The inhibitor of IkappaBalpha phosphorylation BAY 11-7082 prevents NMDA neurotoxicity in mouse hippocampal slices

Neuroscience Letters 377 (2005) 147–151

The inhibitor of IkappaBalpha phosphorylation BAY 11-7082 prevents NMDA neurotoxicity in mouse hippocampal slices F. Goffia , F. Boronia , M. Benaresea , I. Sarnicoa , A. Benettib , P.F. Spanoa , M. Pizzia,∗ a

Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, Viale Europa, 11, 25123 Brescia, Italy b Division of Pathology, University of Brescia, 25123 Brescia, Italy Received 8 November 2004; received in revised form 25 November 2004; accepted 29 November 2004

Abstract NF-␬B is a nuclear transcription factor involved in the control of fundamental cellular functions including cell survival. Among the many target genes of this factor, both pro- and anti-apoptotic genes have been described. To evaluate the contribution of NF-␬B activation to excitotoxic insult, we analysed the effect of IkappaBalpha (I␬B␣) phosphorylation blockade on glutamate-induced toxicity in adult mouse hippocampal slices. By using immunocytochemical and EMSA techniques, we found that (i) acute exposure of hippocampal slices to NMDA induced nuclear translocation of NF-␬B, (ii) NMDA-mediated activation of NF-␬B was prevented by BAY 11-7082, an inhibitor of I␬B␣ phosphorylation and degradation, and (iii) BAY 11-7082-mediated inhibition of NF-␬B activation was associated with neuroprotection. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: NMDA; BAY 11-7082; NF-␬B; Neuroprotection

In response to the binding of extracellular ligands to the cell surface receptors, multiple transcription factors are activated in the cytosol and then translocate to the nucleus where they exert positive or negative control over cellular genes. Among them, NF-␬B transcription factor is one of the more involved in the control of cell survival [18]. It consists of homo- or heterodimeric complexes belonging to the Rel family which include five distinct members, namely p50/p105, p52/p100, p65 (RelA), RelB and c-Rel. In their latent status, NF-␬B proteins are complexed to a family of inhibitory I␬B proteins. Cellular activation results in I␬B phosphorylation, ubiquitination and proteolitical degradation, thus allowing NF-␬B dimer to translocate to the nucleus where it binds to the -␬B consensus sequence of the target genes [7]. Originally described to regulate the expression of genes involved in immunity and in inflammation processes, NF-␬B proteins have also been found to regulate central nervous system (CNS) physiology [23]. Their constitutive nuclear localization in certain subsets

∗

Corresponding author. Tel.: +39 030 3717501; fax: +39 030 3701157. E-mail address: [email protected] (M. Pizzi).

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.11.088

of neurons suggests that these proteins may participate in normal brain functions, such as synaptic transmission and neuronal plasticity [15,21]. On the other hand, growing evidence suggest the involvement of NF-␬B in both acute and chronic neurodegenerative disorders such as brain ischemia [4,5,29], spinal cord trauma [1], Parkinson’s disease [11], Alzheimer’s disease [31] and CNS viral infections. Nevertheless, the precise role of NF-␬B in neurodegeneration is still controversial. Whether NF-␬B proteins inhibit or promote neuron viability, might depend on the cell type, the developmental stage, the environmental factors, the nature of NF-␬B-inducing stimuli and the composition of NF-␬B homo/heterodimers. Therefore, data supporting a pro-survival effect of this transcription factor [13,17,19,20] contrast studies demonstrating a deathpromoting role of NF-␬B proteins [22,26,27,29]. At the present, understanding the molecular mechanisms involved in the activation of NF-␬B is very important. NF␬B induction can be abrogated using NF-␬B inhibitors which may function by various mechanisms including the inhibition of I␬B phosphorylation, inhibition of I␬B proteasomemediated degradation, and inhibition of translocation of activated NF-␬B dimers to the nucleus. BAY 11-7082 ((E)3-[(4-

