Neurosteroids are precursors or metabolites of steroid hormones which exert profound effects on brain function mainly via non-genomic mechanisms. They regulate in allosteric way GABAA receptors, NMDA receptors, sigma receptors and voltage-dependent calcium channels (1-5). Neurosteroids can be synthesized de novo in the central nervous system from cholesterol, e.g. pregnenolone (PGL) or dehydroepiandrosterone (DHEA), but some of them are derived from hormones transported to the brain from peripheral glands, e.g allopregnanolone (Allo) or allotetrahydrodeoxycorticosterone (THDOC) (6). Excitatory neurosteroids, PGL and DHEA have been shown to stimulate learning and memory, but under some conditions they facilitate seizures propagation and neurotoxicity (7-9). On the other hand, inhibitory neurosteroids such as allopregnanolone and THDOC possess sedative, anxiolytic, anesthetic and anticonvulsant properties (10-14).

Neurosteroids are also considered to be endogenous neuroprotectans, however, no simple relation exists between their action on inhibitory and excitatory amino acid receptors and their ability to ameliorate neuronal degeneration. Thus, both the most potent positive modulator of GABAA receptor - Allo, and the positive modulator of NMDA receptor – pregnenolone sulfate (PGLS), under some conditions protect neurons against damaging agents (15, 16). Moreover, a controversy exists about the putative neuroprotective or neurotoxic effects of DHEA and PGL and their sulfate forms. Indeed, PGLS aggravates the NMDA-induced neurotoxicity in cortical slice cultures, but it also protects mouse hippocampal neurons against glutamate and amyloid beta protein toxicity and promotes recovery after spinal cord injury (17-19). A majority of data indicate that DHEA and DHEAS prevent hippocampal and cortical neuronal degeneration induced by glutamate receptor agonists, oxidative stress, corticosterone, and hypoxia (20-22). In contrast, other data show proapoptotic effects of DHEAS (23) or lack of neuroprotective DHEA effect on glutamate-induced toxicity in hippocampal neurons (24). Recently, we found that DHEAS and to a lesser extent DHEA, PGL and PGLS inhibited the hydrogen peroxide toxicity in human neuroblastoma SH-SY5Y cells, whereas Allo had no effect at all (25). The above-mentioned neurosteroids attenuated also staurosporine-induced apoptosis, DHEAS, DHEA and PGL being the most potent in this respect (25). Although SH-SY5Y cells are widely used for studying neurodegenerative processes, the presence of functional glutamate receptors in some passages of this cell line is doubtful (26 - 28). Therefore, in the present study, we evaluated effects of neurosteroids in primary cortical neurons of mice at concentrations at which they inhibited staurosporine-evoked apoptosis in SH-SY5Y model. The mechanism of neuroprotective effects of neurosteroids is poorly understood, however, our previous study implicated phosphatidylinositol-3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK) - mitogen-activated protein kinase (MAPK) in DHEAS and DHEA action on staurosporine-induced decrease in viability of SH-SY5Y cells (25). Thus, in the present study, we examined whether the same kinases participate in neuroprotective action of neurosteroids in primary cortical neurons. Since SH-SY5Y cells lack of functional NMDA receptors (28), and controversy exists about neurosteroid effects on excitatory amino acid-induced neuronal damage (15, 16, 19, 24, 29) in the present study we also evaluated effects of DHEAS, DHEA, PGL and Allo on NMDA- and glutamate-evoked toxicity in primary cortical cell cultures.


MATERIALS AND METHODS
Chemicals

Neurobasal A medium, fetal bovine serum (FBS) and supplement B27 were from Gibco (Invitrogen, Poisley, UK). The Hoechst 33342 was supplied by Molecular Probes (Eugene, Oregon, USA). The Cytotoxicity Detection Kit was from Roche Diagnostics (Mannheim, Germany). PD 98059, an inhibitor of ERK-MAPK kinase pathway, was obtained from Tocris (UK). All other reagents were from Sigma (Sigma-Aldrich Chemie GmbH, Germany).

Primary neuronal cultures

The experiments were conducted on primary cultures of mouse cortical neurons. Neuronal tissues were taken from Swiss mouse embryos at day 15/16 of gestation, and were cultured essentially as described previously (30, 31). Briefly, pregnant females were anesthetized with CO2 vapor, killed by cervical dislocation, and subjected to cesarean section in order to dissect fetal brains. Animal care followed official governmental guidelines and all efforts were made to minimize the number of animals used and their suffering. The dissected tissues were minced separately into small pieces, then digested with trypsin (0.1% for 15 min at room temperature (RT), triturated in the presence of 10% fetal bovine serum and DNAse I (170 Kunitz units per ml), and finally centrifuged for 5 min at 350xg. The cells were suspended in Neurobasal medium supplemented with B27 and plated at a density of 1.5x105 cells per cm2 onto polyornithine (0.1 mg per ml)-coated multiwell plates. This procedure typically yields cultures that contain >90% neurons and <10% supporting cells. The protocol for generating the primary neuronal cultures complied with local and international guidelines on the ethical use of animals. The cultures were then maintained at 37°C in a humidified atmosphere containing 5% CO2 for 7 or 12 days prior to experimentation.

