M. Leskiewicz

The attenuating effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in human neuroblastoma SH-SY5Y cells.

Memantine, a clinically used N-methyl-D-aspartate (NMDA)-receptor antagonist, has been shown to prevent apoptotic neuronal damage connected with the over-activity of NMDA receptors. In the present study, we examined the effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in the SH-SY5Y cell line which does not possess functional NMDA receptors. Electrophysiological recordings and toxicity studies showed no response to NMDA-evoked currents in this cell line, irrespective of the stage of its neuronal differentiation. Memantine (0.1-2 microM) attenuated staurosporine-induced apoptosis as evidenced by reversal of the changes in mitochondrial membrane potential (DeltaPsi(m)) and decreased caspase-3 activity, lactate dehydrogenase (LDH) release and DNA fragmentation. Wortmannin (10 nM) and LY 294002 (10 microM) (inhibitors of phosphatidylinositol-3-kinase, PI3-K) reversed the inhibitory effect of memantine on the staurosporine-induced LDH release, suggesting that the PI3-K/Akt prosurvival pathway is a possible target for antiapoptotic action of memantine. Memantine at low micromolar concentrations also attenuated salsolinol- and doxorubicin-induced LDH release and DNA fragmentation, but only in the case of salsolinol was this effect accompanied by a decrease in caspase-3 activity. The present data indicate that memantine attenuates the toxic effects of various proapoptotic agents and the cytoprotective effect of memantine does not seem to be connected with its action on NMDA receptor but rather with its influence on intracellular pathways engaged in cellular survival/apoptotic processes.

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.

Effects of neurosteroids on hydrogen peroxide- and staurosporine-induced damage of human neuroblastoma SH-SY5Y cells

Neurosteroids are important regulators of central nervous system function and may be involved in processes of neuronal cell survival. This study was undertaken to test the effect of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), pregnenolone (PGL), pregnenolone sulfate (PGLS), and allopregnanolone (Allo) on hydrogen peroxide‐ and staurosporine‐induced toxicity in SH‐SY5Y cells. It has been found that DHEAS inhibited the hydrogen peroxide toxicity in a concentration‐dependent manner, whereas DHEA was active only at higher doses. PGL and PGLS showed neuroprotective effects only at the lowest concentration. Allo had no significant effect on hydrogen peroxide‐evoked lactate dehydrogenase release and at the highest concentration aggravated its toxic effects. Next part of this study evaluated neurosteroid effects on staurosporine‐induced apoptosis. DHEAS, DHEA, and PGL significantly antagonized effects of staurosporine on both caspase‐3 activity and mitochondrial membrane potential. PGLS and Allo inhibited the staurosporine‐induced changes in both apoptotic parameters only at the lowest concentration. Antiapoptotic properties of neurosteroids were positively verified by Hoechst staining. Furthermore, as shown by calcein assay, DHEA, DHEAS, and PGL increased viability of staurosporine‐treated cells, and these effects were attenuated by specific inhibitors of phosphatidylinositol 3‐kinase (PI3‐K) and extracellular signal‐regulated protein kinase (ERK)‐mitogen activated protein kinase (MAPK). These data indicate that neurosteroids prevent SH‐SY5Y cell damage related to oxidative processes and activation of mitochondrial apoptotic pathway. Moreover, neuroprotective effects of DHEA, DHEAS seem to depend on PI3‐K and ERK/MAPK signaling pathways. It can be suggested that, at physiological concentrations, all studied neurosteroids participate in the inhibition of neuronal apoptosis, but with various potencies.

Neuroprotective effects of (24R)-1,24-dihydroxycholecalciferol in human neuroblastoma SH-SY5Y cell line.

The active form of Vitamin D(3) has been reported to prevent neuronal damage caused by a variety of insults, however, it may also induce undesirable hypercalcemic effects. In the present study, we evaluated effects of (24R)-1,24-dihydroxycholecalciferol (PRI-2191) on hydrogen peroxide- and excitatory amino acid-induced neuronal damage in human neuroblastoma (SH-SY5Y) cell line. Exposure of SH-SY5Y cells to N-methyl-d-aspartate (NMDA; 5mM), kainate (0.2mM) and hydrogen peroxide (0.1-1mM) significantly enhanced lactate dehydrogenase release. Furthermore, the neurotoxic effects of hydrogen peroxide was dependent on c-Jun N-terminal kinase (JNK)- and p38- mitogen-activated protein kinase (MAPK) activity. Both secosteroids at nanomolar concentrations inhibited neuronal damage, but their efficacy varied depending on the toxic agent. PRI-2191 was equipotent as 1alpha,25-dihydroxyVitamin D(3) in protecting SH-SY5Ycells against NMDA toxicity, and had stronger effect against hydrogen peroxide-induced damage, but was less efficient against kainate-induced injury. The obtained results suggest potential usefulness of PRI 2191 in the treatment of neurodegenerative diseases.