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methylphenyl)-sulfonil]-2-propenenitrile) is an irreversible inhibitor of I␬B␣ phosphorylation which results in the downregulation of the cytokine-induced NF-␬B activation [24,32] and apoptosis [12,14,28]. The aim of this work was to investigate the role of BAY 11-7082 as possible neuroprotective agent preventing I␬B␣ degradation and NF-␬B nuclear translocation. NF-␬B is activated by various neuronal signals including glutamate. Stimulation of glutamate receptors leads to I␬B␣ degradation and NF-␬B induction in both hippocampal pyramidal neurons and cultured cerebellar granule cells [10,26]. This is associated with neuronal cell death [9,26], as shown by the neuroprotective activity of p65 antisense oligonucleotide [26]. Our working hypothesis was that BAY 11-7082, by its capability to abrogate I␬B␣ degradation, might also decrease neuron susceptibility to the cytotoxic stimuli. The role of NF-␬B activation in excitotoxic cell death was evaluated in acutely dissected hippocampal slices from adult mice. Hippocampal slices were prepared from 40-day-old mice (CD1, Charles River). Hippocampi were rapidly removed, cut at a thickness of 600 ␮m by a 752 M vibroslice according to the method of Gathwaite and Garthwaite [6] with minor modifications [25]. Experiments were performed as described previously [9,26]. Briefly, hippocampal slices were preincubated at 37 ◦ C for 15 min and with 100 ␮M NMDA (Sigma) for an additional 15 min. Slices were washed and incubated in fresh buffer to recover for 30 min. When tested, BAY 11-7082 (Biomol Research Laboratories) was present from the preincubation time and was maintained for the entire experimental period. Finally, slices were fixed and embedded in paraffin for tissue sectioning. Sections (5 ␮m) were stained with methylene blue and azur II and examined by light microscopy. To quantify the cell loss, adjacent cells were counted in cell layer fields taken from CA1 region in each slice. These fields measured 1.5 × 104 ␮m2 . Living neurons appeared homogeneous and compact with a blue cytoplasm and a brighter nucleus while lesioned cells were oedematous, contained white vacuoles and dark shrinking nuclei. The percentage of cell survival was calculated by the ratio between living cells and the total cell number [26]. For immunostaining analysis, hippocampal sections were deparaffinized, re-hydrated in a series of ethanol concentrations and incubated in methanol containing 10% hydrogen peroxide for l0 min, and with Triton-X (0.2% in PBS) for an additional 20 min. Overnight incubation with polyclonal antibodies against p65 (1 ␮g/ml, sc-372-G, Santa Cruz Biotechnology) was performed at +4 ◦ C. Biotinylated anti-rabbit immunoglobulins (1:300, Dako E0353) and an ABComplex/HRP (Dako K0355) were used for detection, following the manufacturer’s instructions. Terminal deoxynucleotidyltransferase-mediated dUTP nick endlabeling (TUNEL) was performed using the kit purchased from Roche Molecular Biochemicals according to the manufacturer’s instructions. In order to get biochemical evidence for effective ␬Bbinding activity, we analyzed nuclear extracts by elec-

Fig. 1. (A, B) Effect of NMDA on neuron survival in the CA1 region of hippocampal slices. After a 15-min exposure to NMDA (100 ␮M) hippocampal slices were left to recover for 30 min in fresh buffer. (C, D) TUNEL labeling of CA1 neurons indicates the presence of DNA fragmentation in the nuclei of slices exposed to NMDA. (A) and (C) vehicle treated slices. (B) and (D) slices exposed to NMDA.

