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. 2006 Apr 22;26(4-6):383–403. doi: 10.1007/s10571-006-9045-9

CREB and NF-κB Transcription Factors Regulate Sensitivity to Excitotoxic and Oxidative Stress Induced Neuronal Cell Death

Jian Zou 1, Fulton Crews 1,✉
PMCID: PMC11520752  PMID: 16633891

Abstract

1. Glutamate–NMDA receptor excitotoxicity and oxidative stress are two common mechanisms associated with most neurodegenerative diseases. We hypothesize that the vital state of neurons is regulated in part by two key transcription factors, CREB and NF-κB. To test this hypothesis we used hippocampal–entorhinal cortex slice cultures.

2. Glutamate neurotoxicity and oxidative stress neurotoxicity, using hydrogen peroxide (H2O2) are both associated with a decrease in CREB DNA binding and an increase in NF-κB DNA binding.

3. Agents that modulate CREB and NF-κB DNA-binding activity alter neurotoxicity. Rolipram, a phosphodiesterase IV inhibitor, increased CREB DNA binding activity and decreased toxicity, whereas TNFα, increased NF-κB DNA-binding activity and increased neurotoxicity to both glutamate and H2O2. Ethanol decreased CREB and increased NF-κB DNA-binding activity and increased neurotoxicity to both glutamate and H2O2.

4. Brain-derived neurotrophic factor (BDNF) is a transcriptionally regulated trophic factor whose expression follows sensitivity to toxicity suggesting it is one of the transcriptionally regulated factors that contributes to neuronal vitality secondary to the balance of CREB–NF-κB-activated transcription. Together these studies suggest that neurotoxicity through glutamate–NMDA receptors or oxidative stress is dependent upon CREB and NF-κB DNA transcription that regulates vitality of neurons.

KEY WORDS: CREB, NF-κB, glutamate, oxidative stress, TNFα, ethanol, slices

INTRODUCTION

Transcription factors, such as cAMP responsive element binding protein (CREB) and nuclear factor κB (NF-κB), regulate expression for a diversity of CNS genes. CREB and CREB-dependent gene expression have been implicated in promoting neuronal survival and protecting neurons from excitotoxicity and apoptosis through the transcription of prosurvival factors (Lonze and Ginty, 2002; Mantamadiotis et al., 2002). Glutamate NMDA receptors regulate synaptic plasticity and can be coupled to CREB, however, high levels of NMDA receptor activation and NMDA receptor-mediated calcium flux, trigger signals that lead to either rapid or delayed neuronal death and are associated with decreased CREB activation. Alternatively, NF-κB is widely known for its ubiquitous roles in inflammatory and immune responses, as well as in control of cell division and apoptosis (O’Neill and Kaltschmidt, 1997; Mattson and Camandola, 2001; Hinoi et al., 2002). In the central nervous system, NF-κB proteins are ubiquitously expressed in neurons and glia (Kaltschmidt et al., 1993; Mattson and Camandola, 2001) where, in addition to regulating physiological processes, they participate in neurodegeneration (O’Neill and Kaltschmidt, 1997; Grilli and Memo, 1999; Pizzi et al., 2002; Schneider et al., 1999). NF-κB proinflammatory transcription is associated with induction of oxidative enzymes such as NADPH oxidase, COX2, iNOS, activation of the immune response, through induction of cytokines and particularly TNFα, a proinflammatory cytokine that activates NF-κB and induces its own synthesis as a component of microglial activation. Thus, CREB and NF-κB are important brain transcription factors that regulate diverse sets of genes that in general appear opposite with CREB being prominent in plasticity and growth, whereas, NF-κB oxidative inflammatory gene induction is generally associated with degeneration.

Julius Axelrod would always encourage scientists to do experiments. He often suggested that once you had the general idea of what the field was thinking you should stop reading, form an exciting testable hypothesis and do the experiments. We hypothesize that neurodegeneration is regulated not by the nature of the insult, but by the vitality of the neuronal transcriptome, that is the genes being actively transcribed at the time of the insult. More specifically, we hypothesized that the relative activity of CREB and NF-κB are fundamental to determining the sensitivity of neurons to neurotoxic insults. To test this hypothesis we used hippocampal–entorhinal cortex slice cultures that contain the neuronal connections and multiple cell types of intact brain. We found that glutamate neurotoxicity is predominantly mediated through activation of NMDA receptors, and is blocked by antagonist MK-801 as well as by agents that increase CREB DNA binding. Oxidative stress neurotoxicity, using hydrogen peroxide (H2O2), is blocked by the antioxidants, BHT and glutathione, but not by MK-801. Each type of neurotoxicity was associated with a decrease in CREB DNA binding and an increase in NF-κB DNA binding. Further, each form of neurotoxicity was reduced by agents that increase CREB and decrease NF-κB DNA-binding activities, and increased by agents that increase NF-κB DNA and decrease CREB DNA binding. Together these studies suggest that neurotoxicity through glutamate–NMDA receptor excitotoxicity or oxidative stress is dependent upon the CREB and NF-κB DNA-binding activity ratios that regulate the vitality of neurons.