Treatment

The cortical cells were treated with DHEA (0.1 and 1 µM), DHEAS (0.1 and 1 µM), PGL (0.1 and 1 µM) or Allo (0.1 µM) one hour before adding cell-damaging agents. All concentrations of neurosteroids were chosen from our previous study in SH-SY5Y cells, where they showed neuroprotective action on staurosporine-evoked cell damage (25). Apoptosis was induced by treatment of 7 div neurons with 0.5 µM staurosporine for 14 and 24 hours. Caspase-3 inhibitor, AcDEVD-CHO (10 µM) was added to cell cultures just before staurosporine in order to verify engagement of caspase-3 dependent apoptotic pathway in staurosporine-induced cell death. Excitotoxicity was evoked by adding glutamate (1 mM) or NMDA (200 µM) to the cells at 7 and 12 DIV for 24 hr. Dizocilpine (MK-801, 1 µM), an NMDA receptor antagonist was added to cell cultures at the same time as glutamate or NMDA.

In the next part of the study, cells were treated with inhibitors of PI3-K, wortmannin (10 nM) and LY 294002 (1 µM), and with an inhibitor of ERK-MAPK pathway, PD 98059 (5 µM). The concentrations of inhibitors were chosen on the basis of our previous report (25) and were adapted to primary neuronal cell cultures system in order to avoid cell death induced by higher concentrations of these protein kinases inhibitors (our unpublished observations). Thirty minutes after the addition of protein kinase inhibitors, cultures were treated with neurosteroids and one hour later staurosporine (0.5 µM) was added for the next 24 h.

Wortmannin, LY 294002, PD 98059, staurosporine and AcDEVD-CHO were dissolved in DMSO and neurosteroids in ethanol/water mixture. The chemicals were present in cultures at a final concentration of 0.1% and the control cultures were supplemented with the same amount of an appropriate vehicle.

Identification of apoptotic cells

In order to reveal the characteristic morphology associated with apoptosis, such as nuclear condensation and cell shrinkage, we stained cells with Hoechst 33342 dye as described previously (32). Briefly, cortical neurons were seeded onto polyornithine (0.1 mg/ml)-coated coverslips in 24-well plates and cultured essentially for 7 days. Twenty four hours after treatment with neurosteroids and staurosporine, cells were fixed for 20 min with 4% paraformaldehyde and exposed to Hoechst 33342 (0.8 µg/ml) for 25 min. Nuclear condensation and cell body shrinkage were evaluated under a Nikon fluorescence microscope. Cells that had bright condensed, fragmented nuclei after Hoechst’s staining were considered to be apoptotic. The number of cells with apoptotic morphology was counted in six randomly chosen fields per one coverslip (150 - 200 cells); two coverslips per one treatment group from 3 separate experiments (n = 6) were evaluated. The data were calculated as a percentage of apoptotic nuclei compared to the total number of cells and presented in histograms as the mean ± S.E.M.

Assessment of caspase-3 activity

An assay of caspase-3 protease activity in the samples treated for 14 and 24 hr with apoptotic agents in 96-well plates was performed as previously described (32). For confirming the specificity of the reaction, a cell-permeable inhibitor of caspase-3 (Ac-DEVD-CHO, 10 µM) was used during cell treatment. After the cells were treated with agents, the culture medium was removed and the cells were frozen and stored at -20°C till conducting proper measurement. Cells were refrozen and lysed in Caspase Assay Buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM dithiothreitol) and then incubated at 37°C with a colorimetric substrate preferentially cleaved by caspase-3, Ac-DEVD-pNA (N-acetyl-asp-glu-val-asp p-nitro-anilide). The amounts of p-nitroanilide were monitored continuously over 60 min with a plate reader (Multiscan, ThermoLabsystems, Finland). The absorption was measured at 405 nm. The data were normalized to the absorbance in vehicle-treated cells and expressed as the percentage of absorbance ± S.E.M. established from n = 6 wells per one experiment from 3 separate experiments. The absorbance of blanks, determined as no-enzyme control, was subtracted from each value.

Measurement of lactate dehydrogenase (LDH) activity

In order to estimate cell death, the level of lactate dehydrogenase (LDH) released from damaged cells into the culture media was measured at 14 and 24 hr after the cells were treated with staurosporine or 24 hr after glutamate- and NMDA-treatment. A colorimetric assay was applied, according to which the amount of formazan salt, formed after the conversion of lactate to pyruvate and then by a reduction of tetrazolium salt, is proportional to the LDH activity in the sample. Cell-free culture supernatants were collected from each well and incubated with the appropriate reagent mixture according to the supplier’s instructions (Cytotoxicity Detection Kit, Roche) at RT for 60 min. The intensity of the red color formed in the assay and measured at a wavelength of 490 nm was proportional to the LDH activity and to the number of damaged cells. Data were normalized to the activity of LDH released from vehicle-treated cells (100%) and expressed as a percent of the control ± S.E.M. established from n=6 wells per one experiment from 3 separate experiments.

