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1.
Stroke ; 55(3): 747-756, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38288607

RESUMO

BACKGROUND: Intravenous injection of alteplase, a recombinant tPA (tissue-type plasminogen activator) as a thrombolytic agent has revolutionized ischemic stroke management. However, tPA is a more complex enzyme than expected, being for instance able to promote thrombolysis, but at the same time, also able to influence neuronal survival and to affect the integrity of the blood-brain barrier. Accordingly, the respective impact of endogenous tPA expressed/present in the brain parenchyma versus in the circulation during stroke remains debated. METHODS: To address this issue, we used mice with constitutive deletion of tPA (tPANull [tPA-deficient mice]) or conditional deletion of endothelial tPA (VECad [vascular endothelial-Cadherin-Cre-recombinase]-Cre∆tPA). We also developed parabioses between tPANull and wild-type mice (tPAWT), anticipating that a tPAWT donor would restore levels of tPA to normal ones, in the circulation but not in the brain parenchyma of a tPANull recipient. Stroke outcomes were investigated by magnetic resonance imaging in a thrombo-embolic or a thrombotic stroke model, induced by local thrombin injection or FeCl3 application on the endothelium, respectively. RESULTS: First, our data show that endothelial tPA, released into the circulation after stroke onset, plays an overall beneficial role following thrombo-embolic stroke. Accordingly, after 24 hours, tPANull/tPANull parabionts displayed less spontaneous recanalization and reperfusion and larger infarcts compared with tPAWT/tPAWT littermates. However, when associated to tPAWT littermates, tPANull mice had similar perfusion deficits, but less severe brain infarcts. In the thrombotic stroke model, homo- and hetero-typic parabionts did not differ in the extent of brain damages and did not differentially recanalize and reperfuse. CONCLUSIONS: Together, our data reveal that during thromboembolic stroke, endogenous circulating tPA from endothelial cells sustains a spontaneous recanalization and reperfusion of the tissue, thus, limiting the extension of ischemic lesions. In this context, the impact of endogenous parenchymal tPA is limited.


Assuntos
Acidente Vascular Cerebral , AVC Trombótico , Animais , Camundongos , Modelos Animais de Doenças , Células Endoteliais , Endotélio , Camundongos Knockout , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismo
2.
BMC Biol ; 20(1): 218, 2022 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-36199089

RESUMO

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.


Assuntos
Parvalbuminas , Ativador de Plasminogênio Tecidual , Agrecanas/metabolismo , Animais , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Matriz Extracelular/metabolismo , Fibrinolisina/metabolismo , Interneurônios/fisiologia , Camundongos , Parvalbuminas/metabolismo , Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo
3.
Acta Neurochir (Wien) ; 164(2): 499-505, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35094147

RESUMO

BACKGROUND: Subarachnoid hemorrhage (SAH) can lead to acute hydrocephalus (AH). AH pathophysiology is classically attributed to an obstruction of the arachnoid granulations by blood. Recent findings in rodents suggest that after intraventricular hemorrhage, AH is related to cerebrospinal fluid (CSF) hypersecretion by the choroid plexus (CP), as it can be reduced by intracerebroventricular (ICV) injection of bumetanide. OBJECTIVE: Here, we investigated if and how CSF hypersecretion and/or CSF outflow disorders contribute to post-SAH hydrocephalus. METHODS: Ninety-four Wistar rats were used. SAH was induced by the endovascular perforation technique. The presence of AH was confirmed by magnetic resonance imaging (MRI), and rats with AH were randomly assigned to 4 groups: control group, superior sagittal sinus (SSS) thrombosis to block CSF reabsorption, ICV injection of saline, and ICV injection of bumetanide to decrease CSF secretion. Clinical outcome was evaluated with a neuroscore. A second MRI was performed 24 h later to evaluate the ventricular volume. RESULTS: Fifty percent of rats that survived SAH induction had AH. Their ventricular volume correlated well to the functional outcome after 24 h (r = 0.803). In rats with AH, 24 h later, ventricular volume remained equally increased in the absence of any further procedure. Similarly, ICV injection of saline or SSS thrombosis had no impact on the ventricular volume. However, ICV injection of bumetanide reduced AH by 35.9% (p = 0.002). CONCLUSION: In rodents, post-SAH hydrocephalus is may be due to hypersecretion of CSF by the CP, as it is limited by ICV injection of bumetanide. However, we cannot exclude other mechanisms involved in post-SAH acute hydrocephalus.


