Nuclear factors-κB (NF-κB) are widely expressed in developing and mature central nervous system,1 where they regulate the adaptive responses to environmental factors stimulus-dependently.2, 3 The NF-κB family of dimeric transcription factors include p50, p52, p65 or RelA, RelB and c-Rel proteins, which use the Rel homology domain for dimerization and DNA binding.4 In resting cells, NF-κB factors interact with inhibitory IκB proteins. Cellular stimulation results in phosphorylation, ubiquitination and degradation of IκB, allowing NF-κB dimers to translocate to the nucleus where they regulate transcriptional function. Besides regulating long-term changes to neuronal function in response to synaptic transmission,5, 6 NF-κB factors participate in pathological events associated with neurodegeneration.2, 7, 8, 9 Increased NF-κB activation has been observed in neurons exposed to trauma and ischemia,9, 10, 11, 12 as well as in the brains of patients with Parkinson's or Alzheimer's disease.13, 14, 15 However, accumulating evidence has pointed to NF-κB as a survival determinant for neurons.16, 17, 18 It has been shown that NF-κB mediates neuroprotection elicited by tumor necrosis factor in hippocampal cells19, 20 and promotes neuron resistance to excitotoxicity21, 22 and amyloid β (Aβ)-induced apoptosis.23

We hypothesized that the opposite regulation of neuron survival by NF-κB in response to proapoptotic or antiapoptotic stimuli might depend on the activation of distinct combinations of subunits resulting in transcriptional regulation of diverse subsets of genes dictating the cell response.24, 25, 26 By studying glutamate and interleukin-1β (IL-1β), we showed that the neurotoxic effect produced by N-methyl-D-aspartate receptor activation in cerebellar granule cells and hippocampal slices was associated with activation of NF-κB dimers composed of p50 and p65 proteins, whereas neuroprotection elicited by IL-1β correlated with activation of c-Rel-containing dimers. Targeting p65 expression with antisense oligodeoxynucleotides prevented glutamate-mediated cell death, while targeting c-Rel abolished IL-1β neuroprotection.27 Our present study sheds light on the prosurvival role of c-Rel factor in neuronal cells and shows that activation of the c-Rel-dependent antiapoptotic pathway reduces neuronal vulnerability to Aβ 1–40 peptide. We demonstrate that neuroprotective activity of agonists at metabotropic glutamate receptor type 5 (mGlu5) against Aβ-mediated apoptosis relies on c-Rel activation. The post-transcriptional silencing of c-Rel gene by RNA interference technique, as its deletion in c-Rel−/− neurons, abolished mGlu5-mediated neuroprotection as well as manganese superoxide dismutase (MnSOD) and Bcl-XL expression. Conversely, c-Rel overexpression, or the application of TAT-Bcl-XL protein, reduced Aβ-mediated apoptosis.


Activation of mGlu5 receptors prevented Aβ toxicity and induced NF-κB activation in mouse cortical neurons and in human SK-N-SH cells

The treatment of primary cultures of mouse cortical neurons with different concentrations of 3HPG, a group I mGlu receptor agonist, prevented neuronal cell death induced by 48 h exposure to 5 μM Aβ. By using MTT reduction as an index of neuron viability we found about 55% of cell survival in Aβ-treated cells and more than 90% in cultures co-exposed to 50 μM 3HPG (Figure 1b). The data obtained by MTT reduction were very similar to those found by counting TUNEL positive cells (Figure 1a). The TUNEL staining of neuronal cells showed a large number of nuclei displaying chromatin fragmentation in Aβ-treated cultures (46±3% of total cells), but nearly no staining in cultures co-exposed to 3HPG (5±2% of total cells), as in control culture (4±3% of total cells). The neuroprotective effect of 3HPG was prevented by the selective mGlu5 receptor antagonist MPEP used at 5 μM concentration, and was reproduced by the selective mGlu5 receptor agonist CHPG at 1 mM concentration (Figure 3b, d). These data indicated that mGlu5 subtype of mGlu receptors expressed in mouse cortical neurons28 is mainly responsible for the neuroprotection elicited by 3HPG against Aβ toxicity. To verify this phenomenon in another neuronal model, we used SK-N-SK cells differentiated by retinoic acid treatment. We found that neuronal SK-N-SH cells, besides expressing functional N-methyl-D-Aspartate receptors,29 expressed mGlu1 and mGlu5 receptors. The RT-PCR analysis revealed the expression of both mGlu1 and mGlu5 receptor mRNAs in SK-N-SH cells, when compared to human thalamus templates (Figure 1c). The immunoblot analysis of total cell extracts confirmed the presence of the receptor proteins (Figure 1d). The application of 3HPG at 50 μM concentration prevented the release of cytochrome c into the cytosol (Figure 1e) as well as the toxic effect produced by 24 h exposure to Aβ peptide (Figure 1f). The neuroprotective response of 3HPG was blocked by MPEP, confirming that mGlu5 receptor signalling had a role in modulating the cell vulnerability to Aβ peptide in different types of neuronal cells. No modification of cell survival was produced by MPEP alone in the two neuronal cultures (Figure 1b, f).

