RESUMEN
Tissue plasminogen activator (tPA) is a serine protease expressed in several brain regions and reported to be involved in the control of emotional and cognitive functions. Nevertheless, little is known about the structure-function relationships of these tPA-dependent behaviors. Here, by using a new model of constitutive tPA-deficient mice (tPAnull), we first show that tPA controls locomotor activity, spatial cognition and anxiety. To investigate the brain structures involved in these tPA-dependent behavioral phenotypes, we next generated tPAflox mice allowing conditional tPA deletion (cKO) following stereotaxic injections of adeno-associated virus driving Cre-recombinase expression (AAV-Cre-GFP). We demonstrate that tPA removal in the dentate gyrus of the hippocampus induces hyperactivity and partial spatial memory deficits. Moreover, the deletion of tPA in the central nucleus of the amygdala, but not in the basolateral nucleus, induces hyperactivity and reduced anxiety-like level. Importantly, we prove that these behaviors depend on the tPA present in the adult brain and not on neurodevelopmental disorders. Also, interestingly, our data show that tPA from Protein kinase-C delta-positive (PKCδ) GABAergic interneurons of the lateral/ capsular part of adult mouse central amygdala controls emotional functions through neuronal activation of the medial central amygdala. Together, our study brings new data about the critical central role of tPA in behavioral modulations in adult mice.
Asunto(s)
Núcleo Amigdalino Central , Proteína Quinasa C-delta/metabolismo , Animales , Ansiedad , Trastornos de Ansiedad , Núcleo Amigdalino Central/metabolismo , Neuronas GABAérgicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
BACKGROUND: Perineuronal nets (PNNs) are specialized extracellular matrix structures mainly found around fast-spiking parvalbumin (FS-PV) interneurons. In the adult, their degradation alters FS-PV-driven functions, such as brain plasticity and memory, and altered PNN structures have been found in neurodevelopmental and central nervous system disorders such as Alzheimer's disease, leading to interest in identifying targets able to modify or participate in PNN metabolism. The serine protease tissue-type plasminogen activator (tPA) plays multifaceted roles in brain pathophysiology. However, its cellular expression profile in the brain remains unclear and a possible role in matrix plasticity through PNN remodeling has never been investigated. RESULT: By combining a GFP reporter approach, immunohistology, electrophysiology, and single-cell RT-PCR, we discovered that cortical FS-PV interneurons are a source of tPA in vivo. We found that mice specifically lacking tPA in FS-PV interneurons display denser PNNs in the somatosensory cortex, suggesting a role for tPA from FS-PV interneurons in PNN remodeling. In vitro analyses in primary cultures of mouse interneurons also showed that tPA converts plasminogen into active plasmin, which in turn, directly degrades aggrecan, a major structural chondroitin sulfate proteoglycan (CSPG) in PNNs. CONCLUSIONS: We demonstrate that tPA released from FS-PV interneurons in the central nervous system reduces PNN density through CSPG degradation. The discovery of this tPA-dependent PNN remodeling opens interesting insights into the control of brain plasticity.
Asunto(s)
Parvalbúminas , Activador de Tejido Plasminógeno , Agrecanos/metabolismo , Animales , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Matriz Extracelular/metabolismo , Fibrinolisina/metabolismo , Interneuronas/fisiología , Ratones , Parvalbúminas/metabolismo , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/metabolismoRESUMEN
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.