MATERIALS AND METHODS

Brain Slice Culture

All protocols followed in this study were approved by the Institutional Animal Care and Use Committee and were in accordance with National Institute of Health regulation for the care and use of animals in research. Organotypic hippocampal–entorhinal complex (HEC) slice cultures were prepared according to the techniques of Stoppini et al. (1991) with slight modifications. Briefly, the hippocampal–entorhinal complexes were dissected from 8-day-old rats in Gey's buffer and sectioned transversely at 400 μm with McIllwain tissue chopper. The slices were transferred on a 30-mm diameter membrane tissue insert (Millicell-CM, Millipore, Bedford, MA), 6–8 slices per membrane, and cultured with medium containing 75% MEM with 25 mM HEPES and Hank's salts (Gibco, USA)+25% horse serum (Gibco)+5.5 g/L glucose+2 mM l-glutamine. The cultures were maintained in a humidified 5% CO2 incubator at 36.5°C for at least 2 weeks prior to any treatment.

Treatments of the Slice Cultures

All treatments were initiated in the slices after 2 weeks in cultures. For ethanol alone or combined treatments, the cultures were maintained in a dessicator containing 300 mL water that was saturated with same concentration of ethanol. TNFα (R&D System, USA) was simultaneously added into the culture with glutamate and/or ethanol as concentration and duration as indicated. For treatments of antioxidant BHT (LKT Lab., MN), glutathione and rolipram (Sigma, USA), the cultures were pretreated with those agents for 40 min and followed by combined treatments as indicated. All slices were removed at the end point of experiments as indicated for further processing.

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extraction of HEC slices was performed using Clontech Mercury TransFactor Kits. Briefly, the slices were removed from the tissue membrane and incubated in lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, pH 7.9, plus protease inhibitor cocktail) for 15 min and then disrupted with a narrow-gauge needle. After centrifuged, the supernatant of cytosolic fraction was removed and the pellet was disrupted again in extraction buffer and the supernatants of nuclear fraction were collected after centrifugation. The protein concentrations were determined by Bradford reagent kit (BioRad). EMSA was performed using double-stranded oligonuleotides containing consensus core sequences for CREB (5′-AGAGATTGCCTGA-CGTCAGAGAGCTAG-3′) and NF-κB (5′-AGTTGAGGCGACTTT-CCCAGGC-3′) (Promega). The probe was labeled with [γ-32P] ATP using T4 polynucleotide kanase. The equal amounts of nuclear extract protein were incubated with probe for 20 min. Bound and free probes were separated by electrophoresis on a 4% acrylamide gel. Gels were exposed to X-ray films for different periods to obtain autoradiograms most adequate for subsequent densitometry quantification. The optical density of densitometry analysis is obtained with the Bioquan imaging program.

Assessment of Neuronal Cell Death

Neuronal cell death was accessed using uptake of the fluorescent exclusion dye propidium iodide (PI). PI is a polar compound which is impermeable to a cell with an intact cell membrane but penetrates damaged cell membranes. Inside the cells it binds nuclear DNA to generate the brightly red fluorescence. This method has been characterized as accurately measuring neuronal degeneration in organotypic brain slice cultures (Noraberg et al., 1999). In the present study, PI was added into the culture medium at a concentration of 5 μg/mL and PI fluorescence images were captured at different time points as indicated with an inverting Axiotvert 100 microscopy and analyzed with AxioVision 3.1 software. Using an interactive drawing tool the hippocampal CA1 field was outlined as regions of interest (ROI), and mean PI fluorescent intensity of ROI was then determined by the program (Zou and Crews, 2005).

RNA Isolation, Reverse Transcription and Real-Time Quantitative RT-PCR

After treatments, the slices were removed, rinsed with cold PBS and immediately followed by total RNA purification using the RNeasy Mini Kit (Qiagen Inc., CA). Total RNA was quantified by spectrophotometry at 260 nm. For the reverse transcription, 2 μg of total RNA was used to synthesize the first strand cDNA using random primers (Invitrogen, CA) and reverse transcriptase Moloney murine leukemia virus (Invitrogen, CA). Total of 1 μL of the first strand cDNA solution was used for RT-PCR. The primer sequences for real time RT-PCR designed by Integrated DNA Technologies (IDT, IA). The following primers were used: for BDNF, forward 5′-GTGACARTATTAGCGAGTGGG-3′ and reverse 5′-GGGTAGTTCGGCA TTGC-3′ and for β-actin, forward 5′-CTACAATGAGCTGCGTGTGGC-3′ and reverse 5′-CAGGTCCAGACGCAGGATGGC-3′. SYBER Green Supermix (Bio-Rad) was used as a RT-PCR solution. The real-time RT-PCR was run with initial activation for 10 min at 95°C and followed with 40 cycles of denaturation (95°C, 40 s), annealing (57°C, 45 s) and extension (72°C, 40 s). All experiments were run in triplicate. The threshold cycle (C T) of each target product was determined and normalized to internal standard β-actin. Difference in C T values (ΔC T) of two genes was calculated Inline graphic, and data are expressed the percentage or fold difference compared to control. The RT-PCR products were confirmed by using 1.5% agarose gel.

Statistical Analysis

All data are expressed as mean±SEM values from the indicated number of experiments. Data are analyzed by one-way ANOVA and student's t-test. Variations are considered to be statistically significant at a p value of <0.05.