Measurements of cell viability

Cell viability assessments were done after 24 hr of treatment with staurosporine in 96-well plates. Cell damage was quantified using a tetrazolium salt colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). In summary, MTT was added to each well (at a final concentration of 0.15 mg/ml) and incubated for 1hr at 37°C, before the dye was then solubilized by 0.1 N HCl in isopropanol, with the absorbance of each sample measured at 570 nm in a 96-well plate-reader (Multiscan, Thermo Labsystems, Finland). Data were normalized to the intensity of the absorbance in vehicle-treated cells (100%) and expressed as a percentage of the control ± S.E.M. established from n = 6 wells per one experiment from 3 separate experiments.

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was determined as described by Menon et al. (33). After 24-h treatment with staurosporine, the cells were harvested, incubated with 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, Cell Technology USA) at 37°C for 15 min and after washing, the red (excitation 550 nm, emission 600 nm) and green fluorescence (excitation 485 nm, emission 535 nm) was measured in a fluorescence plate reader (Fluoroscan Ascent, Labsystem). JC-1 accumulated in mitochondrial matrix of healthy cells in a red aggregated form, whereas in apoptotic cells, in which the mitochondrial membrane potential decreased, it was stained in the cytoplasm in a green fluorescent monomeric form. The results were calculated as the ratio of red fluorescence to green fluorescence from n=6 wells per one experiment from 3 separate experiments.

Phospho-ERK determination by Western blot

Twenty four hours after treatment with staurosporine the cells were washed twice with phosphate-buffered saline and were lysed with RIPA lysis buffer (Sigma-Aldrich) with 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 0.2 nM okadaic acid, 1 mM sodium fluoride and 10 µg/ml of each leupeptin, aprotinin and pepstatin A. The lysates were collected and centrifuged at 3700´g for 15 min at 4° C and supernatants were collected. The cell lysates (equal amount of protein) and the buffer (100 mM Tris-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.005% bromophenol blue, pH = 6.8) were mixed and boiled for 3 min before loading on the gel. Proteins were separated by SDS-PAGE (4% stacking gel, 10 % resolving gel under constant voltage (60 V in stacking gel; 120 V in resolving gel), and were transferred electrophoretically to the PVDF membrane (Roche Diagnostic GmbH) at a 60 V constant current for 2 h. The membranes were washed twice with the Tris-buffered saline (TBS), pH=7.5, blocked in a 1% blocking solution (Roche Diagnostic GmbH) for 1 h, and incubated overnight at 4°C with the primary antibody anti-phospho-ERK1/2 (Santa Cruz Biotechnology, Inc.) and anti-ERK (Santa Cruz Biotechnology, Inc.). The blots were washed: twice with TBS containing a 0.1 % Tween-20 (TBST); twice with a 0.5 % blocking solution in TBS, and were than incubated with a horseradish peroxidase-linked secondary antibody (anti-rabbit/anti-mouse IgG; 40 mU/ml; Santa Cruz Biotechnology, Inc.) for 1 h at a room temperature. Afterwards, the membranes were washed four times with large volumes of TBST, and immunoblots were visualized with a chemiluminescence detection kit (Roche Diagnostic GmbH). The semiquantitative analysis of band intensity was performed using FujiLas 1000 and FujiGauge software. Results (mean ± S.E.M.) are expressed as a relative optical density units from n = 4.

Reactive oxygen species (ROS) assay

Twenty four hours after glutamate treatment, the cultured cells were washed twice with physiological buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1.2 mM NaHPO4, 5 mM glucose and 20 mM HEPES, pH 7.4). 2’7’-dichlorodihydrofluorescein diacetate (100µM DCDHF-DA) (Molecular Probes, USA) in physiological buffer was placed on the cortical cells for 30 min in 5% CO2/95% air at 37°C. DCDHF-DA is hydrolyzed inside the cells to form 2’7’-dichlorodihydrofluorescein which emits fluorescence when it is oxidized to 2’7’-dichlorofluorescein (DCF). Thus, the fluorescence emitted by DCF directly reflects the overall oxidative status of a cell (34). After the incubation, cell cultures were washed three times with buffer. The fluorescence was monitored for 60 min using a microplate spectrofluorometer (Fluoroscan Ascent, Labsystem). The emission was recorded at 535 nm after exciting at 485 nm. All data were calculated and normalized with respect to the increase in fluorescence of controls from n=6 wells per one experiment from 3 separate experiments.

Data analysis

Data from biochemical measurements after normalization as a percentage of control ± S.E.M. was analyzed using Statistica software. The analysis of variance (ANOVA) and post hoc Tukey test for multiple comparisons were used to show statistical significance with assumed P<0.05.


RESULTS
Effects of neurosteroids on staurosporine-induced apoptosis in primary cortical cell cultures

Treatment of cortical neurons with 0.5 µM staurosporine, in time-dependent manner induced lactate dehydrogenase (LDH) release and caspase-3 activity, which reached a maximal increase by about 60% and 110% of control value after 24 and 14 hr, respectively (Fig. 1). Parallelly, a 30 % reduction in the cell viability was observed after 24 hr of staurosporine-exposure as compared to control cultures (Table 1). None of the studied neurosteroids given alone for 24 h affected the cell viability, basal level of caspase-3 activity and LDH release (data not shown). DHEA, DHEAS and PGL (0.1 and 1 µM), but not Allo (0.1 µM), attenuated the staurosporine-evoked LDH release by about 25%, and this effect was observed after 14 (not shown) and 24 h (Fig. 2A) after induction of apoptosis. An increase in the cell viability (about 10 - 15 %) was also noted after treatment with DHEA, DHEAS and PGL but not with Allo (Table 1). The data showed also attenuation (by about 20%) in the number of apoptotic nuclei after the treatment only with excitatory neurosteroids (Fig. 3) although no effect on staurosporine-induced caspase-3 activity was noticed (Fig. 2B). AcDEVD-CHO (10 µM), a cell-permeable inhibitor of caspase-3, reduced staurosporine-stimulated caspase-3 activity to the basal level (Fig. 2B), but only partially (about 25%) attenuated staurosporine-evoked LDH release (Fig. 2A). Moreover, we showed that staurosporine-evoked decrease in the mitochondrial membrane potential in cortical cells measured after 5 and 24 hr of incubation with chemicals was not blocked by tested neurosteroids (Table 2).