Asunto(s)
Corteza Cerebral/embriología , Células Ependimogliales/metabolismo , Neurogénesis/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Movimiento Celular/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiologíaRESUMEN
Hyperfibrinolysis is a systemic condition occurring in various clinical disorders such as trauma, liver cirrhosis, and leukemia. Apart from increased bleeding tendency, the pathophysiological consequences of hyperfibrinolysis remain largely unknown. Our aim was to develop an experimental model of hyperfibrinolysis and to study its effects on the homeostasis of the blood-brain barrier (BBB). We induced a sustained hyperfibrinolytic state in mice by hydrodynamic transfection of a plasmid encoding for tissue-type plasminogen activator (tPA). As revealed by near-infrared fluorescence imaging, hyperfibrinolytic mice presented a significant increase in BBB permeability. Using a set of deletion variants of tPA and pharmacological approaches, we demonstrated that this effect was independent of N-methyl-D-aspartate receptor, low-density lipoprotein-related protein, protease-activated receptor-1, or matrix metalloproteinases. In contrast, we provide evidence that hyperfibrinolysis-induced BBB leakage is dependent on plasmin-mediated generation of bradykinin and subsequent activation of bradykinin B2 receptors. Accordingly, this effect was prevented by icatibant, a clinically available B2 receptor antagonist. In agreement with these preclinical data, bradykinin generation was also observed in humans in a context of acute pharmacological hyperfibrinolysis. Altogether, these results suggest that B2 receptor blockade may be a promising strategy to prevent the deleterious effects of hyperfibrinolysis on the homeostasis of the BBB.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Bradiquinina/fisiología , Permeabilidad Capilar/fisiología , Fibrinolisina/fisiología , Fibrinólisis/fisiología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Bradiquinina/metabolismo , Antagonistas del Receptor de Bradiquinina B2/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/genética , Fibrinolisina/metabolismo , Fibrinólisis/efectos de los fármacos , Fibrinólisis/genética , Hidrodinámica , Ratones , Ratones Transgénicos , Receptor de Bradiquinina B2/genética , Receptor de Bradiquinina B2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Although tissue plasminogen activator (tPA) is known to promote neuronal remodeling in the CNS, no mechanism of how this plastic function takes place has been reported so far. We provide here in vitro and in vivo demonstrations that this serine protease neutralizes inhibitory chondroitin sulfate proteoglycans (CSPGs) by promoting their degradation via the direct activation of endogenous type 4 disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4). Accordingly, in a model of compression-induced spinal cord injury (SCI) in rats, we found that administration of either tPA or its downstream effector ADAMTS-4 restores the tPA-dependent activity lost after the SCI and thereby, reduces content of CSPGs in the spinal cord, a cascade of events leading to an improved axonal regeneration/sprouting and eventually long term functional recovery. This is the first study to reveal a tPA-ADAMTS-4 axis and its function in the CNS. It also raises the prospect of exploiting such cooperation as a therapeutic tool for enhancing recovery after acute CNS injuries.
Asunto(s)
Proteínas ADAM/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Procolágeno N-Endopeptidasa/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Activador de Tejido Plasminógeno/farmacología , Proteína ADAMTS4 , Animales , Axones/efectos de los fármacos , Axones/fisiología , Células Cultivadas , Femenino , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neurocano , Neuropéptidos/farmacología , Inhibidor 1 de Activador Plasminogénico/farmacología , Ratas , Ratas Wistar , Recuperación de la Función , Inhibidores de Serina Proteinasa/farmacología , Serpinas/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Compresión de la Médula Espinal/tratamiento farmacológico , Compresión de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Activador de Tejido Plasminógeno/antagonistas & inhibidores , NeuroserpinaRESUMEN
Recombinant tissue-type plasminogen activator (r-tPA/Actilyse) stands as the prevailing pharmacological solution for treating ischemic stroke patients, of whom because their endogenous circulating tPA alone is not sufficient to rescue reperfusion and to promote favorable outcome. Beyond the tPA contributed by circulating endothelial cells and hepatocytes, neurons also express tPA, sparking debates regarding its impact on neuronal fate ranging from pro-survival to neurotoxic properties. In order to investigate the role of neuronal tPA during brain injuries, we developed models leading to its conditional deletion in neurons, employing AAV9-pPlat-GFP and AAV9-pPlat-Cre-GFP along with tPA floxed mice. These models were subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity or thromboembolic ischemic stroke in mice. Initially, we established that our AAV9 constructs selectively transduce neurons, bypassing other brain cell types. Subsequently, we demonstrated that tPA-expressing neurons exhibit greater resistance against NMDA-induced excitotoxicity compared to tPA negative neurons. The targeted removal of tPA in neurons heightened the susceptibility of these neurons to cell death and prevented a paracrine neurotoxic effect on tPA non-expressing neurons. Under ischemic conditions, the self-neuroprotective influence of tPA encompassed both excitatory (GFP+/Tbr1+) and inhibitory (GFP+/GABA+) neurons. Our data indicate that endogenous neuronal tPA is a protective or deleterious factor against neuronal death in an excitotoxic/ischemic context, depending on whether it acts as an autocrine or a paracrine mediator.