Assuntos
Hidrocefalia , Hemorragia Subaracnóidea , Animais , Bumetanida/farmacologia , Bumetanida/uso terapêutico , Plexo Corióideo , Hidrocefalia/tratamento farmacológico , Hidrocefalia/etiologia , Ratos , Ratos Wistar , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/tratamento farmacológico
4.
Int J Mol Sci ; 22(24)2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34948279

RESUMO

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.


Assuntos
Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feto , Camundongos , Cultura Primária de Células , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-met/fisiologia , Receptor Cross-Talk/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tecidual/fisiologia
5.
Circulation ; 136(7): 646-660, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28487393

RESUMO

BACKGROUND: Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N-Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization. METHODS: Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection. RESULTS: We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis. CONCLUSIONS: We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.


Assuntos
Acetilcisteína/uso terapêutico , Fibrinolíticos/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Tromboembolia/tratamento farmacológico , Acetilcisteína/farmacologia , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Cloretos/toxicidade , Modelos Animais de Doenças , Compostos Férricos/toxicidade , Fibrinolíticos/farmacologia , Infarto da Artéria Cerebral Média/etiologia , Masculino , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Ristocetina/farmacologia , Tromboembolia/induzido quimicamente , Trombose/prevenção & controle , Ativador de Plasminogênio Tecidual/uso terapêutico , Fator de von Willebrand/química , Fator de von Willebrand/metabolismo
6.
Brain ; 140(1): 146-157, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28031221

RESUMO

SEE SUN ET AL DOI101093/AWW306 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: About 20% of patients with ischaemic stroke have a preceding transient ischaemic attack, which is clinically defined as focal neurological symptoms of ischaemic origin resolving spontaneously. Failure to diagnose transient ischaemic attack is a wasted opportunity to prevent recurrent disabling stroke. Unfortunately, diagnosis can be difficult, due to numerous mimics, and to the absence of a specific test. New diagnostic tools are thus needed, in particular for radiologically silent cases, which correspond to the recommended tissue-based definition of transient ischaemic attack. As endothelial activation is a hallmark of cerebrovascular events, we postulated that this may also be true for transient ischaemic attack, and that it would be clinically relevant to develop non-invasive in vivo imaging to detect this endothelial activation. Using transcriptional and immunohistological analyses for adhesion molecules in a mouse model, we identified brain endothelial P-selectin as a potential biomarker for transient ischaemic attack. We thus developed ultra-sensitive molecular magnetic resonance imaging using antibody-based microparticles of iron oxide targeting P-selectin. This highly sensitive imaging strategy unmasked activated endothelial cells after experimental transient ischaemic attack and allowed discriminating transient ischaemic attack from epilepsy and migraine, two important transient ischaemic attack mimics. We provide preclinical evidence that combining conventional magnetic resonance imaging with molecular magnetic resonance imaging targeting P-selectin might aid in the diagnosis of transient ischaemic attack.


Assuntos
Ataque Isquêmico Transitório/metabolismo , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , Selectina-P/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Células Endoteliais , Ataque Isquêmico Transitório/diagnóstico por imagem , Masculino , Camundongos , Acidente Vascular Cerebral/diagnóstico por imagem
7.
Biochim Biophys Acta ; 1862(3): 395-402, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-26493446

RESUMO

The plasminogen activation (PA) system consists in a group of proteases and protease inhibitors regulating the activation of the zymogen plasminogen into its proteolytically active form, plasmin. Here, we give an update of the current knowledge about the role of the PA system on different aspects of neuroinflammation. These include modification in blood-brain barrier integrity, leukocyte diapedesis, removal of fibrin deposits in nervous tissues, microglial activation and neutrophil functions. Furthermore, we focus on the molecular mechanisms (some of them independent of plasmin generation and even of proteolysis) and target receptors responsible for these effects. The description of these mechanisms of action may help designing new therapeutic strategies targeting the expression, activity and molecular mediators of the PA system in neurological disorders involving neuroinflammatory processes. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.


