Functional Occurrence of the Interaction of Tissue Plasminogen Activator With the NR1 Subunit of N -Methyl- d -Aspartate Receptors During Stroke

Abstract

Background and Purpose-Despite its fibrinolytic effect, tissue-type plasminogen activator (tPA) displays deleterious effects in the brain, including proexcitotoxicity, that can reduce the overall benefit from thrombolysis during stroke. We have proposed that tPA potentiates excitotoxicity by interacting with and cleaving the aminoterminal end of the NR1 subunit of N-methyl-D-aspartate receptors, leading to an increased calcium influx, Erk1/2 activation, and neurotoxicity. Because this mechanism is debated, our aim was to demonstrate its in vivo occurrence and relevance. Because tPA is released under ischemic conditions, we hypothesized that if it indeed processes NR1, then the released fragment should reactivate the immune system in animals that had been immunized long before with recombinant aminoterminal end of the NR1. This effect should be exacerbated in ischemic animals thrombolysed with recombinant tPA. Methods-At a time when specific antibodies could not be detected any longer, mice previously vaccinated with recombinant aminoterminal end of the NR1 were subjected to thromboembolic stroke induced by injecting thrombin in the middle cerebral artery alone or with intravenous thrombolysis. Results-Stroke performed 1 year after active immunization induced the reappearance of antibodies against the aminoterminal end of the NR1 in the plasma, an effect significantly increased when ischemia was followed by recombinant tPA-induced reperfusion. Moreover, immunization preventing the interaction of tPA with aminoterminal end of the NR1 reduced ischemic brain damages and extended the therapeutic window of tPA-induced thrombolysis. Conclusion-We

GluN2D Subunit-Containing NMDA Receptors Control Tissue Plasminogen Activator-Mediated Spatial Memory

“…Importantly, these antibodies do not alter NMDA-induced basal neurotransmission (toxicity, calcium influx), but they specifically block the potentiating effect of tPA on these receptors. Accordingly, we have shown that this strategy of blockage of the tPA/GluN1 interaction leads to behavioral deficits in Swiss mice and also prevents the pro-neurotoxicity of tPA both in vitro and in vivo (Benchenane et al, 2007;Macrez et al, 2010). In addition, in recent studies, we evidenced both in vitro and in vivo that the pro-neurotoxic effect of tPA was mediated by GluN2D subunitcontaining NMDAR (Baron et al, 2010;Jullienne et al, 2011), suggesting that, although tPA interacts with the GluN1 subunit, this mechanism occurs preferentially in GluN2D subunitcontaining NMDAR.…”

“…Because we have identified the exact location of the interaction of tPA within GluN1 in cultured cortical neurons (Fernández-Monreal et al, 2004), we performed active immunization leading to the production of antibodies recognizing the ATD of GluN1 (Benchenane et al, 2007;Macrez et al, 2010). Importantly, these antibodies do not alter NMDA-induced basal neurotransmission (toxicity, calcium influx), but they specifically block the potentiating effect of tPA on these receptors.…”

“…Interestingly, a strategy preventing in vivo this tPA/GluN1 interaction in mice [active immunization against the amino-terminal domain (ATD) of the NMDAR GluN1 subunit] led to a reduced sensitivity to ischemic and excitotoxic neuronal death and impaired cognitive functions, such as spatial memory deficits (Benchenane et al, 2007;Macrez et al, 2010). More recently, we have evidenced by pharmacological and molecular approaches that tPA selectively promotes NMDAR signaling and subsequent neurotoxicity through GluN2D subunit-containing NMDARs (Baron et al, 2010;Jullienne et al, 2011).…”

The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration

“…In neurons, tPA can trigger cell signaling through a direct interaction with the NMDA-R, independently of LRP1 (Nicole et al, 2001;Macrez et al, 2010;Ng et al, 2012;Mantuano et al, 2013). To determine whether the NMDA-R signals in response to tPA, independently of LRP1 in Schwann cells, we treated Schwann cells with 12 nM EI-tPA in the presence or absence of RAP for up to 60 min.…”

“…LRP1 ligands have been implicated in neuronal survival (Hayashi et al, 2007;Fuentealba et al, 2009) and in neurite outgrowth (Mantuano et al, 2008a,b;Shi et al, 2009;Yoon et al, 2013). Unlike other LRP1 ligands, tPA interacts directly with the NMDA-R, independently of LRP1, triggering cell signaling, albeit with delayed kinetics (Nicole et al, 2001;Macrez et al, 2010;Ng et al, 2009;Mantuano et al, 2013). NR1 subunit cleavage is reported to be involved in the mechanism by which tPA activates the NMDA-R; however, NR1 cleavage might not be necessary (Nicole et al, 2001;Mantuano et al, 2008a,b).…”