Figure 1
figure 1

The activation of mGlu5 receptors prevents Aβ toxicity in primary cultures of mouse cortical neurons and in neuronal SK-N-SH cells. (a) TUNEL staining in cortical neurons exposed to Aβ with or without 3HPG for 48 h. (b) Cell survival was measured as MTT reduction in cortical neurons exposed to Aβ with or without 3HPG (1–50 μM) for 48 h. MPEP completely prevented the neuroprotection by 3HPG, while per se it did not modify the cell viability. (c) RT-PCR analysis of the expression of mGlu1 (292 bp band) and mGlu5 receptors (513 bp band) or β-actin (270 bp band) in templates from SK-N-SH neuronal cells and human thalamic nucleus (T). (d) Cell extracts from SK-N-SH cells and rat hippocampus (H) were immunoblotted against mGlu1 and mGlu5 receptor antibodies. Blots reprobed with an anti β-tubulin antibody indicated equal amounts of proteins in the different lanes (data not shown). (e) 3HPG attenuated the release of cytochrome c induced by exposure of SK-N-SH neuronal cells to Aβ for 24 h. (f) Cell survival was measured in differentiated SK-N-SH cells exposed to Aβ with or without 3HPG for 24 h. The 3HPG effect was prevented by MPEP. MPEP alone did not modify cell viability. Similar results were obtained in three separate experiments run in quadruplicate. *P0.05 versus corresponding control value

Figure 3
figure 3

Targeting c-Rel expression abolished mGlu5 receptor-mediated neuroprotection. (a) Primary cortical neurons were transfected with siRNA cognate to c-Rel gene (c-Rel siRNA) or with a negative control (non-siRNA) for 3 h, as described in Material and Methods. The cell extracts were prepared 72 h later and immunoblotted with c-Rel antibody. Blots probed with p50, p65 antibodies indicated specificity of c-Rel targeting by siRNA. Blots re-probed with an anti-β-tubulin antibody indicated equal amounts of proteins in the different lanes. (b) At 2 days after siRNA transfection, primary cortical neurons were exposed to 5 μM Aβ with or without 50 μM 3HPG or 1 mM CHPG for 48 h. The protective effect of 3HPG and CHPG, observed in non-siRNA treated cells, was suppressed in cells exposed to c-Rel siRNA. (c) Immunoblot analysis of c-Rel expression in extracts from primary cortical neurons prepared from WT or c-Rel−/− mice. (d) Cortical neurons from WT or c-Rel−/−mice were exposed to Aβ with or without mGlu5 receptor agonists for 48 h as in (b). Data represent the means±S.E.M. of three separate experiments run in triplicate. *P0.05 versus corresponding vehicle-treated samples

Then, we investigated the effect produced by mGlu5 receptor stimulation on the activity of NF-κB transcription factor. An ELISA-based analysis of DNA-binding interactions for the different NF-κB factors was performed in nuclear extracts prepared from cortical neurons (Figure 2b). By this procedure we obtained results similar to those found by EMSA and super shift analysis (Figure 2a). A 20-min application of 50 μM 3HPG or 1 mM CHPG to cortical cells activated NF-κB, as shown by the nuclear translocation of NF-κB factors p50, p65 and c-Rel (Figure 2b). Besides, in line with previous evidence,15, 30, 31 20-min application of the Aβ peptide induced NF-κB, but restricted to p50 and p65 subunits. An ELISA-based assay specific for human cells confirmed that both 3HPG and CHPG application to SK-N-SH neurons activated p50, p65 and c-Rel. No activation of RelB or p52 factors was detected (Figure 2c). To elucidate the specific composition of NF-κB complexes, we carried out co-immunoprecipitation studies. Using antibodies against p50, p65 and c-Rel, we identified the p50/p65, p50/c-Rel and p65/c-Rel dimers in nuclear extracts from unstimulated cells (Figure 2d). Control antibodies to IgG alone were unable to immunoprecipitate any NF-κB proteins. The CHPG treatment increased the nuclear amount of the c-Rel-containing complexes p50/c-Rel and p65/c-Rel, while it left unchanged the nuclear content of p50/p65. Although this approach did not allow us to detect the activation of p50/p50 homodimer, it could be excluded by the lack of a lower band totally supershifted by p50 antibody in the EMSA analysis of NF-κB-DNA complexes. These results indicated that while Aβ peptide activates dimers of p50 and p65 subunits, the mGlu5 receptor signalling leads to selective activation the NF-κB dimers p50/c-Rel and p65/c-Rel.

Figure 2
figure 2

The stimulation of mGlu5 receptors activates NF-κB factors in mouse cortical neurons and neuronal SK-N-SH cells. (a) EMSA analysis was performed in nuclear extracts from cortical cells treated with vehicle or 50 μM 3HPG for 20 min, followed by incubation in fresh medium for an additional 30 min. Competition with 100-fold excess of APPκB demonstrated specificity of binding (arrow). The molecular composition of NF-κB complexes was investigated by incubating nuclear extracts in the presence of antibodies raised against p50, p65 and c-Rel. (b) EMSA results were confirmed by an ELISA-based analysis of DNA-binding interactions for the different NF-κB factors. Nuclear extracts were from cortical cells exposed to 50 μM 3HPG, 1 mM CHPG and 5 μM Aβ in cortical cells. (c) ELISA-based analysis of human NF-κB factors in nuclear extracts from SK-N-SH cells exposed to 50 μM 3HPG and 1 mM CHPG. Absorbance data represent the means of three separate experiments. *P0.05 versus corresponding control value. (d) Co-immunoprecipitation analysis of NF-κB complexes in nuclear extracts from SK-N-SH cells. The efficiency of immunoprecipitations was confirmed by immunoblot analysis with antibodies raised against the immunoprecipitated proteins (data not shown). The antibody to p50 immunoprecipitated p65 and c-Rel. The p65 antibody immunoprecipitated p50 and c-Rel, while the c-rel antibody immunoprecipitated p65 and p50. The antibody to IgG did not immunoprecipitate c-Rel or other NF-κB factors (data not shown). Treatment with 1 mM CHPG enhanced the nuclear translocation of NF-κB complexes p50/c-Rel and p65/c-Rel, but not p50/p65

c-Rel silencing or c-Rel knockout suppressed the mGlu5-mediated neuroprotection in cortical cells