Fig. 1. Time course of staurosporine (0.5 µM) effect on caspase-3 activity and LDH release in primary cultures of mouse neocortical neurons on 7 day in vitro (DIV). Cells were treated with staurosporine or with vehicle for 14 or 24 h. Each point represents an average value taken from n = 6 platings ± S.E.M from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following ANOVA analysis of variance (***P<0.001 vs control cultures).

Table 1. The effect of neurosteroids (DHEA, DHEAS, PGL and Allo) on staurosporine-induced reduction in cell viability in primary cultures of neocortical cells on 7 day in vitro

Cells were treated with DHEA (0.1 and 1 µM), DHEAS (0.1 and 1 µM), PGL (0.1 and 1 µM) and Allo (0.1 µM) and one hour later staurosporine (0.5 µM) was added to the culture medium and cells were further incubated with chemicals for 24 h. Data was normalized to the MTT reduction obtained from vehicle-treated cells (100%) and expressed as a percent of the control ± S.E.M. established from n = 6 wells per one experiment from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following a one-way analysis of variance (***P<0.001 vs control culture; #P<0.05 vs staurosporine-treated cells).

Fig. 2. The effects of DHEA, DHEAS, PGL and Allo on staurosporine (st, 0.5 µM)-induced LDH-release (A) and caspase-3 activity (B) in primary cultures of mouse neocortical neurons on 7 DIV. Cells were treated with staurosporine or with vehicle for 24 h. Each bar represents an average value taken from n = 6 platings ± S.E.M from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following ANOVA analysis of variance (***P<0.001 versus control cultures; #P<0.05 and ###P<0.001 vs staurosporine-treated cells).

Fig. 3. Photomicrographs of attenuating effects of DHEA, DHEAS and PGL on staurosporine-induced DNA fragmentation evaluated by Hoechst 33342 staining. Histograms show the number of cells with apoptotic morphology which was counted in six randomly chosen fields per one coverslip; two coverslips per one experimental group from 3 separate experiments and expressed as a percentage of apoptotic nuclei compared to total number of cells. The significance of differences between the means was evaluated by the Tukey test following ANOVA analysis of variance (***P<0.001 vs control culture; #P<0.05 and ###P<0.001 vs staurosporine-treated cells).

Table 2. The effect of neurosteroids (DHEA, DHEAS, PGL and Allo) on staurosporine-induced decrease in mitochondrial membrane potential in primary cultures of neocortical cells on 7 day in vitro

Cells were treated with DHEA (0.1 and 1 µM), DHEAS (0.1 and 1 µM), PGL (0.1 and 1 µM) and Allo (0.1 µM) and one hour later staurosporine (0.5 µM) was added to the culture medium and cells were further incubated with chemicals for 5 or 24 h. Mitochondrial membrane potential was measured with JC-1 dye (details in Materials and Methods). Data was expressed as a red to green fluorescence ratio ± S.E.M. established from n = 6 wells per one experiment from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following a one-way analysis of variance (***P<0.001 vs control culture).

Effects of selective inhibitors of protein kinases on neurosteroid-exerted neuroprotective action on staurosporine-induced LDH release

In the next part of the study, the effects of selective inhibitors of protein kinases, involved in processes of neuronal survival, on neuroprotective action of neurosteroids in the staurosporine model have been evaluated. The inhibitors of PI3-K: wortmannin (10 nM) and LY 294002 (1 µM) and inhibitor of ERK-MAPK: PD 98059 (5 µM) given alone did not affect the cell viability both in control and staurosporine-treated cell culture (Table 3). Both of the PI3-K inhibitors had no effect on neurosteroid inhibitory action on staurosporine-induced LDH release. On the other hand, the ERK-MAPK inhibitor attenuated partially the protective effects of DHEA and DHEAS and almost completely blocked PGL-mediated neuroprotection (Table 3).

Table 3. The influence of wortmannin, LY294002 and PD98059 on the protective effect of DHEA, DHEAS and PGL against staurosporine-induced cell death of 7DIV neocortical neurons

Cells were treated with wortmannin (10 nM) and LY294002 (1µM), an inhibitors of PI3-K and with PD 98059 (5µM), an inhibitor of ERK/MAPK pathway. Thirty minutes after addition of protein kinase inhibitors, cultures were treated with neurosteroids (1 µM). One hour later staurosporine (0.5 µM) was added and cells were cultured for 24 hours. Cell death was estimated by LDH relase assay and obtained data was normalized to the activity of LDH released from vehicle-treated cells (100%) and expressed as a percent of the control ± S.E.M.established from n=6 wells per one experiment from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following a one-way analysis of variance (***P<0.001 vs control culture; #P<0.05 vs staurosporine-treated cells; ^ P<0.05, ^^ P<0.01 vs cells treated with staurosporine and neurosteroid).

