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. 2022 Jul 28:16:838419.
doi: 10.3389/fncel.2022.838419. eCollection 2022.

Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity

Brian C Baculis  1   2 , Harish Kesavan  2 , Amanda C Weiss  1   2 , Edward H Kim  2 , Gregory C Tracy  2 , Wenhao Ouyang  1   2 , Nien-Pei Tsai  1   2 , Hee Jung Chung  1   2   3   4
Affiliations

Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity

Brian C Baculis et al. Front Cell Neurosci. .

Abstract

Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes including KCNQ3 which encodes Kv7.3. Neuronal Kv7 channels are mostly heterotetramers of Kv7.2 and Kv7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in Kv7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reduces KCNQ3 transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decrease KCNQ3 and BDNF transcripts as well as the density of Kv7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and Kv7.3 density at the AIS during homeostatic scaling of AP firing rate.

Keywords: ERK; Kv7; ankyrin-G; axon initial segment; homeostatic plasticity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Prolonged activity blockade transiently reduces ERK1/2 activity before decreasing TrkB activity. Cultured hippocampal neurons (DIV 10) were treated for 24 h, 36 h, and 48 h with TTX (1 μM) to block their activity or its vehicle control (0.1% H2O), and subjected to immunoblot analysis for pTrkB (pTrkATyr674/675/pTrkBTyr706/707) and TrkB (A,B) or pERK1/2 (pERK1Thr202/Tyr204/pERK2Thr185/Tyr187) and ERK1/2 (C,D). GAPDH served as a loading control. (A,B) TTX treatment for 48 h decreased pTrkB level. (A) Representative immunoblots. (B) Quantification of pTrkB and total TrkB immunodensities. The immunodensity ratios (pTrkB/GAPDH and TrkB/GAPDH) were normalized to vehicle control. Number of culture dishes used: pTrkB (24 h: H2O = 6, TTX = 6; 36 h: H2O = 6, TTX = 6; 48 h: H2O = 13, TTX = 13), TrkB (24 h: H2O = 6, TTX = 6; 36 h: H2O = 6, TTX = 6; 48 h: H2O = 17, TTX = 20). (C,D) TTX treatment for 36 h decreased pERK1/2 level. (C) Representative immunoblots. (D) Quantification of pERK1/2 and total ERK1/2 immunodensities. The immunodensity ratios (pERK1/2/GAPDH and ERK1/2/GAPDH) were normalized to vehicle control. Number of culture dishes used: pERK1/2 (24 h: H2O = 6, TTX = 6; 36 h: H2O = 6, TTX = 6; 48 h: H2O = 12, TTX = 12), ERK1/2 (24 h: H2O = 6, TTX = 6; 36 h: H2O = 6, TTX = 6; 48 h: H2O = 12, TTX = 12). The Student’s t-test was used (***p < 0.005). Data shown represent the mean ± SEM with individual data points.
FIGURE 2
FIGURE 2
Prolonged pharmacological inhibition of ERK1/2 decreased mRNA and protein expression of Kv7.3. (A,B) Cultured hippocampal neurons (DIV 10) were treated for 48 h with vehicle control (0.1% v/v DMSO) or 20 μM U0126. Immunoblot analysis was performed with antibodies for pERK1/2 (pERK1Thr202/Tyr204/pERK2Thr185/Tyr187), ERK1/2, and GAPDH (n = 6 per treatment). (A) Representative immunoblots. (B) Quantification of pERK1/2 and total ERK1/2 immunodensities normalized to vehicle control. (C) Both TTX and U0126 treatment for 48 h decreased BDNF and KCNQ3 expression. Cultured hippocampal neurons (DIV 10) treated for 48 h with 1 μM TTX or its vehicle control (0.1% H2O), and 20 μM U0126 or its vehicle control (0.1% DMSO). QPCR was performed using the cDNA which was prepared from 2 μg of total RNA isolated from cultured neurons and validated primers for Kcna1, Kcna4, Kcnq3, Bdnf, Camk4, and Gapdh (n = 5 per treatment). Data were analyzed using the comparative threshold cycle (Ct) method and Gapdh internal control gene. Following normalization to Gapdh cDNA levels (which is reflected in the ΔCt values), the relative mRNA quantification (RQ) of the fold change for each condition compared to reference control was determined using the following equation: RQ = 2(– Δ Ct)/2(– Δ Ct reference). The RQ data is shown as mean ± SEM. One-way ANOVA with Tukey post hoc was used (***p < 0.005). (D,E) ERK1/2 inhibition for 48 h decreased Kv7.3 and Kv7.2 expression. Hippocampal cultured neurons (DIV 10) were treated for 48 h with 20 μM U0126 or its vehicle control (0.1% v/v DMSO) and subjected to immunoblot analyses with the verified antibodies for Kv7.3 (Supplementary Figure 4) and antibodies for Kv7.2 and GAPDH (n = 6 per treatment). (D) Representative Immunoblot and quantification of Kv7.3. (E) Representative Immunoblot and quantification of Kv7.2. The immunodensity ratios (Kv7.3/GAPDH and Kv7.2/GAPDH) were normalized to vehicle control. Data shown represent the mean ± SEM with individual data points. The Student’s t-test was used (*p < 0.05).
FIGURE 3
FIGURE 3
Prolonged activity blockade decreases Kv7.3 and ankyrin-G expression at the AIS. Cultured hippocampal neurons (DIV 11–12) were treated for 48 h with 1 μM TTX or its vehicle control (0.1% H2O) and subjected to immunocytochemistry with antibodies for the AIS marker ankyrin-G, somatodendritic marker MAP2, or Kv7.3. (A) Representative images of ankyrin-G and MAP2. (B) Representative images of ankyrin-G and Kv7.3. (C) Quantification of the background-subtracted raw integrated fluorescent intensity of ankyrin-G-positive segment in the axon, which was normalized to vehicle control, and the length of the ankyrin-G-positive segment. (D) Quantification of the background-subtracted raw integrated fluorescent intensity of Kv7.3 within the ankyrin-G-positive segment, which was normalized to vehicle control. Data shown represent the mean ± SEM with individual data points (H2O = 62 neurons from 46 images, TTX = 61 neurons from 43 images). Images were collected from 2 independent experiments. The Student’s t-test was used (*p < 0.05). Scale bar = 20 μm.
FIGURE 4
FIGURE 4
Prolonged pharmacological inhibition of ERK1/2 decreases Kv7.3 and ankyrin-G expression at the AIS and the AIS length. Cultured hippocampal cultured neurons (DIV 11–12) were treated for 48 h with 20 μM U0126 or its vehicle control (0.1% DMSO) and subjected to immunocytochemistry with antibodies for the AIS marker ankyrin-G, somatodendritic marker MAP2, or Kv7.3. (A) Representative images of ankyrin-G and MAP2. (B) Representative images of ankyrin-G and Kv7.3. (C) Quantification of the background-subtracted raw integrated fluorescent intensity of ankyrin-G-positive segment in the axon, which was normalized to vehicle control, and the length of the ankyrin-G-positive segment. (D) Quantification of the background-subtracted raw integrated fluorescent intensity of Kv7.3 within the ankyrin-G-positive segment, which was normalized to vehicle control. Data shown represent the mean ± SEM with individual data points (DMSO = 132 neurons from 69 images, U0126 = 126 neurons from 53 images). Images were collected from 2 independent experiments. The Student’s t-test was used (***p < 0.005). Scale bar = 20 μm.
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