Improving stroke outcomes in hyperglycemic mice by modulating tPA/NMDAR signaling to reduce inflammation and hemorrhages.

ABSTRACT: The pharmacological intervention for ischemic stroke hinges on intravenous administration of the recombinant tissue-type plasminogen activator (rtPA, Alteplase/Actilyse) either as a standalone treatment or in conjunction with thrombectomy. However, despite its clinical significance, broader use of rtPA is constrained because of the risk of hemorrhagic transformations (HTs). Furthermore, the presence of diabetes or chronic hyperglycemia is associated with an elevated risk of HT subsequent to thrombolysis. This detrimental impact of tPA on the neurovascular unit in patients with hyperglycemia has been ascribed to its capacity to induce endothelial N-methyl-D-aspartate receptor (NMDAR) signaling, contributing to compromised blood-brain barrier integrity and neuroinflammatory processes. In a mouse model of thromboembolic stroke with chronic hyperglycemia, we assessed the effectiveness of rtPA and N-acetylcysteine (NAC) as thrombolytic agents. We also tested the effect of blocking tPA/NMDAR signaling using a monoclonal antibody, Glunomab. Magnetic resonance imaging, speckle contrast imaging, flow cytometry, and behavioral tasks were used to evaluate stroke outcomes. In hyperglycemic animals, treatment with rtPA resulted in lower recanalization rates and increased HTs. Conversely, NAC treatment reduced lesion sizes while mitigating HTs. After a single administration, either in standalone or combined with rtPA-induced thrombolysis, Glunomab reduced brain lesion volumes, HTs, and neuroinflammation after stroke, translating into improved neurological outcomes. Additionally, we demonstrated the therapeutic efficacy of Glunomab in combination with NAC or as a standalone strategy in chronic hyperglycemic animals. Counteracting tPA-dependent endothelial NMDAR signaling limits ischemic damages induced by both endogenous and exogenous tPA, including HTs and inflammatory processes after ischemic stroke in hyperglycemic animals.

Targeting NMDA Receptors at the Neurovascular Unit: Past and Future Treatments for Central Nervous System Diseases.

The excitatory neurotransmission of the central nervous system (CNS) mainly involves glutamate and its receptors, especially N-methyl-D-Aspartate receptors (NMDARs). These receptors have been extensively described on neurons and, more recently, also on other cell types. Nowadays, the study of their differential expression and function is taking a growing place in preclinical and clinical research. The diversity of NMDAR subtypes and their signaling pathways give rise to pleiotropic functions such as brain development, neuronal plasticity, maturation along with excitotoxicity, blood-brain barrier integrity, and inflammation. NMDARs have thus emerged as key targets for the treatment of neurological disorders. By their large extracellular regions and complex intracellular structures, NMDARs are modulated by a variety of endogenous and pharmacological compounds. Here, we will present an overview of NMDAR functions on neurons and other important cell types involved in the pathophysiology of neurodegenerative, neurovascular, mental, autoimmune, and neurodevelopmental diseases. We will then discuss past and future development of NMDAR targeting drugs, including innovative and promising new approaches.

Phosphorylation of neuroligin-2 by PKA regulates its cell surface abundance and synaptic stabilization.

The trans-synaptic adhesion molecule neuroligin-2 (NL2) is essential for the development and function of inhibitory synapses. NL2 recruits the postsynaptic scaffold protein gephyrin, which, in turn, stabilizes γ-aminobutyric acid type A receptors (GABAARs) in the postsynaptic domain. Thus, the amount of NL2 at the synapse can control synaptic GABAAR concentration to tune inhibitory neurotransmission efficacy. Here, using biochemistry, imaging, single-particle tracking, and electrophysiology, we uncovered a key role for cAMP-dependent protein kinase (PKA) in the synaptic stabilization of NL2. We found that PKA-mediated phosphorylation of NL2 at Ser714 caused its dispersal from the synapse and reduced NL2 surface amounts, leading to a loss of synaptic GABAARs. Conversely, enhancing the stability of NL2 at synapses by abolishing PKA-mediated phosphorylation led to increased inhibitory signaling. Thus, PKA plays a key role in regulating NL2 function and GABA-mediated synaptic inhibition.

