Agnieszka Basta-Kaim

Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid.

Autism is a severe behavioral disorder characterized by pervasive impairments in social interactions, deficits in verbal and non-verbal communication, and stereotyped behaviors, with a four times higher incidence in boys than in girls. The core symptoms are frequently accompanied by a spectrum of neurobehavioral and immunological derangements, including: aberrant sensitivity to sensory stimulation, anxiety, and decreased cellular immune capacity. Recently, a new potential rodent model of autism induced by prenatal exposure to valproic acid (VPA rats) has been proposed. In order to determine if gender has an influence on alterations observed in VPA rats, male and female rats have been evaluated in a battery of behavioral, immunological, and endocrinological tests. A plethora of aberrations has been found in male VPA rats: lower sensitivity to pain, increased repetitive/stereotypic-like activity, higher anxiety, decreased level of social interaction, increased basal level of corticosterone, decreased weight of the thymus, decreased splenocytes proliferative response to concanavaline A, lower IFN-gamma/IL-10 ratio, and increased production of NO by peritoneal macrophages. Female VPA rats exhibited only increased repetitive/stereotypic-like activity and decreased IFN-gamma/IL-10 ratio. Sexual dimorphism characteristics for measured parameters have been observed in both groups of animals, except social interaction in VPA rats. Our results confirm existence of similarities between the observed pattern of aberrations in VPA rats and features of disturbed behavior and immune function in autistic patients, and suggest that they are gender-specific, which is intriguing in light of disproportion in boys to girls ratio in autism.

The effect of antidepressant drugs on the HPA axis activity, glucocorticoid receptor level and FKBP51 concentration in prenatally stressed rats.

Dysregulation of hypothalamic-pituitary-adrenal (HPA) axis activity is thought to be an important factor in pathogenesis of depression. In animals, stress or glucocorticoids given in prenatal period lead to long-lasting behavioral and neuroendocrine changes similar to those observed in depressed patients. However, molecular basis for HPA disturbances in animals exposed to prenatal stress - a model of depression - have been only partially recognized. Therefore, in the present study we investigated the effect of prenatal stress on behavioral changes, blood corticosterone level, concentrations of glucocorticoid receptor (GR) and its cochaperone, FKBP51, in the hippocampus and frontal cortex in adult rats. It has been found that prenatally stressed rats display high level of immobility in the Porsolt test and anxiety-like behavior. The HPA axis hyperactivity in theses animals was evidenced by corticosterone hypersecretion at the end of the light phase and 1h following acute stress. Western blot study revealed that GR level was significantly elevated in the hippocampus but not in the frontal cortex of prenatally stressed rats, whereas concentration of FKBP51 was decreased only in the former brain structure. Chronic treatment with imipramine, fluoxetine, mirtazapine and tianeptine have diminished both behavioral and biochemical alterations observed in this animal model of depression. These data indicate that the increase in hippocampal GR level and low concentration of FKBP51 in the frontal cortex may be responsible for enhanced glucocorticoid action in depression.

Effect of mild chronic stress, as a model of depression, on the immunoreactivity of C57BL/6 mice

Numerous studies correlate the state of depression with some abnormalities in the immune response, such as increased numbers of white blood-cells, alterations in sub-populations of leucocytes, suppression of cytotoxic activity of natural-killer cells, increased levels of some autoantibodies and acute-phase proteins. Some of these changes have been attributed to autoimmunological reactions. While the possibilities to evaluate some reactions in depressed patients are limited, an animal model of depression could well simulate this clinical situation, and the chronic mild state of stress is a well accepted one. After undergoing stress for three-weeks, C57BL/6 mice demonstrate in the present study a decrease in thymus weight, as well as increased interleukin-1 and decreased interleukin-2 production. Splenocytes of the depressed mice exert a decrease in natural-killer-cell activity, in the proliferative response to Concanavalin-A, interleukin-1 and anti-CD3 monoclonal antibodies and an increase in the proliferative response to lipopolysaccharides and pokeweed mitogens. Our results also suggest that chronic stress-induced activation of suppressor cells in the spleen, due to elimination of CD8+ cells, increase the proliferation of splenocytes in response to mitogens of T cells.