Synthetic siRNAs can be readily introduced into primary cortical neurons and effectively inhibit the expression of endogenous genes.32 A double-stranded siRNA targeting c-Rel expression (c-Rel-siRNA) or a control non-siRNA were designed as described in Material and Methods and added to 8 DIV cortical cells. The treatment with a fluorescein-conjugated siRNA showed a transfection efficiency of about 80% (data not shown). After 3 h, the medium was changed to Neurobasal and the c-Rel expression was evaluated by immunoblot analysis (Figure 3a) and immunocytochemistry (data not shown). The RNA interference significantly reduced c-Rel expression time-dependently. The decrease of c-Rel was clearly evident at 48 h (data not shown) and 72 h (Figure 3a). The c-Rel siRNA did not decrease the cellular amounts of p50, p65 or β-tubulin, indicating the specificity of c-Rel silencing. Indeed, the amounts of p50 and p65 appeared slightly increased in c-Rel silenced neurons, suggesting that their expression could compensate the lack of c-Rel in siRNA-treated cells. The siRNA treatment did not affect per se the neuron viability. At 48 h after application of siRNA, cells were exposed for an additional 48 h to Aβ with or without mGlu5 receptor agonists. As shown in Figure 3b, the c-Rel targeted cells were vulnerable to Aβ toxicity, as were cells treated with non-siRNA used as a negative control. Conversely, the c-Rel targeted cells became totally resistant to mGlu5 receptor-mediated neuroprotection. A higher vulnerability trend was observed in c-Rel silenced neurons co-exposed to Aβ and 3HPG or CHPG. Parallel experiments were carried out in primary cultures of cortical neurons prepared from c-Rel−/− embryos (Figure 3c). As reported in Figure 3d, the Aβ-mediated toxicity was preserved in c-Rel−/− cells, while the neuroprotective activity of mGlu5 receptor agonists was completely lost. These results support the observation that c-Rel factor is dispensable for Aβ toxicity, but also absolutely necessary for mGlu5 receptor-mediated neuroprotection.

Overexpression of c-Rel reduced SK-N-SH cell vulnerability to Aβ exposure

As knocking down c-Rel expression abolished neuroprotective response mediated by mGlu5 receptor stimulation, we investigated the effect of direct c-Rel activation on neuron vulnerability. SK-N-SH neuronal cells were transiently transfected with expression plasmid coding for human c-Rel protein, pSG-cRel, or with control pSG5 expression vector. At 24 h after transfection, the overexpression of c-Rel was monitored by immunoblot analysis in total cell extracts (Figure 4a). The cells were exposed to Aβ for an additional 24 h. As reported in Figure 4b, control cultures and pSG5 transfected cells showed a similar decrease of cell survival after Aβ exposure. Conversely, c-Rel overexpressing cells showed a reduced vulnerability to Aβ toxicity (Figure 4b). Counting TUNEL positive cells in pSG5 and pSG-cRel-transfected cultures confirmed the protective effect of c-Rel against Aβ toxicity (Figure 4c).

Figure 4
figure 4

c-Rel overexpression counteracted Aβ-mediated toxicity in SH-N-SH neuronal cells. (a), SK-N-SH cells were transfected with pSG-cRel or pSG5 vector for 24 h. Cell extracts, which were immunoblotted with c-Rel antibody, revealed higher c-Rel expression in pSG-cRel-transfected cells. Blots re-probed with an anti β-tubulin antibody indicated equal amounts of proteins in the different lanes. (b) At the end of the transfection period, control cells and transfected cultures were exposed to Aβ peptide dissolved in fresh serum-free medium for additional 24 h. Cell viability was measured by MTT reduction assay. (c) TUNEL positive cells were measured in sister cultures transfected with pSG5 or pSG-cRel and exposed for 24 h to Aβ peptide. The Hoechst staining revealed similar number of cells in the wells exposed to the different treatments. Data represent the means±S.E.M. of three separate experiments run in triplicate. *P0.05 versus corresponding vehicle-treated samples

CHPG induced Bcl-XL and MnSOD expression in SK-N-SH cells by induction of c-Rel

Bcl-XL and MnSOD are two prosurvival genes under the transcriptional control of NF-κB c-Rel factor in non-neuronal cells.33, 34, 35 To demonstrate the participation of c-Rel in neuroprotection elicited by mGlu5 receptor activation, we investigated the effect of CHPG on MnSOD and Bcl-XL expression in neuronal cells pretreated with control non-siRNA or c-Rel-siRNA. As shown in Figure 5a, the RT-PCR analysis revealed increased amounts of both MnSOD and Bcl-XL mRNAs in SK-N-SH cells exposed for 1 or 4 h to 1 mM CHPG (Figure 5a). Accordingly, the relative protein contents appeared increased 4 h after CHPG application (Figure 5c). Treatment with c-Rel-siRNA efficiently downregulated c-Rel expression (Figure 5c) and prevented the increase of both MnSOD and Bcl-XL by CHPG. A relative estimate of PCR products was carried out by evaluating the ratio between MnSOD or Bcl-XL and corresponding β-actin bands for each template (Figure 5b). Relative variations in protein amounts were measured as ratio between MnSOD or Bcl-XL bands and corresponding β-tubulin (Figure 5d).

