosphorylation-related research in identifying molecular biomarkers of depressogenic vulnerability, at least in women. (C) 2013 Elsevier Ireland Ltd. All rights reserved.