Feedback mechanism in depolarization-induced sustained activation of extracellular signal-regulated kinase in the hippocampus

Abstract

Phosphorylation plays important roles in several processes including synaptic plasticity and memory. The critical role of extracellular signal-regulated kinase (ERK) in these processes is well established. ERK is activated in a sustained manner by different stimuli. However, the mechanisms of sustained ERK activation are not completely understood. Here we show that KCl depolarization-induced sustained ERK activation in the hippocampal slices is critically dependent on protein synthesis and transcription. In addition, the sustained ERK activation requires receptor tyrosine kinase(s) activity. In support of a role for a growth factor in sustained ERK activation, KCl depolarization enhances the level of brain-derived neurotrophic factor (BDNF). Furthermore, BDNF antibody blocks KCl-induced sustained ERK activation. These results suggest a positive feed-back loop in which depolarization-induced BDNF maintains ERK activation in the sustained phase.

Figure 1. KCl depolarization induces sustained ERK activation.

The hippocampal slices were treated with KCl and harvested either immediately (A, Immediate) or 1 h (B, Sustained) after the treatment. The sample blots (A1, B1) and quantified summary data (A2, B2; n = 4 in both sets) show that ERK was activated immediately after KCl treatment and ERK activation was sustained for 1 h after the treatment. Asterisks denote significant difference (p<0.05). In this and others figures, the immediate and sustained ERK activation was examined simultaneously from the slices of the same animal and both samples were resolved on the same gel, but have been shown separately for clarity.

Figure 2. KCl-induced sustained ERK activation requires protein synthesis and transcription.

ERK activation in the hippocampal slices was examined 1 h (Sustained) or immediately (Immediate) after KCl treatment. (A) Whereas ERK activation was observed in the control slices 1 h after KCl stimulation, emetine (Emet) completely blocked KCl-induced ERK activation at this time point. (B) The KCl-induced immediate ERK activation was not affected by emetine treatment (KCl and Emet + KCl, p>0.30). Compared to control, emetine alone had no significant effects on ERK activation (Sustained, p>0.90, Immediate, p>0.75). Representative blots are shown in A1 and B1 and summary data are shown in A2 and B2 (n = 7 in both sets). (C) The hippocampal slices were treated with emetine for 1.5 h (same total time of treatment as in the case of sustained ERK activation analysis) before stimulation with KCl. Slices were harvested after KCl treatment and processed for ERK activation analysis. Representative blots (C1) and quantified summary data (C2; n = 3) show that emetine had no effect on immediate ERK activation even with prolonged treatment with emetine. There was no significant difference between KCl and KCl + emetine groups (p>0.93). (D, E) Representative blots (D1 and E1) and summary data (D2 and E2; n = 6 in both sets) show that ERK activation 1 h after KCl stimulation was blocked by the RNA synthesis inhibitor, actinomycin D (Act D; D), but the immediate ERK activation after KCl treatment was unaffected (KCl and Act D + KCl, p>0.42) by actinomycin D (E). Compared to control, actinomycin D alone treatment did not significantly affect basal level of ERK phosphorylation (Sustained, p>0.45, Immediate, p>0.67). Asterisks denote significant difference (p<0.05).

Figure 3. KCl-induced sustained ERK activation requires receptor tyrosine kinase activity.

ERK activation in the hippocampal slices was examined 1 h (A, sustained) or immediately (B, immediate) after KCl treatment. Representative blots are shown in A1 and B1, and summary data are shown in A2 and B2 (n = 7 in both sets). ERK was activated in the control slices 1 h after KCl treatment. But, the receptor tyrosine kinase inhibitor (K252a) completely blocked ERK activation at this time point. The ERK activation immediately after the KCl stimulation was unaffected by K252a treatment (KCl and K252a + KCl, p>0.63). Compared to control, K252a did not affect the basal level of ERK phosphorylation (Sustained, p>0.08, Immediate, p>0.41). Asterisks denote significant difference (p<0.05).

Figure 4. KCl depolarization increases BDNF levels and BDNF antibody blocks KCl-induced sustained ERK activation.

(A) The hippocampal slices were treated with KCl and the level of mature BDNF protein was examined 1 h after the treatment. Sample Western blots of BDNF and GAPDH proteins are shown in (A1) and quantified summary data are shown in (A2; n = 4). KCl treatment increased BDNF protein level. (B) BDNF antibody blocks KCl-induced sustained ERK activation. The activation of ERK was examined in the hippocampal slices 1 h after KCl treatment. The sample blots (B1) and quantified summary data (B2, n = 6) show that the KCl-induced sustained ERK activation was blocked by BDNF antibody. Compared to control, the BDNF antibody alone did not affect basal ERK activation (p>0.91). Asterisks denotes significant difference (p<0.05).