Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR

Abstract

Tissue-type plasminogen activator (tPA) is a pleiotropic serine protease of the central nervous system (CNS) with reported neurotrophic and neurotoxic functions. Produced and released under its single chain form (sc), the sc-tPA can be cleaved by plasmin or kallikrein in a two chain form, tc-tPA. Although both sc-tPA and tc-tPA display a similar fibrinolytic activity, we postulated here that these two conformations of tPA (sc-tPA and tc-tPA) could differentially control the effects of tPA on neuronal survival. Using primary cultures of mouse cortical neurons, our present study reveals that sc-tPA is the only one capable to promote N-methyl-D-aspartate receptor (NMDAR)-induced calcium influx and subsequent excitotoxicity. In contrast, both sc-tPA and tc-tPA are capable to activate epidermal growth factor receptors (EGFRs), a mechanism mediating the antiapoptotic effects of tPA. Interestingly, we revealed a tPA dependent crosstalk between EGFR and NMDAR in which a tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and to subsequent neurotrophic effects.

Figure 1

sc-tPA and tc-tPA differentially influence NMDAR signaling. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting of sc-tPA and tc-tPA prepared as described in the Materials and Methods section (100 ng per lane). (b) SDS-PAGE followed by immunoblotting of sc-tPA and tc-tPA. sc-tPA was added on cultured cortical neurons 13 DIV for 1 h either alone or in the presence of plasmin or aprotinin. (c) Calcium video imaging performed on primary cultures of cortical neurons (12 DIV). After control NMDA stimulations (2 × 25 μM, 30 s) used as baseline, neurons were incubated for 45 min in the presence of buffer (control, n=90 cells), sc-tPA or tc-tPA at 300 nM (sc-tPA, n=85 cells; tc-tPA, n=78 cells) prior to a second set of NMDA stimulations (2 × 25 μM, 30 s). Percentages of potentiation or inhibition after incubation are calculated for each cell. (d) Percentage of potentiation or inhibition after incubation for each group (mean±S.E.M.; *P<0.0001 Kruskal–Wallis test followed by Mann–Whitney test; ^#P<0.0001 Wilcoxon test comparison of preincubation and postincubation responses)

Figure 2

Both sc-tPA and tc-tPA at 300 nM can modulate EGFR signaling. (a) Representative immunoblots for phospho-EGFR (A–C: tyrosine 1173; D–F: tyrosine 992) and total EGFR on neurons (12–13 DIV) after treatments with EGF (50 ng/ml), sc-tPA or tc-tPA (300 nM) during 15 min. (B, C, E and F) Quantifications of phosphorylated EGFRs and total EGFRs were compared with control (mean±S.E.M.; N=3 or 4 experiments; *P<0.05). (g) Confocal images of endogenous NMDAR–EGFR complexes in cortical neurons. (H) Quantification of NMDAR–EGFR complexes detected by Proximity Ligation Assay (PLA). ***P-value<0.001; Mann–Whitney U-test, N=3. (I) Cross-immunoprecipitation assays demonstrating that EGFRs and NMDARs form stable complexes in cultured neurons. (J) After two NMDA stimulations used as baseline, neurons were incubated for 45 min with buffer (control, n=85 cells) or EGF 50 ng/ml (EGF, n=93 cells) prior to a second set of NMDA stimulations. Percentages of potentiation or inhibition after incubation are calculated for each cell. (K) Percentage of potentiation or inhibition after incubation for each group (mean±S.E.M.; *P<0.0001 Kruskal–Wallis test followed by Mann–Whitney U-test; ^#P<0.0001 Wilcoxon test comparison of preincubation and postincubation responses)

Figure 3

sc-tPA-promoted neuronal calcium influx is dependent on its interaction with NMDARs. Calcium video imaging performed on cortical neurons. (a) After two control NMDA stimulations used as baseline, neurons were incubated for 45 min in the presence of either buffer (control, n=104 cells), sc-tPA at 300 nM (sc-tPA n=111 cells) or GluN1 antibody at 10 μg/ml (n=99 cells) alone or in combination (sc-tPA+GluN1 antibody; n=109 cells) prior to a second set of NMDA stimulations. Percentages of potentiation or inhibition after treatment are calculated for each cell. (b) Percentages of potentiation or inhibition after treatment are calculated for each cell and reported as percentages of responsiveness for each group. (c) In the same protocol, neurons were incubated for 45 min in the presence of buffer (control, n=75 cells), sc-tPA at 300 nM (sc-tPA, n=77 cells) or AG1478 at 5 μM (AG1478, n=77 cells) alone or in combination (sc-tPA+AG1478, n=90 cells) prior to a second set of NMDA stimulations (2 × 25 μM, 30 s). (d) Percentages of responsiveness for each group (mean±S.E.M.; *P<0.0001 Kruskal–Wallis test followed by Mann–Whitney test; ^#P<0.0001 Wilcoxon test comparison of preincubation and postincubation responses). NS: not significant

