Antiepileptic action of c-Jun N-terminal kinase (JNK) inhibition in an animal model of temporal lobe epilepsy

Abstract

Several phosphorylation signaling pathways have been implicated in the pathogenesis of epilepsy arising from both genetic causes and acquired insults to the brain. Identification of dysfunctional signaling pathways in epilepsy may provide novel targets for antiepileptic therapies. We previously described a deficit in phosphorylation signaling mediated by p38 mitogen-activated protein kinase (p38 MAPK) that occurs in an animal model of temporal lobe epilepsy, and that produces neuronal hyperexcitability measured in vitro. We asked whether in vivo pharmacological manipulation of p38 MAPK activity would influence seizure frequency in chronically epileptic animals. Administration of a p38 MAPK inhibitor, SB203580, markedly worsened spontaneous seizure frequency, consistent with prior in vitro results. However, anisomycin, a non-specific p38 MAPK activator, significantly increased seizure frequency. We hypothesized that this unexpected result was due to activation of a related MAPK, c-Jun N-terminal kinase (JNK). Administration of JNK inhibitor SP600125 significantly decreased seizure frequency in a dose-dependent manner without causing overt behavioral abnormalities. Biochemical analysis showed increased JNK expression and activity in untreated epileptic animals. These results show for the first time that JNK is hyperactivated in an animal model of epilepsy, and that phosphorylation signaling mediated by JNK may represent a novel antiepileptic target.

Keywords: antiepileptic drug; c-Jun N-terminal kinase (JNK); p38 mitogen activated kinase (p38 MAPK); phosphorylation; pilocarpine; temporal lobe epilepsy.

Fig. 1

Experimental protocol and characteristics of epilepsy animal model. A. Experimental protocol. Adult animals underwent pilocarpine induction and then were implanted with epidural EEG electrodes and an i.c.v. cannula connected to a vehicle pump at week 5 post-SE. A 10-day continuous baseline video-EEG monitoring period began at week 6, followed by a change to a drug-containing pump, and repeat 10-day monitoring. Following this monitoring period, the animals were sacrificed. B. EEG tracing during a typical Racine class 5 seizure. Shown are right (R) and left (L) epidural cortical electrode channels, with behavioral changes and Racine classifications noted during the course of the electrographic seizure. The typical convulsive seizure in this model is readily apparent from the onset of a high amplitude, high frequency electrographic discharge that gradually increases in amplitude followed by a waning phase. C. Seizure frequency in animals treated throughout weeks 6–9 with a saline pump. Traces show average C3–5 seizure frequency during the two 14-day periods for six animals. Animals with increased seizure frequency are depicted in red; those with decreased seizure frequency in blue. There was variability in the baseline seizure frequency, but little change in seizure frequency in individual animals between weeks 6–7 and 8–9. D. Distribution of Racine classifications and seizure durations for all seizures recorded as shown in Fig. 1C. C3–5 seizures with major motor manifestations (C3–4) or convulsions (C5) were the predominant seizure types. The median seizure duration was 50 sec.

Fig. 2

Inhibition of p38 MAPK increases seizure frequency. A. Plot of seizure frequencies for individual animals under control and drug-treated conditions. Inhibition of p38 MAPK with SB increased spontaneous seizure frequency in eight of nine animals. B. Histograms of aggregate seizure durations for the SB-treated and control groups are shown.

Fig. 3

Nonspecific activation of p38 MAPK with anisomycin increases seizure frequency. A. Plot of seizure frequencies for individual animals under control and drug-treated conditions. Non-specific activation of p38 MAPK with ANI increased spontaneous convulsive seizure frequency in all four treated animals, in one case precipitating SE. B. Histograms of seizure duration are shown under control and ANI treatment.

Fig. 4

JNK inhibition reduces seizure frequency. A. Plot of seizure frequencies for individual animals under control and drug-treated conditions. Inhibition of JNK with SP delivered at 14 μg/d (SP 14) decreased spontaneous convulsive seizure frequency in six of seven animals. B. Histograms of seizure duration are shown under control and lower-dose JNK inhibition conditions. C. Inhibition of JNK with SP delivered at 28 μg/d (SP 28) decreased spontaneous convulsive seizure frequency in all seven animals from a separate experimental series. D. Histograms of seizure duration are shown under control and higher-dose JNK inhibition conditions.

Fig. 5

Summary of treatment results. Shown is the ratio of seizure frequency as a percentage of baseline frequency for each animal (circles), with treatments that on average were proconvulsive depicted in red and those that were antiepileptic in blue. Mean and 95% CIs for each group are shown, along with statistical significance level of the change in seizure frequency (* = p<.05; ** = p<.01).

Fig. 6

JNK inhibition does not affect behavior. Results of open-field behavioral testing are shown for naive controls, chronically epileptic animals that were untreated, and epileptic animals treated with SP at 28 μg/d. A. Mean speed of locomotion and time spent mobile was similar in all groups. B. Untreated epileptic animals showed decreased anxiety-like behavior as shown by increased time spent in the center of the field, compared to naive controls. There were no differences in these measures between treated and untreated epileptic animals.

Fig. 7

JNK signaling is hyperactivated in chronic epilepsy. A. Summary data showing increased JNK activation in hippocampal tissue from naive animals after in vitro treatment with ANI compared to untreated tissue. Shown are elevated phospho-JNK (pJNK) and phospho- to total JNK levels in ANI-exposed tissue, while total JNK levels remained unchanged. Above are representative blots against pJNK and total JNK in ANI-exposed and naive controls. Arrows denote 54 and 46 kDa bands. B. In hippocampal tissue from chronically epileptic animals, pJNK levels are increased compared to those from naive, non-epileptic controls, along with modestly activated total JNK levels, and an increased ratio of phospho- to total JNK expression. C. In vitro ANI treatment of hippocampal tissue from naive animals produced a significant increase in the fraction of phosphorylated c-Jun, while producing a decrease in the level of total c-Jun expression compared to untreated tissue. D. Phosphorylated c-Jun was increased in tissue from chronically epileptic animals compared to that in naive, nonepileptic animals, and similarly to ANI-exposed tissue, demonstrated a decrease in total c-Jun levels.