RESULTS

Decreased CREB and Increased NF-κB DNA-Binding Activities by Glutamate, TNFα and Ethanol are Highly Neurotoxic

We hypothesize that CREB and NF-κB are reciprocally regulated in a manner that regulates neuronal vulnerability to neurotoxic insults. To investigate the role of these transcription factors in glutamate toxicity we used an organotypic hippocampal–entorhinal cortex (HEC) slice culture that preserves the cellular organization and cell–cell interconnections of entorhinal cortex and hippocampus. A variety of agents were used to modulate transcription factors to investigate the effects on neurotoxicity, TNFα, rolipram and ethanol. TNFα is known to activate NF-κB through specific receptors in brain. Rolipram, a phosphodiesterase inhibitor, is known to increase CREB activation (MacKenzie and Houslay, 2000). Ethanol has been previously shown to modulate CREB in brain (Pandey et al., 2005) and NF-κB in liver (Nanji et al., 2001). CREB DNA binding was used as an index of CREB activation. CREB DNA binding was not altered by glutamate, there was a trend for a reduction by TNFα alone and ethanol alone, however, the combination of glutamate, TNFα and ethanol showed a substantial decrease in CREB DNA binding (Fig. 1). A more detailed investigation of the effects of various concentrations of ethanol alone indicated that ethanol in a dose-dependent manner progressively blunted CREB DNA binding while increasing NF-κB binding (Fig. 2) with CREB being reduced by approximately half and NF-κB approximately doubled at the highest ethanol concentrations studied. Thus, ethanol decreases CREB DNA binding while increasing NF-κB DNA binding.

Fig. 1.

Fig. 1.

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Reduction of CREB DNA binding by combined treatment of glutamate–TNFα–ethanol. HEC slices were prepared and cultured as described in the methods. Slices were treated with glutamate (3.3 mM) in the absence or simultaneous presence of TNFα (20 ng/mL) and/or ethanol (100 mM) for 24 h and removed for processing nuclear extracts after PI images were captured for analysis of neuronal cell death. EMSA was performed with 10 μg of nuclear extracts from the slices in each lane and a representative image from three experiments was shown in Lanes: 1. Control; 2. TNFα (20 ng/mL); 3. Ethanol (100 mM); 4. Glutamate 3.3 mM; 5. Glutamate+TNFα; 6. Glutamate+Ethanol; 7. Glutamate+TNFα+Ethanol. Image at left is one of three, with optical density measures bar graph on right, mean±standard error of the mean of three independent experiments of the integrated optical density quantified with BioQuant imaging as described in the methods. (* p<0.02 and ** p<0.03 compared to control, respectively).

Fig. 2.

Fig. 2.

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Ethanol increases CREB and decreases NF-κB DNA binding. HEC slices were treated with various concentrations of ethanol for 24 h and then removed for processing of nuclear extracts. EMSA was performed with 10 μg of nuclear extracts in each lane. Treatments are shown at the top of each lane. Specificity of CREB and NF-κB binding was confirmed with 70-fold excessive unlabeled probe (not shown). Bar graph shows the integrated optical density quantified with BioQuant imaging as described in the methods. Shown are the mean±standard error of the mean of five independent experiments (n=5, * p<0.05). Ethanol dose-dependently decreases CREB but increases NF-κB DNA binding activities (E25=25 mM ethanol, E50=50 mM ethanol, etc.).

TNFα is a proinflammatory cytokine known to activate NF-κB transcription inducing the synthesis of additional proinflammatory proteins. Treatment with TNFα increased NF-κB DNA binding more than 10 fold far greater than the effect of ethanol (Fig. 3). Interestingly, glutamate tended to slightly enhance the TNFα stimulated increase in NF-κB DNA binding, whereas the combination of glutamate and ethanol with TNFα increased NF-κB DNA binding by about 40% more than TNFα alone, i.e. about 14-fold more than basal NF-κB-DNA binding (Fig. 3). Thus, combinations of glutamate with TNFα and ethanol dramatically decrease CREB DNA binding and markedly increase NF-κB DNA binding. Neurotoxicity determinations indicate that neither TNFα or ethanol alone show any marked toxicity on their own, whereas glutamate at 1 or 3 mM shows concentration dependent increases in neurotoxicity. Previous studies have indicated that the ED50 for glutamate neurotoxicity ranges between 3–10 mM in HEC slices (Zou and Crews, 2005). Glutamate neurotoxicity was significantly potentiated by the presence of TNFα and further potentiated by combined treatment with ethanol and TNFα by 41–68% (p<0.001) and 18–30% (p<0.03) at 1 and 3 mM glutamate, respectively (Fig. 4). NMDA glutamate receptor antagonist MK-801 blocked all glutamate and/or ethanol–TNFα–glutamate neurotoxicity (data not shown), suggesting all neurotoxicity is through activation of the glutamate-NMDA receptor. In our previous study, we have demonstrated the correlative relationship between increased NF-κB DNA-binding activity and TNFα enhanced glutamate neurotoxicity since blockade of TNFα-induced activation of NF-κB by antioxidant BHT reduces TNFα potentiation of glutamate induced neuronal cell death (Zou and Crews, 2005). We further demonstrated that ethanol–TNFα–glutamate neurotoxicity is significantly blocked by antioxidant BHT that reduces TNFα activation of NF-κB (Fig. 5). Collectively, these studies support the hypothesis that decreased CREB DNA binding increases neuronal vulnerability to glutamate neurotoxicity and implicate NF-κB activation as enhancing glutamate toxicity.

Fig. 3.

Fig. 3.