trophoretic mobility gel shift assay (EMSA). Nuclear extracts from hippocampal slices were prepared essentially as described by Brooks et al. [2]. Five micrograms of nuclear extracts were combined with 20,000 cpm (0.1 ng) of ␥-32 Plabeled ␬B-oligonucleotides in lipage buffer (17.5 mM Hepes pH 7.5, 103 mM NaCl, 5 mM KCl, 1 mM DTT, 0.35 mM EDTA, 10% glycerol) containing 0.5 ␮g of poly(dI dC) in a total volume of l0 ␮l. Reactions were carried out for 20 min at room temperature, and protein–DNA complexes were resolved on non-denaturing 4% polyacrilamide gels in 1× Tris–glycine–EDTA buffer. Gels were then dried and subjected to autoradiography at room temperature. Oligonucleotide and its complementary strand were synthesized and purified by M-Medical Genenco-Life Science. For gel shift analysis, double-stranded oligonucleotide was end-labelled with ␥-32 P-ATP (ICN, specific activity >7000 Ci/mmol) and T4 polynucleotide kinase (Boehringer Mannheim) to obtain a specific activity of more than 108 cpm/␮g. The sequence was as follow: APP1 (2257 to 2234 of the APP 5 regulatory region) 5 -TAGAGACGGGGTTTCACCGTGTTA-3 . Fifteen minutes exposure of the hippocampal slices to the glutamate receptor agonist NMDA (100 ␮M), followed by a recovery period of 30 min, damaged about 70% of CA1 pyramidal neurons (Fig. 1). Control slices (Fig. 1A) were characterized by the presence of homogeneous and compact neurons. By contrast, slices treated with 100 ␮M NMDA (Fig. 1B) exhibited dark and pycnotic neurons, showing distorted somata and condensed nuclei intermitted with oedematosus and vacuolated cells. Lesioned neurons showed inter-nucleosomal DNA fragmentation as revealed by the TUNEL assay (Fig. 1D). To investigate the activation of NF-␬B after NMDA exposure, NF-␬B p65 immunoreactivity was first examined in the

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Fig. 3. Prevention of excitotoxic effect of NMDA in mouse hippocampal slices by BAY 11-7082. (A) Upper panel: control; middle panel: 100 ␮M NMDA; lower panel: 100 ␮M NMDA plus 100 ␮M BAY 11-7082. Cell viability was evaluated in CA1 region of hippocampal sections. (B) Quantitative analysis of cell loss in the CA1 hippocampal region taken from three different experiments run in septuplicate. Data were analysed by Wilcoxon’s rank sum test. P ≤ 0.05 was considered statistically significant. Fig. 2. (A) Immunocytochemical analysis of NF-␬B p65 subunit in CA1 region of mouse hippocampal slices. Upper panel: control; middle panel: 100 ␮M NMDA; lower panel: 100 ␮M NMDA plus 100 ␮M BAY 11-7082. (B) Effect of BAY 11-7082 on NMDA-induced APP-␬B binding activity in mouse hippocampal slices. EMSA was obtained by incubating 5 ␮g of nuclear extracts with ␥-32 P-end labelled APP1 oligonucleotide probe. Hippocampal sections were untreated (lane 1) or treated with NMDA in the absence (lane 2) or presence of BAY 11-7082 (lane 3).

hippocampal slices (Fig. 2A). Weak and variable immunostaining for NF-␬B p65 subunit was observed in hippocampal neurons of the control slices. By contrast, prominent staining was seen in the neurons of slices exposed to 100 ␮M NMDA. The immunoreactivity to p65 was particularly localized in the nucleus, confirming the translocation of NF-␬B after NMDA receptor activation. BAY 11-7082 at 100 ␮M concentration, prevented the nuclear translocation of p65 elicited by NMDA. In order to find biochemical evidence of effective ␬B-binding activity of nuclear proteins, we analysed nuclear extracts by electrophoretic mobility gel shift assay (EMSA) (Fig. 2B). Nuclear proteins were isolated from the CA1 area of hippocampal slices at the end of the experimental procedure and tested for their capability to specifically interact with the APP-␬B site. In agreement with the immunocytochemical finding, NMDA produced an up-regulation of the APP-␬B binding activity (Fig. 2B, lane 2). BAY 11-7082 prevented the NMDA-induced increase of NF-␬B binding (Fig. 2B, lane 3). Since NMDA-mediated neuronal damage was associated with activation of NF-␬B, we tested BAY 11-7082 for its potential to prevent neuronal cell loss in CA1 field of the hippocampus. Results are shown in Fig. 3A. Application of BAY 11-7082 to hippocampal slices prevented NMDA toxicity occurring in CA1 region. BAY 11-7082 induced a significative cell rescue at 20 ␮M concentration (40% of neuroprotection)

