Silvia Balosso

Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures

Brain inflammation is a major factor in epilepsy, but the impact of specific inflammatory mediators on neuronal excitability is incompletely understood. Using models of acute and chronic seizures in C57BL/6 mice, we discovered a proconvulsant pathway involving high-mobility group box-1 (HMGB1) release from neurons and glia and its interaction with Toll-like receptor 4 (TLR4), a key receptor of innate immunity. Antagonists of HMGB1 and TLR4 retard seizure precipitation and decrease acute and chronic seizure recurrence. TLR4-defective C3H/HeJ mice are resistant to kainate-induced seizures. The proconvulsant effects of HMGB1, like those of interleukin-1β (IL-1β), are partly mediated by ifenprodil-sensitive N-methyl-d-aspartate (NMDA) receptors. Increased expression of HMGB1 and TLR4 in human epileptogenic tissue, like that observed in the mouse model of chronic seizures, suggests a role for the HMGB1-TLR4 axis in human epilepsy. Thus, HMGB1-TLR4 signaling may contribute to generating and perpetuating seizures in humans and might be targeted to attain anticonvulsant effects in epilepsies that are currently resistant to drugs.

The role of cytokines in the pathophysiology of epilepsy.

Recent findings in experimental models and in the clinical setting highlight the possibility that inflammatory processes in the brain contribute to the etiopathogenesis of seizures and to the establishment of a chronic epileptic focus. Prototypical inflammatory cytokines such as IL-1 beta, TNF-alpha and IL-6 have been shown to be overexpressed in experimental models of seizures in brain areas of seizure generation and propagation, prominently by glia and to a lesser extent by neurons. Cytokines receptors are also upregulated, and the related intracellular signalling is activated, in both cell populations highlighting autocrine and paracrine actions of cytokines in the brain. Cytokines have been shown to profoundly affect seizures in rodents; in particular, IL-1 beta is endowed of proconvulsant activity in a large variety of seizure models. The recent demonstration of functional interactions between cytokines and classical neurotransmitters such as glutamate and GABA, suggest the possibility that these interactions underlie the cytokine-mediated changes in neuronal excitability, thus promoting seizure phenomena and the associated neuropathology. These findings point out at novel glio-neuronal communications in diseased conditions and highlight potential new targets for therapeutic intervention.

IL-1 receptor/Toll-like receptor signaling in infection, inflammation, stress and neurodegeneration couples hyperexcitability and seizures.

Increasing evidence supports the involvement of immune and inflammatory processes in the etiopathogenesis of seizures. In particular, activation of innate immune mechanisms and the subsequent inflammatory responses, that are induced in the brain by infection, febrile seizures, neurotrauma, stroke are well documented conditions associated with acute symptomatic seizures and with a high risk of developing epilepsy. A decade ago, pharmacological experiments showed that elevated brain levels of the anti-inflammatory molecule IL-1 receptor antagonist reduced seizures in epilepsy models. This observation, together with the evidence of in situ induction of inflammatory mediators and their receptors in experimental and human epileptogenic brain tissue, established the proof-of-concept evidence that the activation of innate immunity and inflammation in the brain are intrinsic features of the pathologic hyperexcitable tissue. Recent breakthroughs in understanding the molecular organization of the innate immune system first in macrophages, then in the different cell types of the CNS, together with pharmacological and genetic studies in epilepsy models, showed that the activation of IL-1 receptor/Toll-like receptor (IL-1R/TLR) signaling significantly contributes to seizures. IL-1R/TLR mediated pro-excitatory actions are elicited in the brain either by mimicking bacterial or viral infections and inflammatory responses, or via the action of endogenous ligands. These ligands include proinflammatory cytokines, such as IL-1beta, or danger signals, such as HMGB1, released from activated or injured cells. The IL-1R/TLR signaling mediates rapid post-translational changes in voltage- and ligand-gated ion channels that increase excitability, and transcriptional changes in genes involved in neurotransmission and synaptic plasticity that contribute to lower seizure thresholds chronically. The anticonvulsant effects of inhibitors of the IL-1R/TLR signaling in various seizures models suggest that this system could be targeted to inhibit seizures in presently pharmaco-resistant epilepsies.

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

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.

Glia as a source of cytokines: implications for neuronal excitability and survival

In the last decade, preclinical studies have provided a better characterization of the homeostatic and maladaptive mechanisms occurring either during the process of epileptogenesis or after the permanent epileptic state has emerged. Experimental evidence supported by clinical observations highlighted the possibility that brain inflammation is a common factor contributing, or predisposing, to the occurrence of seizures and cell death, in various forms of epilepsy of different etiologies. Expression of proinflammatory cytokines, as a hallmark of brain inflammation, has been demonstrated in glia in various experimental models of seizures and in human epilepsies. Experimental studies in rodents with perturbed cytokine systems indicate that these inflammatory mediators can alter neuronal excitability and affect cell survival by activating transcriptional and posttranslational intracellular pathways. This paper will provide an overview on the current knowledge in this field to discuss mechanistic hypotheses into the study of pathogenesis of epilepsy and recognize new potential therapeutic options.

