Abstracts

Rationale: Hypoxic-ischemic encephalopathy (HIE) accounts for 50-60% of neonatal seizures. Phenobarbital (PB), which prolongs the opening of GABA receptors, has poor anti-seizure efficacy during the neonatal period. Despite this, PB is the most widely used first-line anti-seizure drug to treat neonatal seizures. The electroneutral cation-chloride transporter KCC2 mediates chloride extrusion; an age-dependent up-regulation of KCC2 enables the shift of GABAergic signaling from depolarizing to hyperpolarizing. In a newly characterized mouse model of HIE, ischemia-induced TrkB pathway activation has been shown to lead to early down-regulation of KCC2. Previously we have demonstrated that a small-molecule TrkB antagonist ANA12 rescued both the post-ischemic KCC2 down-regulation and age-dependent PB-resistance to ischemic seizures at P7 in CD-1 pups. We therefore hypothesized that a small-molecule TrkB partial agonist, LM22A-4 (Massa et al. 2010), would either aggravate or have no effect on post-ischemic KCC2 degradation and PB-resistant seizures. Methods: Postnatal day 7 (PB-resistant) or 10 (PB-responsive) CD-1 mouse pups of both sexes underwent unilateral carotid ligation without transection. Ligated pups were randomly chosen for intraperitoneal (I.P.) injections of 0.25mg/kg LM22A-4 in 5% DMSO either one hour before (pre-ligation) or immediately after ligation (post-ligation). After 1h of baseline vEEG recording, a loading dose of PB (25 mg/kg I.P. in saline) was administered. Following 2h of continuous vEEG recordings, the pups were returned to the dam. Twenty-four hours post-ligation, brains were harvested and processed for western blot analyses to investigate TrkB pathway modulation by LM22A-4. Results: Surprisingly, LM22A-4 was efficacious in rescuing PB-resistance (Fig. 1), similar to ANA12, a small-molecule TrkB antagonist. At P7, post-treatment with LM22A-4 was an efficacious anti-seizure agent in females but not males (n=5 per sex per treatment; vs PB alone; female p0.99; Figure). LM22A-4 pre-treatment was also only effective in females (vs PB alone; female p=0.02, male p=0.17). At P10, LM22A-4 pre-treatment significantly improved PB efficacy (p=0.03). At P7, both LM22A-4 treatments significantly reduced the ischemia-induced phosphorylation of TrkB at T817, a site known to mediate post-ischemic KCC2 degradation via PLC?. LM22A-4 pre-treatment at P7 also rescued ischemia-induced KCC2 S940 dephosphorylation, a phosphorylation site known to stabilize KCC2 in the membrane. Conclusions: BDNF-dependent TrkB activation following excitotoxic injury has been shown to activate specific downstream targets that lead to KCC2 hypofunction (Rivera et al., 2004). LM22A-4 prevented post-ischemic KCC2 downregulation; therefore, LM22A-4 may prevent post-ischemic TrkB-BDNF binding by occupying the loop-II domain of TrkB. Prevention of BDNF mediated TrkB activation alone following an excitotoxic injury may be sufficient to prevent emergence of PB-resistant seizures in neonates. Funding: Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R21HD073105 (SDK)