Physiological and pathological implications of the tPA/NR1 interaction: An immunization-based investigation

Tissue-type plasminogen activator (tPA) is thought to be involved in several brain functions and dysfunctions. Its vascular thrombolytic activity led to its use for ischemic stroke treatment. However, increasing evidence suggests that tPA potentiates excitotoxic neuronal death. Accordingly, we have recently shown that tPA can directly modulate N-methyl-D-aspartate (NMDA) receptor signalling by cleaving the arginine 260 within the amino-terminal domain of the NR1 subunit (ATD-NR1). The deleterious effects of tPA during excitotoxic neuronal death suggests that blocking its effect on NMDA-signalling might improve stroke treatment without hindering its beneficial fibrinolytic action. To validate this postulate, and to investigate the relevance of tPA-mediated control of NMDA receptor signalling in vivo, we developped a protocol of immunization in mice against ATD-NR1 including the interaction domain with tPA. Complete Freund's adjuvant (CFA) or recombinant ATD-NR1 (amino acids 19-371) dissolved in CFA were injected intraperitonally once a week during four weeks. By immunoblotting, we show that sera harvested from immunized mice recognized recombinant ATD-NR1, while sera from control mice (CFA only) did not. We then postulated that the antibodies produced in the immune response might have a therapeutic value by physically preventing the interaction between tPA and NR1. We have thus performed excitotoxic lesions by injecting NMDA into the striatum of control and immunized mice, with or without intravenous injection of tPA (1 mg/kg). In CFA-injected animals, while NMDA alone led to an excitotoxic lesion of around 13 mm³, tPA, when injected intravenously, potentiated twice the lesions (29.5 mm³). In contrast, intravenous tPA was unable to potentiate NMDA-induced excitotoxic injury in ATD-NR1-vaccinated mice. No difference was observed between NMDA-induced lesion in naïve, CFA-, or ATD-NR1- vaccinated mice. We then addressed the relevance of this tPA/NMDA receptor interaction in brain physiology. Since tPA is thought to modulate some cognitive functions, we tested the immunized mice in a hippocampal-dependent spatial memory task (Y maze) that does not require previous learning. Locomotor activity was not different between naïve, CFA-injected and ATD-NR1-immunized mice. However, spatial memory was clearly impaired in ATD-NR1-immunized mice as compared to control or CFA-injected mice. This deficit was transient, since it disappeared 5 weeks after the last injection. Mice were also tested in an amygdala-dependent social discrimination task. Social memory was also impaired in immunized mice as compared to control or CFA-injected mice. The binding of endogenous tPA to NR1 is thus physiologically relevant, suggesting that the proteolytic activity of tPA is critical for some normal functions of NMDA receptors in the adult brain. In conclusion, this is the first in vivo demonstration that the ability of tPA to potentiate NMDA receptor signalling is critical, both during physiological and pathological brain conditions. Moreover, the immunization protocol developped here opens therapeutic and fundamental scientific interesting perspectives.