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TNFα induced NF-κB DNA binding is increased in the presence of glutamate and ethanol. HEC slices were prepared and cultured as described in the methods. Slices were treated with TNFα (20 ng/mL) and glutamate (3.3 mM) in the absence or presence of ethanol (100 mM) for 8 h and then removed for processing nuclear extracts. EMSA was performed with 10 μg of nuclear extracts in each lane and a representative EMSA image shown. Lanes: 1. Control; 2. TNFα (20 ng/mL); 3. Ethanol (100 mM); 4. Glutamate 3.3 mM; 5. Glutamate+TNFα; 6. Glutamate+ethanol; 7. Glutamate+TNFα+ethanol. Bar graph shows the integrated optical density (mean±standard error) quantified from 3 independent experiments with BioQuant imaging program as described in the methods. Although ethanol and glutamate increase NF-kB DNA binding (Lane 3 and 4, * p<0.05 compared to Control), TNFα is many fold more efficacious (Lane 2 and 5,** p<0.0001 compared to Control). The presence of ethanol combined with TNFα and glutamate produces the most prominent induction of NF-kB DNA binding activity, which is correlated with maximal cell death. (*** p<0.0001 compared to Control; p<0.007 compared to TNF+glutamate).

Fig. 4.

Fig. 4.

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Potentiation of glutamate induced neuronal cell death by ethanol and TNFα. HEC slices were treated with glutamate (1 and 3.3 mM) in the absence or simultaneous presence of TNFα (20 ng/mL) and/or ethanol (100 mM) for 24 h. Representative PI images of HEC slices are shown from the groups as indicated. The brighter the PI labeling means more cell death (bar=500 μm). The bar graph shows the quantitative data measured from CA1 areas of the slices and expressed as the mean±standard error of the mean of 9–12 slices per group. Similar results were found with two other experiments of comparable design. (* p<0.001 compared to Control; ** p<0.004 compared to corresponding groups of Glut 1 and 3.3 mM, respectively; *** p<0.01 compared to corresponding groups of Glut+TNFα, respectively).

Fig. 5.

Fig. 5.

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Antioxidant BHT inhibits NF-κB-DNA binding and TNFα-Ethanol potentiation of glutamate toxicity. HEC slices were pretreated with antioxidant BHT and followed by TNFα in the absence or presence of glutamate or other combinations for 8 h. EMSA was performed with 10 μg of nuclear extracts in each lane. Lanes: 1. Control; 2. TNFα (20 ng/mL); 3. TNFα (100 ng/mL); 4. TNFα 100 ng/mL+BHT100 μM; 5. TNFα 100 ng/mL+BHT300 μM; 6. TNFα 100 ng/mL+BHT500 μM; 7. TNFα 100 ng/mL+AP-5 50 μM; 8. Glutmate 3.3 mM; 9. TNFα 20 ng/mL+glutamate; 10. TNFα 20 ng/mL+glutamate+BHT300 μM. The bar graph shows the quantitative data measured from CA1 areas of slices that were treated with different combinations as indicated and expressed as the mean±standard error of the mean of 9–15 slices per group. The experiment was repeated with comparable design and similar results. Antioxidant BHT significantly reduced TNFα-enhanced glutamate neurotoxicity (* p<0.001 compared to Glut3.3 mM; ** p<0.0001 compared to Glut+TNFα and G+T+EtOH).

Increased CREB DNA Binding Activity Blocks Glutamate and Glutamate–TNFα–Ethanol Neurotoxicity

The discovery that glutamate in combination with ethanol and TNFα decreased CREB DNA binding and increased glutamate neurotoxicity prompted experiments to determine if alterations in CREB activation would reduce neuronal sensitivity to excitotoxicity. Rolipram is a type IV phosphodiesterase (PDE) inhibitor that increases cAMP and phospho–CREB levels, the activated form of CREB that binds DNA (Nakagawa et al., 2002). We found that rolipram dose-dependently caused a robust increase of CREB DNA binding (Fig. 6). Interestingly, in slices pretreated with rolipram that have greatly increased CREB DNA binding, glutamate toxicity is dramatically inhibited (Fig. 6). To determine if rolipram similarly inhibited TNFα and ethanol potentiation of glutamate, experiments were done with combinations of these agents. As found previously glutamate-TNFα-ethanol markedly reduced CREB DNA binding and enhanced glutamate toxicity. Rolipram reversed the decreased CREB DNA binding with combined glutamate–TNFα–ethanol and reversed neurotoxicity (Fig. 7). These studies indicate that increased levels of CREB DNA binding reduce sensitivity to glutamate neurotoxicity and the enhanced toxicity found with glutamate–TNFα–ethanol.

Fig. 6.

Fig. 6.

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Rolipram induced increase in CREB DNA binding activity and neuroprotection in HEC slices. HEC slices were treated with PDE inhibitor rolipram for 24 h and nuclear extracts prepared. EMSA was performed with 10 μg of nuclear extracts in each lane. Lanes: 1. Control; 2. Rolipram 1 μM and 3. Rolipram 10 μM. Rolipram dose-dependently increases CREB DNA Binding. The bar graph shows the quantitation of neurotoxicity from CA1 areas of slices that were treated with glutamate in the absence or presence of 10 μM Rolipram for 24 h and expressed as the mean±standard error of the mean of 9–12 slices per group. The similar results were obtained from other 2 experiments with comparable design. (* p<0.0001 compared to glutamate).

Fig. 7.

Fig. 7.