Figure 5
figure 5

Targeting c-Rel expression abolished the CHPG-mediated increase of MnSOD and Bcl-XL. SK-N-SH cells were transfected with c-Rel siRNA or with non-siRNA for 3 h. The cells were returned to a fresh medium and 72 h later they were exposed to 1 mM CHPG for 1 or 4 h. (a) RT-PCR analysis of the expression of MnSOD (446 bp band), Bcl-XL (129 bp band) or β-actin (270 bp band) at 1 or 4 h of exposure to CHPG. (b) Data from the densitometry analysis of PCR products are expressed as ratio of MnSOD or Bcl-XL to β-actin. (c) Protein cell extracts were immunoblotted with MnSOD, Bcl-XL, c-Rel or β-tubulin antibodies. (d) Data from densitometry analysis of MnSOD or Bcl-XL immunoblots are expressed as ratio of MnSOD or Bcl-XL to β-tubulin. Columns represent the means±S.E.M. of three experiments *P0.05 versus corresponding control value

To verify the direct prosurvival role for at least one of these proteins on Aβ-mediated cell death, we investigated the effect of TAT-Bcl-XL obtained by the fusion of the TAT protein transduction domain with the antiapoptotic Bcl-XL protein.36 As shown in Figure 6a, the treatment with TAT-Bcl-XL at concentrations ranging from 100 to 300 nM completely rescued SK-N-SH cells from Aβ neurotoxicity. Conversely, no protection against the Aβ peptide was found in cells pretreated with a control TAT-GFP protein. Finally, to confirm the participation of Bcl-XL in the CHPG-mediated neuroprotection, we silenced the Bcl-XL expression in SK-N-SH neuronal cells by using a specific Bcl-XL siRNA.37 The siRNA application reduced the protein content of Bcl-XL after 24 and 48 h (Figure 6b), without significantly modify the basal cell survival (Figure 6d). The exposure to Aβ peptide induced the apoptosis of neuronal cultures pretreated with non-siRNA or Bcl-XL siRNA, as shown by the analysis of cytochrome c release (Figure 6c) and MTT reduction (Figure 6d). However, the protective effect of CHPG elicited in control cells was abolished in Bcl-XL silenced cultures. These results lead to the conclusion that the cell resistance to Aβ toxicity induced by stimulation of mGlu5 receptors relies on c-Rel-mediated activation of Bcl-XL.

Figure 6
figure 6

Induction of Bcl-XL preserved cell viability from Aβ toxicity. (a) SK-N-SH cells were exposed to Aβ with or without different concentrations of TAT-Bcl-XL for 24 h. To exclude the possibility that the TAT domain itself could cause inhibition of Aβ toxicity, we assessed the effect of a TAT-GFP protein. (b) SK-N-SH cells were transfected with non-siRNA or with Bcl-XL siRNA. The level of Bcl-XL protein was strongly reduced 48 h after transfection. (c) At 24 h after Bcl-XL siRNA application, the SK-N-SH cells were exposed to 5 μM Aβ with or without 1 mM CHPG. The release of cytochrome c was evaluated 24 h later. (d) Cell survival was measured as MTT reduction in SK-N-SH cells silenced for Bcl-XL and exposed for 48 h to Aβ with or without CHPG. Data represent the means±S.E.M. of three separate experiments run in triplicate. *P0.05 versus corresponding vehicle-treated samples


Our data show that stimulation of mGlu5 receptors rescues neuronal cells from Aβ-induced apoptosis. This effect requires activation of NF-κB transcription factor c-Rel and involves Bcl-XL and MnSOD induction.

Though the mechanisms underlying neurodegeneration in Alzheimer's disease remain unclear, a growing body of evidence implicates Aβ in a pivotal role.38, 39 A matter of controversy is whether the pathological cascade is initiated by accumulation of Aβ in the extracellular space or by its intraneuronal generation. However, demonstration that wild-type neuronal tissue, when grafted into brain of APP-transgenic mice, also develops amyloid deposits and signs of neurodegeneration,40 implicates the pathological contribution of extracellular Aβ. A lot of evidence has proved the proapoptotic effect of the two main constituents of amyloid plaques, Aβ 1-40 and Aβ 1-42, in cultured neurons,41, 42, 43, 44, 45 and their capability to induce accumulation of intracellular Aβ.46, 47 Although Aβ 1–42 is considered the form initially deposited in diffuse plaques, the subsequent deposition of Aβ 1–40 characterizes the formation of fibril-rich neuritic plaques with surrounding cytopathology.38

