Selective blockade of acid-sensing ion channel 1a can provide substantial hippocampal neuroprotection

Abstract

Background: Acid-sensing ion channel 1a (ASIC1a) is the only member of the ASIC family where Ca²⁺ osmosis has been reported, and it is highly expressed in neurons of the central nervous system. This study aimed to investigate whether ASIC1a is trafficked to the plasma membrane and regulated by the Rho/ROCK and PI3K signaling pathways in temporal lobe epilepsy (TLE). In addition, further research is required to determine whether selective ASIC1a blockade is a viable therapeutic strategy for TLE.

Methods: The localization and expression levels of ASIC1 and mRNA levels of ASIC1a were detected when the Rho/ROCK and PI3K signaling pathways were activated and inhibited in glutamate (Glu)-induced cell. Meanwhile, we analyzed the location and expression of ASIC1 using Western blotting and immunofluorescence in brain tissue samples from TLE patients, kainic acid (KA)-treated rats, and Glu-induced primary hippocampal neurons. Currently, no specific ASIC1a antibody is available, so the ASIC1 antibody was used in this study, as in previous studies. Furthermore, we evaluated the HT22 cell survival rate, mitochondrial damage, apoptosis, and autophagy to examine whether selective blocking ASIC1a (PcTx1) could play a neuroprotective role.

Results: First, the Rho/ROCK and PI3K signaling pathways affect the regulation of the expression and localization of ASIC1, especially the mRNA levels of ASIC1a in the Glu-induced HT22 cell injury model. Second, the high expression of ASIC1 in epilepsy patients was verified in all three sample types, and the phenomenon of its transport from the cytoplasm to the cell membrane/mitochondria was confirmed. Finally, although ASIC1 has a limited epileptogenic effect in the acute phase of epilepsy in vivo, selective blockade of ASIC1a using PcTx1 provided significant hippocampal neuroprotection and reduced mitochondrial damage, apoptosis, and cellular autophagy in vitro.

Interpretation: This study is a systematic report concerning ASIC1a in temporal lobe epilepsy, including in vivo and in vitro experiments addressing both the acute and chronic phases. It provides foundational research for proposing ASIC1a as a new target for epilepsy treatment.

Keywords: ASIC1a; PI3K; Rho/ROCK; hippocampus; neuroprotection; temporal lobe epilepsy.

FIGURE 1

Changes in ASIC1 co‐locate with Rho fluorescence after the administration of Rho agonists/inhibitors in HT22 cells. Control group: A1‐A4; Rho activator group: B1‐B4; Rho inhibitor group: C1‐C4. DAPI (blue, cell nucleus), ASIC1 (green), and Rho (red) immunofluorescence staining was performed on the control group (Con, HT22 cells), Rho activator group (HT22 cells with 1 μg/mL Rho activator for 4 h), and Rho inhibitor group (HT22 cells with 1 μg/mL Rho inhibitor for 4 h).

FIGURE 2

(A) Western blotting indicated that the expression levels of Rho in total protein and total membrane protein changed significantly over time after adding Glu. (B) Relative expression of Rho in total protein. (C) Ratio of the relative expression of Rho in total membrane proteins to relative expression in total proteins. (D–F) Changes in the expression levels of ASIC1 in total protein and total membrane protein fractions of primary cortical neurons after treatment with 5 mM Glu for different durations. The value of total protein minus total membrane protein was plasmosin protein. Statistical chart of the gray value: ASIC1 is normalized against β-actin and the optical density of the control group. (G) Changes in ASIC1a mRNA expression levels after the administration of different drug treatments (1 μg/mL Rho activator, 1 μg/mL Rho inhibitor, and 10 μM ROCK inhibitor Y-27632). (H–J) Changes in ASIC1 expression after treatment with different drugs (1 μg/mL Rho activator, 1 μg/mL Rho inhibitor, and 10 μM ROCK inhibitor Y-27632). (K) Changes in mRNA expression levels of cellular ASIC1a after treatment with different PI3K inhibition (15 μM LY294002 or 60 μM 3-MA). (L,M) Changes in ASIC1 protein expression levels after treatment with different PI3K inhibitors (15 μM LY294002 or 60 μM 3-MA). The optical density of each lane was represented by mean ± SD, and single-factor analysis of variance and the Tukey method were used for comparison between groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