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Increased CREB DNA binding activity correlates with reduction of neuronal cell death induced by Glutamate-TNFα-Ethanol in HEC slices. HEC slices were prepared and cultured as described in the methods. Slices were treated with glutamate in absence or presence of TNFα (20 ng/mL), ethanol (100 mM, pretreatment 40 min) and/or rolipram (pretreatment 40 min). In one set of experiments the slices were removed after treatment for 8 h for nuclear extractions preparations. EMSA was performed with 10 μg of nuclear extraction proteins in each lane. Lanes: 1. Control; 2. Rolipram 500 nM; 3. Rolipram 5 μM; 4. TNFα+ethanol +glutamate; 5. TNFα+ethanol+glutamate+Rolipram 5 μM; 6. TNFα+ ethanol+ glutamate+Rolipram 500 nM and lane P:-Probe only. Neuronal cell death was determined from PI images captured after treatment for 24 h and showed in bar graph. Shown are the mean±standard error of 8–12 slices in each groups. The experiments were at least repeated with similar designs and results (* p<0.001 comparing to Glutamate; ** p<0.05 compared to TNFα+Glutamate; *** p<0.0001 comparing to TNFα+Ethanol+Glutamate).

Oxidative Neurotoxicity is Regulated by CREB and NF-κB DNA-Binding Activities

To further explore the relationship between CREB DNA binding activity, NF-κB DNA binding and neuronal vitality, we studied neuronal vulnerability to oxidative stress induced by hydrogen peroxide (H2O2). H2O2 is a model oxidant that has been used to study oxidative-stress-induced cell death in several experimental models, including PC12 cells (Yamakawa et al., 2000; Lee et al., 2005b), glioma cell lines (Datta et al., 2002) and human neuroblastoma SH-SY5Y cells (Zhang et al., 1997) as well as in primary neuronal cell cultures (Lee et al., 2004). Oxidative stress has been suggested to play a role in neurodegeneration and excitotoxicity as well as activation of the transcription factor NF-κB. Treatment with H2O2 for 12–24 h caused disseminated cell death in a time- and dose-dependent manner throughout the HEC slice with profound cell death in hippocampal CA1 (Fig. 8). H2O2-induced cell death was potentiated by TNFα approximately 20–30%. Ethanol did not potentiate or reduce H2O2-induced cell death, but it did potentiate H2O2–TNFα toxicity (Fig. 9). CREB DNA binding was markedly decreased by H2O2 treatment, which was further decreased by the addition of TNFα and ethanol to approximately 20% of basal levels (Fig. 10). Determinations of NF-κB DNA binding surprisingly indicated that neurotoxic concentrations of H2O2 did not increase NF-κB DNA binding. TNFα and TNFα–ethanol in combination did increase NF-κB DNA binding (Fig. 11). Interestingly, the H2O2–TNFα–ethanol combination produces the greatest reductions in CREB DNA binding and increases in NF-κB DNA binding in association with the greatest neurotoxicity. Glutathione, the antioxidant, blocks TNFα–ethanol–H2O2 increased NF-κB DNA-binding activity, but not the MAP Kinase inhibitor PD98589 (Fig. 11). MAP Kinase can regulate CREB transcription in some systems. We found using western blotting that H2O2 caused a sustained activation of MAPK as indicated by increased phosphorylated ERK 1/2 (data not shown), however, the specific MAPK inhibitor PD98589 did not alter cell death induced by ethanol–TNFα–H2O2, which may be explained by the fact that PD98589 failed to reduce NF-κB activation or activate CREB (Fig. 11). These studies indicate that H2O2 oxidative stress induced neurotoxicity is associated with decreased CREB DNA binding and increased NF-κB DNA binding similar to glutamate neurotoxicity.

Fig. 8.

Fig. 8.

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Time course and concentration-dependent studies of H2O2 toxicity in HEC slices. HEC slices were prepared and cultured as described in the methods. The slices were treated with H2O2 and PI images captured in hippocampal CA1. Shown are the mean±standard error of 8–10 slices per group. Similar results were found with two other experiments of comparable design. H2O2 induced cell death in concentration- (A) and time- (B) dependent manner.

Fig. 9.

Fig. 9.

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Potentiation of H2O2 toxicity by TNFα and ethanol. HEC slices were treated with H2O2 (50 μM) with or without treatments as indicated for 24 h. Shown are the mean±standard error of the mean of 8 to 10 slices per group from a representative experiment of two experiments with similar designs (* p<0.01 comparing with H2O2; ** p<0.05 comparing with H2O2+TNFα; *** p<0.001 comparing to corresponding group).

Fig. 10.

Fig. 10.

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Reduction of CREB DNA binding activity by treatment with H2O2 and different combinations in HEC slices. HEC slices were prepared and cultured as described in the methods. Slices were treated with H2O2 (50 μM) in the absence or simultaneous presence of TNFα (20 ng/mL) and/or ethanol (100 mM) for 12 h and removed for processing nuclear extracts after PI images were captured for analysis of neuronal cell death. EMSA was performed with 10 μg of nuclear extracts from the slices in each lane. Lanes: 1. Control; 2. H2O2 (50 μM); 3. H2O2+Ethanol (100 mM); 4. H2O2+TNFα (20 ng/mL); 5. TNFα+Ethanol+H2O2; 6. TNFα+Ethanol+H2O2+GSH and 7. TNFα+Ethanol+H2O2+PD98589 (50 μM). Bar graph shows the integrated optical density quantified with BioQuant imaging program as described in the methods. Shown are the mean±standard error of the mean of triplicate EMSA from two independent experiments (* p<0.03 comparing to control).

