Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival.

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

Distant Space Processing is Controlled by tPA-dependent NMDA Receptor Signaling in the Entorhinal Cortex.

In humans, spatial cognition and navigation impairments are a frequent situation during physiological and pathological aging, leading to a dramatic deterioration in the quality of life. Despite the discovery of neurons with location-specific activity in rodents, that is, place cells in the hippocampus and later on grid cells in the entorhinal cortex (EC), the molecular mechanisms underlying spatial cognition are still poorly known. Our present data bring together in an unusual combination 2 molecules of primary biological importance: a major neuronal excitatory receptor, N-methyl-D-aspartate receptor (NMDAR), and an extracellular protease, tissue plasminogen activator (tPA), in the control of spatial navigation. By using tPA-deficient mice and a structure-selective pharmacological approach, we demonstrate that the tPA-dependent NMDAR signaling potentiation in the EC plays a key and selective role in the encoding and the subsequent use of distant landmarks during spatial learning. We also demonstrate that this novel function of tPA in the EC is reduced during aging. Overall, these results argue for the concept that encoding of proximal versus distal landmarks is mediated not only by different anatomical pathways but also by different molecular mechanisms, with the tPA-dependent potentiation of NMDAR signaling in the EC that plays an important role.

Neuroendothelial NMDA receptors as therapeutic targets in experimental autoimmune encephalomyelitis.

Multiple sclerosis is among the most common causes of neurological disability in young adults. Here we provide the preclinical proof of concept of the benefit of a novel strategy of treatment for multiple sclerosis targeting neuroendothelial N-methyl-D-aspartate glutamate receptors. We designed a monoclonal antibody against N-methyl-D-aspartate receptors, which targets a regulatory site of the GluN1 subunit of N-methyl-D-aspartate receptor sensitive to the protease tissue plasminogen activator. This antibody reverted the effect of tissue plasminogen activator on N-methyl-D-aspartate receptor function without affecting basal N-methyl-D-aspartate receptor activity (n = 21, P < 0.01). This antibody bound N-methyl-D-aspartate receptors on the luminal surface of neurovascular endothelium in human tissues and in mouse, at the vicinity of tight junctions of the blood-spinal cord barrier. Noteworthy, it reduced human leucocyte transmigration in an in vitro model of the blood-brain barrier (n = 12, P < 0.05). When injected during the effector phase of MOG-induced experimental autoimmune encephalomyelitis (n = 24), it blocked the progression of neurological impairments, reducing cumulative clinical score (P < 0.001) and mean peak score (P < 0.001). This effect was observed in wild-type animals but not in tissue plasminogen activator knock-out animals (n = 10). This therapeutic effect was associated to a preservation of the blood-spinal cord barrier (n = 6, P < 0.001), leading to reduced leucocyte infiltration (n = 6, P < 0.001). Overall, this study unveils a critical function of endothelial N-methyl-D-aspartate receptor in multiple sclerosis, and highlights the therapeutic potential of strategies targeting the protease-regulated site of N-methyl-D-aspartate receptor.

New thrombolytic strategy providing neuroprotection in experimental ischemic stroke: MMP10 alone or in combination with tissue-type plasminogen activator.

AIMS: Early reperfusion with tissue-type plasminogen activator (tPA) is an effective therapeutic strategy to treat acute ischemic stroke, but only 1/3 of tPA-treated patients recover and are free from disability. tPA has also shown neurotoxicity in experimental models of cerebral ischemia. Considering that MMP-10 improves stroke injury, we have examined the therapeutic and protective effect of MMP10 and tPA/MMP10 as clot-dissolving and neuroprotective agent in an experimental model of ischemic stroke and studied in vitro the molecular pathways involved in MMP10-mediated effects. METHODS AND RESULTS: Cerebral ischemia was induced by the local injection of thrombin into the middle cerebral artery followed by reperfusion with MMP10 (6.5 µg/kg) and tPA (10 mg/kg) alone or in combination with MMP10. Cell cultures were also performed to determine the effect of MMP10 and tPA/MMP10 on brain endothelial cells and neurons. tPA/MMP10 significantly reduced the infarct size in the ischemic stroke model compared with tPA alone (P < 0.05). In vitro, MMP10 reduced the tPA-promoted endothelial ionic permeability, preserved the expression of claudin-5 and decreased ERK1/2 activation. Moreover, combination of tPA/MMP10 prevented tPA-mediated neuronal excitotoxicity and calcium influx. These effects were reversed by blocking MMP10 activity with a monoclonal antibody. CONCLUSION: These results show that MMP10, either alone or in combination with tPA, might represent a new strategy for thrombolysis in ischemic stroke, providing higher protection against cerebrovascular damage.

Tissue-type plasminogen activator controls neuronal death by raising surface dynamics of extrasynaptic NMDA receptors.

N-methyl-d-aspartate receptors (NMDARs) are ion channels whose synaptic versus extrasynaptic localization critically influences their functions. This distribution of NMDARs is highly dependent on their lateral diffusion at the cell membrane. Each obligatory subunit of NMDARs (GluN1 and GluN2) contains two extracellular clamshell-like domains with an agonist-binding domain and a distal N-terminal domain (NTD). To date, the roles and dynamics of the NTD of the GluN1 subunit in NMDAR allosteric signaling remain poorly understood. Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator (tPA), well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic NMDARs. This process explains the previously reported ability of tPA to promote NMDAR-mediated calcium influx. In parallel, we developed a monoclonal antibody capable of specifically blocking the interaction of tPA with the NTD of the GluN1 subunit of NMDAR. Using this original approach, we demonstrate that the tPA binds the NTD of the GluN1 subunit at a lysine in position 178. Accordingly, when applied to mouse neurons, our selected antibody (named Glunomab) leads to a selective reduction of the tPA-mediated surface dynamics of extrasynaptic NMDARs, subsequent signaling and neurotoxicity, both in vitro and in vivo. Altogether, we demonstrate that the tPA is a ligand of the NTD of the obligatory GluN1 subunit of NMDAR acting as a modulator of their dynamic distribution at the neuronal surface and subsequent signaling.

Impacts of tissue-type plasminogen activator (tPA) on neuronal survival.

Tissue-type plasminogen activator (tPA) a serine protease is constituted of five functional domains through which it interacts with different substrates, binding proteins, and receptors. In the last years, great interest has been given to the clinical relevance of targeting tPA in different diseases of the central nervous system, in particular stroke. Among its reported functions in the central nervous system, tPA displays both neurotrophic and neurotoxic effects. How can the protease mediate such opposite functions remain unclear but several hypotheses have been proposed. These include an influence of the degree of maturity and/or the type of neurons, of the level of tPA, of its origin (endogenous or exogenous) or of its form (single chain tPA versus two chain tPA). In this review, we will provide a synthetic snapshot of our current knowledge regarding the natural history of tPA and discuss how it sustains its pleiotropic functions with focus on excitotoxic/ischemic neuronal death and neuronal survival.