For our study we used two different cell-based models of Aβ-mediated toxicity, the primary cultures of mouse cortical cells and differentiated human SK-N-SH cells. In both neuronal cell types, activation of mGlu5 receptors prevented the cell loss produced by exposure to the Aβ peptide. The precise role of group I mGlu receptors, including mGlu1 and mGlu5 subtypes, in regulating neuronal cell death is still debated. Most studies, addressed to evaluating poorly selective group I receptor ligands in models of excitotoxic or ischemia-induced injury, produced conflicting results.48 The recent availability of more selective compounds has generated evidence suggesting that activation of mGlu1, but not mGlu5, is likely to play a major role in neurodegeneration secondary to brain ischemia.49 The role of mGlu5 receptors is less clear, possibly because we lack mGlu5 agonists suitable for in vivo studies and most mGlu5 antagonists were found to block NMDA receptors, at high concentrations.50, 51, 52, 53 By means of either pharmacological blockers or strategies targeting the receptor expression, it was shown that endogenous activation of mGlu5 receptors elicits antiapoptotic effect and sustains neuronal survival during early brain development.54, 55 However, endogenous stimulation of mGlu5 receptors also contributes to neurotoxicity induced by the Aβ 25–35 soluble fragment in cultured neurons56 and amplifies nigro-striatal damage induced by methamphetamine or MPTP.57, 58 More homogeneous but opposite evidence rise from studies investigating the effects of exogenous stimulation of mGlu5 receptors by selective agonists. It was shown that CHPG is neuroprotective in a rat model of cerebral ischemia when administered intraventricularly.52 Activation of mGlu5 receptors protects neurons from oxidative stress,59 as well as from neurotoxicity mediated by glutamate and by a variety of proapoptotic agents60, 61, 62 including the Aβ peptide.63 It can be argued that, while endogenous stimulation of mGlu5 receptors, possibly associated with the activation of other glutamate receptor subtypes, is detrimental, selective exogenous stimulation of mGlu5 by pharmacological agonists leads to neuroprotection. This is in line with studies indicating that under sustained activation, the function of group I metabotropic receptors in the control of glutamate transmission and cell death can switch from a facilitating to an inhibiting mode.64, 65, 66, 67 Transient stimulation of receptors can exacerbate neuron vulnerability. Tonic stimulation of receptors induces the desensitization of the facilitatory activity, switching the receptor function to the inhibitory activity and neuroprotection. Although not completely elucidated, the signalling pathway responsible for that switch in function involves protein kinase C activation.65

We have here demonstrated that neuroprotection elicited by mGlu5 agonists was associated with activation of NF-κB factor c-Rel. In line with our previous evidence that peculiar composition of NF-κB dimers may contribute to establish neuronal cell fate in response to environmental stimuli,27 and according to Akama et al.,68 we found that the neurotoxic Aβ peptide activated only p50 and p65 subunits. Prevention of p50/p65 translocation, by a decoy oligonucleotide containing the κB regulatory sequence, prevented the Aβ toxicity (data not shown). Conversely, by co-immunoprecipitation studies we demonstrated that the mGlu5 agonists activated p50/c-Rel and p65/c-Rel dimers. The activation of c-Rel-containing dimers was an absolute requirement for prosurvival effect elicited by mGlu5 receptor agonists. This was demonstrated by targeting c-Rel expression through either RNA interference or gene knockout techniques. Both conditions completely suppressed the prosurvival effect of mGlu5 agonists, while they did not affect the Aβ-induced cell death. The involvement of c-Rel activation in NF-κB-preserved cell survival was further confirmed by overexpressing c-Rel in cultured neurons. The overexpression of c-Rel reproduced the neuroprotective effect of mGlu5 agonists in SK-N-SH cells, as previously found for the antiapoptotic response of nerve growth factor in sympathetic neurons17, 69 or insulin-like growth factor-1 in cerebellar granule cells.70

Consistent with our evidence, Movsesyan et al.,63 recently showed that mGlu5 receptor activation prevents the Aβ-mediated apoptosis in rat cortical cells and reduces cytochrome c translocation, caspase-3 activation and apoptosis-inducing factor release. We have here demonstrated that neuroprotection by mGlu5 agonists was associated with increased expression of the antioxidant enzyme MnSOD and the antiapoptotic protein Bcl-XL. MnSOD is a mitochondrial protein that catalyzes the dismutation of the anion superoxide radical to oxygen and hydrogen peroxide, a mechanism that may be responsible for cell resistance to apoptogenic burst of reactive oxygen species produced under Aβ exposure.30, 71 Neuronal cells overexpressing MnSOD exhibit nearly complete resistance to apoptosis induced by Aβ.71, 72 Bcl-XL is a bcl-x gene product generated, together with Bcl-XS, by alternative splicing of bcl-x transcript.73 It is generally accepted that Bcl-XL preserves cell survival by controlling mitochondrial membrane ion permeability and cytochrome c release.74 Additionally, it acts downstream of cytochrome c release through binding to the apoptosis protease-activating factor-1 that prevents procaspase-9 and subsequent caspase-3 activation.75 Both MnSOD and Bcl-XL are expressed in neuronal tissues where their relative expression level can determine cell commitment to apoptosis.19, 76, 77 We found that the intracellular increase of Bcl-XL, through exogenous application of a fusion protein of Bcl-XL with TAT protein transduction domain,36 completely prevented the Aβ-mediated cell death, as already reported for staurosporine-, glutamate- or ischemia-induced apoptosis.78, 79 Both MnSOD and Bcl-XL genes were shown to be transcriptional targets of c-Rel33, 34, 35 in non-neuronal cells. Though the transcriptional regulation of Bcl-XL and MnSOD by c-Rel has never been investigated in neurons, the p50/c-Rel dimers were found to bind to bcl-x gene promoter during brain ischemia. The p50/cRel activation appeared to be area-specific and correlated with increased Bcl-XL expression and higher neuronal resistance to hypoxic insult.80 Indeed, we found that the silencing of c-Rel abolished the increases of both MnSOD and Bcl-XL induced by CHPG. Besides, the silencing of Bcl-XL suppressed the neuroprotective activity of CHPG. These results give compelling evidence about the primary role of c-Rel in the antiapoptotic program activated by CHPG, which includes the expression of Bcl-XL and MnSOD.