A new animal model of (chronic) depression induced by repeated and intermittent lipopolysaccharide administration for 4 months

Chronic activation of immune-inflammatory and oxidative and nitrosative stress (O&NS) pathways plays an important role in the pathophysiology of clinical depression. Increased IgA responses directed against LPS of gram-negative bacteria, indicating increased bacterial translocation, may be one of the drivers underpinning these pathways. There is a strong association between signs of bacterial translocation and chronicity of depression and O&NS, but not pro-inflammatory cytokines. The aims of the present study were to: (1) develop a new neurobehavioral model of (chronic) depression (anhedonic behavior) that may reflect chronic LPS stimulation and is associated with increased oxidative stress, and (2) to delineate the effects of fluoxetine on this new depression model. We established that in female mice repeated LPS injections once daily for 5 days (from 750 μg/kg to a maximal dose 1250 μg/kg; increasing doses for the first three days which were then gradually decreased on day 4 and 5) at a one-month interval and this repeated for 4 consecutive months induced chronic anhedonia (estimated by the preference to drink a 1% sucrose) lasting for at least 7 weeks. Chronic LPS administration significantly decreased thymus weight, proliferative activity of splenocytes, production of interferon (IFN)γ and interleukin-(IL)10, and increased superoxide and corticosterone production. Treatment with fluoxetine for 3 weeks abolished the neurobehavioral effects of LPS. The antidepressant effect of fluoxetine was accompanied by increased production of IL-10 and reduced superoxide and corticosterone production. Our results suggest that repeated intermittent LPS injections to female mice may be a useful model of chronic depression and in particular for the depressogenic effects of long standing activation of the toll-like receptor IV complex.

Prenatal stress is a vulnerability factor for altered morphology and biological activity of microglia cells

Several lines of evidence suggest that the dysregulation of the immune system is an important factor in the development of depression. Microglia are the resident macrophages of the central nervous system and a key player in innate immunity of the brain. We hypothesized that prenatal stress (an animal model of depression) as a priming factor could affect microglial cells and might lead to depressive-like disturbances in adult male rat offspring. We investigated the behavioral changes (sucrose preference test, Porsolt test), the expression of C1q and CD40 mRNA and the level of microglia (Iba1 positive) in 3-month-old control and prenatally stressed male offspring rats. In addition, we characterized the morphological and biochemical parameters of potentially harmful (NO, iNOS, IL-1β, IL-18, IL-6, TNF-α, CCL2, CXCL12, CCR2, CXCR4) and beneficial (insulin-like growth factor-1 (IGF-1), brain derived neurotrophic factor (BDNF)) phenotypes in cultures of microglia obtained from the cortices of 1–2 days old control and prenatally stressed pups. The adult prenatally stressed rats showed behavioral (anhedonic- and depression-like) disturbances, enhanced expression of microglial activation markers and an increased number of Iba1-immunopositive cells in the hippocampus and frontal cortex. The morphology of glia was altered in cultures from prenatally stressed rats, as demonstrated by immunofluorescence microscopy. Moreover, in these cultures, we observed enhanced expression of CD40 and MHC II and release of pro-inflammatory cytokines, including IL-1β, IL-18, TNF-α and IL-6. Prenatal stress significantly up-regulated levels of the chemokines CCL2, CXCL12 and altered expression of their receptors, CCR2 and CXCR4 while IGF-1 production was suppressed in cultures of microglia from prenatally stressed rats. Our results suggest that prenatal stress may lead to excessive microglia activation and contribute to the behavioral changes observed in depression in adulthood.

Antipsychotic drugs inhibit the human corticotropin-releasing-hormone gene promoter activity in neuro-2A cells-an involvement of protein kinases.

