Targeting Cytokine Systems to Achieve Neuroprotection: Experimental Models of Seizures and Neurodegeneration

This study investigated whether IL-1β and TNF-α have a role in the pathophysiology of seizures by altering neuronal excitability and cell survival and also probed new targets to achieve an effective anticonvulsive action. The first set of experiments evaluated if acute and spontaneous seizures can be effectively inhibited by blocking the brain production of IL-1β using a selective inhibitor of caspase-1, the key enzyme specifically involved in the production of the releasable and biologically active form of IL-1β. Caspase-1 inhibition significantly reduced the number and the total time spent in seizures, indicating that this treatment represents an effective and novel anticonvulsive strategy. The second set of experiments investigated the intracellular pathway involved in the IL-1β proconvulsant actions which appear to occur via a novel non-transcriptional pathway: namely, the IL-1β-mediated activation of neutral-sphingomyelinase, the production of ceramide and the subsequent phosphorylation of Src-family of tyrosine kinases and the target receptor protein NR2B subunit of the NMDA receptor, resulting in the potentiation of NMDA function. This fast post-translational effect of IL-1β represents a novel and non-conventional pathway by which inflammatory molecules produced in epileptic tissue can affect neurotransmission. Then, this work demonstrated that a brief pro-inflammatory stimulus, characterized by the activation of microglia and subsequent lasting release of critical concentrations of IL-1β, primes neuronal vulnerability to a subsequent excitotoxic insult, highlighting one mechanism by which a pre-existent pro-inflammatory state may increase hippocampal neuronal susceptibility to the excitotoxic damage associated with seizures. Thus, pharmacological approaches specifically targeted to block the overproduction of IL-1β and its functions in diseased conditions may represent new, nonconventional strategies for the treatment of seizure disorders, which are refractory to classical anticonvulsant treatments. The second part of this thesis demonstrated that TNF-α significantly decreases epileptic activity by specifically acting on neuronal p75 receptors. The actions of TNF-α on neuronal excitability strictly depend on whether p55 or p75 receptors are preferentially involved and appear to be mediated also by changes in the assembly of glutamate receptor subunits. These novel functional glia-neuronal interactions add important insights into the mechanism of ictogenesis and seizure-associated neuronal cell death, highlighting innovative pharmacological strategies to block the activation of cytokine-mediated signalling in diseased conditions

CXCL1-CXCR1/2 signaling is induced in human temporal lobe epilepsy and contributes to seizures in a murine model of acquired epilepsy

Abstract CXCL1, a functional murine orthologue of the human chemokine CXCL8 (IL-8), and its CXCR1 and CXCR2 receptors were investigated in a murine model of acquired epilepsy developing following status epilepticus (SE) induced by intra-amygdala kainate. CXCL8 and its receptors were also studied in human temporal lobe epilepsy (TLE). The functional involvement of the chemokine in seizure generation and neuronal cell loss was assessed in mice using reparixin (formerly referred to as repertaxin), a non-competitive allosteric inhibitor of CXCR1/2 receptors. We found a significant increase in hippocampal CXCL1 level within 24 h of SE onset that lasted for at least 1 week. No changes were measured in blood. In analogy with human TLE, immunohistochemistry in epileptic mice showed that CXCL1 and its two receptors were increased in hippocampal neuronal cells. Additional expression of these molecules was found in glia in human TLE. Mice were treated with reparixin or vehicle during SE and for additional 6 days thereafter, using subcutaneous osmotic minipumps. Drug-treated mice showed a faster SE decay, a reduced incidence of acute symptomatic seizures during 48 h post-SE, and a delayed time to spontaneous seizures onset compared to vehicle controls. Upon reparixin discontinuation, mice developed spontaneous seizures similar to vehicle mice, as shown by EEG monitoring at 14 days and 2.5 months post-SE. In the same epileptic mice, reparixin reduced neuronal cell loss in the hippocampus vs vehicle-injected mice, as assessed by Nissl staining at completion of EEG monitoring. Reparixin administration for 2 weeks in mice with established chronic seizures, reduced by 2-fold on average seizure number vs pre-treatment baseline, and this effect was reversible upon drug discontinuation. No significant changes in seizure number were measured in vehicle-injected epileptic mice that were EEG monitored in parallel. Data show that CXCL1-IL-8 signaling is activated in experimental and human epilepsy and contributes to acute and chronic seizures in mice, therefore representing a potential new target to attain anti-ictogenic effects

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

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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 option

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

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)-03B2 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-103B2, by using selective inhibitors of interleukin-converting enzyme (ICE/caspase-1) or through caspase-1 gene deletion. Methods: Caspase-1 was selectively blocked by using pralnacasan or VX-765. IL-103B2 release was induced in mouse organotypic hippocampal slice cultures by proinflammatory stimuli [lipopolysaccaride (LPS) + adenosine triphosphate (ATP)] and measured with enzyme-linked immunosorbent assay (ELISA). IL-103B2 production during seizures was measured in the rat hippocampus by Western blot. Seizures were induced in freely moving mice and rats by intrahippocampal injection of kainic acid and recorded by EEG analysis. Results: Caspase-1 inhibition reduced the release of IL-103B2 in organotypic slices exposed to LPS+ATP. Administration of pralnacasan (intracerebroventricular, 50 03BCg) or VX-765 (intraperitoneal, 252013200 mg/kg) to rats blocked seizure-induced production of IL-103B2 in the hippocampus, and resulted in a twofold delay in seizure onset and 50% reduction in seizure duration. Mice with caspase-1 gene deletion showed a 70% reduction in seizures and an approximate fourfold delay in their onset. Conclusions: Inhibition of caspase-1 represents an effective and novel anticonvulsive strategy, which acts by selectively reducing the brain availability of IL-103B2