Abstracts

Rationale: BDNF is initially expressed in a precursor form (proBDNF) and cleaved to form mature BDNF (mBDNF). Stimulation of primary neurons in cell culture with either recombinant proBDNF or mBDNF induces JAK/STAT activation, specifically phosphorylation of STAT3. Following status epilepticus (SE), increases in BDNF regulate a wide variety of cell signaling pathways including pro-survival and cell death machinery in a receptor-specific manner. ProBDNF preferentially binds to the p75 neurotrophin receptor (NTR), while mBDNF is the major ligand of the tropomysin related kinase receptor (TrkB). LM11A-31, a small molecule ligand that binds to p75NTR promotes survival signaling and inhibits cell death. Recent studies have shown that inhibition of p75NTR prevents BDNF-induced activation of the JAK/STAT pathway in cultured hippocampal neurons. In the current study, we examined the effect of upstream regulation of p75NTR signaling (using LM11A-31) vs. a direct inhibitor of STAT3 phosphorylation (using WP-1066) on SE-induced STAT3 phosphorylation and subsequent progression of epileptogenesis. Methods: Rats were injected with 200 mg/kg of LM11A-31 or vehicle at time of pilocarpine injection and again at onset of SE then sacrificed 1 h and 3 h later for tissue and plasma harvesting. A second set of animals received 50 mg/kg WP1066 at SE onset and the same dose 45 minutes later. Continuous video-EEG was collected from animals treated with LM11A-31, WP1066, and vehicle using the same drug administration protocols to evaluate effects on daily seizure frequency for one month following SE. Results: LM11A-31 produces a transient decrease in STAT3 phosphorylation in hippocampus after SE. There was significantly less LM11A-31 present in the brain and plasma 3 h after SE when compared to levels 1 h after SE. A similarly rapid pharmacokinetic clearance was observed with the pSTAT3 inhibitor. LM11A-31 reduced SE-induced phosphorylation of STAT3 by ~75% 1 h after SE (SE, n=5; SE+LM11A, n=5; p = 0.002), but not 3 h after SE (SE, n = 4; SE+LM11A, n=4). Similar to LM11A-31, the pSTAT3 inhibitor significantly decreased pSTAT3 immunoreactivity by ~58% at 1 h after SE (SE, n=4; SE+WP1066, n=4; p<0.0001). Neither treatment affected electrophysiological parameters of SE or seizure latency. The pSTAT3 inhibitor slowed the progression of epileptogenesis (i.e., reduced the daily seizure frequency over time; SE+vehicle, n=14; SE+WP1066, n=8). LM11A-31 treated rats had cumulative seizure frequency similar to vehicle-treated rats (SE+vehicle, n=6; SE+LM11A-31, n=6). Conclusions: Despite early inhibition of STAT3 phosphorylation by both LM11A-31 and the pSTAT3 inhibitor, upstream modulation of the JAK/STAT pathway using LM11A-31 did not inhibit the progression of epileptogenesis, while direct block of STAT3 phosphorylation successfully inhibits epilepsy progression. Further studies are required to understand the role of p75NTR activation in epileptogenesis and identify potential cell-specific pSTAT3-related mechanisms responsible for seizure generation.