Fig. 11.

Fig. 11.

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NF-κB DNA binding induced by combined H2O2, TNFα and ethanol. HEC slices were treated with H2O2 alone or with agents as indicated for 12 h and nuclear extracts prepared. NF-κB DNA binding activity by EMSA was performed with 10 μg of nuclear extracts in each lane. Lanes: 1. Control; 2. H2O2 (100 μM); 3. H2O2+Ethanol (100 mM); 4. H2O2+TNFα; 5. H2O2+TNFα+Ethanol; 6. H2O2+TNFα+Ethanol+GSH and 7. H2O2+ TNFα+ Ethanol+PD98589. Bar graph shows the integrated optical density quantified with BioQuant imaging program as described in the methods. Shown are the mean±standard error of the mean of triplicate from two independent experiments (* p<0.05 compared to control; ** p<0.05 compared to group of H2O2+TNFα and *** p<0.03 comparing to H+T+E).

Modulation of CREB and NF-κB DNA-Binding Activities Regulates H2O2 Oxidative Stress Induced Neurotoxicity

We further investigated the effects of inhibitors of NF-κB DNA binding and enhancers of CREB DNA binding on H2O2 oxidative stress induced neurotoxicity. The antioxidants, BHT and glutathione, markedly reduced H2O2 oxidative stress induced neurotoxicity (Fig. 12). Similarly rolipram, which we found to markedly increase CREB DNA binding activity, completely blocked H2O2 oxidative stress induced neurotoxicity. Interestingly, MK-801, the glutamate-NMDA receptor antagonist that blocked all glutamate toxicity failed to change H2O2 oxidative stress induced neurotoxicity (Fig. 12). Taken together these studies suggest that H2O2 oxidative stress induced neurotoxicity does not involve glutamate, but does involve changes in NF-κB DNA and CREB DNA binding consistent with their regulating sensitivity to H2O2 oxidative stress induced neurotoxicity similar to glutamate–NMDA neurotoxicity.

Fig. 12.

Fig. 12.

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Inhibition of H2O2 toxicity by antioxidants and Rolipram. HEC slices were treated with H2O2 in the absence or presence of MK-801 (30 μM), glutathione (GSH, 500 μM), BHT (300 μM) and rolipram (5 μM) for 12 h and then PI images were captured in hippocampal CA1. Representative psudocolor color PI images of HEC slices are shown in Control (A); H2O2 (100 μM) (B); H2O2+BHT (C) and H2O2+GSH (D). H2O2-induced cell death is significantly reduced by GSH and BHT (bar=500 μm). The bar graph shows the quantitative data measured from CA1 expressed as the mean±standard error of the mean of 6–10 slices per group. The experiments were done in triplicate with comparable design and similar results. (* p<0.001 comparing to corresponding groups).

Brain-Derived Neurotrophic Factor (BDNF) is Transcriptionally Regulated as a Neuroprotective Gene

BDNF has been shown to protect neurons from glutamate–NMDA excitotoxicity (Crews et al., 1999) and is transcriptionally regulated by CREB (Finkbeiner et al., 1997; Shieh and Ghosh, 1999; Fukuchi et al., 2005). To determine if BDNF was reciprocally regulated by NF-κB and CREB, we measured BDNF mRNA expression using RT-PCR analysis. Glutamate, TNFα, ethanol, and the combinations of all three that reduced CREB and enhanced NF-κB DNA-binding activities all reduced BDNF mRNA expression at early time points (Fig. 13). Although the magnitude of the BDNF mRNA changes varied with treatments and did not exactly correspond with the level of neurotoxicity, the transcription of many other genes likely contribute to overall neuronal vitality. Similarly H2O2 oxidative stress induced neurotoxicity and treatments that increase NF-κB DNA-binding activity reduced BDNF mRNA expression. Neurotoxicity is complex and BDNF likely contributes to sensitivity to neurotoxicity among many other proteins that regulate toxicity. In general, both decreased CREB and increased NF-κB DNA-binding activities reduced BDNF mRNA expression and increased neurotoxicity regardless of primary insult, e.g. oxidation or glutamate-NMDA excitotoxicity. These studies further support the role of CREB and NF-κB in regulating sensitivity to multiple neurotoxic insults.

Fig. 13.

Fig. 13.

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Alterations in CREB target gene BDNF in HEC slices. HEC slices were prepared and cultured as described in the methods. Slices were treated with glutamate or H2O2 (50 μM) in the absence or simultaneous presence of TNFα (20 ng/mL) and/or ethanol (100 mM) for 4 h and removed for processing BDNF RT-PCR as described in the methods. Shown are the mean±SEM of the mean of triplicate RT-PCR measurements in a representative experiment. BDNF mRNA expression is significantly decreased with treatments, however, combined treatments shows no further reduction in BDNF mRNA expression compared to corresponding groups (* p<0.05 comparing to Control). RT-PCR products were confirmed with 1.5% argrose gel.