DISC1 Regulates Mitochondrial Trafficking in a Miro1-GTP-Dependent Manner.

The disrupted in schizophrenia 1 (DISC1) protein is implicated in major mental illnesses including schizophrenia and bipolar disorder. A key feature of psychiatric disease is aberrant synaptic communication. Correct synaptic transmission is dependent on spatiotemporally regulated energy provision and calcium buffering. This can be achieved by precise distribution of mitochondria throughout the elaborate architecture of the neuron. Central to this process is the calcium sensor and GTPase Miro1, which allows mitochondrial trafficking by molecular motors. While the role of Miro1-calcium binding in mitochondrial transport is well described, far less is known regarding the functions of the two GTPase domains. Here, we investigate the effects of a psychiatric disease-associated mutation in DISC1 on mitochondrial trafficking. We show that this DISC1 mutation impairs Miro1's ability to transport mitochondria. We also demonstrate the necessity of the first Miro1 GTPase domain in determining direction of mitochondrial transport and the involvement of DISC1 in this process. Finally, we describe the effects of mutant DISC1 on positioning of mitochondria at synapses.

Axonal autophagosome maturation defect through failure of ATG9A sorting underpins pathology in AP-4 deficiency syndrome.

Adaptor protein (AP) complexes mediate key sorting decisions in the cell through selective incorporation of transmembrane proteins into vesicles. Little is known of the roles of AP-4, despite its loss of function leading to a severe early onset neurological disorder, AP-4 deficiency syndrome. Here we demonstrate an AP-4 epsilon subunit knockout mouse model that recapitulates characteristic neuroanatomical phenotypes of AP-4 deficiency patients. We show that ATG9A, critical for autophagosome biogenesis, is an AP-4 cargo, which is retained within the trans-Golgi network (TGN) in vivo and in culture when AP-4 function is lost. TGN retention results in depletion of axonal ATG9A, leading to defective autophagosome generation and aberrant expansions of the distal axon. The reduction in the capacity to generate axonal autophagosomes leads to defective axonal extension and de novo generation of distal axonal swellings containing accumulated ER, underlying the impaired axonal integrity in AP-4 deficiency syndrome.Abbreviations: AP: adaptor protein; AP4B1: adaptor-related protein complex AP-4, beta 1; AP4E1: adaptor-related protein complex AP-4, epsilon 1; ATG: autophagy-related; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GFAP: glial fibrillary acidic protein; GOLGA1/Golgin-97/GOLG97: golgi autoantigen, golgin subfamily a, 1; GOLGA2/GM130: golgi autoantigen, golgin subfamily a, 2; HSP: hereditary spastic paraplegia; LC3/MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK8IP1/JIP1: mitogen-acitvated protein kinase 8 interacting protein 1; NEFH/NF200: neurofilament, heavy polypeptide; RBFOX3/NeuN (RNA binding protein, fox-1 homolog [C. elegans] 3); SQSTM1/p62: sequestosome 1; TGN: trans-Golgi network; WIPI2: WD repeat domain, phosphoinositide interacting protein 2.

The adaptor proteins HAP1a and GRIP1 collaborate to activate the kinesin-1 isoform KIF5C.

Binding of motor proteins to cellular cargoes is regulated by adaptor proteins. HAP1 and GRIP1 are kinesin-1 adaptors that have been implicated individually in the transport of vesicular cargoes in the dendrites of neurons. We find that HAP1a and GRIP1 form a protein complex in the brain, and co-operate to activate the kinesin-1 subunit KIF5C in vitro Based upon this co-operative activation of kinesin-1, we propose a modification to the kinesin activation model that incorporates stabilisation of the central hinge region known to be critical to autoinhibition of kinesin-1.

SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling.

