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Background: Regulation of cerebral blood flow (CBF) directly influence brain functions and dysfunctions and involves complex mechanisms, including neurovascular coupling (NVC). It was suggested that the serine protease tissue-type plasminogen activator (tPA) could control CNV induced by whisker stimulation in rodents, through its action on N-methyl-d-Aspartate receptors (NMDARs). However, the origin of tPA and the location and mechanism of its action on NMDARs in relation to CNV remained debated. Methods: Here, we answered these issues using tPANull mice, conditional deletions of either endothelial tPA (VECad-CreΔtPA) or endothelial GluN1 subunit of NMDARs (VECad-CreΔGluN1), parabioses between wild-type and tPANull mice, hydrodynamic transfection-induced deletion of liver tPA, hepatectomy and pharmacological approaches. Results: We thus demonstrate that physiological concentrations of vascular tPA, achieved by the bradykinin type 2 receptors-dependent production and release of tPA from liver endothelial cells, promote NVC, through a mechanism dependent on brain endothelial NMDARs. Conclusions: These data highlight a new mechanism of regulation of NVC involving both endothelial tPA and NMDARs. [ABSTRACT FROM AUTHOR]

NMDA type glutamate receptors (NMDAR5) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GC5) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca2+ influx in immature GCs before synapse formation. The VEGF receptor FIki forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flkl/NR2B coclusters on the cell surface increases. Stimulation of FIki by VEGF activates Src-family kinases. which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca2+ influx in GC5. These findings identify VEGF as a modulator of NMDAR5 before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR). [ABSTRACT FROM AUTHOR]

Cessation of chronic ethanol consumption can increase the sensitivity of the brain to excitotoxic damages. Cannabinoids have been proposed as neuroprotectants in different models of neuronal injury, but their effect have never been investigated in a context of excitotoxicity after alcohol cessation. Here we examined the effects of the pharmacological activation/inhibition of the endocannabinoid system in an in vitro model of chronic ethanol exposure and withdrawal followed by an excitotoxic challenge. Ethanol withdrawal increased N-methyl-D-aspartate (NMDA)-evoked neuronal death, probably by altering the ratio between GluN2A and GluN2B NMDA receptor subunits. The stimulation of the endocannabinoid system with the cannabinoid agonist HU-210 decreased NMDA-induced neuronal death exclusively in ethanol-withdrawn neurons. This neuroprotection could be explained by a decrease in NMDA-stimulated calcium influx after the administration of HU-210, found exclusively in ethanol-withdrawn neurons. By contrast, the inhibition of the cannabinoid system with the CB1 receptor antagonist rimonabant (SR141716) during ethanol withdrawal increased death of ethanol-withdrawn neurons without any modification of NMDA-stimulated calcium influx. Moreover, chronic administration of rimonabant increased NMDA-stimulated toxicity not only in withdrawn neurons, but also in control neurons. In summary, we show for the first time that the stimulation of the endocannabinoid system is protective against the hyperexcitability developed during alcohol withdrawal. By contrast, the blockade of the endocannabinoid system is highly counterproductive during alcohol withdrawal. [ABSTRACT FROM AUTHOR]

Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (KD=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment.Journal of Cerebral Blood Flow & Metabolism (2008) 28, 1212–1221; doi:10.1038/jcbfm.2008.14; published online 12 March 2008 [ABSTRACT FROM AUTHOR]

Tissue-plasminogen activator (t-PA) is now available for the treatment of thrombo-embolic stroke but adverse effects have been reported in some patients, particularly hemorrhaging. In contrast, the results of animal studies have indicated that t-PA could increase neuronal damage after focal cerebral ischemia. Here we report for the first time that t-PA potentiates signaling mediated by glutamatergic receptors by modifying the properties of the N-methyl-D-aspartate (NMDA) receptor. When depolarized, cortical neurons release bio-active t-PA that interacts with and cleaves the NR1 subunit of the NMDA receptor. Moreover, the treatment with recombinant t-PA leads to a 37% increase in NMDA-stimulated fura-2 fluorescence, which may reflect an increased NMDA-receptor function. These results were confirmed in vivo by the intrastriatal injection of recombinant-PA, which potentiated the excitotoxic lesions induced by NMDA. These data provide insight into the regulation of NMDA-receptor-mediated signaling and could initiate therapeutic strategies to improve the efficacy of t-PA treatment in man. [ABSTRACT FROM AUTHOR]

Fine-tuning of NMDA glutamatergic receptor signalling strategically controls crucial brain functions. This process depends on several ligands and modulators, one of which unexpectedly includes the serine protease tissue-type plasminogen activator (tPA). In vitro, tPA increases NMDA-receptor-mediated calcium influx by interacting with, and then cleaving, the NR1 subunit within its N-terminal domain. Owing to lack of in vivo evidence of the relevance and contribution of this mechanism in physiological and pathological brain processes, active immunisation was developed here in mice, to allow transient and specific prevention of the interaction of tPA with the NR1 subunit. Immunisation significantly reduced the severity of ischemic and excitotoxic insults in the mouse brain. Cognitive function was altered in some, but not all behavioural tasks affected in tPA-deficient mice. Our data demonstrate that in vivo, tPA controls neurotoxicity and the encoding of novel spatial experiences by binding to and cleaving the NMDA receptor NR1 subunit. Interesting therapeutic possibilities for several brain pathologies that involve excitotoxicity may now be envisaged. [ABSTRACT FROM AUTHOR]

Tissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-D-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59-64). Here, we show that tPA forms a direct complex with the aminoterminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the ATD of NR1 and to modulate the NMDA receptor function. [ABSTRACT FROM AUTHOR]