Ivana Elaković

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.

Lymphocyte glucocorticoid receptor expression level and hormone-binding properties differ between war trauma-exposed men with and without PTSD.

OBJECTIVE Posttraumatic stress disorder (PTSD) has been shown to be associated with altered glucocorticoid receptor (GR) activity. We studied the expression and functional properties of the receptor in peripheral blood mononuclear cells (PBMCs) from non-traumatized healthy individuals (healthy controls; n=85), and war trauma-exposed individuals with current PTSD (n=113), with life-time PTSD (n=61) and without PTSD (trauma controls; n=88). The aim of the study was to distinguish the receptor alterations related to PTSD from those related to trauma itself or to resilience to PTSD. METHODS Functional status of the receptor was assessed by radioligand binding and lysozyme synthesis inhibition assays. The level of GR gene expression was measured by quantitative PCR and immunoblotting. RESULTS Current PTSD patients had the lowest, while trauma controls had the highest number of glucocorticoid binding sites (Bmax) in PBMCs. Hormone-binding potential (Bmax/KD ratio) of the receptor was diminished in the current PTSD group in comparison to all other study groups. Correlation between Bmax and KD that normally exists in healthy individuals was decreased in the current PTSD group. Contrasting Bmax data, GR protein level was lower in trauma controls than in participants with current or life-time PTSD. CONCLUSIONS Current PTSD is characterized by reduced lymphocyte GR hormone-binding potential and by disturbed compensation between Bmax and hormone-binding affinity. Resilience to PTSD is associated with enlarged fraction of the receptor molecules capable of hormone binding, within the total receptor molecule population in PBMCs.

Brain region- and sex-specific modulation of mitochondrial glucocorticoid receptor phosphorylation in fluoxetine treated stressed rats: Effects on energy metabolism

Antidepressants affect glucocorticoid receptor (GR) functioning partly through modulation of its phosphorylation but their effects on mitochondrial GR have remained undefined. We investigated the ability of chronic fluoxetine treatment to affect chronic stress-induced changes of mitochondrial GR and its phosphoisoforms (pGRs) in the prefrontal cortex and hippocampus of female and male rats. Since mitochondrial GR regulates oxidative phosphorylation, expression of mitochondrial-encoded subunits of cytochrome (cyt) c oxidase and its activity were also investigated. Chronic stress caused accumulation of the GR in mitochondria of female prefrontal cortex, while the changes in the hippocampus were sex-specific at the levels of pGRs. Expression of mitochondrial COXs genes corresponded to chronic stress-modulated mitochondrial GR in both tissues of both genders and to cyt c oxidase activity in females. Moreover, the metabolic parameters in stressed animals were affected by fluoxetine therapy only in the hippocampus. Namely, fluoxetine effects on mitochondrial COXs and cyt c oxidase activity in the hippocampus seem to be conveyed through pGR232 in females, while in males this likely occurs through other mechanisms. In summary, sex-specific regulation of cyt c oxidase by the stress and antidepressant treatment and its differential convergence with mitochondrial GR signaling in the prefrontal cortex and hippocampus could contribute to clarification of sex-dependent vulnerability to stress-related disorders and sex-specific clinical impact of antidepressants.

Effects of fluoxetine on plasticity and apoptosis evoked by chronic stress in rat prefrontal cortex

The prefrontal cortex is the brain region sensitive to detrimental effects of stress and even mild stress can rapidly impair its function. Aside from initiating proadaptive neuroplastic changes in the prefrontal cortex, chronic stress may also increase vulnerability of cortical neurons to apoptosis. Understanding the mechanism of plasticity and apoptotic processes is of immense importance for therapy of stress-related psychiatric disorders. In this study we tested whether molecular alterations in the prefrontal cortex, which occurred upon chronic social isolation, could be influenced by a prolonged fluoxetine treatment. We analyzed the expression of synaptic plasticity and apoptotic molecular markers in the prefrontal cortex of young-adult male Wistar rats exposed to 6-week social isolation with and without fluoxetine treatment during the last 3 weeks. Compartmental redistribution of NFκB transcription factor, involved in regulation of plasticity and apoptosis, was also examined. The level of synaptosomal polysialic neural cell adhesion molecule (PSA-NCAM) was increased in the prefrontal cortex of isolated rats as compared to untreated controls. Treatment with fluoxetine reduced the PSA-NCAM level only in isolated animals. In addition, mitochondrial Bax protein was elevated by chronic social isolation, while fluoxetine failed to abolish this effect. In spite of elevated Bcl-2 in the mitochondria, the calculated Bax/Bcl-2 ratio and concomitant absence of NFκB activation pointed to initiation of apoptotic signaling in the prefrontal cortex. The results imply that fluoxetine influences plasticity in the prefrontal cortex of chronically isolated rats and fails to prevent stress-induced initiation of apoptosis in this brain structure.