Effects of neurosteroids on staurosporine-induced decrease in ERK-MAPK phosphorylation.

Twenty four-hour incubation of the primary cortical cells with staurosporine resulted in ca. 25% decrease in phospho-ERK2 level (Fig. 4). Exposure to staurosporine did not alter the amount of phospho-ERK1 level and total levels of ERK1 and ERK2. DHEA, DHEAS and PGL given alone had no effect on phospho-ERK1/ 2 and total ERK 1/2 concentration (data not shown) but significantly attenuated the staurosporine-induced decrease in phospho-ERK2 (Fig. 4). The effects of investigated neurosteroids on staurosporine-induced changes in phospho-ERK2 level were reversed by the ERK-MAPK inhibitor, PD98059 (5 µM) (Fig.4).

Fig. 4. The effects of DHEA, DHEAS, PGL and ERK-MAPK inhibitor, PD98059 (5 µM), on staurosporine-induced changes in phospho-ERK level. Results (mean ± S.E.M.) are expressed as a relative optical density units from n = 4. The significance of differences between the means was evaluated by the Tukey test following ANOVA analysis of variance (* P<0.05 vs cells control culture; #P<0.05 vs staurosporine-treated; ^ P<0.05 vs cells treated with a neurosteroid and staurosporine).

Effects of neurosteroids on glutamate- and NMDA-evoked toxicity in primary cortical cells

As revealed by LDH release assay, glutamate (1 mM) evoked cell death of cortical neurons in both, 7 and 12 DIV cultures, with higher toxic action at 7 DIV. In contrast, 7 DIV neuronal cultures were more resistant to NMDA (200 µM) toxic action than 12 DIV cells (Table 4). DHEA, DHEAS and PGL, but not Allo, partially attenuated glutamate-induced cell death in both 7 and 12 DIV cortical neurons (Fig. 5 A, B). Additionally, it was found that glutamate enhanced production of free radicals, and this effect was attenuated by NMDA receptor antagonist (MK-801), but not by neurosteroids under study (Table 5). There was no effect of the treatment with neurosteroids on NMDA-stimulated LDH release at 12 DIV cortical cells (Fig. 6). Moreover, an antagonist of NMDA receptor, MK-801 (1 µM) almost completely inhibited glutamate and NMDA-mediated toxicity in neuronal cell cultures (Fig. 5 A, B; Fig. 6).

Table 4. The effect of glutamate (1 mM) or NMDA (200 µM) on LDH release in primary cultures of mouse neocortical cells on 7 and 12 day in vitro (DIV)

Cells were treated with glutamate (1 mM) or NMDA (200 µM) and incubated with chemicals for 24 h. Cell death was estimated by LDH release assay and data was normalized to the activity of LDH released from vehicle-treated cells (100%) and expressed as a percent of the control ± S.E.M. established from n = 6 wells per one experiment from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following a one-way analysis of variance (***P<0.001 vs control culture; ^^^ P<0.001 12DIV cells vs 7 DIV ones).

Table 5. The effect neurosteroids (DHEA, DHEAS, PGL and Allo) on glutamate-induced increase in reactive oxygen level in primary cultures of neocortical cells on 7 day in vitro

Cells were treated with DHEA (0.1 and 1 µM), DHEAS (0.1 and 1 µM), PGL (0.1 and 1 µM) and Allo (0.1 µM) and one hour later staurosporine (0.5 µM) was added to the culture medium and cells were further incubated with chemicals for 24 h. Reactive oxygen species level was measured with DCF fluorescence (details in Materials and Methods). Data was normalized to the ROS level obtained from vehicle-treated cells (100%) and expressed as a percent of the control ± S.E.M. established from n = 6 wells per one experiment from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following a one-way analysis of variance (***P<0.001 vs control culture, #P<0.05 vs glutamate-treated cells).

Fig. 5. The effects of DHEA, DHEAS, PGL and Allo on cell death induced by glutamate (1 mM) in 7 (A) and 12 DIV (B) primary cultures of mouse neocortical neurons. Cell death was estimated by LDH release assay after 24 h of treatment with chemicals. Each bar represents an average value taken from n = 6 platings ± S.E.M. from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following ANOVA analysis of variance (***P<0.001 vs control cultures; #P<0.05, ##P<0.01 and ###P<0.001 vs glutamate-treated cells).

Fig. 6. The effects of DHEA, DHEAS, PGL and Allo on cell death induced by NMDA (200 µM) in 12 DIV neurons. Cell death was estimated by LDH release assay after 24 h of treatment with chemicals. Each bar represents an average value taken from n = 6 platings ± S.E.M. from 3 separate experiments. The significance of differences between the means was evaluated by the Tukey test following ANOVA analysis of variance (***p<0.001 vs control cultures; ###p<0.001 vs NMDA-treated cells).

