| Objective: Micro RNAs(mi RNAs), small noncoding RNAs, have been increasingly recognised as post-transcriptional regulators involved in the pathogenesis of epilepsy. Brain-derived neurotrophic factor(BDNF) and its receptor tropomyosin related kinase type B(Trk B) play a crucial role in epileptogenesis. Completely prevention of epileptogenesis was identified in the kindling model in mice with a conditional deletion of Trk B in the central nervous system. It has been reported that mi R-204 directly targeted and downregulated Trk B protein in a variety of cancers to suppress tumorigenesis, but its effect on epileptogenesis is unknown. Insight into the Trk B signaling pathway regulated by mi R-204 in epileptogenesis may provide valuable clues to the underlying mechanisms as well as a novel target for antiepileptic therapy. The aim of this study is to investigate the contribution of mi R-204 in epileptogenesis and to elucidate the regulation role of mi R-204 in Trk B signaling pathway using Mg2+-free model in hippocampal neuronal cultures. Aberrant expression of mi R-132 was been recently reported in our previous study. The further study is to investigate the possibility of mi R-132 involved in epileptic seizure and its underlying mechanisms.Methods: 1. Induction of epileptiform discharges model by Mg2+-free treatment of hippocampal neuronal cultures for 3h. 2. The mechanism of inhibiting epileptiform discharges through Trk B signaling regulated by mi R-204.(1) The change of mi R-204 expression in the Mg2+-free model was assayed using real-time quantitative polymerase chain reaction(q RT-PCR).(2) To examine the effect of mi R-204 in Trk B m RNA expression, overexpression of mi R-204 was carried out by transfection.(3) There are four major downstream signaling pathways activated by Trk B, including Akt, PLCγ1, ERK1/2 and CREB. The activation of signaling molecules was tested using western blotting and evaluated by the ratio of phoshorylated form to the total amount of protein.(4) Whole-cell current–clamp techniques were performed to observe the effect of mi R-204 on the epileptiform discharges.(5) The effect of mi R-204 on voltage-gated calcium channel(VGCC) was investigated by whole-cell patch clamp recordings. 3. The mechanism of epileptiform discharges through BDNF regulated by mi R-132. Aberrant expression of mi R-132 was been recently reported in our previous study. To investigate the possibility of mi R-132 involved in epileptic seizure and its underlying mechanisms, further study on mi R-132 and BDNF was carried out.(1) The changes of BDNF m RNA expression in the Mg2+-free model with or without mi R-132 transfection were assayed using q RT-PCR.(2) Whole-cell current–clamp techniques were performed to observe the respective and common effect of mi R-132 and BDNF on the epileptiform discharges.(3) Whole-cell patch clamp recordings were performed to observe the respective and common effect of mi R-132 and BDNF on the activation of VGCC.Results: 1. Mi R-204 suppressed epileptiform discharges through Trk B signaling.(1) Mi R-204 was downregulated in the Mg2+-free model(P < 0.001, n = 6).(2) The expression of Trk B m RNA was significantly reduced in the Mg2+-free model overexpressing mi R-204(P < 0.001, n = 6), but there was no difference in Trk B m RNA expression between Mg2+-free group and control(P > 0.05, n = 6).(3) No significant increase of Akt activation was detected in the Mg2+-free model, but the activation of PLCγ1, ERK1/2 and CREB was increased in the Mg2+-free model(P < 0.001, n = 6). Overexpression of mi R-204 decreased ERK1/2(P < 0.01, n = 6) and CREB activation(P < 0.001, n = 6) but had no effect on PLCγ1 activation(P > 0.05, n = 6) in the Mg2+-free model.(4) Mi R-204 inhibited epileptiform discharges in the Mg2+-free model. Whole-cell current-clamp recordings revealed normal baseline activity consisting of sporadic firing of action potentials in control neurons, while continuous high-frequency epileptiform discharges in the Mg2+-free model. Spike frequency was significantly decreased by mi R-204 overexpression(P < 0.001, n = 14).(5) Mi R-204 suppressed the enhancement of VGCC in the Mg2+-free model. The maximal Ca2+ current density of VGCC was significantly increasesd in the Mg2+-free model(P < 0.001, n = 10). A decrease in half-activation potential(V1/2) of steady-state activation curve and an increase in half-inactivation potential(V1/2) of steady-state inactivation curve were observed in the Mg2+-free model(P < 0.01, n = 10). The maximal current density in the Mg2+-free model was reduced by mi R-204 transfection(P < 0.001, n = 10). An increase in V1/2 of steady-state activation curve(P < 0.05, n = 10) and a decrease in V1/2 of steady-state inactivation curve(P < 0.01, n = 10) were induced by mi R-204. 2. Mi R-132 aggravated epileptiform discharges via suppression of BDNF in the Mg2+-free model.(1) BDNF m RNA was significantly increased in the Mg2+-free model(P < 0.001, n = 6). BDNF m RNA was upregulated by mi R-132 transfection in the control group(P < 0.001, n = 6). In sharp contrast, BDNF m RNA expression was significantly reduced by mi R-132 transfection in the Mg2+-free model(P < 0.001, n = 6).(2) The epileptiform discharges in the Mg2+-free model were suppressed by BDNF, but aggravated by mi R-132. The epileptiform discharges frequency was increased in the Mg2+-free model transfected with mi R-132(P < 0.01, n = 14). When the epileptic neurons were treated with BDNF, epileptiform discharges frequency was decreased(P < 0.001, n = 14), but mi R-132 enhanced epileptiform discharges to a level similar with that of Mg2+-free model.(3) The VGCC activation in the Mg2+-free model was suppressed by BDNF, but enhanced by mi R-132. The maximal Ca2+ current density of VGCC was significantly increasesd by mi R-132 transfecion in the Mg2+-free model(P < 0.01, n = 10). When the epileptic neurons were treated with BDNF, the maximal Ca2+ current density(P < 0.05, n = 10) and V1/2 of steady-state inactivation curve weredecreased(P < 0.01, n = 10), the V1/2 of steady-state activation curve was increased(P < 0.001, n = 10). However, mi R-132 enhanced the VGCC activation in the Mg2+-free + BDNF group to a level similar with that of Mg2+-free model.Conclusion: 1. Mi R-204 suppressed epileptiform discharges through Trk B signaling.(1) Mi R-204 was downregulated in the Mg2+-free model.(2) The expression of Trk B m RNA was downregulated by mi R-204.(3) The PLCγ1 and ERK1/2-CREB signaling pathways were activated in the Mg2+-free model, of which the ERK1/2-CREB pathway could be inhibited by mi R-204.(4) Mi R-204 suppressed epileptiform discharges and VGCC activation in the Mg2+-free model. 2. Mi R-132 aggravated epileptiform discharges via suppression of BDNF in the Mg2+-free model(1) BDNF m RNA was downregulated by mi R-132 in the Mg2+-free model.(2) BDNF suppressed epileptiform discharges and VGCC activation in the Mg2+-free model, but mi R-132 had opposite effects. |
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