Antipsychotic drugs can regulate transcription of some genes, including those involved in regulation of hypothalamic–pituitary–adrenal (HPA) axis, whose activity is frequently disturbed in schizophrenic patients. However, molecular mechanism of antipsychotic drug action on the corticotropin-releasing hormone (CRH) gene activity has not been investigated so far. This study was undertaken to examine the influence of conventional and atypical antipsychotic drugs on the CRH gene promoter activity in differentiated Neuro-2A cell cultures stably transfected with a human CRH promoter fragment linked to the chloramphenicol acetyltransferase (CAT) reporter gene. It has been found that chlorpromazine (0.1–5.0 μM), haloperidol (0.5–5.0 μM), clozapine (1.0–5.0 μM), thioridazine (1.0–5.0 μM), promazine (5.0 and 10 μM), risperidone (5.0 and 10.0 μM), and raclopride (only at the highest used concentrations, ie 30 and 100 μM) present in culture medium for 5 days inhibited the CRH-CAT activity. Sulpiride and remoxipride had no effect. Since CRH gene activity is most potently enhanced by cAMP/protein kinase A pathway, the effect of antipsychotics on the forskolin-induced CRH-CAT activity was determined. Chlorpromazine (1.0–5.0 μM), haloperidol (1.0–5.0 μM), clozapine (1.0–5.0 μM), thioridazine (3.0 and 5.0 μM), and raclopride (30 and 100 μM), but not promazine, sulpiride, risperidone, and remoxipride, inhibited the forskolin-stimulated CRH gene promoter activity. A possible involvement of protein kinases in chlorpromazine and clozapine inhibitory action on CRH activity was also investigated. It was found that wortmannin (0.01 and 0.02 μM), an inhibitor of phosphatidylinositol 3-kinase (PI3-K), significantly attenuated the inhibitory effect of chlorpromazine and clozapine on CRH gene promoter activity. In line with these results, a Western blot study showed that these drugs increased phospho-Ser-473 Akt level, had no effect on total Akt, and decreased glycogen synthase kinase-3β level. Additionally, we found that clozapine decreased protein kinase C (PKC) level and that its action on CRH activity was attenuated by PKC activator (TPA, 0.1 μM). The obtained results indicate that inhibition of CRH gene promoter activity by some antipsychotic drugs may be a molecular mechanism responsible for their inhibitory action on HPA axis activity. Clozapine and chlorpromazine action on CRH activity operates mainly through activation of the PI3-K/Akt pathway. Moreover, PKC-mediated pathway seems to be involved in clozapine action on CRH gene activity.

Rapamycin induces of protective autophagy in vascular endothelial cells exposed to oxygen-glucose deprivation.

The protective potential of rapamycin has been reported in a few experimental models of brain ischemia, both in vivo and in vitro. Although the precise cellular processes underlying the neuroprotective effects of rapamycin in experimental models of stroke remain unknown, the current experimental data suggest that the mechanism of action of the drug may result from the mTOR-mediated autophagy induction. However, it is unclear whether the activation of autophagy acts as a pro-death or pro-survival factor in vascular endothelial cells in ischemic brain damage. It seems to be very important, since stroke affects not only neurons and astrocytes but also microvessels. In the present study, we used human umbilical vein endothelial cells (HUVEC) subjected to ischemia-simulating conditions (combined oxygen and glucose deprivation, OGD) for 6h to determine potential effect of rapamycin-induced autophagy on HUVEC damage. The drug at concentrations of 100 and 1000nM increased the expression of Beclin 1 and LC3-II together with a significant increase in the p62 degradation in ischemic HUVEC. Treatment with rapamycin in OGD significantly increased the cell viability, indicating that the drug exerts cytoprotective effect. The inhibition of Beclin 1 by siRNAs significantly attenuated the expression of autophagy-related proteins and reduced HUVEC viability following OGD and rapamycin treatment. Our findings demonstrated that toxicity of simulated ischemia conditions were enhanced in HUVEC when autophagy was blocked, and that rapamycin effectively prevented OGD-evoked damage by induction of protective autophagy via inhibition of the mTOR pathway.

The effect of chronic treatment with imipramine on the immunoreactivity of animals subjected to a chronic mild stress model of depression.

A depression-like state was induced in Wistar rats by chronic (3-week) exposure to very mild, unpredictable stress, which led to diminished food consumption and diminished preference for sweet drinks (anhedonia). Anhedonia was then abolished by 5 weeks of daily administration of imipramine to the continually stressed animals. One day after the last drug injection and stressful event, a statistically significant decrease in the proliferative activity of splenocytes to Con A stimulation in vitro was observed in those animals. Eight weeks of stress (without antidepressant therapy) affected likewise, but in a less potent and non-significant manner, the activity of splenocytes. Administration of imipramine alone for a period of 5 weeks did not modify the activity of these cells.

