RESUMEN
The extracellular space (ECS) plays a central role in brain physiology, shaping the time course and spread of neurochemicals, ions, and nutrients that ensure proper brain homeostasis and neuronal communication. Astrocytes are the most abundant type of glia cell in the brain, whose processes densely infiltrate the brain's parenchyma. As astrocytes are highly sensitive to changes in osmotic pressure, they are capable of exerting a potent physiological influence on the ECS. However, little is known about the spatial distribution and temporal dynamics of the ECS that surrounds astrocytes, owing mostly to a lack of appropriate techniques to visualize the ECS in live brain tissue. Mitigating this technical limitation, we applied the recent SUper-resolution SHadow Imaging technique (SUSHI) to astrocyte-labeled organotypic hippocampal brain slices, which allowed us to concurrently image the complex morphology of astrocytes and the ECS with unprecedented spatial resolution in a live experimental setting. Focusing on ring-like astrocytic microstructures in the spongiform domain, we found them to enclose sizable pools of interstitial fluid and cellular structures like dendritic spines. Upon experimental osmotic challenge, these microstructures remodeled and swelled up at the expense of the pools, effectively increasing the physical interface between astrocytic and cellular structures. Our study reveals novel facets of the dynamic microanatomical relationships between astrocytes, neuropil, and the ECS in living brain tissue, which could be of functional relevance for neuron-glia communication in a variety of (patho)physiological settings, for example, LTP induction, epileptic seizures or acute ischemic stroke, where osmotic disturbances are known to occur.
Asunto(s)
Astrocitos , Encéfalo/diagnóstico por imagen , Isquemia Encefálica , Espacio Extracelular , Humanos , Accidente CerebrovascularRESUMEN
It is well--established that Rab11-dependent recycling endosomes drive the activity-dependent delivery of AMPA receptors (AMPARs) into synapses during long-term potentiation (LTP). Nevertheless, the molecular basis for this specialized function of recycling endosomes is still unknown. Here, we have investigated RAB11FIP2 (FIP2 hereafter) as a potential effector of Rab11-dependent trafficking during LTP in rat hippocampal slices. Surprisingly, we found that FIP2 operates independently from Rab11 proteins, and acts as a negative regulator of AMPAR synaptic trafficking. Under basal conditions, FIP2 associates with AMPARs at immobile compartments, separately from recycling endosomes. Using shRNA-mediated knockdown, we found that FIP2 prevents GluA1 (encoded by the Gria1 gene) AMPARs from reaching the surface of dendritic spines in the absence of neuronal stimulation. Upon induction of LTP, FIP2 is rapidly mobilized, dissociates from AMPARs and undergoes dephosphorylation. Interestingly, this dissociation of the FIP2-AMPAR complex, together with FIP2 dephosphorylation, is required for LTP, but the interaction between FIP2 and Rab11 proteins is not. Based on these results, we propose a retention-release mechanism, where FIP2 acts as a gate that restricts the trafficking of AMPARs, until LTP induction triggers their release and allows synaptic delivery.
Asunto(s)
Proteínas Portadoras/metabolismo , Espinas Dendríticas/metabolismo , Potenciación a Largo Plazo/fisiología , Proteínas de la Membrana/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Animales , Proteínas Portadoras/genética , Endosomas/metabolismo , Femenino , Hipocampo/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratas , Ratas Wistar , Receptores AMPA/genéticaRESUMEN
Hippocampal synaptic plasticity involves both membrane trafficking events and intracellular signaling, but how these are coordinated is far from clear. The endosomal transport of glutamate receptors in and out of the postsynaptic membrane responds to multiple signaling cascades triggered by synaptic activity. In this work, we have identified adaptor protein containing a plekstrin homology domain, phosphotyrosine-binding domain and leucine zipper motif 1 (APPL1) as a crucial element linking trafficking and signaling during synaptic plasticity. We show that APPL1 knockdown specifically impairs PI3K-dependent forms of synaptic plasticity, such as long-term potentiation (LTP) and metabotropic-glutamate-receptor-dependent long-term depression (mGluR-LTD). Indeed, we demonstrate that APPL1 is required for the activation of the phosphatidylinositol triphosphate (PIP3) pathway in response to LTP induction. This requirement can be bypassed by membrane localization of PI3K and is related to phosphoinositide binding. Interestingly, inhibitors of PDK1 (also known as PDPK1) and Akt have no effect on LTP expression. Therefore, we conclude that APPL1 gates PI3K activation at the plasma membrane upon LTP induction, which is then relayed by downstream PIP3 effectors that are different from PDK1 and Akt.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Potenciación a Largo Plazo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Animales , Espinas Dendríticas/metabolismo , Activación Enzimática , Hipocampo/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Dominios Proteicos , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , ARN Interferente Pequeño/metabolismo , Ratas Wistar , Transducción de Señal , Sinapsis/metabolismo , Transmisión SinápticaRESUMEN
Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15µm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.
