miRNA-187-3p-Mediated Regulation of the KCNK10/TREK-2 Potassium Channel in a Rat Epilepsy Model.

Regulatory RNAs play a key role in the regulation of protein expression patterns in neurological diseases. Here we studied the regulation of miRNAs in a chronic rat model of temporal lobe epilepsy. The analysis was focused on a putative link with pharmacoresponsiveness as well as the functional implications of the regulation of a selected miRNA. The findings did not reveal a difference in hippocampal miRNA expression between phenobarbital responders and nonresponders. However, when comparing rats following status epilepticus with control rats we identified 13 differentially expressed miRNAs with miRNA-187-3p being most strongly regulated. mRNAs encoding KCNK10/TREK-2 as well as DYRK2 were confirmed as targets of miRNA-187-3p. Expression of the potassium channel protein KCNK10/TREK-2 negatively correlated with hippocampal miRNA-187-3p expression and proved to be upregulated in the chronic phase of the epilepsy model. In conclusion, our data do not suggest a relevant impact of miRNA expression patterns on pharmacoresponsiveness. However, we confirmed regulation of miRNA-187-3p and demonstrated that it impacts the expression of the two-pore domain potassium channel protein KCNK10/TREK-2. Considering evidence from brain ischemia models, KCNK10/TREK-2 upregulation might serve a protective function with a beneficial impact on astrocytic potassium and glutamate homeostasis.

Synergism of perampanel and zonisamide in the rat amygdala kindling model of temporal lobe epilepsy.

OBJECTIVE: Anticonvulsive monotherapy fails to be effective in one third of patients with epilepsy resulting in the need for polytherapy regimens. However, with the still limited knowledge, drug choices for polytherapy remain empirical. Here we report experimental data from a chronic epilepsy model for the combination of perampanel and zonisamide, which can render guidance for clinical studies and individual drug choices. METHODS: The anticonvulsant effects of the combination of perampanel and zonisamide were evaluated in a rat amygdala kindling model. Furthermore, the potential for motor impairment was evaluated. The type of interaction was quantitatively assessed based on isobolographic analysis. RESULTS: When administered alone, zonisamide dose-dependently increased the afterdischarge threshold in fully kindled rats. Moreover, data confirmed efficacy of perampanel to inhibit seizure initiation and progression with an impact on propagation of activity from the focus. Pronounced threshold increases were observed following administration of a constant zonisamide dosage combined with different doses of perampanel. Isobolographic analysis of drug responses, which is based on individual drug dose-effect data, revealed a synergistic interaction substantiating the high efficacy of the combination. Furthermore, rotarod data indicated that the combination has a favorable tolerability profile when zonisamide is coadministered with low doses of perampanel. Plasma concentration analysis argued against a pharmacokinetic interaction as a basis for the synergism. SIGNIFICANCE: The findings clearly indicate a pronounced synergistic anticonvulsant effect for the combination of the noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist perampanel with zonisamide, which modulates voltage-sensitive sodium channels and T-type calcium currents. Consequently, polytherapy using these two antiepileptic drugs might be efficacious for clinical management of partial-onset seizures. The findings indicate that the impact of dose ratios on tolerability needs be taken into account. With regard to conclusions about the extent of the synergism and its implications further antiepileptic drug combinations need to be evaluated allowing direct comparison.

Proteomic profiling of epileptogenesis in a rat model: Focus on inflammation.

Detailed knowledge about the patterns of molecular alterations during epileptogenesis is a presupposition for identifying targets for preventive or disease-modifying approaches, as well as biomarkers of the disease. Large-scale differential proteome analysis can provide unique and novel perspectives based on comprehensive data sets informing about the complex regulation patterns in the disease proteome. Thus, we have completed an elaborate differential proteome analysis based on label-free LC-MS/MS in a rat model of epileptogenesis. Hippocampus and parahippocampal cortex tissues were sampled and analyzed separately at three key time points chosen for monitoring disease development following electrically-induced status epilepticus, namely, the early post-insult phase, the latency phase, and the chronic phase with spontaneous recurrent seizures. We focused the bioinformatics analysis on proteins linked to immune and inflammatory responses, because of the emerging evidence of the specific pathogenic role of inflammatory signalings during epileptogenesis. In the early post-insult and the latency phases, pathway enrichment analysis revealed an extensive over-representation of Toll-like receptor signaling, pro-inflammatory cytokines, heat shock protein regulation, and transforming growth factor beta signaling and leukocyte transendothelial migration. The inflammatory response in the chronic phase proved to be more moderate with differential expression in the parahippocampal cortex exceeding that in the hippocampus. The data sets provide novel information about numerous differentially expressed proteins, which serve as interaction partners or modulators in key disease-associated inflammatory signaling events. Noteworthy, a set of proteins which act as modulators of the ictogenic Toll-like receptor signaling proved to be differentially expressed. In addition, we report novel data demonstrating the regulation of different Toll-like receptor ligands during epileptogenesis. Taken together, the findings deepen our understanding of modulation of inflammatory signaling during epileptogenesis providing an excellent and comprehensive basis for the identification of target and biomarker candidates.

Microglial ROS production in an electrical rat post-status epilepticus model of epileptogenesis.

Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiological factors contributing to epileptogenesis. Considering the development of combined anti-inflammatory and antioxidant treatment strategies with antiepileptogenic potential, a characterization of the time course of microglial reactive oxygen species generation during epileptogenesis is of major interest. Thus, we isolated microglia cells and analyzed the generation of reactive oxygen species by flow cytometric analysis in an electrical rat post-status epilepticus model. Two days post status epilepticus, a large-sized cell cluster exhibited a pronounced response with excessive production of reactive oxygen species upon stimulation with phorbol-myristate-acetate. Neither in the latency phase nor in the chronic phase with spontaneous seizures a comparable cell population with induction of reactive oxygen species was identified. We were able to demonstrate in the electrical rat post-status-epilepticus model, that microglial ROS generation reaches a peak after the initial insult, is only marginally increased in the latency phase, and returns to control levels during the chronic epileptic phase. The data suggest that a combination of anti-inflammatory and radical scavenging approaches might only be beneficial during a short time window after an epileptogenic brain insult.