The evidence that c-Rel-containing dimers are required for neuroprotection and induction of antiapoptotic genes is in line with the view that specific combination of NF-κB subunits within the NF-κB complex is responsible for activating specific subsets of genes.24, 26 Though, an additional level of regulation of NF-κB-target genes, which may involve post-translational modification of nuclear NF-κB subunits and protein–protein interaction with other promoter-bound factors, has recently emerged.26 As recently reviewed,81 both phosphorylation and acetylation of NF-κB factors are required to generate a fully active NF-κB complex. Phosphorylation of p65 has been found to promote the recruitment of the transcriptional coactivators, p300/CPB (CREB binding protein) and p300/CPB-associated factor, which, in turn, acetylate both p65 and histones surrounding the promoter of NF-κB-target genes. It results in chromatin remodelling and increased transcriptional response. Likewise, phosphorylation of c-Rel is necessary for NF-κB-mediated transactivation and modulation of cell apoptosis in response to tumor necrosis factor-α.82 It can be inferred that post-translational modifications, together with cooperation with other transcription factors, may integrate the signalling cascades activated by extracellular stimuli, to provide specificity and versatility in the regulation of genes and cellular responses by distinct NF-κB factors.

The specific role of c-Rel in neuronal pathophysiology emerges from our data and from a recent study dealing with molecular mechanisms of memory formation. Through a bioinformatics analysis and the use of c-Rel−/− mice, it was shown that c-Rel-mediated gene transcription is specifically required for the consolidation of long-term memory in the hippocampus.6 Thus, clarifying the signalling cascade responsible for c-Rel activation, or for post-translational modifications implicated in the interaction of c-Rel with the transcriptional machinery in the brain, will unravel new potential targets for pharmacological treatment of neurodegenerative diseases and memory dysfunction.

Materials and Methods

Cell culture

Primary cultures of mouse cortical neurons

Fifteen-day embryonic mice were taken with cesarean section from anesthetized pregnant dams. C57/BL6 mice were purchased from Charles River, Italia, and c-Rel−/− mice (background strain C57/BL6) were provided by H-C Liou.83 Cerebral cortices were isolated and dissociated by manual dispersion with a fire-polished Pasteur pipette. Cells were plated at a density of 1.5 × 105 cells/cm2, in 8 cm2 culture dishes (NUNC) for Western blot, EMSA and ELISA analyses, and in 2 cm2 tissue culture dishes for viability studies. Culture dishes were coated with 10 μg/ml poly-L-lysine. The cells were plated in Neurobasal medium (Invitrogen Corporation) supplemented with 2% B27 (Invitrogen Corporation), 0.5 mM L-glutamine and 50 U/ml penicillin/streptomicin (Invitrogen Corporation). At 3 days after plating, 50% of the medium was changed with fresh medium and subsequently 50% of the medium was changed twice a week, until 11 days in vitro.

SK-N-SH cell culture

The human SK-N-SH neuroblastoma cell line was purchased from American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM as previously described.29 Cells were plated at a density of 25 × 103/cm2 in 8 cm2 dishes (NUNC) and neuronally differentiated by addition of 50 μM RA for 12–15 days.

Beta-amyloid toxicity

Soluble Aβ 1-40 (Bachem, Switzerland) was dissolved in water to a final concentration of 2.5 mM, divided into aliquots and frozen. Before being used it was left to aggregate for a week, at 37°C. For our experiments, 11 DIV mouse cortical neurons or differentiated SK-N-SH cells were preincubated at 37°C for 5 min with the following ligands for mGlu receptors (Tocris, UK), (S)-3-hydroxy-phenyl-glycine (3HPG) or (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG). When present, 2-methyl-6-phenylethynyl-pyridine (MPEP) was co-administered. TAT-Bcl-XL and TAT-GFP (green fluorescent protein),36 kindly provided by Dr. S Cheng (Weill Medical College of Cornell University, New York, USA), were dissolved in serum-free medium and added to SK-N-SH cells during Aβ exposure. The Aβ 1–40 was then added at a concentration of 5 μM and incubation was carried out for 48 h in a Neurobasal/B27 medium for cortical neurons, or for 24 h in a serum-free medium for SK-N-SH cells. At the end of these periods, cell viability was determined by various techniques. The conversion of tetrazolium bromide (MTT) (Sigma) to purple formazan was measured. The reaction product obtained after 1 h of cell incubation with 0.5 mg/ml MTT was analyzed spectrophotometrically at 540 nm with an automated microplate reader. MTT is a water-soluble tetrazolium salt that is converted to insoluble purple formazan by dehydrogenase enzymes. As active mitochondrial dehydrogenases of living cells, but not of dead cells, cause MTT conversion, this method is used to measure cell viability according to Hansen et al.84 Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) was performed using the kit purchased by Roche Molecular Biochemicals according to the manufacturer's instructions. The TUNEL positive cells were counted in eight different fields for each well taken from experiments that were run in triplicate. The ratio between TUNEL positive cells and Hoechst labelled neurons was calculated. The release of cytochrome c was measured according to Movsesyan et al.,63 by immunoblot analysis of cytosolic extracts.