Asunto(s)
Hipocampo , Receptores AMPA , Sinapsis , Animales , Ratones , Hipocampo/metabolismo , Hipocampo/citología , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Proteínas de la Membrana/metabolismo , Oro/química , Microscopía Electrónica/métodos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismoRESUMEN
Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.
Asunto(s)
Colorantes Fluorescentes , Neuronas , Homólogo 4 de la Proteína Discs Large/genética , Homólogo 4 de la Proteína Discs Large/metabolismo , Neuronas/metabolismo , Colorantes Fluorescentes/metabolismo , Sinapsis/metabolismoRESUMEN
The strength of excitatory synaptic transmission depends partly on the number of AMPA receptors (AMPARs) at the postsynaptic surface and, thus, can be modulated by membrane trafficking events. These processes are critical for some forms of synaptic plasticity, such as long-term potentiation and long-term depression (LTD). In the case of LTD, AMPARs are internalized and dephosphorylated in response to NMDA receptor activation. However, the fate of the internalized receptors upon LTD induction and its relevance for synaptic function is still a matter of debate. Here we examined the functional contribution of receptor recycling versus degradation for LTD in rat hippocampal slices, and their correlation with receptor dephosphorylation. We observed that GluA1 undergoes sequential dephosphorylation and degradation in lysosomes after LTD induction. However, this degradation does not have functional consequences for the regulation of synaptic strength, and therefore, for the expression of LTD. In contrast, the partition of internalized AMPARs between Rab7-dependent trafficking (toward lysosomes) or Rab11-dependent endosomes (recycling back toward synapses) is the key factor determining the extent of synaptic depression upon LTD induction. This sorting decision is related to the phosphorylation status of GluA1 Ser845, the dephosphorylated receptors being those preferentially targeted for lysosomal degradation. Altogether, these new data contribute to clarify the fate of AMPARs during LTD and emphasize the importance of membrane sorting decisions to determine the outcome of synaptic plasticity.
Asunto(s)
Hipocampo/citología , Depresión Sináptica a Largo Plazo/fisiología , Lisosomas/metabolismo , Neuronas/ultraestructura , Receptores AMPA/metabolismo , Sinapsis/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Animales Recién Nacidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biofisica , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Estimulación Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/fisiología , Leupeptinas/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Masculino , N-Metilaspartato/farmacología , Técnicas de Cultivo de Órganos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Estadísticas no Paramétricas , Transfección/métodosRESUMEN
Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPARs) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminal. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP-GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.