Gender-specific response of brain corticosteroid receptors to stress and fluoxetine.

Gender-related differences in dexamethasone binding to corticosteroid receptors (CR) and in glucocorticoid receptor (GR) protein level in the pituitary, hypothalamus, hippocampus and prefrontal cortex were studied before and after antidepressant fluoxetine administration to both unstressed and rats exposed to a chronic social isolation stress. Untreated males, in comparison to females, displayed higher hormone-binding capacity of both GR and mineralocorticoid receptor (MR) in the hippocampal cytosol, as well as higher GR protein level in the pituitary cytosol. In both genders, dexamethasone binding to MR exceeded that to GR. While fluoxetine treatment and social isolation had no effect on GR activity, the influence on MR was gender-specific. Fluoxetine facilitated MR hormone-binding only in females, increasing the MR/GR activity ratio. In contrast, after a 6-week isolation of males, MR binding capacity was diminished and MR/GR ratio inverted in favor of GR. In addition, fluoxetine induced elevation of cytosolic GR protein level in the pituitary and hypothalamus, the latter change being gender-specific. The results point to gender-related differences in the CRs functioning and suggest that both MR and GR may contribute to well-known sexual dimorphism in vulnerability to stress and stress-related disorders and in the outcome of antidepressant treatment.

Mineralocorticoid receptor and heat shock protein expression levels in peripheral lymphocytes from war trauma-exposed men with and without PTSD

Alterations in the number and functional status of mineralocorticoid (MR) and glucocorticoid receptors (GR) may contribute to vulnerability to posttraumatic stress disorder (PTSD). Corticosteroid receptors are chaperoned by heat shock proteins Hsp90 and Hsp70. We examined relations between corticosteroid receptor and heat shock protein expression levels, and related them with war trauma exposure, PTSD and resilience to PTSD. Relative levels of MR, Hsp90 and Hsp70 were determined by immunoblotting in lymphocytes from war trauma-exposed men with current PTSD (current PTSD group, n=113), with life-time PTSD (life-time PTSD group, n=61) and without PTSD (trauma control group, n=88), and from non-traumatized healthy controls (healthy control group, n=85). Between-group differences in MR, Hsp90 and Hsp70 levels and in MR/GR ratio were not observed. The level of MR was correlated with both Hsp90 and Hsp70 levels in trauma control and healthy control groups. On the other hand, GR level was correlated only with Hsp90 level, and this correlation was evident in current PTSD and trauma control groups. In conclusion, PTSD and exposure to trauma are not related to changes in lymphocyte MR, Hsp90 or Hsp70 levels, but may be associated with disturbances in corticosteroid receptors interaction with heat shock proteins.

