Abstracts

Rationale: c-Jun N-terminal kinases (JNK1-3) are members of the stress-activated kinase family that are encoded by three genes, Mapk8-10.  Abnormal JNK activation has been implicated in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. We recently discovered that JNK is hyperactivated in an animal model of chronic epilepsy, and that pharmacological inhibition of JNK exerts an antiepileptic effect (Tai TY et al, Neuroscience, 2017). Currently, we do not know the role JNK plays during the development of chronic epilepsy; thus, this project aims to understand the timeline of JNK activation. Methods: We measured both activated (phosphorylated; pJNK) and total JNK (tJNK) levels by immunoblotting hippocampal tissue homogenates both from chronically epileptic rats induced via status epilepticus (SE) and from age-matched controls. We measured JNK levels at four time points after SE onset: one hr, when animals are in SE; one day, when animals are seizure-free; one wk, when animals begin to have spontaneous seizures; and 6 wks, when animals are chronically epileptic. Results: JNK isoforms separate into two electrophoretic bands: 54 kDa and 46 kDa. In both bands, we discovered significant increases in pJNK levels at the chronic epilepsy stage (54 kDa: 160 ± 26% of naive levels, p < 0.05; 46 kDa: 125 ± 17%, p < 0.05), while there were no significant changes in pJNK levels at the other three earlier time points. We saw no significant changes in the levels of total JNK for either band at all time points. We also measured changes in the fractional activation of JNK by quantifying pJNK/tJNK. In the 54 kDa band, we discovered significant increases in pJNK/tJNK at one hr and one day, no change at one wk, and a significant increase in chronic epilepsy. In the 46 kDa band, there was no change in pJNK/tJNK at one hr, but significant increases were observed at all later timepoints. We then asked how JNK isoforms segregated into the 54 and 46 kDa bands. We determined that JNK1 segregates primarily in the 46 kDa band, JNK2 is present nearly equally in both bands, and JNK3 (which is brain-specific) is predominately in the 54 kDa band. Conclusions: We conclude that in chronic epilepsy there are substantial increases in activated JNK present in the 54 kDa (containing JNK3 and JNK2) and 46 kDa (containing JNK1 and JNK2) bands, consistent with our previous studies. The increase in fractional activation of JNK (pJNK/tJNK) during epileptogenesis suggests upregulation of upstream signaling mechanisms. Further experiments are underway to quantify the activation of JNK isoforms in chronic epilepsy and their alterations during the development of chronic epilepsy. Funding: National Institutes of Health NS050229 (to NPP). Mary Gates Scholarship (to ANP).