(data not shown) and a maximal effect at 100 ␮M concentration (70% of neuroprotection). No apparent modification of neuron viability was observed after exposure of slices to BAY 11-7082 (100 ␮M) alone. The cell loss count in the CA1 region of hippocampal slices after the different treatments is shown in Fig. 3B. This study demonstrates that an inhibitor of I␬B␣ phosphorylation preserves neuron viability from the NMDAmediated excitotoxic injury. NF-␬B/Rel proteins have been shown to be fundamental regulators of cell death and cell survival in a variety of experimental settings. At the present it is not clear, however, how cells translate NF-␬B activation in gene programs promoting death or survival. It might depend on the particular system under investigation, the type of stimulus, the different combination of NF-␬B subunits which could activate different sets of target genes [18,26]. Recently, two new inhibitors of I␬B␣ phosphorylation and subsequent NF-␬B activation, namely BAY 11-7082 and 117085 have been identified. Both drugs inhibit TNF␣-induced expression of endothelian-leukocyte adhesion molecules in human endothelian cells. BAY 11-7085 was also tested in vivo and it was demonstrated to be a potent anti-inflammatory drug. It reduces the oedema formation in a rat carrageenan paw oedema assay and reduces paw swelling in a rat adjuvant arthritis model [24]. The precise molecular target of these drugs is not yet clear. The inhibition of NF-␬B phosphorylation may result from direct blockade of an inducible I␬B␣ kinase or may be due to inhibition of a signalling event upstream of the I␬B␣ kinase. Furthermore, these drugs might induce phosphatase activities involved in the regulation of I␬B␣ phosphorylation [3]. In order to understand whether the disruption of NF-␬B/I␬B regulatory pathway by these agents can promote neuronal survival, we tested

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the effect of BAY 11-7082 in acutely dissected mouse hippocampal slices. We found that the stimulation of NMDA receptor induces a characteristic cell injury which is associated with activation of p50/p65 dimer [26]. The application of BAY 11-7082 at 100 ␮M concentration prevented NF-␬B activation and preserved the hippocampal neuron viability from NMDA-mediated toxicity. Interestingly, when used at 10 ␮M concentration in cultured cerebellar neurons, Bay 11-7082 was inactive on glutamate toxicity but inhibited NF-␬B activation and neuroprotection elicited by pigment epithelium-derived factor [33]. In line with this we found no prevention of glutamate-mediated toxicity in cultured cerebellar cells exposed to 10 ␮M Bay 11-7082, but a significant protection with exposures shorter than 60 min at concentrations lower than 2 ␮M (data not shown). This might reflect a possible differential affinity of the I␬B␣ phosphorylation inhibitor for the NF-␬B complexes involved in the opposite regulation of neuron survival [26]. Lower concentrations may target p50/p65 dimers acutely activated by neurotoxic insults, while higher concentrations may affect the constitutive or long lasting activation of NF-␬B complexes involved in neuroprotection. The higher concentrations of Bay 11-7082 required to inhibit glutamate toxicity in the hippocampal slices, compared to cultured neurons, may very well depend on the thickness of the brain sections used as well as on the wide presence of glial cells. Our data are consistent with evidence showing that NF-␬B activation by glutamate may trigger neuronal cell death [9,26,29,30]. A possible role of NF-␬B and p53 in the glutamate-induced neuronal apoptosis has been suggested [8]. It was found that NF-␬B proteins promote an apoptotic response in rat striatal neurons exposed to excitotoxic insult through up-regulation of c-Myc and p53 [27]. The ability of inhibitors of I␬B␣ degradation to prevent glutamate-mediated neurotoxicity has been already proposed. The neuroprotection by salycilates correlated with their capability to inhibit NF-␬B activation [9] by interfering with a pathway leading to phosphorylation or degradation of I␬B [34]. Likewise, calpain inhibitors, by blocking calpain-dependent mechanism, including the cleavage of I␬B␣, prevented glutamate toxicity [30]. Thus, the cell transfection with a super-repressor mutant form of I␬B␣ increased the neurons resistance to the oxidative stress [16]. Our data, by showing the neuroprotective properties of BAY 11-7082, indicate that selective inhibition of I␬B␣ phosphorylation may represent a suitable strategy for a wide therapeutic intervention covering, besides the inflammatory and neoplastic diseases, the neurodegenerative disorders.

Acknowledgements This work was supported by grants from the Italian Ministry of Education, University and Scientific ResearchCOFIN 2002, and FIRB 2002; Centre of Study and Research on Ageing, Brescia; MURST Centre of Excellence for Inno-

vative Diagnostics and Therapeutics (IDET) of Brescia University.

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