Tumor necrosis factor-α inhibits seizures in mice via p75 receptors

Brain inflammatory reactions have been described in various neurological disorders, including epilepsy. Although there is clear evidence that cytokines affect neuroglial functions and blood–brain barrier permeability, scarce information is available on the functional consequences of brain inflammation on seizures. We studied the role of tumor necrosis factor‐α (TNF)‐α and its p55 and p75 receptors in seizure modulation. We found that intrahippocampal injection of murine recombinant TNF‐α potently inhibits seizure in mice while human recombinant TNF‐α, which shows strong specificity for mouse p55 receptors, was ineffective. p75 receptors were detected in mouse hippocampal neurons, whereas p55 receptors were absent. Transgenic mice with a perturbed TNF‐α system showed profound alterations in seizure susceptibility: astrocytic overexpression of TNF‐α was associated with reduced seizures, whereas mice lacking TNF‐α p75 or both p55 and p75, receptors showed prolonged seizures. Mice deficient in p55 receptor only showed reduced seizures; and both p75 and TNF receptor–associated factor 2 protein levels were upregulated in their hippocampi. Our findings show that increased brain levels of TNF‐α result in significant inhibition of seizures in mice, and this action is mediated by neuronal p75 receptors. This evidence highlights a novel function of TNF‐α in brain and indicates a new system for anticonvulsive intervention. Ann Neurol 2005

Inflammation and prevention of epileptogenesis.

CNS injuries such as trauma, stroke, viral infection, febrile seizures, status epilepticus occurring either in infancy or during a lifetime are considered common risk factors for developing epilepsy. Long term CNS inflammation develops rapidly after these events, suggesting that a pro-inflammatory state in the brain might play a role in the development of the epileptic process. This hypothesis is corroborated by two main lines of evidence: (1) the upregulation of pro-inflammatory signals during epileptogenesis in brain areas of seizure onset/generalization; (2) pharmacological targeting of specific pro-inflammatory pathways after status epilepticus or in kindling shows antiepileptogenic effects. The mechanisms by which pro-inflammatory molecules might favor the establishment of chronic neuronal network hyperexcitability involve both rapid, non-transcriptional effects on glutamate and GABA receptors, and transcriptional activation of genes involved in synaptic plasticity. This emerging evidence predicts that pharmacological interventions targeting brain inflammation might provide a key to new antiepileptic drug design.

Inactivation of Caspase-1 in Rodent Brain : A Novel Anticonvulsive Strategy

Summary:  Purpose: Cytokines and related inflammatory mediators are rapidly synthesized in the brain during seizures. We previously found that intracerebral administration of interleukin‐1 (IL‐1)‐β has proconvulsant effects, whereas its endogenous receptor antagonist (IL‐1Ra) mediates potent anticonvulsant actions in various models of limbic seizures. In this study, we investigated whether seizures can be effectively inhibited by blocking the brain production of IL‐1β, by using selective inhibitors of interleukin‐converting enzyme (ICE/caspase‐1) or through caspase‐1 gene deletion.

The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y2 and not Y5 receptors

Neuropeptide Y (NPY) potently inhibits glutamate release and seizure activity in rodent hippocampus in vitro and in vivo, but the nature of the receptor(s) mediating this action is controversial. In hippocampal slices from rats and several wild‐type mice, a Y2‐preferring agonist mimicked, and the Y2‐specific antagonist BIIE0246 blocked, the NPY‐mediated inhibition both of glutamatergic transmission and of epileptiform discharges in two different slice models of temporal lobe epilepsy, stimulus train‐induced bursting (STIB) and 0‐Mg2+ bursting. Whereas Y5 receptor‐preferring agonists had small but significant effects in vitro, they were blocked by BIIE0246, and a Y5 receptor‐specific antagonist did not affect responses to any agonist tested in any preparation. In slices from mice, NPY was without effect on evoked potentials or in either of the two slice seizure models. In vivo, intrahippocampal injections of Y2‐ or Y5‐preferring agonists inhibited seizures caused by intrahippocampal kainate, but again the Y5 agonist effects were insensitive to a Y5 antagonist. Neither Y2‐ nor Y5‐preferring agonists affected kainate seizures in mice. A Y5‐specific antagonist did not displace the binding of two different NPY ligands in WT or mice, whereas all NPY binding was eliminated in the mouse. Thus, we show that Y2 receptors alone mediate all the anti‐excitatory actions of NPY seen in the hippocampus, whereas our findings do not support a role for Y5 receptors either in vitro or in vivo. The results suggest that agonists targeting the Y2 receptor may be useful anticonvulsants.

Interleukin-1 type 1 receptor/Toll-like receptor signalling in epilepsy: The importance of IL-1beta and high-mobility group box 1

Abstract.  Maroso M., Balosso S., Ravizza T., Liu J., Bianchi M.E., Vezzani A. (Mario Negri Institute for Pharmacological Research, Milano; and San Raffaele University and Research Institute Milano; Italy). Interleukin‐1 type 1 receptor/Toll‐like receptor signalling in epilepsy: the importance of IL‐1beta and high‐mobility group box 1 (Symposium). J Intern Med 2011; 270: 319–326.