DISCUSSION

CREB–NF-κB DNA Binding Regulate Neuronal Vitality

One of the most exciting discoveries reported here is that modulation of CREB DNA and NF-κB DNA binding can dramatically alter the response of neurons to both glutamate and H2O2 oxidative stress induced neurotoxicity. We found that rolipram dramatically increased CREB DNA binding and completely reversed neuronal cell death by glutamate–NMDA receptor activation and H2O2 oxidative stress induced neurotoxicity, as well as totally blocking the highly neurotoxic combinations of TNFα–ethanol–H2O2 and TNFα–ethanol–glutamate. CREB represents a family of protein transcription factors implicated in playing a central role in long term learning and memory (Silva et al., 1998) as well as a variety of behaviors (Carlezon et al., 2005). Overexpression of dominant-negative CREB promotes survival in melanoma cells (Jean et al., 1998) and mediates nerve growth factor survival in sympathetic neurons (Riccio et al., 1999) whereas CREB mutants that can not be phosphorylated reduce survival of granuloma cells (Somers et al., 1999) supporting a variety of studies suggesting that CREB increases neuronal survival of insults (Walton and Dragunow, 2000). Models of stroke in hippocampus have found that transient ischemia produces transient phosphorylation of CREB in CA1, but prolonged phosphorylation in DG, that corresponds with a delayed loss of CA1 neurons and a sparing of DG neurons (Hu et al., 1999; Walton et al., 1999). Induction of neuroprotection by ischemic tolerance to toxicity requires CRE-CREB-mediated gene expression (Hara et al., 2003) and ischemic preconditioning require CREB-dependent transcription (Mabuchi et al., 2001). Recent studies have linked increased CREB phosphorylation to synaptic prosurvival NMDA receptors and neurotoxic extrasynaptic receptors to decreased CREB (Hardingham and Bading, 2002). Thus a variety of studies have linked CREB to resistance to insults. Our findings extend these studies to both glutamate–NMDA receptor excitotoxicity and H2O2 oxidative stress induced neurotoxicity, as well as highly neurotoxic combinations. Increased CREB DNA binding is associated with completely blunted neurotoxicity to all combinations of insults studied. The MAPKase pathway is associated with phosphorylation and activation of CREB (Lee et al., 2005a). We found activation of P-MAPK with H2O2 treatment (unpublished data) and reduced CREB DNA binding suggesting uncoupling of P-CREB activation by the MAPK pathway, perhaps due to activation of CREB shut off pathways (Hardingham and Bading, 2002; Lee et al., 2005a). Regardless of the signaling mechanisms, together these studies suggest that CREB DNA binding regulates neuronal vitality and sensitivity to insults with high levels of CREB DNA binding able to block a variety of insults.

NF-κB is a family of transcription factors involved in promoting inflammatory cascades (Ghosh et al., 1998). NF-κB DNA binding was modestly altered by glutamate or H2O2 oxidative stress induced neurotoxicity, however, TNFα and the highly neurotoxic combinations of TNFα–ethanol–H2O2 and TNFα–ethanol–glutamate increased NF-κB DNA binding and increased neurotoxicity. Ischemia induced brain damage is reduced by soluble TNFα receptor (Nawashiro et al., 1997), consistent with TNFα induced NF-κB DNA binding contributing to glutamate–NMDA excitotoxicity in focal ischemia. We found that the antioxidants BHT and GSH reduced H2O2 oxidative stress induced neurotoxicity and NF-κB-DNA binding, but not the decrease in CREB DNA binding. Previous studies have shown that BHT can block NF-κB DNA binding induced by combinations of glutamate–TNFα and block TNFα potentiated glutamate neurotoxicity, but not the glutamate component of the neurotoxicity (Zou and Crews, 2005). Other studies have found that antioxidants, including BHT, block NF-κB activation (Flohe et al., 1997; Shrivastava and Aggarwal, 1999), although antioxidants that block NF-κB activation may act by unique mechanisms (Hayakawa et al., 2003). We did not see direct activation of NF-κB DNA binding by H2O2, however, both BHT and GSH reduce TNFα–ethanol–H2O2 NF-κB DNA binding and reduce TNFα–ethanol–H2O2 induced cell death. BHT and GSH may reduce H2O2 induced cell death in part by reducing the oxidative stress as well as increasing NF-κB DNA binding and decreasing CREB- NF-κB DNA binding. Taken together, these studies suggest that increased NF-κB DNA binding by TNFα and ethanol potentiate both glutamate–NMDA and H2O2 oxidative stress induced neurotoxicity.

Ethanol treatment provides insight into the interaction of CREB DNA binding and NF-κB DNA binding on neuronal vitality. Alcoholism is associated with neurodegeneration (Crews et al., 2004) consistent with ethanol induced neurotoxicity. Further, alterations in CREB family transcription factors have been implicated in drug addiction (Lonze and Ginty, 2002; Nestler, 2002). Ethanol exposure for 24 h modestly decreased CREB DNA binding activity and increased NF-κB DNA binding activity, although it did not cause measurable neuronal death or alter the death induced by either glutamate or H2O2. However, TNFα robustly activated NF-κB DNA binding with little direct effect on CREB DNA binding and increased glutamate and H2O2 cytotoxicity. When combined with TNFα, ethanol dramatically enhanced neuronal cell death induced by glutamate and H2O2 consistent with the ethanol induced modest decrease in CREB DNA binding contributing to increased neurotoxicity. Pharmacologic manipulation increasing CREB DNA binding and reducing NF-κB DNA binding levels reverses cell death induced by ethanol–TNFα–glutamate and ethanol–TNFα–H2O2 consistent with ethanol acting by modifying these transcription factors. Thus, ethanol decreased CREB DNA binding and increased NF-κB-DNA binding activities promote neurotoxicity consistent with the balance of these transcription factors regulating sensitivity to cell death induced by glutamate and H2O2.