FIGURE 3

Expression and localization of ASIC1 in temporal lobe tissue of TLE patients and KA-treated rats. DAPI (blue, cell nucleus), calponin 3 (green, nerve fiber), and ASIC1 (red) immunofluorescence staining was performed on normal temporal lobe brain tissue (NC, normal control group: normal temporal cortex tissue removed incidentally during surgery in non-epileptic patients) and surgical resection specimens from temporal lobe cortex tissue of TLE patients.

FIGURE 4

Expression and localization of ASIC1 were dynamically changed in KA-treated rats and Glu-induced primary hippocampal neurons. (A) Changes in the expression levels of ASIC1a in total protein and mitochondrial protein of hippocampal tissue at different time points after epileptic seizures. (B) Relative expression of ASIC1 at different time points. (C) Relative expression of mito-ASIC1 at different time points. (D) Ratio of ASIC1 expression in mitochondrial protein to total protein at different time points. The optical density of each lane was represented by mean ± SD, and single-factor analysis of variance and the Tukey method were used for comparison between groups. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001. mito-ASIC1, ASIC1 in mitochondrial protein.

FIGURE 5

Role of PcTx1 in kainic acid-treated rats and Glu-induced primary hippocampal neurons. (A) Nissl staining in the CA3 region of hippocampus (400×): the neurons in the sham group were arranged neatly and had a normal morphology; the neurons in the KA group, CA 3 area, died and were clearly lost, and the remaining neurons were disorderly distributed. Neuronal loss in the PcTx1 group was not significantly different from that in the KA group. (B) CCK-8 was used to evaluate the cell viability of HT22 under different Glu concentrations. (C) Cell viability of HT22 treated with 5 mM Glu was evaluated by CCK-8 at different time points. (D) Effect of PcTx1 (2 nM) on HT22 cell survival (Glu: 5 mM; 6 h). (B–D)*, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p <0.0001. Data represent the absorbance calculated using the corresponding formula and are expressed as the mean ± SD.

FIGURE 6

Blocking ASIC1a can alleviate mitochondrial damage. MitoTracker (red, mitochondria), ASIC1 (green), and DAPI (blue) immunofluorescence staining were performed on the control group (Con, HT22 cells: (A1-A4)), Glu group (Glu-induced HT22 cells: (B1-B4)), and Glu + PcTx1 group (Glu-induced HT22 cells with PcTx1: (C1-C4)). HT22 cells in each group were treated with the same amount of DMEM medium, 5 mM Glu, and 5 mM Glu + 2 nM PcTx1 for 6 h.

FIGURE 7

PcTx1 decreased mitochondrial apoptosis and autophagy of HT22 cells induced by Glu. (A) JC-1 staining group (A1-A3): the transformation of JC-1 from red fluorescence (polymer) to green fluorescence (monomer) can be used as an early detection index of apoptosis. The Annexin V-EGFP staining group (A4-A6) revealed green fluorescence in apoptotic cells. Dead cells exhibit red and green fluorescence, while living cells show almost no fluorescence. Blocking ASIC1a was found to attenuate Glu‐induced apoptosis in both sets of staining. (B,D) Western blotting results showed that the ratio of LC3BⅡ to LC3BⅠ in HT22 cells changed after treatment with 5 mM Glu at different times. (C, E) Western blotting results showed that blocking ASIC1a attenuated the Glu‐induced elevation of the ratio of LC3BⅡ to LC3BⅠ in HT22 cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001. Data represent the absorbance calculated using the corresponding formula and are expressed as the mean ± SD.