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

Summary Real-time RT PCR has been recognized as an accurate, reliable and sensitive method for quantifying gene transcription. However, several steps preceding PCR represent critical points and source of inaccuracies. These steps include cell processing, RNA extraction, RNA storage, assessment of RNA concentration and cDNA synthesis. To compensate for potential variability introduced by the procedure, normalization of target gene expression has been established. Accurate normalization has become an absolute prerequisite for the correct quantification of gene expression. Several strategies are in use for the normalization of data, including normalization to sample size, to total RNA or to an internal reference. Among these, the use of housekeeping genes as an internal (endogenous) control is the most common approach. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper reference gene for normalization have become increasingly stringent. The aim of this paper is to discuss the concept of normalization in mRNA quantification, as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. By showing that the use of inappropriate endogenous control might lead to incorrect results and misinterpretation of experimental data, we are joining the creators of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in an attempt to convince scientists that proper validation of potential reference genes is an absolute prerequisite for correct normalization and, therefore, for providing accurate and reliable data by quantitative real-time RT PCR gene expression analyses. Kratak sadržaj RT-PCR je prepoznat kao precizna, pouzdana i osetljiva metoda za kvantifikaciji! transkripcije gena. Medutim, ovoj metodi prethodi nekoliko koraka koji predstavljaju kritične tačke i izvor potencijalnih grešaka. Ovi korači uključuju obradu čelijskog materijala, ekstrakciju i čuvanje RNK, odredivanje koncentracije RNK i sintezu cDNK. Da bi se kompenzovala potencijalna varijabilnost na- stala tokom procedure, uvedena je normalizacija ekspresije ciljnih gena. Precizna normalizacija je postala apsolutni pre- duslov za tačnu kvantifikaciju ekspresije gena. Postoji neko- liko strategija za normalizaciju eksperimentalnih podataka, uključujuči normalizaciju u odnosu na veličinu uzorka, ukup- nu RNK ili internú kontrolu (referencu). Kao interna (endo- gena) kontrola najčešče se koriste geni sa stabilnom ekspre- sijom. Imajuči u vidu veliku osetljivost, reproducibilnost i veliki dinamički opseg PCR metode, zahtevi za odgovara- jučim referentnim genima koji če se koristiti za normalizaciju podataka postali su veoma restriktivni. Cilj ovog rada je da razjasni koncept normalizacije i prokomentariše nekoliko sta- tističkih algoritama koji su razvijeni kako bi pomogli u valida- ciji potencijalnih referentnih gena. Pokazujuči da koriščenje neodgovarajučih referentnih gena (endogenih kontrola) može da dovede do netačnih rezultata i pogrešne inter- pretacije eksperimentalnih podataka, mi se priključujemo tvorcima uputstva MIQE (eng. Minimum Information for Publication of Quantitative Real-Time PCR Experiments) u pokúšajú da ubedimo naučnu javnost da je ispravna validacija potencijalnih referentnih gena apsolutni preduslov za tačnu normalizaciju i, shodno tome, preduslov za dobijanje tačnih i pouzdanih podataka u analizi ekspresije gena metodom kvantitativnog PCR-a u reálnom vremenu.

Dietary fructose-related adiposity and glucocorticoid receptor function in visceral adipose tissue of female rats

AbstractPurpose Excessive fructose intake coincides with the growing rate of obesity and metabolic syndrome, with women being more prone to these disorders than men. Findings that detrimental effects of fructose might be mediated by glucocorticoid regeneration in adipose tissue only indirectly implicated glucocorticoid receptor (GR) in the phenomenon. The aim of the present study was to elucidate whether fructose overconsumption induces derangements in GR expression and function that might be associated with fructose-induced adiposity in females.MethodsWe examined effects of fructose-enriched diet on GR expression and function in visceral adipose tissue of female rats. Additionally, we analyzed the expression of genes involved in glucocorticoid prereceptor metabolism [11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and hexose-6-phosphate dehydrogenase], lipolysis (hormone-sensitive lipase) and lipogenesis (sterol regulatory element binding protein 1 and peroxisomal proliferator-activated receptor γ).ResultsFructose-fed rats had elevated energy intake that resulted in visceral adiposity, as indicated by increased visceral adipose tissue mass and its share in the whole-body weight. GR hormone binding capacity and affinity, as well as the expression of GR gene at both mRNA and protein levels were reduced in visceral adipose tissue of the rats on fructose diet. The glucocorticoid prereceptor metabolism was stimulated, as evidenced by elevated tissue corticosterone, while the key regulators of lipolysis and lipogenesis remained unaffected by fructose diet.ConclusionsThe results suggest that the 11βHSD1-mediated elevation of intracellular corticosterone may induce GR downregulation, which may be associated with failure of GR to stimulate lipolysis in fructose-fed female rats.

Sexually dimorphic functional alterations of rat hepatic glucocorticoid receptor in response to fluoxetine

Gender-related differences in the expression and functional properties of the hepatic glucocorticoid receptor were studied before and after antidepressant fluoxetine administration to both unstressed and rats exposed to a chronic social isolation stress. Some of the receptors functional properties, including hormone-binding capacity (B(max)), hormone-binding potency (B(max)/K(D) ratio) and the DNA-binding ability, were found to be sexually dimorphic. Fluoxetine treatment (5mg/kg body mass, 21day, intraperitoneally) induced a decrease in B(max) and in the amount of Hsp70 co-immunoprecipitated with the glucocorticoid receptor only in males, and stimulated the association of the receptor with Hsp90 in females. When applied during the last three weeks of the 6-week isolation, fluoxetine parallelly elevated B(max) and the receptor protein level in female animals, while in males diminished B(max) and inhibited association of the receptor with Hsp70. Binding of dexamethasone-receptor complexes both to DNA-cellulose and to isolated liver nuclei did not appear to be a target for fluoxetine action. The results point to sex-related differences in the glucocorticoid receptor functioning and in its response to fluoxetine, and suggest that these differences may contribute to well known sexual dimorphism in the sensitivity to stress, to stress-related disorders and to antidepressant treatment.