GABAA receptors mediate fast inhibitory transmission in the brain, and their number can be rapidly up- or downregulated to alter synaptic strength. Neuroligin-2 plays a critical role in the stabilization of synaptic GABAA receptors and the development and maintenance of inhibitory synapses. To date, little is known about how the amount of neuroligin-2 at the synapse is regulated and whether neuroligin-2 trafficking affects inhibitory signaling. Here, we show that neuroligin-2, when internalized to endosomes, co-localizes with SNX27, a brain-enriched cargo-adaptor protein that facilitates membrane protein recycling. Direct interaction between the PDZ domain of SNX27 and PDZ-binding motif in neuroligin-2 enables membrane retrieval of neuroligin-2, thus enhancing synaptic neuroligin-2 clusters. Furthermore, SNX27 knockdown has the opposite effect. SNX27-mediated up- and downregulation of neuroligin-2 surface levels affects inhibitory synapse composition and signaling strength. Taken together, we show a role for SNX27-mediated recycling of neuroligin-2 in maintenance and signaling of the GABAergic synapse.

Tissue-Type Plasminogen Activator Controlled Corticogenesis Through a Mechanism Dependent of NMDA Receptors Expressed on Radial Glial Cells.

Modifications of neuronal migration during development, including processes that control cortical lamination are associated with functional deficits at adult stage. Here, we report for the first time that the lack of the serine protease tissue-type Plasminogen Activator (tPA), previously characterized as a neuromodulator and a gliotransmitter, leads to an altered cortical lamination in adult. This results in a neuronal migration defect of tPA deficient neurons which are stopped in the intermediate zone at E16. This phenotype is rescued by re-expressing a wild-type tPA in cortical neurons at E14 but not by a tPA that cannot interact with NMDAR. We thus hypothetized that the tPA produced by cortical neuronal progenitors can control their own radial migration through a mechanism dependent of NMDAR expressed at the surface of radial glial cells (RGC). Accordingly, conditional deletion of tPA in neuronal progenitors at E14 or overexpression of a dominant-negative NMDAR that cannot bind tPA in RGC also delayed neuronal migration. Moreover, the lack of tPA lead to an impaired maturation and orientation of RGC. These data provide the first demonstration that the neuronal serine protease tPA is an actor of a proper corticogenesis by its ability to control NMDAR signaling in RGC.

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.

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.

Tissue Plasminogen Activator Expression Is Restricted to Subsets of Excitatory Pyramidal Glutamatergic Neurons.

Although the extracellular serine protease tissue plasminogen activator (tPA) is involved in pathophysiological processes such as learning and memory, anxiety, epilepsy, stroke, and Alzheimer's disease, information about its regional, cellular, and subcellular distribution in vivo is lacking. In the present study, we observed, in healthy mice and rats, the presence of tPA in endothelial cells, oligodendrocytes, mastocytes, and ependymocytes, but not in pericytes, microglial cells, and astrocytes. Moreover, blockage of the axo-dendritic transport unmasked tPA expression in neurons of cortical and hippocampal areas. Interestingly, combined electrophysiological recordings, single-cell reverse transcription polymerase chain reaction (RT-PCR), and immunohistological analyses revealed that the presence of tPA is restricted to subsets of excitatory pyramidal glutamatergic neurons. We further evidenced that tPA is stored in synaptobrevin-2-positive glutamatergic synaptic vesicles. Based on all these data, we propose the existence of tPA-ergic neurons in the mature brain.

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.

Urokinase versus Alteplase for intraventricular hemorrhage fibrinolysis.

Intraventricular hemorrhage (IVH) is the most severe form of stroke with intraventricular fibrinolysis (IVF) as a hopeful treatment. Urokinase (uPA) and tissue-type plasminogen activator (tPA) are used for IVF in Human. No clinical trial has evaluated the differential impact of these two fibrinolytics for IVF. Thus, we decided here to compare the use of these two fibrinolytics in a pre-clinical study. IVH was induced in rats by injection of collagenase type VII within the brain parenchyma followed by an IVF. Rats were randomized to receive uPA, tPA or saline within the ventricle, and cerebrospinal fluid was aspirated. Hematoma and ventricular volumes, brain water contents, inflammation and neurological deficits were measured at day three post-treatments. We also performed in vitro studies, in which neuronal cultures were subjected to an excitotoxic paradigm in the presence of either uPA or tPA. In the IVH model, we showed that although both uPA and tPA led to reduced ventricular volumes, only uPA significantly improved functional recovery. These results could be explained by the fact that uPA, in contrast of tPA, fails to promote inflammatory processes and neurotoxicity. Our study provides evidence supporting the use of uPA for fibrinolysis of IVH. A clinical trial could be warranted if tPA failed to improve outcomes in human IVH.