DISCUSSION
This study showed that excitatory neurosteroids (DHEA, DHEAS and PGL) protected primary cortical neurons against staurosporine and glutamate toxicity, but they failed to affect NMDA-induced cell damage. Regarding the effects of neurosteroids on staurosporine-induced decrease in cell viability, these data are in agreement with our previous study performed on neuroblastoma SH-SY5Y cells (25). Surprisingly, in primary cortical cell cultures the neurosteroid-induced inhibition of staurosporine toxicity was not accompanied by attenuation of caspase-3 activity or changes in mitochondrial membrane potential as previously observed in the model of SH-SY5Y cells. This strongly suggests that in primary cortical neuronal cell culture antiapoptotic effects of neurosteroids in staurosporine model, as evidenced by Hoechst staining, are caspase-3-independent. Although staurosporine is the best known activator of mitochondrial pathway of apoptosis with downstream induction of caspase-9 and caspase-3, it can also activate apoptosis via intracellular translocation of apoptosis inducing factor (AIF) from mitochondria into cytoplasm and finally into nucleus where it causes fragmentation of DNA (35, 36). Moreover, activation of extramitochondrial apoptotic signaling pathways in mixed glia/neuronal cerebrocortical culture by staurosporine was reported (37). Interestingly, Yan et al. (38) found that antagonism of DHEAS for dexamethasone-induced apoptosis in mouse thymocytes was also caspase-3- and caspase-6-independent. However, a possible role of AIF pathway in neurosteroid antiapoptotic effects should be verified in further studies. On the other hand, regarding mechanism of neurosteroid action on staurosporine-induced apoptosis, an involvement of ERK-MAPK and PI3-K should be seriously taken into account. Indeed, these kinase pathways were shown to participate in DHEA and DHEAS antiapoptotic action in SH-SY5Y cells (25). In contrast to SH-SY5Y cells, in primary cortical neurons ERK-MAPK, but not PI3-K seems to participate in the mechanism of DHEA, DHEAS and PGL. Thus, the specific inhibitor of ERK-MAPK, PD98059, diminished the DHEA, DHEAS and PGL action on staurosporine-induced LDH release. Furthermore, the Western blot study showed that staurosporine decreased the level of active, phosphorylated form of ERK2 and this effect was attenuated by DHEA, DHEAS and PGL. The role of ERKs in cell death depends on the cell type and toxic agent, but usually activation of ERK1/2 increases neuronal survival. ERK-MAPK has been found to phosphorylate and inactivate some pro-apoptotic proteins, such as Bad and Bim (39). ERK1/2 may protect cells via phosphorylation of cAMP response element binding protein (CREB) or via inhibition of proapoptotic kinase GSK-3b (40, 41). Apart from ERK-MAPK, the mechanism of neuroprotective effect of excitatory neurosteroids may involve the activation of NF-B, PI3-K/Akt pathway, antioxidant effects, inhibition of glucose-6 phosphate dehydrogenase and facilitation of JNK3-MAPK translocation to nucleus (24, 42).

Virtually, in contrast to primary hippocampal cells, there are only a few data regarding effects of neurosteroids on primary cortical neuronal degeneration. Studies conducted by Shirakawa et al. (15, 19) on cortical slice culture showed that DHEA, DHEAS and PGL had no effect on NMDA- and AMPA-induced cytotoxicity, whereas PGLS even enhanced the toxicity in this model. They also found that in primary cortical neurons, the glutamate-induced neuronal death was exacerbated by PGLS. Lack of neurosteroid effects on NMDA-induced cortical neuronal damage found in our study is in agreement with the above-mentioned report. On the other hand, a synthetic homologue of pregnanolone sulfate, 3-ol-5ß-pregnan-20-one hemisuccinate (35ßHS) in high micromolar concentrations inhibited NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons (29). Interestingly, in contrast to NMDA-evoked cell death in our study, DHEA, DHEAS and PGL inhibited the neurotoxic effects of glutamate on both 7 and 12 DIV, which underlines differences in the mechanism of neurosteroid action on toxic effects evoked by glutamate or its more specific receptor agonists. Our data exclude interference of neurosteroids with glutamate-induced production of free radicals, especially because this effect, as evidenced by MK-801 experiment, seems to be mediated by NMDA receptor. An engagement of other ionotropic (AMPA or KA) glutamate receptors is rather unlikely since our previous data showed that neurosteroids (Allo, DHEA, DHEAS and PGL) at used in the present study concentrations did not affect [3H]-glutamate, [3H]-AMPA and [3H]-MK-801 binding or glutamate uptake in rat hippocampus (43). In contrast to excitatory neurosteroids, Allo showed no effect on staurosporine-, glutamate-, and NMDA-evoked toxicity. These findings were unexpected since some reports indicated that allopregnanolone possessed ability to prevent neuronal damage. For instance, it was reported that Allo prevented in vivo brain tissue damage evoked by hypoxia/ischemia (44), whereas in vitro studies showed the inhibitory effect of Allo on glutamate-, NMDA-, and kainate-induced toxicity in various cell cultures (16, 45 - 48). It should be stressed that in our study the concentration of Allo was close to the physiological level (0.1 µM), whereas in the study of Xilouri and Papazafiri (16) a significant antiapoptotic effect of this neurosteroids on NMDA-evoked toxicity in P19 neurons could be observed only at much higher concentrations (10 and 20 µM) and this effect was prevented by inhibitor of PI3-K and by an antagonist of GABAA receptor.

