A novel non-transcriptional pathway mediates the proconvulsive effects of interleukin-1beta

Abstract

Interleukin-1beta (IL-1beta) is overproduced in human and rodent epileptogenic tissue and it exacerbates seizures upon brain application in rodents. Moreover, pharmacological prevention of IL-1beta endogenous synthesis, or IL-1 receptor blockade, mediates powerful anticonvulsive actions indicating a significant role of this cytokine in ictogenesis. The molecular mechanisms of the proconvulsive actions of IL-1beta are not known. We show here that EEG seizures induced by intrahippocampal injection of kainic acid in C57BL6 adult mice were increased by 2-fold on average by pre-exposure to IL-1beta and this effect was blocked by 3-O-methylsphingomyelin (3-O-MS), a selective inhibitor of the ceramide-producing enzyme sphingomyelinase. C2-ceramide, a cell permeable analog of ceramide, mimicked IL-1beta action suggesting that ceramide may be the second messenger of the proconvulsive effect of IL-1beta. The seizure exacerbating effects of either IL-1beta or C2-ceramide were dependent on activation of the Src family of tyrosine kinases since they were prevented by CGP76030, an inhibitor of this enzyme family. The proconvulsive IL-1beta effect was associated with increased Tyr(418) phosphorylation of Src-family of kinases indicative of its activation, and Tyr(1472) phosphorylation of one of its substrate, the NR2B subunit of the N-methyl-d-aspartate receptor, which were prevented by 3-O-MS and CGP76030. Finally, the proconvulsive effect of IL-1beta was blocked by ifenprodil, a selective NR2B receptor antagonist. These results indicate that the proconvulsive actions of IL-1beta depend on the activation of a sphingomyelinase- and Src-family of kinases-dependent pathway in the hippocampus which leads to the phosphorylation of the NR2B subunit, thus highlighting a novel, non-transcriptional mechanism underlying seizure exacerbation in inflammatory conditions.

Fig. 1

IL-1β and IL-1R1 expression in the mouse hippocampus after kainic acid-induced seizures. Representative photomicrographs of IL-1β (A–B) and IL-1R1 (C–D) immunoreactivity in the CA3 area of the hippocampus, 3 h after seizures induced by intrahippocampal injection of kainic acid (7 ng/0.5 μl; B and D) and in vehicle-injected C57BL6 mice (A, C). IL-1β (A) and IL-1R1 (C) immunostaining was not detectable in control hippocampus. After seizures, IL-1β immunoreactivity is strongly enhanced in GFAP-positive astrocytes (B, yellow signal in inset); IL-1R1 staining was enhanced both in neurons (D, yellow signal in d₁) and astrocytes (D, yellow signal in d₂). Scale bar: A–D 100 μm; insets, 25 μm.

Fig. 2

Effect of C2-ceramide on seizures. (A) Bargrams represent the mean ± SE (n = 12). Dihydroceramide, the inactive analogue of ceramide, (2 μg/0.5 µl) or C2-ceramide (the cell-permeable analogue of ceramide; 0.25–1.0–2.0 μg/0.5 μl) were injected intrahippocampally, 10 min before kainic acid. Significant increases in seizure parameters were observed at 1 and 2 µg C2-ceramide. *P < 0.05; **P < 0.01 versus dihydroceramide by one way ANOVA followed by Tukey test. (B) Representative EEG tracings of freely moving C57BL6 mice injected unilaterally in the hippocampus with kainic acid ± IL-1β (1 ng/0.5 μl) or C2-ceramide (2 μg/0.5 µl). Treatments or vehicles were given 10 min before kainic acid. (a) Baseline recording before kainic acid injection; arrowheads in (b) and (c) include representative ictal episodes recorded in the EEG during 90 min after kainic acid injection ± IL-1β or C2-ceramide; tracings in (d) depict spiking activity in the EEG after termination of seizures. Either IL-1β or C2-ceramide alone did not induce seizures. RHP and LHP are right and left (injected) hippocampus, respectively.

Fig. 3

IL-1β and seizure induced tyrosine phosphorylation of Src-family of kinases and the NR2B subunit of the NMDA receptor: effect of pharmacological treatments. Bargrams show densitometry analysis of the Src kinases and NR2B bands corresponding to their phosphorylated (p) forms (Src-Tyr⁴¹⁸; NR2B-Tyr¹⁴⁷²) in the various experimental groups, 60 min after the onset of kainate-induced seizures (∼70 min after kainate injection), as assessed by western blot analysis of hippocampal homogenates. Vehicle- or IL-1β alone- treated mice were killed 70 min after the injection. Data (means ± SE, n = 5–7) are optical density (O.D.) values of the relevant bands (as depicted in the representative western blot), divided by the corresponding β-actin value (internal standard). Data are expressed as percentage of values measured in corresponding vehicle-treated mice. IL-1β (1 ng/0.5 μl) was injected alone or 10 min before kainic acid; 3-O-MS (3 µg/0.5 µl) and CGP76030 (65 ng/0.5 µl) were injected 20 min before kainic acid (i.e. 10 min before IL-1β). 3-O-MS or CGP76030 alone did not change protein phosphorylation levels as compared to vehicle-injected mice; total NR2B levels were not changed by the various treatments (not shown). *P < 0.05; **P < 0.01 versus vehicle; ^##P < 0.01 versus 3-O-MS+IL-1+KA and versus CGP76030+IL-1+KA (for pNR2B only) by two-way ANOVA followed by Kruskal–Wallis test. Representative western blot bands corresponding to the specific proteins are depicted in B.