Asunto(s)
Accidente Cerebrovascular Isquémico , Síndromes de Neurotoxicidad , Animales , Ratones , Células Endoteliales , N-Metilaspartato/farmacología , Neuronas , Activador de Tejido PlasminógenoRESUMEN
Owing to its ability to generate the clot-dissolving protease plasmin, tissue plasminogen activator (tPA) is the only approved drug for the acute treatment of ischemic stroke. However, tPA also promotes hemorrhagic transformation and excitotoxic events. High mobility group box-1 protein (HMGB-1) is a non-histone transcription factor and a pro-inflammatory cytokine, which has also been shown to bind to both tPA and plasminogen. We thus investigated the cellular and molecular effects through which HMGB-1 could influence the vascular and parenchymal effects of tPA during ischemia. We demonstrate that HMGB-1 not only increases clot lysis by tPA, but also reduces the passage of vascular tPA across the blood-brain barrier, as well as tPA-driven leakage of the blood-brain barrier. In addition, HMGB-1 prevents the pro-neurotoxic effect of tPA, by blocking its interaction with N-methyl-D-aspartate (NMDA) receptors and the attendant potentiation of NMDA-induced neuronal Ca²âº influx. In conclusion, we show in vitro that HMGB-1 can promote the beneficial effects of tPA while counteracting its deleterious properties. We suggest that derivatives of HMGB-1, devoid of pro-inflammatory properties, could be used as adjunctive therapies to improve the overall benefit of tPA-mediated thrombolysis following stroke.
Asunto(s)
Fibrinólisis/efectos de los fármacos , Proteína HMGB1/farmacología , Activador de Tejido Plasminógeno/farmacología , Animales , Biomarcadores/sangre , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Calcio/metabolismo , Bovinos , Células Cultivadas , Técnicas de Cocultivo , Dominios HMG-Box , Proteína HMGB1/metabolismo , Humanos , Ratones , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Activador de Tejido Plasminógeno/metabolismoRESUMEN
The discovery of the neuronal expression of the serine protease tissue-type plasminogen activator (tPA) has opened new avenues of research, with important implications in the physiopathology of the central nervous system. For example, the interaction of tPA with synaptic receptors (NMDAR, LRP1, Annexin II, and EGFR) and its role in the maturation of BDNF have been reported to influence synaptic plasticity and neuronal survival. However, the mechanisms regulating the neuronal trafficking of tPA are unknown. Here, using high-resolution live cell imaging and a panel of innovative genetic approaches, we first unmasked the dynamic characteristics of the dendritic and axonal trafficking of tPA-containing vesicles under different paradigms of neuronal activation or inhibition. We then report a constitutive exocytosis of tPA- and VAMP2-positive vesicles, dramatically increased in conditions of neuronal activation, with a pattern which was mainly dendritic and thus post-synaptic. We also observed that the synaptic release of tPA led to an increase of the exocytosis of VGlut1 positive vesicles containing glutamate. Finally, we described alterations of the trafficking and exocytosis of neuronal tPA in cultured cortical neurons prepared from tau-22 transgenic mice (a preclinical model of Alzheimer's disease (AD)). Altogether, these data provide new insights about the neuronal trafficking of tPA, contributing to a better knowledge of the tPA-dependent brain functions and dysfunctions.
