Interleukin-1 beta impairs brain derived neurotrophic factor-induced signal transduction

Abstract

The expression of IL-1 is elevated in the CNS in diverse neurodegenerative disorders, including Alzheimer's disease. The hypothesis was tested that IL-1 beta renders neurons vulnerable to degeneration by interfering with BDNF-induced neuroprotection. In trophic support-deprived neurons, IL-1 beta compromised the PI3-K/Akt pathway-mediated protection by BDNF and suppressed Akt activation. The effect was specific as in addition to Akt, the activation of MAPK/ERK, but not PLC gamma, was decreased. Activation of CREB, a target of these signaling pathways, was severely depressed by IL-1 beta. As the cytokine did not influence TrkB receptor and PLC gamma activation, IL-1 beta might have interfered with BDNF signaling at the docking step conveying activation to the PI3-K/Akt and Ras/MAPK pathways. Indeed, IL-1 beta suppressed the activation of the respective scaffolding proteins IRS-1 and Shc; this effect might involve ceramide generation. IL-1-induced interference with BDNF neuroprotection and signal transduction was corrected, in part, by ceramide production inhibitors and mimicked by the cell-permeable C2-ceramide. These results suggest that IL-1 beta places neurons at risk by interfering with BDNF signaling involving a ceramide-associated mechanism.

Fig. 1. A, IL-1β abrogates BDNF protection of cortical neurons from trophic support deprivation-induced cell death

The serum-free “trophic” medium, containing B-27, was removed from cultures and after a DMEM wash, cultures were incubated for 36 h in DMEM in the presence or absence of BDNF (10 ng/ml) ± IL-1β (50 ng/ml). When IL-1β was added, there was a 1 h preincubation period in the presence of the cytokine before the removal of the B-27-containing medium. In controls, the B-27-containing medium was removed, but replaced, after a DMEM wash, with B-27-containing medium. Cell survival was assayed by MTT assay; data are mean ± S.E. (n = 3). B, The effect of IL-1 β on neuroprotection by BDNF is concentration-dependent. In both A. and B., significance (p<0.05) was determined by ANOVA (with Fisher's PLSD as the post hoc test) in comparison with either the control cells or neurons treated with BDNF alone (*).

Fig. 2. Activation of the PI3K pathway by BDNF is required for BDNF-mediated neuroprotection

Experimental conditions of deprivation from trophic support were done as described in the legend of Fig. 1. Inhibitors of the PI3-K/Akt and Ras/MAPK pathways, LY294002 (30 μM) and PD98059 (40 μM) respectively, were added to the cultures 30 min prior to the BDNF treatment. Cell survival was assayed by MTT assay; data are mean ± S.E. (n = 3). Significance (p<0.05) determined by ANOVA (with Fisher's PLSD as the post hoc test) in comparison with either control cells or cultures treated with BDNF only is indicated (*).

Fig. 3. IL-1β pretreatment decreased BDNF-induced Akt activation

A, Exposure to BDNF (10 ng/ml, 10 min) increased the amount of P-Akt. Pretreatment with IL-1β (50 ng/ml) suppressed the effect of BDNF on P-Akt, without influencing total Akt (T-Akt) content. B, Quantification of the blots corresponding to that shown in A. Unless otherwise indicated, here and in the other legends, the quantification data for Western blots are expressed relative to the estimates in the BDNF-treated cultures (n =3) and significance (p<0.05) is indicated by *. C, Exposure to IL-1β for 2 h had no significant effect on the level of activated Akt. The competence of the cells is indicated by the elevation of P-Akt content induced by BDNF (10 ng/ml, 10 min). D. The effect of 24 h pretreatment with IL-1β was similar to that of 2 h exposure. E, IL-1ra blocks the suppression by IL-1β of BDNF-induced activation of Akt. F, Quantification of the blots corresponding to E.

Fig. 4. Treatment with IL-1β decreases the BDNF-induced activation of MAPK/ERK, CREB, but not PLCγ

A, Pretreatment with IL-1β (50 ng/ml) for 2 h decreased phosphorylated MAPK/ERK (P-MAPK) levels in cultures stimulated with BDNF(10 ng/ml, 10 min), without influencing total MAPK/ERK (T-MAPK) content. The cytokine on its own did not increase P-MAPK levels (not shown). B, Quantification of blots corresponding to A (n=3). C, Western blots show that exposure to IL-1β (50 ng/ml, 2 h) had no significant effect on the level of activated MAPK/ERK. Cells treated with BDNF (10 ng/ml, 10 min) served as positive control. D, BDNF (10 ng/ml, 10min) -induced tyrosine phosphorylation of PLCγ is unaffected by IL-1β. Tyrosine phosphorylation of PLCγ was examined by immunoprecipitation (IP), followed by Western blotting (WB), as described in Methods. BDNF-induced PLCγ phosphorylation was not influenced significantly by IL-1β pretreatment; in terms of phosphorylated PLCγ levels obtained in the BDNF-exposed cultures, the estimate in the IL-1β-treated cultures was 93.1 ± 5.6% (n = 3). Total PLCγ estimated from cell lysates was not influenced by IL-1β. E, pretreatment with IL-1β at the concentrations used at ≥5 ng/ml for 2 h compromised the increase in the BDNF-induced level of phosphorylated CREB (P-CREB), but had no effect on total CREB (T-CREB) levels. Note that IL-1β on its own had no significant effect on the basal level of P-CREB. F, Quantification of the effect of IL-1β on CREB activation.