Summing up, this study indicates that excitatory neurosteroids (DHEA, DHEAS and PGL), but not the inhibitory Allo, have a distinct antiapoptotic effects in staurosporine model of apoptosis in primary cortical cells and that their action seems to depend on phosphorylation of ERK-MAPK. These compounds inhibit also glutamate, but not NMDA toxicity, which indicates that protective action of neurosteroids depend on the type of toxic agent.

Acknowledgements: This study was supported by grant No. 2 P05A 079 27 from the Ministry of Science and Higher Education, Warsaw, Poland. We would like to thank Ms. B. Korzeniak for her skillful technical assistance.


REFERENCES

1. Majewska MD. Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. Prog Neurobiol 1992; 38: 379-395.
2. Majewska MD. Steroids and ion channels in evolution: from bacteria to synapses and mind. Acta Neurobiol Exp 2007; 67: 219-233.
3. Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM. Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 1986; 232: 1004-1007.
4. Maurice T, Phan VL, Urani A, Kamei H, Noda Y, Nabeshima T. Neuroactive neurosteroids as endogenous effectors for the sigma1 (sigma1) receptor: pharmacological evidence and therapeutic opportunities. Jpn J Pharmacol 1999; 81: 125-155.
5. Irwin RP, Maragakis NJ, Rogawski MA, Purdy RH, Farb DH, Paul SM. Pregnenolone sulfate augments NMDA receptor mediated increases in intracellular Ca2+ in cultured rat hippocampal neurons. Neurosci Lett 1992; 141: 30-34.
6. Mellon SH, Griffin LD, Compagnone NA. Biosynthesis and action of neurosteroids. Brain Res Brain Res Rev 2001; 37: 3-12.
7. Alhaj HA, Massey AE, McAllister-Williams RH. Effects of DHEA administration on episodic memory, cortisol and mood in healthy young men: a double-blind, placebo-controlled study. Psychopharmacology (Berl) 2006; 188: 541-551.
8. Mellon SH. Neurosteroid regulation of central nervous system development. Pharmacol Ther 2007; 116: 107-124.
9. Reddy DS, Kulkarni SK. Proconvulsant effects of neurosteroids pregnenolone sulfate and dehydroepiandrosterone sulphate in mice. Eur J Pharmacol 1998; 345: 55-59.
10. Budziszewska B, Siwanowicz J, Leskiewicz M, Jaworska-Feil L, Lason W. Protective effects of neurosteroids against NMDA-induced seizures and lethality in mice. Eur Neuropsychopharmacol 1998; 8: 7-12.
11. Crawley JN, Glowa JR, Majewska MD, Paul SM. Anxiolytic activity of an endogenous adrenal steroid. Brain Res 1986; 398: 382-385.
12. Czlonkowska AI, Krzascik P, Sienkiewicz-Jarosz H et al. The effects of neurosteroids on picrotoxin-, bicuculline- and NMDA-induced seizures, and a hypnotic effect of ethanol. Pharmacol Biochem Behav 2000; 67: 345-353.
13. Mares P, Mikulecká A, Haugvicová R, Kasal A. Anticonvulsant action of allopregnanolone in immature rats. Epilepsy Res 2006; 70: 110-117.
14. Mendelson WB, Martin JV, Perlis M, Wagner R. Sleep and benzodiazepine receptor subtypes. J Neural Transm 1987; 70: 329-336.
15. Shirakawa H, Katsuki H, Kume T, Kaneko S, Akaike A. Pregnenolone sulphate attenuates AMPA cytotoxicity on rat cortical neurons. Eur J Neurosci 2005; 21: 2329-2335.
16. Xilouri M, Papazafiri P. Anti-apoptotic effects of allopregnanolone on P19 neurons. Eur J Neurosci 2006; 23: 43-54.
17. Gursoy E, Cardounel A, Kalimi M. Pregnenolone protects mouse hippocampal (HT-22) cells against glutamate and amyloid beta protein toxicity. Neurochem Res 2001; 26: 15-21.
18. Guth L, Zhang Z, Roberts E. Key role for pregnenolone in combination therapy that promotes recovery after spinal cord injury. Proc Natl Acad Sci USA 1994; 91: 12308-12312.
19. Shirakawa H, Katsuki H, Kume T, Kaneko S, Ito J, Akaike A. Regulation of N-methyl-D-aspartate cytotoxicity by neuroactive steroids in rat cortical neurons. Eur J Pharmacol 2002; 454: 165-175.
20. Kimonides VG, Khatibi NH, Svendsen CN, Sofroniew MV, Herbert J. Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid-induced neurotoxicity. Proc Natl Acad Sci USA 1998; 95: 1852-1857.
21. Kimonides VG, Spillantini MG, Sofroniew MV, Fawcett JW, Herbert J. Dehydroepiandrosterone antagonizes the neurotoxic effects of corticosterone and translocation of stress-activated protein kinase 3 in hippocampal primary cultures. Neuroscience 1999; 89: 429-436.
22. Bastianetto S, Ramassamy C, Poirier J, Quirion R. Dehydroepiandrosterone (DHEA) protects hippocampal cells from oxidative stress-induced damage. Brain Res Mol Brain Res 1999; 66, 35-41.