Asunto(s)
Ácido Glutámico , Activador de Tejido Plasminógeno , Ratones , Animales , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismo , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Acoplamiento Neurovascular , Activador de Tejido Plasminógeno , Ratones , Animales , N-Metilaspartato/farmacología , Células Endoteliales/metabolismo , Encéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Ratones Noqueados , Hígado/metabolismoRESUMEN
BACKGROUND: In the vascular compartment, the serine protease tissue-type plasminogen activator (tPA) promotes fibrinolysis, justifying its clinical use against vasculo-occlusive diseases. Accumulating evidence shows that circulating tPA (endogenous or exogenous) also controls brain physiopathological processes, like cerebrovascular reactivity, blood-brain barrier (BBB) homeostasis, inflammation and neuronal fate. Whether this occurs by direct actions on parenchymal cells and/or indirectly via barriers between the blood and the central nervous system (CNS) remains unclear. Here, we postulated that vascular tPA can reach the brain parenchyma via the blood-cerebrospinal fluid barrier (BCSFB), that relies on choroid plexus (CP) epithelial cells (CPECs). METHODS: We produced various reporter fusion proteins to track tPA in primary cultures of CPECs, in CP explants and in vivo in mice. We also investigated the mechanisms underlying tPA transport across the BCSFB, with pharmacological and molecular approaches. RESULTS: We first demonstrated that tPA can be internalized by CPECs in primary cultures and in ex vivo CPs explants. In vivo, tPA can also be internalized by CPECs both at their basal and apical sides. After intra-vascular administration, tPA can reach the cerebral spinal fluid (CSF) and the brain parenchyma. Further investigation allowed discovering that the transcytosis of tPA is mediated by Low-density-Lipoprotein Related Protein-1 (LRP1) expressed at the surface of CPECs and depends on the finger domain of tPA. Interestingly, albumin, which has a size comparable to that of tPA, does not normally cross the CPs, but switches to a transportable form when grafted to the finger domain of tPA. CONCLUSIONS: These findings provide new insights on how vascular tPA can reach the brain parenchyma, and open therapeutic avenues for CNS disorders.
Asunto(s)
Plexo Coroideo , Activador de Tejido Plasminógeno , Albúminas/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Plexo Coroideo/metabolismo , Lipoproteínas/metabolismo , RatonesRESUMEN
Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular , Autofagia , Isquemia Encefálica/tratamiento farmacológico , Glucosa/farmacología , Humanos , Hipoxia , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Oxígeno/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Accidente Cerebrovascular/tratamiento farmacológico , Serina-Treonina Quinasas TOR/metabolismo , Terapia Trombolítica , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Tissue-type plasminogen activator (tPA) is the only drug approved for the acute treatment of ischemic stroke but with two faces in the disease: beneficial fibrinolysis in the vasculature and damaging effects on the neurovascular unit and brain parenchyma. To improve this profile, we developed a novel strategy, relying on antibodies targeting the proneurotoxic effects of tPA. METHODS: After production and characterization of antibodies (αATD-NR1) that specifically prevent the interaction of tPA with the ATD-NR1 of N-methyl-d-aspartate receptors, we have evaluated their efficacy in a model of murine thromboembolic stroke with or without recombinant tPA-induced reperfusion, coupled to MRI, near-infrared fluorescence imaging, and behavior assessments. RESULTS: In vitro, αATD-NR1 prevented the proexcitotoxic effect of tPA without altering N-methyl-d-aspartate-induced neurotransmission. In vivo, after a single administration alone or with late recombinant tPA-induced thrombolysis, antibodies dramatically reduced brain injuries and blood-brain barrier leakage, thus improving long-term neurological outcome. CONCLUSIONS: Our strategy limits ischemic damages and extends the therapeutic window of tPA-driven thrombolysis. Thus, the prospect of this immunotherapy is an extension of the range of treatable patients.
Asunto(s)
Anticuerpos/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Receptores de N-Metil-D-Aspartato/inmunología , Accidente Cerebrovascular/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Anticuerpos/inmunología , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Isquemia Encefálica/inmunología , Fibrinolíticos/inmunología , Ratones , Accidente Cerebrovascular/inmunología , Activador de Tejido Plasminógeno/inmunologíaRESUMEN
BACKGROUND INFORMATION: Despite its pro-fibrinolytic activity, tPA (tissue plasminogen activator) is a serine protease known to influence a number of physiological and pathological functions in the central nervous system. Accordingly, tPA was reported to mediate some of its functions in the central nervous system through NMDA (N-methyl-D-aspartate) receptors, LRP (low-density lipoprotein receptor-related protein) or annexin II. RESULTS: We provide here both in vitro and in vivo evidence that tPA could mediate proteolysis and subsequent delocalization of neuronal nitric oxide synthase, thereby reducing endogenous neuronal nitric oxide release. We also demonstrate that although this effect is independent of NMDA receptors, LRP signalling and calpain-mediated proteolysis, it is dependent on the ability of tPA to promote the conversion of plasminogen into plasmin. CONCLUSION: Altogether, these results demonstrate a new function for tPA in the central nervous system, which most likely contributes to its pleiotropic functions.
