Selective hyperactivation of JNK2 in an animal model of temporal lobe epilepsy

Abstract

c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family and are derived from three genes, Jnk1-3. These kinases are involved in cellular responses to homeostatic insults, such as inflammation and apoptosis. Furthermore, increased JNK expression and activation are associated with debilitating neurodegenerative diseases, including Alzheimer's and Parkinson's. We previously reported elevated levels of phosphorylated JNK (pJNK), indicative of JNK hyperactivation, in the CA1 hippocampus of chronically epileptic rats. We also showed that pharmacological inhibition of JNK activity reduced seizure frequency in a dose-dependent fashion (Tai TY et al., Neuroscience, 2017). Building on these observations, the objectives of this current study were to investigate the timeline of JNK activation during epileptogenesis, and to identify the JNK isoform(s) that undergo hyperactivation in the chronic epilepsy stage. Western blotting analysis of CA1 hippocampal homogenates showed JNK hyperactivation only during the chronic phase of epilepsy (6-9 weeks post-status epilepticus), and not in earlier stages of epileptogenesis (1 h, 1 day, and 1 week post-status epilepticus). After enrichment for pJNK by immunoprecipitation, we identified JNK2 as the only significantly hyperactivated JNK isoform, with expression of the 54 kDa pJNK2 variant elevated to a greater extent than the 46 kDa pJNK2 variant. Expression of the total amounts of both JNK2 variants (phosphorylated plus non-phosphorylated) was reduced in epilepsy, however, suggesting that activation of upstream phosphorylation pathways was responsible for JNK2 hyperactivation. Since our prior work demonstrated that pharmacological inhibition of JNK activation had an antiepileptic effect, JNK2 hyperactivation is therefore likely a pathological event that promotes seizure occurrences. This investigation provides evidence that JNK2 is selectively hyperactivated in epilepsy and thus may be a novel and selective antiepileptic target.

Keywords: Epilepsy; Epileptogenesis; JNK; Phosphorylation; Pilocarpine; Status epilepticus.

Fig. 1

Time course of JNK expression during epileptogenesis. A. Example western blot of JNK expression in rat CA1 hippocampus during the chronic epilepsy phase (6–9 weeks post-status epilepticus [post-SE]) compared to age-matched controls. When probed with a primary antibody that recognizes either all phosphorylated JNK isoforms (pJNK) or JNK isoforms regardless of phosphorylation state (total JNK), JNK expression is seen in two electrophoretic bands at 54 and 46 kDa. Shown are four different protein loading amounts in each condition, and a molecular weight standard in the leftmost lane. GAPDH expression was measured as a control for protein loading. B. Time course of pJNK expression during epileptogenesis shows that there is no significant change at times from 1 h to 1 week post-SE. In chronic epilepsy 6–9 weeks post-SE, there is significant elevation of pJNK expression in both 54 and 46 kDa bands compared to control. C. Total JNK expression during epileptogenesis shows no significant change at any time point post-SE. D. The phosphorylated fraction of JNK expression shows significant increases for both 54 and 46 kDa bands in chronic epilepsy and at some intermediate time points during epileptogenesis.

Fig. 2

Segregation of JNK isoforms into 54 and 46 kDa bands. A. Western blots from a single sample probed with a pan-specific antibody against total JNK (all three isoforms, irrespective of phosphorylation state) and with an isoform-specific JNK1 antibody. Shown are three different protein loading amounts in each western, and two lanes with molecular weight standards. The JNK1 isoform migrated predominantly but not completely in the 46 kDa band. B. The JNK2 isoform migrated nearly equally in the 54 and 46 kDa bands. C. The JNK3 isoform was found almost but not entirely in the 54 kDa band. D. Quantification of the percentage of expression of each JNK isoform in the 54 and 46 kDa bands.

Fig. 3

Upregulation of pJNK isoform expression in chronic epilepsy. A. Western blot of immunoprecipitated pJNK from the CA1 hippocampus of chronically epileptic animals and age-matched naïve controls. Shown are three different protein loading amounts under each condition and a lane containing molecular weight standards. Epileptic tissue showed less total pJNK expression than control, most likely due to hippocampal neuronal loss in chronic epilepsy. B. When immunoprecipitated pJNK was probed for expression of the JNK1 isoform, pJNK1 expression normalized to overall pJNK expression was unchanged compared to control conditions. C. Expression of pJNK2 showed a different pattern from pJNK1 and pJNK3, with higher expression in epileptic compared to control conditions. Of note, immunoprecipitation with the anti-pJNK antibody produced two artifactual electrophoretic bands as shown in Fig. 3C. One is the band at a slightly larger molecular weight than the 54 kDa JNK band and is demarcated by the open triangle, while the other is labeled as a 25 kDa band. The identities of these artifactual bands are discussed in Experimental procedures. D. pJNK3 expression, like pJNK1, was unchanged compared to control conditions. E. Quantification of the two pJNK electrophoretic bands, in which the 54 kDa band is significantly decreased in chronic epileptic rats but not the 46 kDa band. (F & G) Quantification of pJNK isoform-specific expression before (F) and after (G) normalization to overall immunoprecipitated pJNK. Only pJNK2 showed increased expression under epileptic conditions, while pJNK1 and pJNK3 showed no significant change. The 54 kDa variant of pJNK2 showed the larger increase in epilepsy.

Fig. 4

Expression of total JNK isoforms in chronic epilepsy. A. Western blot showing total expression of JNK1 isoform (phosphorylated plus non-phosphorylated) in chronic epilepsy and naïve control conditions. JNK1 expression was not significantly changed in the epileptic condition. B. Expression of JNK2 was reduced in epilepsy for both the 54 and 46 kDa variants. C. Expression of only the 54 kDa variant of JNK3 was significantly reduced under epileptic conditions. D. Quantification of JNK isoform expression in the epileptic condition as a percentage of control expression shows significant downregulation of both JNK2 variants as well as the 54 kDa JNK3 variant.