CREB DNA and NF-κB DNA Binding and Cell Phenotype

Hippocampal-entorhinal cortex slice (HEC) cultures contain the neuronal connections and the multiple cell types of intact brain. Similar to other studies, we found that glutamate neurotoxicity is blocked by antagonist MK-801, whereas H2O2 is not blocked suggesting different mechanisms of toxicity. Glutamate-NMDA receptors are localized primarily on neurons and previous studies in neuronal cultures have suggested that CREB regulates neuronal sensitivity to glutamate–NMDA excitotoxicity (Hardingham and Bading, 2002). A variety of studies using histochemistry in vivo suggest that CREB is prominent in neurons of hippocampus in vivo (Walton and Dragunow, 2000; Bison and Crews, 2003), however, most cells contain both CREB and NF-κB making cellular localization difficult in HEC cultures. For example, the secretion of NGF by microglia is thought to be mediated in part by CREB activation (Heese et al., 1997). Rolipram would be expected to increase CREB DNA binding in cells with high signals coupled to activation of the CREB family of proteins. Although neuronal CREB DNA binding is associated with prosurvival signals, additional studies are needed to further understand the relationship between CREB DNA binding and neurotoxicity.

Similar to CREB, the NF-κB transcription factor family is expressed in both neurons and glia (O’Neill and Kaltschmidt, 1997). A variety of stimuli in brain activate NF-κB, particularly TNFα, a proinflammatory cytokine with a wide range of biological actions, but is best characterized as increasing death signals (Venters et al., 2000; Meffert and Baltimore, 2005). TNFα induces NF-κB DNA binding, increasing its own synthesis in macrophage–microglial cells as well as others. In microglial–neuronal cocultures inhibition of TNFα synthesis protects neurons from neuroinflammatory toxicity (Chao et al., 1993). In primary cultures of neurons TNFα protects against NMDA excitotoxicity (Cheng et al., 1994), alternatively TNFα can effectively inhibit prosurvival signals of IGF-1 promoting cell death (Venters et al., 1999). Interestingly, synaptic activity in cultured neurons is sufficient to activate NF-κB family members in neurons (Meffert et al., 2003). In our HEC cultures, we found that TNFα increased NF-κB DNA binding that is supper shifted by P50/P65 antibodies, suggesting mixtures of these members of the NF-κB family are translocated to the nucleus, binding DNA and altering transcription. In TNFα treated HEC slices increased NF-κB-DNA binding was associated with increased p65 immunohistochemical staining in nuclei of elongated shape and morphological location consistent with glial (astrocyte?) nuclei (Zou and Crews, 2005). Together with the finding that the astrocytic glutamate transporter, GLT1, is inhibited by increased NF-κB activation supports a role for TNFα activation of astrocytic NF-κB DNA binding potentiating glutamate neurotoxicity. The site of ethanol modulation is not clear. Ethanol blocks NMDA neurotoxicity in neuronal primary cultures (Chandler et al., 1993), but in our HEC slices ethanol potentiates glutamate neurotoxicity in association with decreased CREB DNA and increased NF-κB DNA binding. In neurons, CaMkinase II is associated NF-κB activation (Meffert and Baltimore, 2005) and potentiation of glutamate that could change the threshold of neurotoxicity. Our EMSA and histochemical studies in whole HEC slices capture the sum of the most robust signals. It is possible that different responses in different brain cell phenotypes are lost in our whole slice analysis of NF-κB DNA binding, however in general our findings indicate increased NF-κB DNA binding is associated with potentiation of glutamate–NMDA neurotoxicity as well as H2O2 oxidative stress induced neurotoxicity that is reduced by antioxidants that reduce NF-κB DNA binding.

Gene Transcription, BDNF and Neuronal Vitality

CREB DNA binding and NF-κB DNA binding do not directly translate to gene transcription. BDNF is an important neurotrophic factor in hippocampus previously found to play an important role in promoting neuronal survival, neuronal differentiation and synaptic plasticity (Huang and Reichardt, 2001). CREB DNA binding sites are known to contribute to activation of BDNF mRNA transcription with activity-dependent calcium-regulated sites also contributing to induction of BDNF (Fukuchi et al., 2005). Previously we found that BDNF treatment of mesolimbic cultures made neurons completely resistant to NMDA neurotoxicity (Crews et al., 1999). We investigated the relationship between neurotoxicity and BDNF mRNA in HEC slices and found that neurotoxic treatments in general reduced BDNF expression. Interestingly, ethanol which directly decreased CREB DNA binding showed a prominent decrease in BDNF mRNA. BDNF acts through receptors that activate CREB creating a positive loop not entirely different from the TNFα–NF-κB loop. Multiple genes and cascades are likely involved in regulating neuronal vitality, however, BDNF appears to play a role in regulating this sensitivity as a factor downstream and upstream of CREB.

In summary, CREB DNA binding and NF-κB DNA binding contribute to sensitivity to both glutamate–NMDA neurotoxicity as well as H2O2 oxidative stress induced neurotoxicity. Combinations of agents that reduce CREB DNA binding activity and enhance NF-κB DNA binding increase neuronal death whereas agents that increase CREB DNA binding activity make neurons resistant to both forms of neurotoxicity. These findings provide the basis for new studies directed at increasing resistance to multiple neurodegenerative insults through promotion of prosurvival transcription and reduction of proinflammatory gene transcription.

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