Asunto(s)
Fibrinolisina/metabolismo , Neuronas/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Ratones , N-Metilaspartato/metabolismo , Neuronas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Factor XII (FXII) is a serine protease that participates in the intrinsic coagulation pathway. Several studies have shown that plasma FXII exerts a deleterious role in cerebral ischemia and traumatic brain injury by promoting thrombo-inflammation. Nevertheless, the impact of FXII on neuronal cell fate remains unknown. OBJECTIVES: We investigated the role of FXII and FXIIa in neuronal injury and apoptotic cell death. METHODS: We tested the neuroprotective roles of FXII and FXIIa in an experimental model of neuronal injury induced by stereotaxic intracerebral injection of N-methyl-D-aspartic acid (NMDA) in vivo and in a model of apoptotic death of murine primary neuronal cultures through serum deprivation in vitro. RESULTS: Here, we found that exogenous FXII and FXIIa reduce brain lesions induced by NMDA injection in vivo. Furthermore, FXII protects cultured neurons from apoptosis through a growth factor--like effect. This mechanism was triggered by direct interaction with epidermal growth factor (EGF) receptor and subsequent activation of this receptor. Interestingly, the "proteolytically" active and two-chain form of FXII, FXIIa, exerts its protective effects by an alternative signaling pathway. FXIIa activates the pro-form of hepatocyte growth factor (HGF) into HGF, which in turn activated the HGF receptor (HGFR) pathway. CONCLUSION: This study describes two novel mechanisms of action of FXII and identifies neurons as target cells for the protective effects of single and two-chain forms of FXII. Therefore, inhibition of FXII in neurological disorders may have deleterious effects by preventing its beneficial effects on neuronal survival.
Asunto(s)
Factor XII , Proteínas Proto-Oncogénicas c-met , Animales , Apoptosis , Coagulación Sanguínea , Factor XIIa , Ratones , NeuronasRESUMEN
Recombinant tissue-type plasminogen activator (tPA) is the fibrinolytic drug of choice to treat stroke patients. However, a growing body of evidence indicates that besides its beneficial thrombolytic role, tPA can also have a deleterious effect on the ischaemic brain. Although ageing influences stroke incidence, complications and outcome, age-dependent relationships between endogenous tPA and stroke injuries have not been investigated yet. Here, we report that ageing is associated with a selective lowering of brain tPA expression in the murine brain. Moreover, our results show that albumin D site-binding protein (DBP) as a key age-associated regulator of the neuronal transcription of tPA. Additionally, inhibition of DBP-mediated tPA expression confers in vitro neuroprotection. Accordingly, reduced levels of tPA in old mice are associated with smaller excitotoxic/ischaemic injuries and protection of the permeability of the neurovascular unit during cerebral ischaemia. Likewise, we provide neuroradiological evidence indicating the existence of an inverse relationship between age and the volume of the ischaemic lesion in patients with acute ischaemic stroke. Together, these results indicate that the relationship among DBP, tPA and ageing play an important role in the outcome of cerebral ischaemia.