Assuntos
Barreira Hematoencefálica/imunologia , Doenças do Sistema Nervoso Central/imunologia , Inflamação/imunologia , Microglia/imunologia , Plasminogênio/imunologia , Animais , Barreira Hematoencefálica/patologia , Doenças do Sistema Nervoso Central/patologia , Fibrina/imunologia , Fibrinolisina/imunologia , Humanos , Inflamação/patologia , Leucócitos/imunologia , Leucócitos/patologia , Microglia/patologia , Ativador de Plasminogênio Tecidual/imunologia
8.
Glia ; 65(12): 1961-1975, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28850711

RESUMO

Myelination is a late developmental process regulated by a set of inhibitory and stimulatory factors, including extracellular matrix components. Accordingly, chondroitin sulfate proteoglycans (CSPGs) act as negative regulators of myelination processes. A disintegrin and metalloproteinase with thrombospondin motifs type 4 (ADAMTS-4) is an extracellular protease capable of degrading CSPGs. Although exogenous ADAMTS-4 has been proven to be beneficial in several models of central nervous system (CNS) injuries, the physiological functions of endogenous ADAMTS-4 remain poorly understood. We first used Adamts4/LacZ reporter mice to reveal that ADAMTS-4 is strongly expressed in the CNS, especially in the white matter, with a cellular profile restricted to mature oligodendrocytes. Interestingly, we evidenced an abnormal myelination in Adamts4-/- mice, characterized by a higher diameter of myelinated axons with a shifting g-ratio. Accordingly, lack of ADAMTS-4 is accompanied by motor deficits and disturbed nervous electrical activity. In conclusion, we demonstrate that ADAMTS-4 is a new marker of mature oligodendrocytes contributing to the myelination processes and thus to the control of motor capacities.


Assuntos
Proteína ADAMTS4/metabolismo , Transtornos dos Movimentos/genética , Oligodendroglia/metabolismo , Proteína ADAMTS4/genética , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio/metabolismo , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Corpo Caloso/ultraestrutura , Modelos Animais de Doenças , Potenciais Somatossensoriais Evocados/genética , Potenciais Somatossensoriais Evocados/fisiologia , Transtornos Neurológicos da Marcha/etiologia , Locomoção/genética , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica , Transtornos dos Movimentos/fisiopatologia , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/patologia , Oligodendroglia/ultraestrutura , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Estatísticas não Paramétricas , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
9.
Stroke ; 46(6): 1641-50, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25922513

RESUMO

BACKGROUND AND PURPOSE: Tissue-type plasminogen activator (tPA) is the only acute treatment for ischemic stroke. Unfortunately, the benefit of tPA-driven thrombolysis is not systematic, and understanding the reasons for this is mandatory. The balance between beneficial and detrimental effects of tPA might explain the limited overall efficiency of thrombolysis. Here, we investigated whether this balance could be influenced by excessive alcohol intake. METHODS: We used a murine model of thromboembolic stroke, coupled to an array of biochemical assays, near-infrared or magnetic resonance imaging scans, 2-photon microscopy, hydrodynamic transfections, and immunohistological techniques. RESULTS: We found that 6 weeks of alcohol consumption (10% in drinking water) worsens ischemic lesions and cancels the beneficial effects of tPA-induced thrombolysis. We accumulate in vivo and in vitro evidence showing that this aggravation is correlated with a decrease in lipoprotein receptor-related protein 1-mediated hepatic clearance of tPA in alcohol-exposed mice. CONCLUSIONS: An efficient liver-driven clearance of tPA might influence the safety of thrombolysis after stroke.


Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Isquemia Encefálica/tratamento farmacológico , Fígado/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/farmacocinética , Consumo de Bebidas Alcoólicas/patologia , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Fígado/patologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Ativador de Plasminogênio Tecidual/farmacologia
10.
Stroke ; 46(2): 477-84, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25503547

RESUMO

BACKGROUND AND PURPOSE: Despite the effectiveness of recombinant tissue-type plasminogen activator (r-tPA) during the acute phase of ischemic stroke, the therapy remains limited by a narrow time window and the occurrence of occasional vascular side effects, particularly symptomatic hemorrhages. Our aim was to investigate the mechanisms underlying the endothelial damage resulting from r-tPA treatment in ischemic-like conditions. METHODS: Microarray analyses were performed on cerebral endothelial cells submitted to r-tPA treatment during oxygen and glucose deprivation to identify novel biomarker candidates. Validation was then performed in vivo in a mouse model of thromboembolic stroke and culminated in an analysis in a clinical cohort of patients with ischemic stroke treated with thrombolysis. RESULTS: The transcription factor NURR1 (NR4A2) was identified as a downstream target induced by r-tPA during oxygen and glucose deprivation. Silencing NURR1 expression reversed the endothelial-toxicity induced by the combined stimuli, a protective effect attributable to reduced levels of proinflammatory mediators, such as nuclear factor-kappa-beta 2 (NF-κ-B2), interleukin 1 alpha (IL1α), intercellular adhesion molecule 1 (ICAM1), SMAD family member 3 (SMAD3), colony stimulating factor 2 (granulocyte-macrophage; CSF2). The detrimental effect of delayed thrombolysis, in conditions in which NURR1 gene expression was enhanced, was confirmed in the preclinical stroke model. Finally, we determined that patients with stroke who had a symptomatic hemorrhagic transformation after r-tPA treatment exhibited higher baseline serum NURR1 levels than did patients with an asymptomatic or absence of cerebral bleedings. CONCLUSIONS: Our results suggest that NURR1 upregulation by r-tPA during ischemic stroke is associated with endothelial dysfunction and inflammation and the enhancement of hemorrhagic complications associated to thrombolysis.


Assuntos
Isquemia Encefálica/sangue , Isquemia Encefálica/tratamento farmacológico , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/sangue , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/sangue , Isquemia Encefálica/diagnóstico , Linhagem Celular , Feminino , Fibrinolíticos/efeitos adversos , Fibrinolíticos/uso terapêutico , Humanos , Inflamação/sangue , Inflamação/induzido quimicamente , Inflamação/diagnóstico , Masculino , Camundongos , Pessoa de Meia-Idade , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Acidente Vascular Cerebral/diagnóstico , Ativador de Plasminogênio Tecidual/efeitos adversos , Resultado do Tratamento
11.
Neurobiol Dis ; 66: 28-42, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24576594

RESUMO

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.


Assuntos
Proteínas ADAM/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Pró-Colágeno N-Endopeptidase/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Ativador de Plasminogênio Tecidual/farmacologia , Proteína ADAMTS4 , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Células Cultivadas , Feminino , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurocam , Neuropeptídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Inibidores de Serina Proteinase/farmacologia , Serpinas/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Compressão da Medula Espinal/tratamento farmacológico , Compressão da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Neuroserpina
12.
Proc Natl Acad Sci U S A ; 108(33): 13782-7, 2011 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-21804034

RESUMO

NMDA type glutamate receptors (NMDARs) 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 (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 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 Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).


Assuntos
Cerebelo/citologia , Neurônios/ultraestrutura , Receptores de N-Metil-D-Aspartato/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Quinases da Família src/metabolismo , Indutores da Angiogênese , Animais , Cálcio/metabolismo , Camundongos , Complexos Multiproteicos , Fosforilação , Receptores de Neurotransmissores , Sinapses , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
13.
Cell Death Dis ; 15(4): 261, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38609369

RESUMO

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.