Transfection with expression plasmids

Transfection of differentiated SK-N-SH cells was carried out according to the manufacturer's instructions with LipofectAMINE 2000 Reagent (LF 2000, Invitrogen Corporation). The day before transfection cultures were incubated with normal growth medium containing serum and without antibiotics. Cells were transfected with expression plasmids encoding c-Rel (pSG-cRel), or with the expression vector pSG585 as a negative control. For each cell well, 1 μg of DNA was diluted into 50 μl of Opti-MEM (Invitrogen Corporation) and 3 μl of LF 2000 Reagent into 50 μl of Opti-MEM. The two solutions were mixed and incubated for 20 min at room temperature to form the transfection complex. After washing the cells with serum-free medium, the transfection complex was added to the cells at a final volume of 1 ml in DMEM without serum and antibiotics. Cells were incubated at 37°C under an atmosphere of 5% CO2, 95% air for 24 h, before undergoing the experiment with the Aβ peptide.

Transfection with siRNA

Double-stranded siRNA corresponding to homologous sequences of human and mouse c-Rel gene was designed as recommended,86 with 5′ phosphate, 3′ hydroxyl, and two base overhangs on each strand. It was synthesized by Qiagen (Qiagen-Xeragon, Germantown, MD, USA). The following gene-specific sequences were used successfully: c-Rel siRNA sense 5′-CCGUGCUCCAAAUACUGCA-3′ and antisense 3′-UGCAGUAUUUGGAGCACGG-5′. Bcl-XL siRNA sense 5′-GAGAAUCACUAACCAGAGATT-3′ and antisense 3′-UCUCUGGUUAGUGAUUCUCTT-5′.37 As a negative control (non-siRNA) the following sequences were used: sense 5′-UUCUCCGAACGUGUCACGU-3′ and antisense 3′-ACGUGACACGUUCGGAGAA-5′. The siRNAs were dissolved in buffer (100 mM potassium acetate, 30 mM HEPES-potassium hydroxide, 2 mM magnesium acetate, pH 7.4) to a final concentration of 20 μM, heated at 90°C for 60 s and incubated at 37°C for 60 min before use, to disrupt any higher aggregates forming during the synthesis. Cell transfection was carried out as follows: for every 2 cm2 dishes, c-Rel siRNA (0.8 μg) or Bcl-XL siRNA (1.2 μg) were condensed with 6 μl RNAiFect Transfection Reagent (Qiagen), for 15 min at room temperature, according to the manufacturer's instructions. To transfect cortical cultures, the transfection complex was diluted in 300 μl of Neurobasal/B27 and added directly to the cells. It was replaced with fresh Neurobasal/B27 3 h later. To transfect SK-N-SH cells, the transfection complex was added in DMEM without serum and antibiotics. It was replaced with complete DMEM 3 h later.

NF-κB activation

Nuclear extract preparation and electrophoretic mobility shift assay (EMSA)

Neuronal cultures were exposed to the test drugs for 20 min and to the fresh medium for additional 30 min. Nuclear extracts were prepared as previously described.27 The protein concentration was assessed by the Bio-Rad Bradford assay according to the manufacturer's instructions. In total, 5 μg of nuclear extracts were combined with 15 fmol of biotin-labelled κB oligonucleotides (APP1, 5′-TAGAGACGGGGTTTCACCGTGTTA-3′) in binding buffer using the LightShift Chemilumineshent EMSA kit (Pierce Biotechnology, Rockford, IL, USA) containing poly(dI·dC) 25 ng/μl and 1 μg bovine serum albumin in a total volume of 20 μl. In competition experiments, 100-fold molar excess of unlabelled competitor oligonucleotides were added together with biotinilated probes. Reactions were carried out for 20 min at room temperature, and protein–DNA complexes were resolved on nondenaturing 4% polyacrylamide gels in 0.5 × TBE buffer. Gels were then transferred electrophoretically onto a nylon Hybond-N+ membrane (Amersham Biociences, UK). The crosslink of transferred DNA was performed by exposing the membrane to 1.2 J/cm2 UV light. The biotin-labelled κB DNA was detected according to the manufacturer's instructions. In supershift experiments, nuclear proteins were incubated with antibodies (5 μg) against various NF-κB subunits for 45 min at room temperature. Then, other components of the reaction mixture were added and incubated for an additional 20 min at room temperature. Rabbit polyclonal antibodies against p50, p65 and c-Rel were from Santa Cruz Biotechnology (Germany).


Binding of human NF-κB subunits to the NF-κB binding consensus sequence was measured with the ELISA-based Trans-Am NF-κB kit (Active Motif, Carlsbad, CA, USA). The mouse NF-κB subunits were analyzed using the Mercury TransFactor kit (BD Biosciences, Clontech, Palo Alto, CA, USA). The Preparation of cell extracts and analysis procedures were performed as recommended by the manufacturer. Aliquots of nuclear extracts (5 μg) were transferred to 96-well plates containing high-density immobilized κB oligonucleotides. The active forms of either the NF-κB subunits in whole-cell extracts were detected using antibodies specific for the subunit bound to the target DNA. The addition of primary antibody was followed by the addition of an HRP-conjugated secondary antibody. Developing solutions were added and the samples were read by spectrophotometry. The specificity of protein–DNA interaction was checked by measuring the ability of soluble wild-type or mutated oligonucleotides to inhibit the binding. Data are expressed as the difference of absorbance observed in the presence of nuclear extracts and that observed in the absence of nuclear extracts. The ELISA-based analysis of DNA-binding activity of diverse NF-κB factors correlated very well with the EMSA analysis.