GluN2D subunit-containing NMDA receptors control tissue plasminogen activator-mediated spatial memory.

Tissue plasminogen activator (tPA) is a serine protease with pleiotropic actions in the CNS, such as synaptic plasticity and neuronal death. Some effects of tPA require its interaction with the GluN1 subunit of the NMDA receptor (NMDAR), leading to a potentiation of NMDAR signaling. We have reported previously that the pro-neurotoxic effect of tPA is mediated through GluN2D subunit-containing NMDARs. Thus, the aim of the present study was to determine whether GluN2D subunit-containing NMDARs drive tPA-mediated cognitive functions. To address this issue, a strategy of immunization designed to prevent the in vivo interaction of tPA with NMDARs and GluN2D-deficient mice were used in a set of behavioral tasks. Altogether, our data provide the first evidence that tPA influences spatial memory through its preferential interaction with GluN2D subunit-containing NMDARs.

Memantine improves safety of thrombolysis for stroke.

BACKGROUND AND PURPOSE: Despite side effects including N-methyl-d-aspartate-mediated neurotoxicity, recombinant tissue-type plasminogen activator (rtPA) remains the only approved acute treatment for ischemic stroke. Memantine, used for treatment of Alzheimer disease, is an antagonist for N-methyl-d-aspartate receptors. We investigated whether memantine could be used as a neuroprotective adjunct therapy for rtPA-induced thrombolysis after stroke. METHODS: In vitro N-methyl-d-aspartate exposure, oxygen and glucose deprivation, and N-methyl-d-aspartate-mediated calcium videomicroscopy experiments were performed on murine cortical neurons in the presence of rtPA and memantine. The therapeutic safety of rtPA and memantine coadministration was evaluated in mouse models of thrombotic stroke and intracerebral hemorrhage. Ischemic and hemorrhagic volumes were assessed by MRI and neurological evaluation was performed by the string test and automated gait analysis. RESULTS: Our in vitro observations showed that memantine was able to prevent the proneurotoxic effects of rtPA in cultured cortical neurons. Although memantine did not alter the fibrinolytic activity of rtPA, our in vivo observations revealed that it blunted the noxious effects of delayed thrombolysis on lesion volumes and neurological deficits after ischemic stroke. In addition, memantine rescued rtPA-induced decrease in survival rate after intracerebral hemorrhage. CONCLUSIONS: Memantine could be used as an adjunct therapy to improve the safety of thrombolysis.

Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo.

BACKGROUND: Tissue plasminogen activator (tPA) exerts multiple functions in the central nervous system, depending on the partner with which it interacts. In particular, tPA acts as a positive neuromodulator of N-methyl-D-aspartate glutamatergic receptors (NMDAR). At the molecular level, it has been proposed that the pro-neurotoxicity mediated by tPA might occur through extrasynaptic NMDAR containing the GluN2D subunit. Thus, selective antagonists targeting tPA/GluN2D-containing NMDAR signaling would be of interest to prevent noxious effects of tPA. RESULTS: Here, we compared three putative antagonists of GluN2D-containing NMDAR and we showed that the new compound UBP145 ((2R*,3S*)-1-(9-bromophenan-threne-3-carbonyl)piperazine-2,3-dicarboxylic acid) is far more selective for GluN2D subunits than memantine and PPDA (phenanthrene derivative (2S*, 3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid). Indeed, in vitro, in contrast to the two other compounds, UBP145 prevented NMDA toxicity only in neurons expressing GluN2D (ie, in cortical but not hippocampal neurons). Furthermore, in cultured cortical neurons, UBP145 fully prevented the pro-excitotoxic effect of tPA. In vivo, we showed that UBP145 potently prevented the noxious action of exogenous tPA on excitotoxic damages. Moreover, in a thrombotic stroke model in mice, administration of UBP145 prevented the deleterious effect of late thrombolysis by tPA. CONCLUSIONS: In conclusion, tPA exerts noxious effects on neurons by acting on GluN2D-containing NMDAR and pharmacological antagonists of GluN2D-containing NMDAR could be used to prevent the ability of tPA to promote neurotoxicity.