Regulation of the human corticotropin-releasing-hormone gene promoter activity by antidepressant drugs in Neuro-2A and AtT-20 cells.

Major depression is frequently associated with hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis. Clinically effective therapy with antidepressant drugs normalizes the disturbed activity of HPA axis, in part, by decreasing corticotropin-releasing hormone (CRH) synthesis, but the mechanism of this action is poorly recognized. In order to find out whether antidepressants directly affect CRH gene promoter activity, we studied their effect on undifferentiated and differentiated Neuro-2A cells, and for comparison the effect of the selected antidepressants on AtT-20 cells was also determined. The cells were stably transfected with a human CRH promoter fragment (−663 to +124 bp) linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The regulation of CRH gene promoter activity is similar in Neuro-2A cells, both intact and differentiated, and in AtT-20 cell line, and cAMP/PKA-dependent pathway plays an important role in the stimulation of CRH gene. It was found that imipramine, amitryptyline, desipramine, fluoxetine, and mianserin, present in the culture medium for 5 days, in a concentration-dependent manner inhibited basal hCRH gene promoter activity in undifferentiated Neuro-2A cells, while other drugs under study (citalopram, tianeptine, moclobemide, venlafaxine, reboxetine, mirtazapine, and milnacipram) were inactive. In the differentiated cells, all examined antidepressants, except moclobemide (no effect) and tianeptine (increase), inhibited hCRH gene transcription. Moreover, in differentiated cells, the drugs acted stronger and were effective at lower concentrations. Forskolin-induced CAT activity was attenuated by imipramine and fluoxetine and to a lesser degree by amitriptyline and desipramine in differentiated cells, whereas other drugs were inactive. Moreover, imipramine and fluoxetine, but not tianeptine, showed moderate inhibitory effect on CRH gene promoter activity also in AtT-20 cell line, commonly used in CRH gene regulation studies. These results indicate that neuron-like differentiated Neuro-2A cells are a better model than pituitary and intact neuroblastoma to investigate the mechanism of psychotropic drug action. Inhibition of CRH gene promoter activity by antidepressant drugs may be a molecular mechanism by which these drugs inhibit the activity of HPA axis.

Chlorpromazine inhibits the glucocorticoid receptor-mediated gene transcription in a calcium-dependent manner.

Antipsychotic drugs can modulate transcription factors and also nuclear receptors, but their action on glucocorticoid receptors (GR)-members of the steroid/thyroid hormone receptor family has not been studied so far. In the present study we investigated effects of various antipsychotics on the glucocorticoid-mediated gene transcription in fibroblast cells, stably transfected with a mouse mammary tumor virus promoter (LMCAT cells). Chlorpromazine (3-100 microM) inhibited the corticosterone-induced gene transcription in a concentration- and time-dependent manner. Clozapine showed a similar, but less potent effect, while haloperidol acted only in high concentrations, and other antipsychotic drugs (sulpiride, raclopride, remoxipride) were without any effect. It was also found that a phorbol ester (an activator of protein kinase C (PKC)) and A-23187 (Ca(2+)-ionophore) attenuated the inhibitory effect of chlorpromazine on the GR-induced gene transcription. An antagonist of the L-type Ca(2+) channel, as well as an inhibitor of phospholipase C (PLC) inhibited the corticosterone-induced gene transcription, but had no effect on the chlorpromazine-induced changes. The involvement of a PKC/PLC pathway in the chlorpromazine action was confirmed by Western blot analysis which showed that the drug in question decreased the PLC-beta(1) protein level, and to a lesser extent that of the PKC-alpha protein in LMCAT cells. The aforementioned data suggest that inhibition of the glucocorticosteroid-induced gene transcription by chlorpromazine and clozapine may be a mechanism by which these drugs block some effects induced by glucocorticoids. The inhibitory effect of chlorpromazine on the corticosterone-induced gene transcription seems to depend on the inhibition of Ca(2+) influx and/or the inhibition of some calcium-dependent enzymes, e.g. phospholipase beta(1).