Assuntos
AVC Isquêmico , Síndromes Neurotóxicas , Animais , Camundongos , Células Endoteliais , N-Metilaspartato/farmacologia , Neurônios , Ativador de Plasminogênio Tecidual
14.
Nat Commun ; 15(1): 8430, 2024 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-39341842

RESUMO

Subarachnoid hemorrhage (SAH) can be associated with neurological deficits and has profound consequences for mortality and morbidity. Cerebral vasospasm (CVS) and delayed cerebral ischemia affect neurological outcomes in SAH patients, but their mechanisms are not fully understood, and effective treatments are limited. Here, we report that urotensin II receptor UT plays a pivotal role in both early events and delayed mechanisms following SAH in male mice. Few days post-SAH, UT expression is triggered by blood or hemoglobin in the leptomeningeal compartment. UT contributes to perimeningeal glia limitans astrocyte reactivity, microvascular alterations and neuroinflammation independent of CNS-associated macrophages (CAMs). Later, CAM-dependent vascular inflammation and subsequent CVS develop, leading to cognitive dysfunction. In an SAH model using humanized UTh+/h+ male mice, we show that post-SAH CVS and behavioral deficits, mediated by UT through Gq/PLC/Ca2+ signaling, are prevented by UT antagonists. These results highlight the potential of targeting UT pathways to reduce early meningeal response and delayed cerebral ischemia in SAH patients.


Assuntos
Macrófagos , Meninges , Receptores Acoplados a Proteínas G , Hemorragia Subaracnóidea , Vasoespasmo Intracraniano , Animais , Masculino , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/metabolismo , Vasoespasmo Intracraniano/metabolismo , Vasoespasmo Intracraniano/etiologia , Camundongos , Macrófagos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Meninges/metabolismo , Humanos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Astrócitos/metabolismo
15.
J Neurosci ; 32(37): 12726-34, 2012 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-22972996

RESUMO

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.


Assuntos
Ácido Glutâmico/metabolismo , Aprendizagem em Labirinto/fisiologia , Memória de Curto Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Percepção Espacial/fisiologia , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Subunidades Proteicas
16.
Neurobiol Dis ; 50: 201-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23103420

RESUMO

Glutamate excitotoxicity is a consolidated hypothesis in neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor mediated effects. In brain microvascular endothelial cell (nBMEC) cultures from neonates, t-PA content and release upon glutamate are higher than in adult (aBMECs) cultures. Owing to the variety of t-PA substrates and receptor targets, the study was aimed at determining the putative roles of endothelial t-PA in the neonatal brain parenchyma under glutamate challenge. Basal t-PA release was 4.4 fold higher in nBMECs vs aBMECs and glutamate was 20 fold more potent to allow Evans blue vascular permeability in neonate microvessels indicating that, under noxious glutamate (50 µM) exposure, high amounts of endothelial t-PA stores may be mobilized and may access the nervous parenchyma. Culture media from nBMECS or aBMECs challenged by excitotoxic glutamate were applied to neuron cultures at DIV 11. While media from adult cells did not evoke more LDH release in neuronal cultures that under glutamate alone, media from nBMECs enhanced 2.2 fold LDH release. This effect was not observed with media from t-PA(-/-) nBMECs and was inhibited by hr-PAI-1. In Cortical slices from 10 day-old mice, hrt-PA associated with glutamate evoked neuronal necrosis in deeper (more mature) layers, an effect reversed by NMDA receptor GluN1 amino-terminal domain antibody capable of inhibiting t-PA potentiation of the receptor. In superficial layers (less mature), hrt-PA alone inhibited apoptosis, an effect reversed by the EGF receptor antagonist AG1478. Applied to immature neurons in culture (DIV5), media from nBMEC rescued 85.1% of neurons from cell death induced by serum deprivation. In cortical slices, the anti-apoptotic effect of t-PA fitted with age dependent localization of less mature neurons. These data suggest that in the immature brain, propensity of vessels to release high amounts of t-PA may not only impact vascular integrity but may also influence neuronal fate, via regulation of apoptosis in immature cells and, as in adult by potentiating glutamate toxicity in mature neurons. The data point out putative implication of microvessels in glutamate neurotoxicity in the development, and justify research towards vessel oriented neuroprotection strategies in neonates.