Fig. 5. IL-1β does not interfere with BDNF-induced activation of TrkB

A, Immunocytochemical detection of TrkB receptors using specific antibodies (red) (a). Neurons were identified using MAP-2 antibodies (b) (green). The merged images (orange) (c) show that almost all neurons express TrkB receptor. In independent experiments (not shown), an additional staining with Hoechst 33342 dye showed that virtually all the live nerve cells expressed TrkB receptors. B TrkB receptors on the neurons are functionally active. Under basal conditions, P-CREB staining using an antibody recognizing Ser-133-phosphorylated CREB was very weak (b), and BDNF (50 ng/ml, 10 min) caused a great increase in immunostaining in virtually all cells (d) (BDNF at 10 ng/ml also induced a similar marked increase in P-CREB labeling in almost all the cells; not shown). Phase contrast images of the corresponding fields are shown in (a) and (c). Magnification: X200. C, Phosphorylation of TrkB was examined using Western blotting with an antibody against Trk phosphorylated at Tyr-490 (P-TrkB). Pretreatment with 50 ng/ml IL-1β had no significant influence on the BDNF-induced P-TrkB content: in terms of P-TrkB levels obtained in the BDNF-exposed culture, the estimate in the IL-1β-pretreated cells was 98 ± 4.5% (n = 3).

Fig. 6. IL-1β interferes with BDNF-induced phosphorylation of the docking proteins IRS-1 and Shc

A, IL-1β pretreatment (50 ng/ml, 2 h) decreased BDNF-induced level of Tyr-phosphorylated IRS-1 analyzed by immunoprecipitation with an IRS-1 antibody, followed by Western blotting with anti-phosphotyrosine antibody (top panel) and with anti-IRS-1 antibody (bottom panel). B, Quantification of blots corresponding to A (n=3). C, IL-1β pretreatment (50 ng/ml, 2 h) decreased BDNF-induced increase in the association between PI3-K and IRS-1 analyzed by immunoprecipitation with an IRS-1 antibody followed by Western blotting with anti-PI3-K antibody (top panel) and with anti-IRS-1 antibody (bottom panel). D, Quantification of blots corresponding to C (n=3). E, IL-1β pretreatment (50 ng/ml, 2 h) decreased BDNF-induced levels of Tyr-phosphorylated Shc isoforms analyzed by immunoprecipitation with an Shc antibody, followed by Western blotting with anti-phosphotyrosine antibody (top panel) and with anti-Shc antibody (bottom panel). The isoforms are indicated as a, b and c (approximate molecular weights, kDa: 66, 52 and 46, respectively). F, Quantification of the effects of pretreatment with IL-1β on Shc activation (n=3).

Fig. 7. Inhibitors of ceramide synthesis attenuate the inhibition by IL-1β of BDNF signaling

A, The de novo pathway inhibitor FB1 (5 μM) attenuated the suppression by IL-1β (50 ng/ml) of Akt activation induced by BDNF (10 ng/ml). B, Quantification of the effect of FB1. Significance (p<0.05) is indicated here and in Fig. 7D and F by * in comparison with IL-1β-pretreated cultures stimulated by BDNF (BDNF+IL-1β. C, Another inhibitor of the de novo pathway myriocin (ISP-1; 50 nM) or the neutral sphingomyelinase inhibitor glutathione (GSH; 2 mM) attenuated IL-1β-mediated suppression of BDNF-induced CREB phosphorylation. D, Quantification of blots corresponding to that shown in C. E, the neutral sphingomyelinase inhibitor GW4869 (10 μM) attenuated IL-1β-mediated suppression of BDNF-induced CREB phosphorylation. F, Quantification of the blots corresponding to that shown in E.

Fig. 8. Inhibitors of ceramide synthesis attenuate the inhibition by IL-1β of BDNF-mediated neuroprotection

The de novo pathway inhibitor ISP-1 (ISP, 100 nM) and the neutral sphingomyelinase inhibitor glutathione (GSH, 2 mM) attenuated IL-1β-mediated suppression of BDNF (10 ng/ml)-mediated protection of cortical neurons from trophic support deprivation-induced cell death. The acidic sphingomyelinase inhibitor desipramine (DES, 10 μM) had no effect on the action of IL-1β. Experimental conditions were as described in Fig. 1. When inhibitors of ceramide synthesis were employed, they were added to the cultures 30 min before exposure to IL-1β (50 ng/ml). Cell survival was assayed using the MTT assay; data are mean ± S.E. (n = 3). Significance (p<0.05) determined by ANOVA (with Fisher's PLSD as the post hoc test) in comparison with cultures treated with BDNF in the presence of IL-1β (*).

Fig. 9. Treatment with the ceramide analogue C2-ceramide decreased BDNF-induced Akt activation and neuroprotection

A, Phosphorylated Akt (P-Akt) levels were determined with an antibody specific to Akt phosphorylated at Ser-473 (P-Akt). Exposure to BDNF (10 ng/ml, 10 min) increased the amount of P-Akt. Pretreatment with the cell-permeable C2-ceramide at 10 μM for 2 h suppressed the effect of BDNF on P-Akt, but had no effect on total Akt (T-Akt) levels. C2-ceramide on its own had no significant effect on P-Akt content. B, Quantification of the C2-ceramide effect. C, C2-ceramide at 10 μM abrogates BDNF protection of cortical neurons from trophic support deprivation-induced cell death. The experiment conditions were as described in Fig. 1. Cell survival was assayed using the MTT assay; data are mean ± S.E. (n = 3). Significance (p<0.05) determined by ANOVA (with Fisher's PLSD as the post hoc test) in comparison with control (B-27) or BDNF-treated cultures (*).