Calponin-3 is associated with epilepsy through the regulation of astrocyte activity

Abstract

Astrocytes contribute in critical ways to the pathophysiology of epilepsy not only through trophic support but also through the regulation of neuronal excitability by modulating glutamate, γ-aminobutyric acid (GABA), adenosine triphosphate (ATP), and adenosine levels. Calponin-3 is an actin-binding protein that is enriched in the brain. We have previously reported that increased calponin-3 expression is correlated with epileptic seizures. In the present study, we revealed that in the hippocampus of epileptic mice models, increased calponin-3 protein expression was correlated with the expression of the astrocytic marker glial fibrillary acidic protein (GFAP). Calponin-3 overexpression in the hippocampus significantly increased susceptibility to epileptic seizures, whereas calponin-3 downregulation was associated with reduced spontaneous recurrent seizures in mice. Furthermore, changes in calponin-3 levels corresponded to astrocyte activation in both mice and cultured human astrocytes and were associated with changes in the protein levels of adenosine kinase (ADK) and equilibrative nucleoside transporter 1 (ENT1), which are two key regulators of adenosine metabolism that have been shown to play critical roles in epileptogenesis. Collectively, our findings suggest that calponin-3 may regulate astrocyte-mediated adenosine metabolism and could represent a potential therapeutic target for epilepsy.

Keywords: adenosine kinase (ADK); astrocyte; calponin‐3; epilepsy; equilibrative nucleoside transporter 1 (ENT1).

FIGURE 1

Elevated Calponin‐3 protein levels are correlated with astrocyte activation in epileptic mouse models. (A, B) WB and statistical analysis of calponin‐3 and GFAP protein levels in the hippocampus of mice with KA‐induced epilepsy (n = 5). (C, D) The correlation between calponin‐3 protein expression and GFAP protein expression is observed in the ipsilateral hippocampus (C) and the contralateral hippocampus (D), (n = 25). (E) Coexpression of calponin‐3 (red) and GFAP (green) in hippocampus of mice with KA‐induced epilepsy (7d, white arrows). Scale bars, 100 μm. (F) Quantification of the percentage of calponin‐3⁺ cells and GFAP⁺ cells (n = 6). (G) Number of calponin‐3⁺/GFAP⁺ cells (n = 6). (H–J) WB and statistical analysis of calponin‐3 and GFAP expression in the hippocampus of mice with pilocarpine‐induced epilepsy (n = 4). (K) Coexpression of calponin‐3 (red) and GFAP (green) in the hippocampus of mice with pilocarpine‐induced epilepsy (7d, white arrows). Scale bars, 50 μm. (L) Quantification of the percentage of calponin‐3⁺ cells and GFAP⁺ cells (n = 6). (M) Number of calponin‐3⁺/GFAP⁺ cells (n = 6). The results are presented as the mean ± SEM. Statistical analysis: F, G, I, J, L, M: Unpaired Student's t test. *p < 0.05, **p < 0.01, ****p < 0.0001. B: One‐way ANOVA followed by Dunnett's multiple comparisons test. Calponin‐3: **p < 0.01, ***p < 0.001; GFAP: ^# p < 0.05, ^## p < 0.01, ^### p < 0.001. C, D: Spearman's rank correlation analysis. Ctrl, control, EP, epilepsy, IF, immunofluorescence, WB, Western blotting. See also Figure S1.

FIGURE 2

Hippocampal calponin‐3 overexpression promotes the susceptibility of mice to epilepsy. (A) Representative WB images and statistical analysis of calponin‐3 expression 3 weeks after AAV‐CNN3 injection (n = 4). (B) Calponin‐3 (green) was successfully upregulated in the bilateral hippocampus. Nucleus (blue). Scale bar, 1 mm. (C1) IF of calponin‐3 (green) and GFAP (red). (C2) IF of calponin‐3 (green) and MAP2 (red). The white arrows show co‐localized cells. Scale bars, 20 μm. (D) Schematic diagram of KA‐induced seizures following the upregulation of calponin‐3. (E) Mortality after KA‐induced SE (n = 12). (F) The latency period of the first spontaneous seizure. (G) Total number of spontaneous seizures. (H) Total number of days with spontaneous seizures (F–H, AAV‐con, n = 11, AAV‐CNN3, n = 9). (I, J, L, M) LFP in the two groups of mice. (I) Epileptiform discharges amplitude and frequency. (J) Corresponding heatmap energy. (L) Number of SLEs (n = 5). (M) During of SLEs (n = 5). (K, N) Interictal ECoG of the two groups. (K1) Typical spike. (N) Frequency of spikes (n = 3). The results are presented as the mean ± SEM. Statistical analysis: A: One‐way ANOVA followed by Dunnett's multiple comparisons test. E: Chi‐square test. F–H, L–N: Unpaired Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001. LTP, local field potential; ECoG, electrocorticography; IF, immunofluorescence; WB, Western blotting.

