Jelena Djordjevic

Acute or chronic stress induce cell compartment-specific phosphorylation of glucocorticoid receptor and alter its transcriptional activity in Wistar rat brain

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 21 day isolation of Wistar rats on the extrinsic negative feedback part of HPA 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 from 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.

Regulation of Inducible Nitric Oxide Synthase (iNOS) and its Potential Role in Insulin Resistance, Diabetes and Heart Failure.

Nitric oxide synthases (NOS) are the enzymes responsible for nitric oxide (NO) generation. NO is a reactive oxygen species as well as a reactive nitrogen species. It is a free radical which mediates several biological effects. It is clear that the generation and actions of NO under physiological and pathophysiological conditions are regulated and extend to almost every cell type and function within the circulation. In mammals 3 distinct isoforms of NOS have been identified: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). The important isoform in the regulation of insulin resistance (IR) is iNOS. Understanding the molecular mechanisms regulating the iNOS pathway in normal and hyperglycemic conditions would help to explain some of vascular abnormalities observed in type 2 diabetes mellitus (T2DM). Previous studies have reported increased myocardial iNOS activity and expression in heart failure (HF). This review considers the recent animal studies which focus on the understanding of regulation of iNOS activity/expression and the role of iNOS agonists as potential therapeutic agents in treatment of IR, T2DM and HF.

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.

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.

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.

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.

Chronic social isolation suppresses proplastic response and promotes proapoptotic signalling in prefrontal cortex of Wistar rats

Successful adaptation to stress involves synergized actions of glucocorticoids and catecholamines at several levels of the CNS, including the prefrontal cortex (PFC). Inside the PFC, hormonal signals trigger concerted actions of transcriptional factors, such as glucocorticoid receptor (GR) and nuclear factor kappa B (NFκB), culminating in a balanced, proadaptive expression of their common genes, such as proplastic NCAM and/or apoptotic Bax and Bcl‐2. In the present study, we hypothesized that chronic stress may compromise the balance between GR and NFκB signals and lead to an altered/maladaptive expression of their cognate genes in the PFC. Our results obtained with Wistar rats exposed to chronic social isolation indicated alterations of the GR relative to the NFκB, in favor of the GR, in both the cytoplasmic and the nuclear compartments of the PFC. Although these alterations did not affect the induction of proplastic NCAM gene, they decreased the NCAM sialylation necessary for plastic response and caused marked relocation of the mitochondrial membrane antiapoptotic Bcl‐2 protein to its cytoplasmic form. Moreover, the compromised PSA‐NCAM plastic response found under chronic stress was sustained after exposure of animals to the subsequent acute stress, whereas the proapoptotic signals were further emphasized. It is concluded that chronic social isolation of Wistar animals leads to a maladaptive response of the PFC, considering the diminishment of its plastic potential and potentiating of apoptosis. Such conditions in the PFC are likely to compromise its ability to interact with other CNS structures, such as the hippocampus, which is necessary for successful adaptation to stress.

Chronic Social Isolation Compromises the Activity of Both Glutathione Peroxidase and Catalase in Hippocampus of Male Wistar Rats

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.

Lymphocyte levels of redox-sensitive transcription factors and antioxidative enzymes as indicators of pro-oxidative state in depressive patients.

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.