RESUMEN
Glutamatergic neurons contain free zinc packaged into neurotransmitter-loaded synaptic vesicles. Upon neuronal activation, the vesicular contents are released into the synaptic space, whereby the zinc modulates activity of postsynaptic neurons though interactions with receptors, transporters and exchangers. However, high extracellular concentrations of zinc trigger seizures and are neurotoxic if substantial amounts of zinc reenter the cells via ion channels and accumulate in the cytoplasm. Tissue plasminogen activator (tPA), a secreted serine protease, is also proepileptic and excitotoxic. However, tPA counters zinc toxicity by promoting zinc import back into the neurons in a sequestered form that is nontoxic. Here, we identify the zinc influx transporter, ZIP4, as the pathway through which tPA mediates the zinc uptake. We show that ZIP4 is upregulated after excitotoxin stimulation of the mouse, male and female, hippocampus. ZIP4 physically interacts with tPA, correlating with an increased intracellular zinc influx and lysosomal sequestration. Changes in prosurvival signals support the idea that this sequestration results in neuroprotection. These experiments identify a mechanism via which neurons use tPA to efficiently neutralize the toxic effects of excessive concentrations of free zinc.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Hipocampo/metabolismo , Espacio Intracelular/metabolismo , Neuronas/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Zinc/metabolismo , Animales , Línea Celular , Células Cultivadas , Femenino , Hipocampo/efectos de los fármacos , Humanos , Técnicas In Vitro , Espacio Intracelular/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Activador de Tejido Plasminógeno/deficiencia , Activador de Tejido Plasminógeno/genéticaRESUMEN
Clustered protocadherins (Pcdhs) are a family of cadherin-like molecules arranged in gene clusters (alpha, beta, and gamma). gamma-Protocadherins (Pcdh-gammas) are involved in cell-cell interactions, but their prominent intracellular distribution in vivo and different knock-out phenotypes suggest that these molecules participate in still unidentified processes. We found using correlative light and electron microscopy that Pcdh-gammaA3 and -gammaB2, but not -gammaC4, -alpha1, or N-cadherin, generate intracellular juxtanuclear membrane tubules when expressed in cells. These tubules recruit the autophagy marker MAP1A/1B LC3 (LC3) but are not associated with autophagic vesicles. Lipidation of LC3 is required for its coclustering with Pcdh-gamma tubules, suggesting the involvement of an autophagic-like molecular cascade. Expression of wild-type LC3 with Pcdh-gammaA3 increased tubule length whereas expression of lipidation-defective LC3 decreased tubule length relative to Pcdh-gammaA3 expressed alone. The tubules were found to emanate from lysosomes. Deletion of the luminal/extracellular domain of Pcdh-gammaA3 preserved lysosomal targeting but eliminated tubule formation whereas cytoplasmic deletion eliminated both lysosomal targeting and tubule formation. Deletion of the membrane-proximal three cadherin repeats resulted in tubes that were narrower than those produced by full-length molecules. These results suggest that Pcdh-gammaA and -gammaB families can influence the shape of intracellular membranes by mediating intraluminal interactions within organelles.
Asunto(s)
Cadherinas/fisiología , Proteínas Asociadas a Microtúbulos/genética , Animales , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Comunicación Celular , Línea Celular , Proteínas Fluorescentes Verdes/genética , Humanos , Riñón/embriología , Proteínas Luminiscentes/genética , Ratones , Microscopía Confocal , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Plásmidos , Ratas , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Clustered protocadherins (Pcdhs) are a family of ~60 cadherin-like proteins (divided into subclasses α, ß, and γ) that regulate dendrite morphology and neural connectivity. Their expression is controlled through epigenetic regulation at a gene cluster encoding the molecules. During neural development, Pcdhs mediate dendrite self-avoidance in some neuronal types through an uncharacterized anti-adhesive mechanism. Pcdhs are also important for dendritic complexity in cortical neurons likely through a pro-adhesive mechanism. Pcdhs have also been postulated to participate in synaptogenesis and connectivity. Some synaptic defects were noted in knockout animals, including synaptic number and physiology, but the role of these molecules in synaptic development is not understood. The effect of Pcdh knockout on dendritic patterning may present a confound to studying synaptogenesis. We showed previously that Pcdh-γs are highly enriched in intracellular compartments in dendrites and spines with localization at only a few synaptic clefts. To gain insight into how Pcdh-γs might affect synapses, we compared synapses that harbored Pcdh-γs versus those that did not for parameters of synaptic maturation including pre- and postsynaptic size, postsynaptic perforations, and spine morphology by light microscopy in cultured hippocampal neurons and by serial section immuno-electron microscopy in hippocampal CA1. In mature neurons, synapses immunopositive for Pcdh-γs were larger in diameter with more frequent perforations. Analysis of spines in cultured neurons revealed that mushroom spines were more frequently immunopositive for Pcdh-γs at their tips than thin spines. These results suggest that Pcdh-γ function at the synapse may be related to promotion of synaptic maturation and stabilization.