Assuntos
Apoptose/fisiologia , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/patologia , Técnicas de Cultura de Órgãos
17.
J Cell Sci ; 124(Pt 12): 2070-6, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21610098

RESUMO

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 Ca²âº 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.


Assuntos
Fibrinólise/efeitos dos fármacos , Proteína HMGB1/farmacologia , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Biomarcadores/sangue , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Cálcio/metabolismo , Bovinos , Células Cultivadas , Técnicas de Cocultura , Domínios HMG-Box , Proteína HMGB1/metabolismo , Humanos , Camundongos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tecidual/metabolismo
18.
Ann Neurol ; 72(4): 536-49, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23109148

RESUMO

OBJECTIVE: Activated microglia play a central role in the inflammatory and excitotoxic component of various acute and chronic neurological disorders. However, the mechanisms leading to their activation in the latter context are poorly understood, particularly the involvement of N-methyl-D-aspartate receptors (NMDARs), which are critical for excitotoxicity in neurons. We hypothesized that microglia express functional NMDARs and that their activation would trigger neuronal cell death in the brain by modulating inflammation. METHODS AND RESULTS: We demonstrate that microglia express NMDARs in the murine and human central nervous system and that these receptors are functional in vitro. We show that NMDAR stimulation triggers microglia activation in vitro and secretion of factors that induce cell death of cortical neurons. These damaged neurons are further shown to activate microglial NMDARs and trigger a release of neurotoxic factors from microglia in vitro, indicating that microglia can signal back to neurons and possibly induce, aggravate, and/or maintain neurologic disease. Neuronal cell death was significantly reduced through pharmacological inhibition or genetically induced loss of function of the microglial NMDARs. We generated Nr1 LoxP(+/+) LysM Cre(+/-) mice lacking the NMDAR subunit NR1 in cells of the myeloid lineage. In this model, we further demonstrate that a loss of function of the essential NMDAR subunit NR1 protects from excitotoxic neuronal cell death in vivo and from traumatic brain injury. INTERPRETATION: Our findings link inflammation and excitotoxicity in a potential vicious circle and indicate that an activation of the microglial NMDARs plays a pivotal role in neuronal cell death in the perinatal and adult brain.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Inflamação/induzido quimicamente , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/agonistas , Animais , Lesões Encefálicas/patologia , Cálcio/metabolismo , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Ácido Ibotênico/toxicidade , Imuno-Histoquímica , Inflamação/patologia , Masculino , Camundongos , Camundongos Knockout , Microglia/efeitos dos fármacos , Microscopia Confocal , Neocórtex/patologia , Técnicas de Patch-Clamp , Espécies Reativas de Oxigênio , Acidente Vascular Cerebral/patologia
19.
Fluids Barriers CNS ; 20(1): 11, 2023 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-36737775

RESUMO

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.


Assuntos
Acoplamento Neurovascular , Ativador de Plasminogênio Tecidual , Camundongos , Animais , N-Metilaspartato/farmacologia , Células Endoteliais/metabolismo , Encéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Camundongos Knockout , Fígado/metabolismo
20.
Stroke ; 43(10): 2774-81, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22879098

RESUMO

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.


Assuntos
Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Memantina/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/efeitos adversos , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Quimioterapia Adjuvante , Antagonistas de Aminoácidos Excitatórios/farmacologia , Fibrinolíticos/efeitos adversos , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Técnicas In Vitro , Imageamento por Ressonância Magnética , Masculino , Memantina/farmacologia , Camundongos , Modelos Animais , N-Metilaspartato/farmacologia , Acidente Vascular Cerebral/patologia , Ativador de Plasminogênio Tecidual/farmacologia , Resultado do Tratamento
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