23. Zhang L, Li B, Ma W et al. Dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) regulate apoptosis during neurogenesis by triggering the Akt signaling pathway in opposing ways. Brain Res Mol. Brain Res 2002; 98: 58-66.
24. Mao X, Barger SW. Neuroprotection by dehydroepiandrosterone-sulfate: role of an NFkappaB-like factor. Neuroreport 1998; 9: 759-763.
25. Leskiewicz M, Regulska M, Budziszewska B, et al. Effects of neurosteroids on hydrogen peroxide- and staurosporine- induced damage of human neuroblastoma SH-SY5Y cells. J Neurosci Res 2008; 86: 1361-1370.
26. Uberti D, Rizzini C, Galli P, et al. Priming of cultured neurons with sabeluzole results in long-lasting inhibition of neurotoxin-induced tau expression and cell death. Synapse 1997; 26: 95-103.
27. Glantz SB, Cianci CD, Iyer R, Pradhan D, Wang KKW, Morrow S. Sequential degradation ofaII and bII spectrin by calpain in glutamate or maitotoxin-stimulated cells. Biochemistry 2007; 46: 502-513.
28. Jantas D, Pytel M, Mozrzymas JW, et al. The attenuating effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in human neuroblastoma SH-SY5Y cells. Neurochem Int 2008; 52: 864-877.
29. Weaver CE Jr, Marek P, Park-Chung M, Tam SW, Farb DH. Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA. 1997; 94: 10450-10454.
30. Brewer GJ. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res 1995; 42: 674-683.
31. Kajta M, Lason W, Kupiec T. Effects of estrone on N-methyl-D-aspartic acid- and staurosporine-induced changes in caspase-3-like protease activity and lactate dehydrogenase-release: time- and tissue-dependent effects in neuronal primary cultures. Neuroscience 2004; 123: 515-526.
32. Jantas-Skotniczna D, Kajta M, Lason, W. Memantine attenuates staurosporine-induced activation of caspase-3 and LDH release in mouse primary neuronal cultures. Brain Res 2006; 1069: 145-153.
33. Menon B, Singh M, Ross RS, Johnson JN, Singh K. beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway. Am J Physiol Cell Physiol 2006; 290: C254-261.
34. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 1999; 27: 612-616.
35. Daugas E, Susin SA, Zamzami N, et al. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 2000; 14: 729-739.
36. Déas O, Dumont C, MacFarlane M, et al. Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes. Immunology 1998; 161: 3375-3383.
37. Budd SL, Tenneti L, Lishank T, Lepton SA. Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons. Proc Natl Acad Sci USA 2000; 97: 6161-6166.
38. Yan CH, Jiang XF, Pei X, Dai YR. The in vitro antiapoptotic effect of dehydroepiandrosterone sulphate in mouse thymocytes and its relation to caspase-3/caspase-6. Cell Mol Life Sci 1999; 56: 543-547.
39. Bonni A, Brunet A, West AE, Datta SR, Takasu MA, Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 1999; 286: 1358-1362.
40. Hetman M, Gozdz A. Role of extracellular signal regulated kinases 1 and 2 in neuronal survival. Eur J Biochem 2004; 271: 2050-2055.
41. Hetman M, Hsuan SL, Habas A, Higgins MJ, Xia Z. ERK1/2 antagonizes glycogen synthase kinase-3beta-induced apoptosis in cortical neurons. J Biol Chem 2002; 277: 49577-49584.
42. Yang NC, Jeng KC, Ho WM, Chou SJ, Hu ML. DHEA inhibits cell growth and induces apoptosis in BV-2 cells and the effects are inversely associated with glucose concentration in the medium. J Steroid Biochem Mol Biol 2000; 75: 159-166.
43. Leskiewicz M, Budziszewska B, Jaworska-Feil L, Kajta M, Lason W. Effect of neurosteroids on glutamate binding sites and glutamate uptake in rat hippocampus. Pol J Pharmacol 1998; 50: 355-360.
44. Sayeed I, Guo Q, Hoffman SW, Stein DG. Allopregnanolone, a progesterone metabolite, is more effective than progesterone in reducing cortical infarct volume after transient middle cerebral artery occlusion. Ann Emerg Med 2006; 47: 381-389.
45. Kajta M, Budziszewska B, Lason W. Allopregnanolone attenuates kainate-induced toxicity in primary cortical neurons and PC12 neuronal cells. Pol J Pharmacol 1999; 51: 531-534.
46. Frank C, Sagratella S. Neuroprotective effects of allopregnenolone on hippocampal irreversible neurotoxicity in vitro. Prog Neuropsychopharmacol Biol Psychiatry 2000; 24: 1117-1126.
47. Lockhart EM, Warner DS, Pearlstein RD, Penning DH, Mehrabani S, Boustany RM. Allopregnanolone attenuates N-methyl-D-aspartate-induced excitotoxicity and apoptosis in the human NT2 cell line in culture. Neurosci Lett 2002; 328: 33-36.
48. Yu R, Xu XH, Sheng MP. Differential effects of allopregnanolone and GABA on kainate-induced lactate dehydrogenase release in cultured rat cerebral cortical cells. Acta Pharmacol Sin 2002; 23: 680-684.