The impact of prenatal stress on insulin-like growth factor-1 and pro-inflammatory cytokine expression in the brains of adult male rats: The possible role of suppressors of cytokine signaling proteins

Stress, inflammation and the reduced expression of neurotrophic factors are risk factors for depression. The objective of this study was to determine if prenatal stress affects IGF-1 - cytokine interactions by influencing suppressors of cytokine signaling (SOCS) in the brains of adult rats, in basal conditions and after acute lipopolysaccharide (LPS) treatment. We demonstrated that prenatal stress leads to depression-like behavior, decreased IGF-1, increased IL-1β, TNF-α and IFN-γ release and disturbed SOCS-1, SOCS-2 and SOCS-3 expression in the hippocampus and frontal cortex of adult offspring. Furthermore, prenatal stress enhances the brain response to LPS-evoked inflammatory challenges.

Prenatal stress affects insulin-like growth factor-1 (IGF-1) level and IGF-1 receptor phosphorylation in the brain of adult rats

It has been shown that stressful events occurring in early life have a powerful influence on the development of the central nervous system. Insulin-like growth factor-1 (IGF-1) promotes the growth, differentiation and survival of both neurons and glial cells and is thought to exert antidepressant-like activity. Thus, it is possible that disturbances in the function of the IGF-1 system may be responsible for disturbances observed over the course of depression. Prenatal stress was used as a valid model of depression. Adult male offspring of control and stressed rat dams were subjected to behavioural testing (forced swim test). The level of IGF-1 in the blood and the expression of IGF-1, IGF-1R, and IRS-1/2 in the hippocampus and frontal cortex using RT-PCR, ELISA and western blotting were measured. In addition the effect of intracerebroventricularly administered IGF-1 and/or the IGF-1R receptor antagonist JB1 in the forced swim test was studied. Prenatally stressed rats showed depressive like behaviour, including increased immobility time as well as decreased mobility and climbing. Intracerebroventricular administration of IGF-1 reversed these effects in stressed animals, whereas concomitant administration of the IGF-1R antagonist JB1 completely blocked the effects. Biochemical analysis of homogenates from the hippocampus and frontal cortex revealed decreases in IGF-1 level and IGF-1R phosphorylation along with disturbances in IRS-1 phosphorylation. These findings reveal that prenatal stress alters IGF-1 signalling, which may contribute to the behavioural changes observed in depression.

Effect of amantadine and imipramine on immunological parameters of rats subjected to a forced swimming test

The activation of cell-mediated immunity and the hypothalamic-pituitary-adrenal axis may play a role in the pathophysiology of depression, especially a treatment-resistant one, and antidepressant treatments may exert their effect by suppressing this activation. In our previous studies we described synergistic, antidepressant-like effects of a combination of amantadine (10 mg/kg) and imipramine (5 mg/kg) - drugs otherwise ineffective when given separately in such doses - in the forced swimming test (FST), an animal model of depression. Moreover, preliminary clinical data show that the above-described combination has beneficial effects on treatment-resistant patients. However, it is still unknown whether these positive effects of combined treatment with amantadine and imipramine on behavioural depressive changes are accompanied with normalization of immunoendocrine parameters. Therefore, the present study was aimed at ascertaining whether the antidepressive effect of a combination of amantadine and imipramine was accompanied with a decrease in some immunoendocrine parameters. The antidepressant activity was accompanied with a reversal of the stress-induced increase in the proliferation of splenocytes in response to concanavalin A (ConA). Imipramine, amantadine and a combination of amantadine and imipramine enhanced the production of the negative immunoregulator IL-10 in rats subjected to the FST. The exposure to the FST produced an increase in plasma corticosterone levels, which was significantly attenuated by pretreatment with imipramine or amantadine (a combination of imipramine and amantadine causes reduction within the margin of error). In summary, the antidepressive efficacy of a combination of amantadine and imipramine given in suboptimal doses may be related to their negative immunoendocrine effects.