FIGURE 3

Calponin‐3 knockdown inhibits spontaneous seizures in mice with KA‐induced epilepsy. (A) Schematic diagram of downregulation of calponin‐3 after KA‐induced seizures. (B) IF of EGFP (green) and DAPI (blue) in the hippocampus of mice. Scale bar, 200 μm. (C) Representative WB images and statistical analysis of calponin‐3 expression after AAV‐KD was injected for 3 weeks (n = 6). (D) IF of calponin‐3 (red) in the hippocampus of AAV‐KD mice. (E) Percentage of calponin‐3⁺ cells in the DG (n = 6). (F) Video monitoring of typical grade 4 and 5 seizures in mice. (G) Interictal ECoG of AAV‐CON and AAV‐KD group. (H–J) Behavioral statistics of mice under video monitoring. (H) Number of spontaneous seizures above grade 4. (I) Duration of each seizure above grade 4. (J) Total number of days of spontaneous seizures (n = 5). (K–N) Statistical analysis of ECoG results. (K) Frequency of spikes. (L–N) Average duration, average amplitude and average power (n = 3). The data are expressed as the mean ± SEM, Statistical analysis: Unpaired Student's t test was performed for comparison between the two groups. (**p < 0.01; ***p < 0.001).

FIGURE 4

Altered calponin‐3 protein levels are directly associated with astrocyte activation. (A–H) Increased reactive astrogliosis was observed following the upregulation of calponin‐3 in the hippocampus of mice. (A) Coexpression of calponin‐3 (green), GFAP (pink) and S100β (red). (B, C) Quantification of the percentage of GFAP⁺ and S100β⁺ cells. (D) Number of GFAP⁺/calponin‐3⁺ cells (n = 6). (E) Number of S100β⁺/calponin‐3⁺ (n = 6). (F, G) Representative WB images and statistical analysis of GFAP and S100β expression. (H) Number of GFAP⁺/S100β⁺ cells (n = 6). (I–P) Following the downregulation of calponin‐3 in the hippocampus of KA mice, the number of reactive astrocytes decreased. (I) Coexpression of calponin‐3 (pink) and GFAP (red). (J) Coexpression of calponin‐3 (red) and S100β (pink). (K, L) Quantification of the percentage of GFAP⁺ and S100β⁺ cells (n = 6). (M, N) Representative WB images and statistical analysis of GFAP and S100β expression (n = 6). (O) Number of GFAP⁺/calponin‐3⁺ cells (n = 6). (P) Number of S100β⁺/calponin‐3⁺ cells (n = 6). The results are presented as the mean ± SEM. Statistical analysis: (A–H) One‐way ANOVA followed by Dunnett's multiple comparisons test, n = 6. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (I–P) Unpaired Student's t test, n = 6. *p < 0.05; **p < 0.01; ***p < 0.001. WB, Western blotting. See also Figure S2.

FIGURE 5

Calponin‐3 regulates the protein levels of ADK and ENT1 in astrocytes. (A–G) ADK and ENT1 expression increased following the upregulation of calponin‐3 in the hippocampus of mice. (A) Coexpression of calponin‐3 (green), GFAP (pink) and ADK (red). The white arrows show calponin‐3⁺/GFAP⁺/ADK⁺ cells. (B) Quantification of the percentage of ADK⁺ cells. (C) Number of ADK⁺/calponin‐3⁺ cells (n = 6). (D–F) Representative WB images and statistical analysis of ADK and ENT1 expression (n = 6). (G) Number of ADK⁺/GFAP⁺ cells (n = 6). (H–M) ADK and ENT1 levels decreased after the downregulation of calponin‐3 in the hippocampus of KA mice. (H) Coexpression of calponin‐3 (pink) and ADK (red). (I) Quantification of the percentage of ADK⁺ cells (n = 6). (J) Number of ADK⁺/calponin‐3⁺ cells (n = 6). (K–M) Representative WB images and statistical analysis of ADK and ENT1 expression (n = 6). The results are presented as the mean ± SEM. Statistical analysis: (A–G) One‐way ANOVA followed by Dunnett's multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (H–M) Unpaired Student's t test. *p < 0.05; ***p < 0.001. WB, Western blotting. See also Figure S3.

FIGURE 6

Calponin‐3 knockdown reduces ENT1 and ADK expression and proliferation in HACs. (A) Coexpression of calponin‐3 (red) and ADK (green). Scale bars, 100 μm. (B) Proliferation of HACs after knockdown of calponin‐3 expression. The red was EdU staining positive cells with proliferative ability, and the blue was Hoechst labeled nucleus. Scale bars, 100 μm. (C) Quantification of the percentage of ADK⁺ cells (n = 3). (D) Proportion of EdU‐positive cells (n = 6). (E–H) Representative WB images and statistical analysis of ADK, ENT1 and GLT‐1 expression (n = 4). The data are expressed as the mean ± SEM, One‐way ANOVA followed by Dunnett's multiple comparisons test was performed. (*p < 0.05; **p < 0.01; ***p < 0.001). WB, Western blotting. See also Figure S4.