Asunto(s)
Envejecimiento/metabolismo , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al ADN/fisiología , Activador de Tejido Plasminógeno/metabolismo , Factores de Transcripción/fisiología , Anciano , Anciano de 80 o más Años , Envejecimiento/patología , Animales , Isquemia Encefálica/patología , Muerte Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen/métodos , Humanos , Imagen por Resonancia Magnética/métodos , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Transcripción/genéticaRESUMEN
The increase of cerebral blood flow evoked by neuronal activity is essential to ensure enough energy supply to the brain. In the neurovascular unit, endothelial cells are ideally placed to regulate key neurovascular functions of the brain. Nevertheless, some outstanding questions remain about their exact role neurovascular coupling (NVC). Here, we postulated that the tissue-type plasminogen activator (tPA) present in the circulation might contribute to NVC by a mechanism dependent of its interaction with endothelial N-Methyl-D-Aspartate Receptor (NMDAR). To address this question, we used pharmacological and genetic approaches to interfere with vascular tPA-dependent NMDAR signaling, combined with laser speckle flowmetry, intravital microscopy and ultrafast functional ultrasound in vivo imaging. We found that the tPA present in the blood circulation is capable of potentiating the cerebral blood flow increase induced by the activation of the mouse somatosensorial cortex, and that this effect is mediated by a tPA-dependent activation of NMDAR expressed at the luminal part of endothelial cells of arteries. Although blood molecules, such as acetylcholine, bradykinin or ATP are known to regulate vascular tone and induce vessel dilation, our present data provide the first evidence that circulating tPA is capable of influencing neurovascular coupling (NVC).
Asunto(s)
Endotelio Vascular/fisiología , Acoplamiento Neurovascular/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Activador de Tejido Plasminógeno/fisiología , Animales , Encéfalo/diagnóstico por imagen , Arterias Cerebrales/fisiología , Circulación Cerebrovascular/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroimagen , Reología , Activador de Tejido Plasminógeno/sangre , Activador de Tejido Plasminógeno/genética , Transfección , UltrasonografíaRESUMEN
The neuronal serine protease tissue-type Plasminogen Activator (tPA) is an important player of the neuronal survival and of the synaptic plasticity. Thus, a better understanding the mechanisms regulating the neuronal trafficking of tPA is required to further understand how tPA can influence brain functions. Using confocal imaging including living cells and high-resolution cell imaging combined with an innovating labeling of tPA, we demonstrate that the neuronal tPA is contained in endosomal vesicles positives for Rabs and in exosomal vesicles positives for synaptobrevin-2 (VAMP2) in dendrites and axons. tPA-containing vesicles differ in their dynamics with the dendritic tPA containing-vesicles less mobile than the axonal tPA-containing vesicles, these laters displaying mainly a retrograde trafficking. Interestingly spontaneous exocytosis of tPA containing-vesicles occurs largely in dendrites.
RESUMEN
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 (K(D)=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.
Asunto(s)
Fibrinolíticos/toxicidad , Receptores de N-Metil-D-Aspartato/metabolismo , Accidente Cerebrovascular/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Células Cultivadas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Activadores Plasminogénicos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Transducción de Señal , Técnicas de Cultivo de Tejidos , Activador de Tejido Plasminógeno/química , Activador de Tejido Plasminógeno/metabolismoRESUMEN
BACKGROUND: The ability of oligodendrocyte progenitor cells (OPCs) to give raise to myelin forming cells during developmental myelination, normal adult physiology and post-lesion remyelination in white matter depends on factors which govern their proliferation, migration and differentiation. Tissue plasminogen activator (tPA) is a serine protease expressed in the central nervous system (CNS), where it regulates cell fate. In particular, tPA has been reported to protect oligodendrocytes from apoptosis and to facilitate the migration of neurons. Here, we investigated whether tPA can also participate in the migration of OPCs during CNS development and during remyelination after focal white matter lesion. METHODS: OPC migration was estimated by immunohistological analysis in spinal cord and corpus callosum during development in mice embryos (E13 to P0) and after white matter lesion induced by the stereotactic injection of lysolecithin in adult mice (1 to 21 days post injection). Migration was compared in these conditions between wild type and tPA knock-out animals. The action of tPA was further investigated in an in vitro chemokinesis assay. RESULTS: OPC migration along vessels is delayed in tPA knock-out mice during development and during remyelination. tPA enhances OPC migration via an effect dependent on the activation of epidermal growth factor receptor. CONCLUSION: Endogenous tPA facilitates the migration of OPCs during development and during remyelination after white matter lesion by the virtue of its epidermal growth factor-like domain.