Western blot assay

SK-N-SH cells and mouse cortical neuron cells were harvested in 100 μl of lysis buffer (pH 6.9) containing 1 mM methyl sulfonyl fluoride, 1 μg/ml leupeptin, 5 μg/ml aprotinin and 1 μg/ml pepstatin. The suspension was sonicated for 30 s at full power and centrifuged at 21 000 × g for 30 min at 4°C. Total proteins present in the supernatant (25 μg proteins/sample) were suspended in 62.5 mM Tris-HCl, 1% SDS, 5% 2-mercaptoethanol, 10% glycerol and 0.02% bromophenol blue (pH 7.5), and resolved by 10% SDS/polyacrylamide gel. The proteins were transferred electrophoretically onto nitrocellulose membrane. Immunodetection was performed by incubating the membrane O/N at 4°C, with the following primary antibodies: polyclonal anti-c-Rel antibody (1 μg/ml, Santa Cruz Biotechnology), polyclonal anti-MnSOD antibody (0.2 μg/ml StressGen Biotechnology Corporation), polyclonal anti-Bcl-XL antibody (4 μg/ml, Santa Cruz Biotechnology), polyclonal anti-mGluR1α antibody (1 μg/ml, Chemicon International, Inc.), polyclonal anti-mGluR5 antiserum specifically recognizing the carboxy-terminal portion mGluR5 (1 : 500),87 kindly provided by Dr. Rainer Kuhn and anti-β-tubulin antibody (1 : 1500, NeoMarkers). The immunoreaction was revealed by 1 h incubation at 4°C with secondary antibodies coupled to horseradish peroxidase (1 : 1500; Santa Cruz Biotechnology) and chemoluminescence detection using ECL Western blotting reagents (Amersham, Italy). Immunoblot of cytochrome c in cytosolic cell extracts was performed with the anticytochrome c monoclonal antibody (sc-13156, 1 : 500, Santa Cruz Biotechnology).


Co-immunoprecipitation studies were carried out in nuclear extracts from SK-N-SH neuronal cells, either untreated or treated with a CHPG, in RIPA buffer composed of 10 mM tris-HCl pH 8, 140 mM NaCl, 0.5% (v/v) Nonidet P-40, 1 mM sodium orthovanodato, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1% protease inhibitor cocktail. The antibodies used for immunoprecipitation and immunoblot analysis were: rabbit anti-p50 (Abcam, Cambridge, Science park Cambridge, UK), goat or rabbit anti-c-Rel (c-Rel-C, Santa Cruz Biotechnology), goat or rabbit anti-p65 (C-20-G, Santa Cruz Biotechnology) and anti-rabbit IgG (Chemicon), as control antiserum. Nuclear extracts (50–100 μg) were incubated at 4°C overnight with 2 μg/0.5 ml of corresponding antibodies. After incubation with the antibody, 25 μl of protein A/G (Santa Cruz Biotechnology) was added to the reaction mixture and rotated for 2 h at 4°C. Immunoprecipitates were collected by centrifuging at 1000 × g for 5 min followed by washing four times with RIPA buffers. Following the final wash, all the liquids that adhered to the protein A/G beads were removed. Samples were then resuspended in the sample loading buffer, subjected to SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and the immunoprecipitated proteins were then detected by Western blotting.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was extracted from RA-differentiated SK-N-SH cells, using QuickPrep Total RNA extraction kit (Amersham) according to the procedures suggested by the manufacturer. Human thalamic RNA was purchased by BD Clontech (Germany) and used as internal control. Reverse transcriptase reaction was performed using ImProm IITM Reverse transcriptase kit (Promega Corp., USA), according to the manufacturer's instructions using 2 μg of RNA as template and a mixture of antisense oligonucleotides (7.8 μM each) for mGlu1 (5′-AGGCCGTCTCATTGGTCTTCA-3′), mGlu5 receptor (5′-GGACCATACTTCATCATCATC-3′)88 and human β-actin (5′-TGATCTTCATTCTGCTGGGTG-3′). For MnSOD and Bcl-XL mRNA analysis, cDNA was synthesized from 2 μg total RNA per sample, using oligo dT primers. PCR was performed in a Perkin-Elmer Model 480 Thermocycler (Emeryville, CA, USA) by incubating 0.2–0.4 μg of the templates with 6.2 μM of specific primers for mGlu1 and mGlu5 receptors,88 for β-actin (sense 5′-GAAGAGCTACGAGCTGCCTGA-3′; antisense 5′-TGATCTTCATTCTGCTGGGTG-3′), 6.6 μM of specific primers for MnSOD (sense 5′-GGCGCCCTGGAACCTCACAT-3′; antisense 5′-ACACATCAATCCCCAGCAGT-3′) according to Bernard et al.,33 and 7.3 μM of specific primers for Bcl-XL (sense 5′-GGATGGCCACTTACCTGA-3′; antisense 5′-CGGTTGAAGCGTTCCTG-3′) according to Aerbajinai et al.89 The templates were heated at 94°C for 5 min. In total, 37 (mGlu1, mGlu5) or 35 (MnSOD, Bcl-XL, β-actin) temperature cycles were conducted as follows: denaturation at 94°C for 1 min, annealing at 56°C (mGlu1, mGlu5, MnSOD, β-actin) or 61°C (Bcl-XL) for 1 min and extension at 72°C for 1 min. Additional extension was carried out at 72°C for 5 min. PCR products were resolved on 1.2% ethidium bromide-stained agarose gel.


Columns in the figures represent means±S.E.M. The cell viability values are means of at least three separate experiments run in triplicate. Statistical significance of the differences was analyzed by Kruskal–Wallis nonparametric ANOVA with adjustment for multiple comparisons.