Unveiling an exceptional zymogen: the single-chain form of tPA is a selective activator of NMDA receptor-dependent signaling and neurotoxicity

Abstract

Unlike other serine proteases that are zymogens, the single-chain form of tissue plasminogen activator (sc-tPA) exhibits an intrinsic activity similar to that of its cleaved two-chain form (tc-tPA), especially in the presence of fibrin. In the central nervous system tPA controls brain functions and dysfunctions through its proteolytic activity. We demonstrated here, both in vitro and in vivo, that the intrinsic activity of sc-tPA selectively modulates N-methyl-D-aspartate receptor (NMDAR) signaling as compared with tc-tPA. Thus, sc-tPA enhances NMDAR-mediated calcium influx, Erk(½) activation and neurotoxicity in cultured cortical neurons, excitotoxicity in the striatum and NMDAR-dependent long-term potentiation in the hippocampal CA-1 network. As the first demonstration of a differential function for sc-tPA and tc-tPA, this finding opens a new area of investigations on tPA functions in the absence of its allosteric regulator, fibrin.

Figure 1

tPA is a mosaic protein consisting of five distinct modules: a finger domain (F), an epidermal growth factor-like domain (EGF), two kringle domains (K1 and K2) and a serine protease proteolytic domain (SP). Secreted sc-tPA can be converted to its two-chain (tc-tPA) form by plasmin. Both sc-tPA and tc-tPA display an equivalent efficacy for plasmin-induced fibrinolysis in the vascular compartment. In contrast, sc-tPA is the primary effector of plasminogen-independent but proteolytically mediated NMDA-induced neurotoxicity, calcium influx and NMDA-dependent Erk(½) activation on cortical neurons. Similarly, sc-tPA modulates NMDA receptor-induced neuronal plasticity by enhancing long-term potentiation on hippocampal slices

Figure 2

sc-tPA and tc-tPA display an equivalent fibrinolytic activity. (a) SDS-PAGE followed by immunoblotting of rtPA, dtPA, sc-tPA and tc-tPA (200 ng per lane). (b and c) Fibrinolytic activity of the different forms of tPA normalized to an International Reference (IRP 98/714) using the half-time for clot lysis

Figure 3

In the absence of fibrin, tc-tPA displays an enzymatic activity higher than sc-tPA. (a and b) Activity of sc-tPA and tc-tPA (60 nM) measured either on a fluorogenic substrate (a) or by plasminogen-casein zymography assay (b). Similar amidolytic activity of sc-tPA and tc-tPA was observed in the presence of a five times higher concentration of sc-tPA as measured with a fluorogenic substrate (c) or by plasminogen-casein zymography assay (d). Activities of rtPA, dtPA, sc-tPA and tc-tPA were normalized to amidolytic activity (c and d) and corresponding concentrations used for the next experiments

Figure 4

Whereas sc-tPA promotes NMDAR-mediated neurotoxicity through its proteolytic activity, tc-tPA does not. (a, b and d) Neuronal death was assessed by measuring LDH release in the bathing media 24 h after a 1 h exposure of primary cultured cortical neurons (14 DIV) to 50 μM NMDA alone or supplemented with either (a) rtPA or dtPA (0.3 μM; n=12, four independent experiments), (b) rtPA, sc-tPA or tc-tPA at 0.3 μM (n=16, four independent experiments), or (d) rtPA, dGGACK-tPA at 0.3 μM (n=12, three independent experiments). Data are presented as the mean value±S.D. of neuronal death in percent relative to control. (c) Incubation of dtPA with the chloromethylketone dGGACK produced an inactive form of sc-tPA as assayed by fluorogenic assay (dGGACK-tPA). (e) NMDA-induced excitotoxic brain lesions measured (thionine staining) 24 h after intrastriatal injections of NMDA (10 mM) either alone or in the presence of either sc-tPA or tc-tPA (45 μM, n=4). Data are presented as the mean values±S.D. of lesion volumes in mm³ (***P<0.01; *P<0.05; ns: not significant)

