Miroslav Adzic

Effects of Chronic Social Isolation on Wistar Rat Behavior and Brain Plasticity Markers

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.

The role of phosphorylated glucocorticoid receptor in mitochondrial functions and apoptotic signalling in brain tissue of stressed Wistar rats.

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 marked 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.

Fluoxetine affects hippocampal plasticity, apoptosis and depressive-like behavior of chronically isolated rats.

Plastic response and successful adaptation to stress are of particular importance in the hippocampus, where chronic stress may cause cell death instead of neural remodeling. Structural modifications that occur both in the brain of depressed patients and animal stress models may be reversed by antidepressants. Since morphological changes induced by stress and/or antidepressants could be mediated by presynaptically located proteins, determining the levels of these proteins may be a useful way to identify molecular changes associated with synaptic plasticity. In this study we analyzed the effects of chronic (six-week) social isolation and long-term (three-week) fluoxetine treatment on molecular markers of plasticity and apoptosis in the hippocampus of Wistar rats. Compartmental redistribution of NFκB transcription factor involved in the regulation of plasticity and apoptosis was also examined. To establish whether social isolation is able to evoke behavioral-like effects, which might be related to the observed molecular changes, we performed the forced swimming test. The results show that synaptosomal polysialic neural cell adhesion molecule (PSA-NCAM), a molecular plasticity marker, was increased in the hippocampus of chronically isolated rats, while subsequent treatment with fluoxetine set it at the control level. In addition, analysis of cytoplasm/mitochondria redistribution of apoptotic proteins Bax and Bcl-2 after exposure to chronic isolation stress, revealed an increase in Bcl-2 protein expression in both compartments, while fluoxetine enhanced the effect of stress only in the mitochondria. The observed alterations at the molecular level were accompanied by normalization of stress-induced behavioral changes by fluoxetine.

Antitumor Effects of a Natural Anthracycline Analog (Aloin) Involve Altered Activity of Antioxidant Enzymes in HeLaS3 Cells

The antiproliferative and cytotoxic potential of the natural anthracycline aloin from Aloe vera was tested on human uterine carcinoma HeLaS3 cells. Aloin showed a pronounced antiproliferative effect at physiological concentration (IC50=97μM), caused cell cycle arrest in the S phase, and markedly increased HeLaS3 cell apoptosis (to 24%). In the concentration range of 20-100 μM, its action was accompanied by remarkable changes in the activity of almost all antioxidant enzymes: MnSOD activity was increased many fold, while CuZnSOD and iNOS activities were inhibited. Moreover, inhibition of CuZnSOD was shown to occur by direct aloin interaction with the enzyme. As catalase activity was not changed, it is suggested that such conditions were responsible for antiproliferative and cytotoxic effects owing to accumulation of H2O2. Aloin alone was a more potent proapoptotic agent than a 2 Gy fractional dose of ionizing radiation or a combination of the two. Compared to other currently used therapeutics, aloin, due to its less undesirable side effects and antimetastatic potential, may prove to be the agent of choice on which clinical protocols for the treatment of human cervical carcinoma should rely in future.

Fluoxetine affects antioxidant system and promotes apoptotic signaling in Wistar rat liver

Selective serotonin reuptake inhibitors (SSRI) are a treatment of choice for stress related disorders including clinical depression and a range of anxiety-related disorders. In the experimental animals, chronic stress paradigms are considered as a model of depression, and in that context are used for examining the effects of different drug treatments. The present research was designed to investigate the effect of SSRI fluoxetine on antioxidant status and apoptotic signaling in Wistar rat liver, which is a central organ for activation and detoxification of many xenobiotics and reactive oxygen species. We also investigated whether chronic fluoxetine treatment exhibits the same effects in the liver of control animals vs. animals stressed by chronic psychosocial isolation. Our results revealed that fluoxetine downregulated the activity of superoxide dismutases and upregulated the activity of glutathione peroxidase in both rat groups, while elevating glutathione reductase activity and total antioxidant status only in stressed animals. These results suggested that fluoxetine interfered with stress-induced pathways of oxidative defense in the liver. In addition, in both experimental groups, fluoxetine induced several hallmarks of apoptosis in the liver, including a decrease in Bcl-2 expression and increased DNA fragmentation. However, apoptotic alterations were more pronounced in stressed animals, suggesting that stress related oxidative damage could have primed apoptotic effects of fluoxetine.

Stress Type Dependence of Expression and Cytoplasmic-Nuclear Partitioning of Glucocorticoid Receptor, Hsp90 and Hsp70 in Wistar Rat Brain

Chronic exposure to stress is associated with different behavioral and neurological syndromes including impaired excitability of nerve cells in hippocampus (HIPPO) and prefrontal cortex (PFC), regions of the brain that are important for adaptation. The successful adaptation to stress involves negative feedback at the level of the hypothalamic-pituitary-adrenal (HPA) axis provided by the glucocorticoid receptor (GR), which is a steroid-dependent transcription factor found in a heterocomplex with heat shock proteins Hsp90 and Hsp70. In Wistar rats, chronic social isolation leads to a significant decrease in serum corticosterone (CORT), probably due to alterations in the GR signaling pathway. We exploited this type of stress, alone or in combination with acute immobilization, to define changes in the expression level and compartmental distribution of GR, Hsp90 and Hsp70 in HIPPO and PFC. The results indicated that in acute and combined stress, when CORT was increased, GR was translocated to the nucleus in both brain structures. Under chronic stress, when CORT was below the control level, GR was retained in the cytoplasm of PFC, and evenly distributed between compartments in HIPPO. Simultaneously, heat shock proteins partitioning in HIPPO seemed to be mainly stress type-independent, while that of PFC was dependent on stress type. Thus, the stress type-specific responses of GR and heat shock proteins were mainly detected in PFC rather than in HIPPO of Wistar rats. The observed alterations in protein expression and cytoplasmic-nuclear partitioning of the GR, Hsp90 and Hsp70 proteins may be related to maladaptive response of the HPA axis under chronic stress.

Alterations in the Nrf2-Keap1 signaling pathway and its downstream target genes in rat brain under stress.

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 compartments of hippocampus and prefrontal cortex (PFC). We also determined mRNA levels of Nrf2-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 (NFκB) 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 naïve animals Nrf2 activity corresponded with 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.

Gender-specific effects of fluoxetine on hippocampal glucocorticoid receptor phosphorylation and behavior in chronically stressed rats

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 naïve 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.

Chronic social isolation is related to both upregulation of plasticity genes and initiation of proapoptotic signaling in Wistar rat hippocampus

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 (NFκB) is considered as an important stress sensor implicated in adaptive synaptic plasticity. Numerous stress-related genes are regulated by both hippocampal GR and NFκB, 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 NFκB 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 NFκB 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 NFκB. 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.

Phosphorylation of leukocyte glucocorticoid receptor in patients with current episode of major depressive disorder.

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<0.001) and, to a less extent, at S211 (p<0.05) in MDD patients compared to controls. Consequently, the pGR-S211/pGR-S226 ratio was decreased (p<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.