Figure 4

AG1478 reverses the inhibitory effect of tc-tPA on NMDAR signaling independently of its interaction with NMDARs. (a) After two NMDA stimulations used as baseline, neurons were incubated for 45 min in the presence of buffer (control, n=74 cells), AG1478 at 5 μM (AG1478, n=84 cells) or tc-tPA at 300 nM alone or in combination (tc-tPA, n=70 cells; tc-tPA+AG1478, n=81 cells) prior to a second set of NMDA stimulations. Percentages of potentiation or inhibition after incubation are calculated for each cell. (b) Percentages of potentiation or inhibition after incubation are calculated for each individual cell and reported as percentages of responsiveness for each group. (c) In the same protocol, neurons were incubated for 45 min in the presence of buffer (control, n=99 cells), GluN1 antibody at 10 μg/ml (GluN1 antibody, n=99 cells) or tc-tPA at 300 nM either alone or in combination tc-tPA 300 nM n=103 cells, tc-tPA+GluN1 antibody n=106 cells) prior to a second set of NMDA stimulations (2 × 25 μM, 30 s). (d) Percentages of potentiation or inhibition after incubation are calculated for each individual cell and reported as percentages of responsiveness for each group (mean±S.E.M.; *P<0.0001, Kruskal–Wallis test followed by Mann–Whitney test; ^#P<0.0001 Wilcoxon test of comparison of preincubation and postincubation responses). NS: not significant

Figure 5

Only high concentrations of active sc-tPA promote excitotoxicity. (a) Cortical neurons (12-13 DIV) were exposed for 1 h to NMDA (50 μM) in the presence of sc-tPA or tc-tPA. Neuronal death was quantified 24 h later. (b and c) Same experiments as in panel (a) were performed with neurons exposed for 24 h to NMDA (10 μM) in the presence of sc-tPA alone or in combination with tPA stop (10 nM, b) or GluN1 antibody (10 μg/ml, c). (mean±S.E.M.; n=3 experiments; 4 wells per condition; *P<0.05, NS: not significant, Kruskal–Wallis test followed by Mann–Whitney test)

Figure 6

Both sc-tPA and tc-tPA rescue neurons from serum deprivation-induced apoptosis. (a) Neuronal death measured after a 24-h exposure to serum deprivation (SD) alone or in the presence of sc-tPA or tc-tPA at 300 nM alone or plus aprotinin (1 μM; mean±S.E.M.; n=3 experiments; *P<0.05). (b and c) Neuronal death measured after a 24-h exposure to SD alone or in the presence of sc-tPA or tc-tPA plus GluN1 antibody (10 μg/ml; b) or AG1478 (5 μM; c) (mean±S.E.M.; n=3 experiments; 4 wells per condition; *P<0.05, NS: not significant, Kruskal–Wallis test followed by Mann–Whitney test)

Figure 7

Both sc-tPA and tc-tPA at 10 nM promote EGFR signaling and are neuroprotective. (a) Representative immunoblots for phospho-EGFR (tyrosine 1173) and total EGFR on neurons after treatments with sc-tPA and tc-tPA (10 nM) during 15 min. (b and c) Quantification of phosphorylated EGFRs and total EGFRs compared with the control condition (n=3 experiments; *P<0.05). (d) After two NMDA stimulations used as baseline, neurons were incubated for 45 min in the presence of buffer (control, n=75 cells), AG1478 at 5 μM (n=77 cells), sc-tPA (10 nM) alone or in combination with AG1478 (sc-tPA, n=78 cells; sc-tPA+AG1478, n=83 cells) or tc-tPA (10 nM) alone or in combination with AG1478 (tc-tPA, n=92 cells; tc-tPA+AG1478, n=92 cells) prior to a second set of NMDA stimulations. Percentages of potentiation or inhibition after incubation are calculated for each cell. (e) Percentages of responsiveness for each group (mean±S.E.M.; *P<0.0001 Kruskal–Wallis test followed by Mann–Whitney test; ^#P<0.0001 Wilcoxon test comparison of preincubation and postincubation responses). (f) Percentages of cells either potentiated, inhbited or without effect for each group. (g) Cortical neurons were subjected to 24-h exposure to NMDA (10 μM) in the presence of either sc-tPA or tc-tPA (10 nM) alone or in combination with AG1478 (5 μM; n=3 experiments; 4 wells per condition; *P<0.05, NS: not significant; Kruskal–Wallis test followed by Mann–Whitney test). (h) Neuronal death measured after a 24-h exposure to either serum deprivation (SD) alone or in the presence of either sc-tPA or tc-tPA at 300 or 10 nM (n=3 experiments; 4 wells per condition experiments; *P<0.05, NS: not significant; Kruskal–Wallis test followed by Mann–Whitney test, mean±S.E.M.)

Figure 8

tPA-dependent crosstalk between EGFRs and NMDARs. sc-tPA-induced potentiation of NMDAR signaling and subsequent neurotoxicity, inhibited by GluN1 antibody. tPA also promotes EGFR signaling and subsequent antiapoptotic effects, independently of its conformation (sc-tPA and tc-tPA), an effect occurring even at low concentrations (down to 10 nM) and inhibited by AG-1478, an inhibitor of EGFR transphosphorylation. tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and subsequent neurotrophic effects