Figure 5

sc-tPA specifically promotes NMDAR-dependent signaling. (a–c) Neuronal death was assessed by measuring LDH release in the bathing media harvested 24 h after a 1-h exposure of primary cultured cortical neurons (14 DIV) to 50 μM NMDA alone or supplemented with increasing concentrations of either (a) sc-tPA or (b) tc-tPA (12 nM/60 nM/120 nM/300 nM) (n=12, three independent experiments) or (c) in the presence of a molar concentration of 60 nM tPA, using differential ratios of sc-tPA and tc-tPA from 40 to 100% (n=16, four independent experiments). Data are presented as the mean value±S.D. of neuronal death in percent relative to control (**P<0.02; *P<0.05; ns: not significant)

Figure 6

sc-tPA promotes NMDAR-dependent neurotoxicity through a plasmin-independent mechanism. (a) LDH released was measured 24 h after a 1-h exposure of primary cultured cortical neurons (14 DIV) to NMDA alone or in the presence of either sc-tPA alone or sc-tPA+plasmin (100 nM; n=16, four independent experiments, insert shows conversion of sc-tPA (A) into tc-tPA (B) after an 1 h exposure to neurons). Primary cultures of cortical neurons were plated in the presence or absence of (b) EACA (25 mM) or (c) aprotinin (1 μM). After 14 DIV, neurons were incubated overnight with 12.5 μM NMDA alone or supplemented with rtPA (0.3 μM, n=12, three independent experiments). Data are presented as the mean value±S.D. of neuronal death in percent relative to control. (d) NMDA-induced excitotoxic brain lesions were measured by MRI as described in the methods section, 24 h after intrastriatal injection of NMDA (10 mM) alone or in combination with either sc-tPA (45 μM), tc-tPA (45 μM), or plasmin (150 nM) in the presence or absence of α2-antiplasmin (500 nM, n=7). Plasmin (150 nM) and α2-antiplasmin (500 nM) were injected alone as controls. Data are presented as the mean values±S.D. of lesion volumes in mm³. (e) Zymography assay performed (20 μg of protein extracts) from perfused striatum of swiss mice receiving an intrastriatal injection of either NMDA (10 mM), NMDA+α2-antiplasmin (100 nM), NMDA+sc-tPA (45 μM), NMDA+α2-antiplasmin+sc-tPA (representative of n=4 animals per group). Plasmin (200 nM), uPA (0.25 IU/ml) and tPA (0.06 IU/ml) were run in parallel as controls. (f) NMDA-induced excitotoxic brain lesion was measured (thionine staining) 24 h after intrastriatal injection of NMDA (10 mM) alone or in combination with sc-tPA (45 μM, n=5) in plasminogen null mice. Data are presented as the mean values±S.D. of lesion volumes in mm³ (***P<0.01; **P<0.02; *P<0.05; ns: not significant)

Figure 7

Whereas sc-tPA promotes both NMDA-induced calcium influx and Erk(½) activation, tc-tPA does not. (a–c) NMDA-induced calcium influx recorded from cultured cortical neurons exposed to NMDA either alone (50 μM) or in the presence of either sc-tPA (a: 0.3 μM, n=126 cells, three independent experiments) or tc-tPA (b: 0.3 μM, n=132 cells, three independent experiments). Data are plotted in c (dtPA: white bar, sc-tPA: gray bar and tc-tPA: black bar). Data were normalized to NMDA alone (slashed bar). Data are represented as mean value±S.D. relative to NMDA. (d) NMDA-induced Erk(½) activation was evaluated by anti phospho-Erk(½) immunoblotting alone or in the presence of sc-tPA and tc-tPA at a molar concentration of 0.3 μM (n=3). Data were normalized to NMDA alone and are represented as mean value±S.D. of phospho-Erk(½) variation relative to control (*P<0.05; ns: not significant)

Figure 8

Whereas sc-tPA modulates neuronal synaptic plasticity through NMDAR signaling, tc-tPA does not. (a) LTP was recorded in the hippocampal CA1 area in the presence or absence of sc-tPA (100 nM, white dots, n=13) and tc-tPA (100 nM, black triangles, n=13). APV is used as a control to block NMDA receptors. (b) LTD induced alone (gray squares, n=9) or in the presence of either sc-tPA (100 nM, white dots, n=10) or tc-tPA (100 nM, black triangles, n=8). In inserts (a and b), fEPSPs recorded before and 50 min after stimulation. Statistical analysis was done for this interval using ANOVA repeated measures (**P<0.02; ***P<0.01)