Protective effects of TRH and its analogues against various cytotoxic agents in retinoic acid (RA)-differentiated human neuroblastoma SH-SY5Y cells

TRH (thyroliberin) and its analogues were reported to possess neuroprotective effects in cellular and animal experimental models of acute and chronic neurodegenerative diseases. In the present study we evaluated effects of TRH and its three stable analogues, montirelin (CG-3703), RGH-2202 and Z-TRH (N-(carbobenzyloxy)-pGlutamyl-Histydyl-Proline) on the neuronally differentiated human neuroblastoma SH-SY5Y cell line, which is widely accepted for studying potential neuroprotectants. We found that TRH and all the tested analogues at concentrations 0.1-50 μM attenuated cell damage induced by MPP(+) (2 mM), 3-nitropropionate (10 mM), hydrogen peroxide (0.5 mM), homocysteine (250 μM) and beta-amyloid (20μM) in retinoic acid differentiated SH-SY5Y cells. Furthermore, we demonstrated that TRH and its analogues decreased the staurosporine (0.5 μM)-induced LDH release, caspase-3 activity and DNA fragmentation, which indicate the anti-apoptotic proprieties of these peptides. The neuroprotective effects of TRH (10 μM) and RGH-2202 (10 μM) on St-induced cell death was attenuated by inhibitors of PI3-K pathway (wortmannin and LY294002), but not MAPK/ERK1/2 (PD98059 and U0126). Moreover, TRH and its analogues at neuroprotective concentrations (1 and 10 μM) increased expression of Bcl-2 protein, as confirmed by Western blot analysis. All in all, these results extend data on neuroprotective properties of TRH and its analogues and provide evidence that mechanism of anti-apoptotic effects of these peptides in SH-SY5Y cell line involves induction of PI3K/Akt pathway and Bcl-2. Furthermore, the data obtained on human cell line with a dopaminergic phenotype suggest potential utility of TRH and its analogues in the treatment of some neurodegenerative diseases including Parkinsons disease.

Antidepressants attenuate the dexamethasone-induced decrease in viability and proliferation of human neuroblastoma SH-SY5Y cells: a involvement of extracellular regulated kinase (ERK1/2).

Excessive glucocorticoid levels in depressed patients have been associated with atrophic changes in some brain regions, but only few studies suggest that some antidepressants can interfere with deleterious effect of glucocorticoids on neuronal cells. The aim of the present study was to examine the effect of dexamethasone (DEX), a synthetic glucocorticoid and some antidepressants from different chemical groups (imipramine, desipramine, amitriptyline, citalopram, fluoxetine, reboxetine and tianeptine) on SH-SY5Y cells cultured in the medium containing steroid-free serum. DEX in concentrations from 1 to 100 μM did not change LDH release but exposure to 10 μM and 100 μM DEX for 24, 48 and 72 h caused a significant reduction in cell viability and proliferation as confirmed by MTT reduction and BrdU ELISA assays, respectively. Twenty four-hour incubation of cells with antidepressants (0.05-10 μM) and DEX (10 μM) showed that imipramine, amitriptyline, desipramine, citalopram and fluoxetine at concentrations from 0.1 up to 1 μM, reboxetine (0.1 μM) and tianeptine (0.05 μM) prevented the DEX-induced decreases in cell viability and proliferation rate. The protective effects of antidepressants were ameliorated by inhibitors of MAPK/ERK1/2, but not PI3-K/Akt pathway as shown for imipramine, fluoxetine and reboxetine. Moreover, Western blot analysis showed the decrease in the activated form of ERK1/2 (p-ERK) after DEX treatment and this effect was inhibited by imipramine. Thus, the reduction in SH-SY5Y cell viability caused by DEX appears to be related to its antiproliferative activity and some antidepressant drugs in low concentrations attenuate this effect by mechanism which involves the activation of MAPK/ERK1/2 pathway.

Stimulatory effect of antidepressant drug pretreatment on progression of B16F10 melanoma in high-active male and female C57BL/6J mice

The effect of a two-week desipramine or fluoxetine (10 mg/kg, i.p.) pretreatment on B16F10 melanoma growth in 3-5 month old female and male C57BL/6J mice differing in behavioral characteristics (high- vs. low-active) was compared. Antidepressant pretreatment increased metastasis formation, shortened the survival, decreased splenocyte anti-tumor natural killer cell cytotoxicity (in vitro), and the pro-inflammatory cytokine IL-12p40, IFN-γ production while it increased anti-inflammatory cytokine IL-10 production in high-active males (desipramine) or females (fluoxetine). The obtained results emphasize a stimulatory effect of pretreatment with antidepressants on progress of B16F10 melanoma that depends on gender and behavioral characteristics of the animal.