Fig. 5. CPE binds to cell surface membrane and activates ERK-BCL2 signaling cascade, and its neuroprotective effect is tyrosine kinase independent.

a [¹²⁵I] CPE binds specifically to HT22 cell surface. HT22 cells were incubated with 25 nM [¹²⁵I] radio-labeled CPE (hot) for 3 h on ice in serum-free binding medium. To demonstrate binding specificity, 900 nM non-radio-labeled CPE (cold) or bovine serum albumin (BSA) was co-incubated with hot CPE to compete and displace the hot CPE binding. The bar graphs show that the [¹²⁵I] CPE binding to HT22 cell surface membrane was displaced by cold CPE but not by BSA. One-way ANOVA followed by Tukey’s post hoc multiple comparison test, [F_(2,6) = 29.11, p = 0.0008]. ⁺p = 0.002 for cold CPE + [¹²⁵I] CPE compared with [¹²⁵I] CPE (hot) treatment; ^#p = 0.001 for BSA + [¹²⁵I] CPE compared with CPE (cold)+ [¹²⁵I] CPE treatment. The experiment was run in triplicates, values are mean ± SEM. b [¹²⁵I] CPE binds HT22 cell surface in a saturated manner. HT22 cells were incubated with different concentrations (1.25–25 nM) of [¹²⁵I] CPE (hot) with or without 900 nM cold CPE for 3 h on ice in serum-free binding medium. The non-specific binding was determined by measuring bound [¹²⁵I] CPE in the presence of excess non-radio-labeled CPE (cold) with the [¹²⁵I] CPE. Note that BSA did not displace total [¹²⁵I] CPE binding. c Graph shows specific binding obtained by subtracting non-specific binding from total [¹²⁵I] CPE binding. d–g CPE and CPE-E342Q activate ERK signaling in HT22^cpe−/− neuronal cells. d HT22 ^cpe−/− cells were treated with 0, 10, 25, and 50 nM WT-CPE for 5 min and pERK 1/2 were analyzed by western blotting. e Bar graphs showing the fold change in phosphorylation of ERK1/2 which was determined by normalization using tERK1/2 as an internal control. One-way ANOVA analysis followed by Tukey’s post hoc multiple comparison test, [F_{(3, 8)} = 9.830, p = 0.0046]. ⁺p = 0.0065 for 50 nM WT-CPE compared with control at 0 nM concentration. N = 3, values are mean ± SEM. f HT22 ^cpe−/− cells were treated with 50 nM CPE-E342Q for 5 min and 15 min and pERK 1/2 were analyzed by western blotting. g Bar graphs showing the fold change in phosphorylation of ERK1/2 which was determined by normalization using tERK1/2 as an internal control. One-way ANOVA analysis followed by Tukey’s post hoc multiple comparison test, [F_(3,8)=61.33, p < 0.0001]. ⁺p < 0.0001 for CPE-E342Q treatment compared with control at 15 min. N = 3, values are mean ± SEM. h–k CPE and CPE-E342Q protect HT22^cpe−/− cells against oxidative stress by regulating BCL2 level. h, j Western blot analysis of BCL2 protein in HT22^cpe−/− cells which were treated with CPE-WT or CPE-E342Q respectively for 6 h followed by 100 μM H₂O₂ for 18–24 h. i, k Bar graphs showing quantification of BCL2 protein after normalization with β-actin which was used as a loading control. Results are expressed as fold change as compared to untreated controls. Similar to WT-CPE protein, CPE-E342Q was also able to prevent the H₂O₂ induced decrease in BCL2 level. For CPE-WT(i): one-way ANOVA analysis followed by Tukey’s post hoc multiple comparison test, [F_{(2, 6)} =19.37, p = 0.0024]. ⁺p = 0.002 for H₂O₂ compared to untreated control, ^#p = 0.0201 for H₂O₂ + CPE compared to H₂O₂ treatment. N = 3, values are mean ± SEM. For CPE-E342Q (k): one-way ANOVA analysis followed by Tukey’s post hoc multiple comparison test, [F_{(2, 6)} = 38.69, p = 0.0004]. ⁺p = 0.0004 for H₂O₂ treatment compared to control, ^#p = 0.002 for H₂O₂ + E342Q compared to H₂O₂ treatment. N = 3, values are mean ± SEM. l, m Effect of ERK inhibitor on CPE induced BCL2 expression. l Western blot analysis of BCL2 protein in HT22^cpe−/− cells which were treated with or without 5 μM MEK 1/2 inhibitor, U0126 for 30 min followed by treatment with 50 nM WT or E342Q recombinant CPE for 6 h. Cells were then treated with 100 μM H₂O₂ for the next 24 h. m Bar graphs showing quantification of BCL2 protein after normalization with β-actin which was used as a loading control. Results are expressed as fold decrease as compared to untreated controls. One-way ANOVA analysis followed by Tukey’s post hoc multiple comparison test, [F_(5,12) = 138.6, p < 0.0001]. ⁺p < 0.0001 for H₂O₂ compared to control, ^#p < 0.0001 for both CPE + H₂O₂ and E342Q + H₂O₂ compared with H₂O₂ treatment, ^@p < 0.0001 for U0126 + CPE + H₂O₂ compared with CPE + H₂O₂ treatment, ^&p < 0.0001 for U0126 + E342Q + H₂O₂ compared with E342Q + H₂O₂ treatment_. N = 3, values are mean ± SEM. n Neuroprotective effect of WT-CPE and CPE-E342Q against H₂O₂-induced cytotoxicity in mouse primary hippocampal neurons is independent of Trk receptor signaling. Bar graphs show that, 1 µM of Trk inhibitor, K-252a, or 100 µM of ANA12, a specific TrkB inhibitor, or 1 µM of FGFR1,2,3 inhibitor, PD166285 did not inhibit the neuroprotective effect of CPE and CPE-E342Q against H₂O₂-induced cytotoxicity assessed by LDH assay. One-way ANOVA analysis followed by Tukey’s post hoc multiple comparison test, [F_(15,32) = 26.93, p < 0.0001]. ⁺p < 0.0001 for H₂O₂ + WT-CPE and H₂O₂ + E342Q compared with H₂O₂ + BSA; ^#p < 0.0001 for H₂O₂ + WT-CPE + ANA12 and H₂O₂ + E342Q + ANA12 compared with H₂O₂ + BSA + ANA12; ^@p < 0.0001 for H₂O₂ + WT-CPE + K252 and H₂O₂ + E342Q + K252 compared with H₂O₂ + BSA + K252; ^&p < 0.0001 for H₂O₂ + WT-CPE + PD and H₂O₂ + E342Q + PD compared with H₂O₂ + BSA + PD; ^%p < 0.0001 for H₂O₂ + WT-CPE + PD + K252 and H₂O₂ + E342Q + PD + K252 compared with H₂O₂ + BSA + PD + K252 treatment. N = 3 independent experiments which were run in triplicate, values are mean ± SEM.