TrkB agonists prevent postischemic emergence of refractory neonatal seizures in mice
Pavel A. Kipnis, Brennan J. Sullivan, Brandon M. Carter, Shilpa D. Kadam
Pavel A. Kipnis, Brennan J. Sullivan, Brandon M. Carter, Shilpa D. Kadam
View: Text | PDF
Research Article Development Neuroscience

TrkB agonists prevent postischemic emergence of refractory neonatal seizures in mice

Abstract

Refractory neonatal seizures do not respond to first-line antiseizure medications like phenobarbital (PB), a positive allosteric modulator for GABAA receptors. GABAA receptor–mediated inhibition is dependent upon electroneutral cation-chloride transporter KCC2, which mediates neuronal chloride extrusion and its age-dependent increase and postnatally shifts GABAergic signaling from depolarizing to hyperpolarizing. Brain-derived neurotropic factor–tyrosine receptor kinase B activation (BDNF–TrkB activation) after excitotoxic injury recruits downstream targets like PLCγ1, leading to KCC2 hypofunction. Here, the antiseizure efficacy of TrkB agonists LM22A-4, HIOC, and deoxygedunin (DG) on PB-refractory seizures and postischemic TrkB pathway activation was investigated in a mouse model (CD-1, P7) of refractory neonatal seizures. LM, a BDNF loop II mimetic, rescued PB-refractory seizures in a sexually dimorphic manner. Efficacy was associated with a substantial reduction in the postischemic phosphorylation of TrkB at Y816, a site known to mediate postischemic KCC2 hypofunction via PLCγ1 activation. LM rescued ischemia-induced phospho–KCC2-S940 dephosphorylation, preserving its membrane stability. Full TrkB agonists HIOC and DG similarly rescued PB refractoriness. Chemogenetic inactivation of TrkB substantially reduced postischemic neonatal seizure burdens at P7. Sex differences identified in developmental expression profiles of TrkB and KCC2 may underlie the sexually dimorphic efficacy of LM. These results support a potentially novel role for the TrkB receptor in the emergence of age-dependent refractory neonatal seizures.

Authors

Pavel A. Kipnis, Brennan J. Sullivan, Brandon M. Carter, Shilpa D. Kadam

×

Figure 4

LM rescued postischemic TrkB/PLCγ1 pathway activation, activated the TrkB/ERK1/2 pathway, and rescued ipsilateral KCC2 degradation.

Options: View larger image (or click on image) Download as PowerPoint
LM rescued postischemic TrkB/PLCγ1 pathway activation, activated the Trk...
All proteins of interest were normalized to housekeeping protein β-actin. Phosphoproteins were also normalized to their respective total protein (see Supplemental Figure 3). The * indicates differences between treatment group and naive controls; the # indicates differences between contralateral and ipsilateral hemispheres within groups. (A) Representative Western blots showing TrkB and p–TrkB-Y816 expression for all treatment groups. (B) Contralateral (L) and ipsilateral (R) TrkB expression 24 hours after ischemic insult. ***P < 0.001 by 1-way ANOVA. #P < 0.05 by 2-tailed t test. (C) Contralateral (L) and ipsilateral (R) p–TrkB-Y816 expression 24 hours after ischemic insult. **P < 0.01, ****P < 0.0001 by 1-way ANOVA. (D) Representative Western blots showing PLCγ1 and p–PLCγ1-Y783 expression. (E) Contralateral (L) and ipsilateral (R) PLCγ1 expression 24 hours after ischemic insult. #P < 0.05 by 2-tailed t test. (F) Contralateral (L) and ipsilateral (R) p–PLCγ1-Y783 expression 24 hours after ischemic insult. *P < 0.05, ***P < 0.001 by 1-way ANOVA. #P < 0.05 by 2-tailed t test. (G) Representative Western blots showing ERK1/2 and p-ERK1/2-T202/Y204 expression. (H) Contralateral (L) and ipsilateral (R) ERK1/2 expression 24 hours after ischemic insult. #P < 0.05 by 2-tailed t test. (I) Contralateral (L) and ipsilateral (R) p-ERK1/2-T202/Y204 expression 24 hours after ischemic insult. *P < 0.05, **P < 0.01 by 1-way ANOVA. (J) Representative Western blots showing KCC2 and p–KCC2-S940 expression. (K) Representative Western blot showing lack of KCC2 and p–KCC2-S940 band in liver samples from P7 pups. (L) Contralateral (L) and ipsilateral (R) KCC2 expression 24 hours after ischemic insult. #P < 0.05, ###P < 0.001 by 2-tailed t test. (M) Contralateral (L) and ipsilateral (R) p–KCC2-S940 expression 24 hours after ischemic insult. #P < 0.05, ##P < 0.01 by 2-tailed t test. Number of mice: n = 13 [6/7] (naive [M/F]), 19 [10/9] (ligate+PB), 13 [5/8] (post-LM), 22 [11/11] (pre-LM). Box-and-whisker plots show quartiles with median with minima and maxima at the bottom and top whiskers, respectively.