Asunto(s)
Proteínas Relacionadas con las Cadherinas/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Neuronas/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructura , Animales , Técnicas de Inactivación de Genes , Hipocampo/metabolismo , Hipocampo/ultraestructura , Microscopía Inmunoelectrónica , Ratas , Ratas Sprague-DawleyRESUMEN
Gamma protocadherins (Pcdh-γs) resemble classical cadherins and have the potential to engage in cell-cell interactions with homophilic properties. Emerging evidence suggests non-conventional roles for some protocadherins in neural development. We sought to determine whether Pcdh-γ trafficking in neurons is consistent with an intracellular role for these molecules. Here we show that, in contrast to the largely surface localization of classical cadherins, endogenous Pcdh-γs are primarily intracellular in rat neurons in vivo and are equally distributed within organelles of subsynaptic dendritic and axonal compartments. A strikingly higher proportion of Pcdh-γ-containing organelles in synaptic compartments was observed at postnatal day 16. To determine the origin of Pcdh-γ-trafficking organelles, we isolated organelles with Pcdh-γ antibody-coupled magnetic beads from brain organelle suspensions. Vesicles with high levels of COPII and endoplasmic reticulum-Golgi intermediate compartment (ERGIC) components were isolated with the Pcdh-γ antibody but not with the classical cadherin antibody. In cultured hippocampal neurons, Pcdh-γ immunolabeling partially overlapped with calnexin- and COPII-positive puncta in dendrites. Mobile Pcdh-γ-GFP profiles dynamically codistributed with a DsRed construct coupled to ER retention signals by live imaging. Pcdh-γ expression correlated with accumulations of tubulovesicular and ER-like organelles in dendrites. Our results are consistent with the possibility that Pcdh-γs could have a unique function within the secretory pathway in addition to their documented surface roles.
Asunto(s)
Cadherinas/metabolismo , Neuronas/metabolismo , Vías Secretoras/fisiología , Vesículas Secretoras/metabolismo , Animales , Proteínas Relacionadas con las Cadherinas , Células Cultivadas , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Vesículas Secretoras/fisiologíaRESUMEN
Gamma-protocadherins (Pcdh-gammas) are good candidates to mediate specificity in synaptogenesis but their role in cell-cell interactions is a matter of debate. We proposed that Pcdh-gammas modify preformed synapses via trafficking of Pcdh-gammas-containing organelles, insertion into synaptic membranes and homophilic transcellular interaction. Here we provide evidence in support of this model. We show for the first time that Pcdh-gammas have homophilic properties and that they accumulate at dendro-dendritic and axo-dendritic interfaces during neuronal development. Pcdh-gammas are maintained in a substantial mobile intracellular pool in dendrites and cytoplasmic deletion shifts the molecule to the surface and reduces the number and velocity of the mobile packets. We monitored Pcdh-gamma temporal and spatial dynamics in transport organelles. Pcdh-gamma organelles bud and fuse with stationary clusters near synapses. These results suggest that Pcdh-gamma-mediated cell-cell interactions in synapse development or maintenance are tightly regulated by control of intracellular trafficking via the cytoplasmic domain.
Asunto(s)
Cadherinas/metabolismo , Neuronas/fisiología , Isoformas de Proteínas/metabolismo , Animales , Proteínas Relacionadas con las Cadherinas , Cadherinas/química , Cadherinas/genética , Diferenciación Celular/fisiología , Línea Celular , Humanos , Uniones Intercelulares/fisiología , Ratones , Neuronas/citología , Orgánulos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sinapsis/metabolismoRESUMEN
BACKGROUND: Accumulating evidence demonstrates a critical involvement of tissue-type plasminogen activator (tPA) in pathological and physiological brain conditions. Determining whether and how vascular tPA can cross the blood-brain barrier (BBB) to enter the brain is thus important, not only during stroke but also in physiological conditions. METHODS AND RESULTS: In the present work, we provide evidence in vivo that intravenous injection of tPA increases NMDA-induced striatal lesion in the absence of BBB leakage. Accordingly, we show that tPA crosses the BBB both after excitotoxic lesion and in control conditions. Indeed, vascular injected tPA can be detected within the brain parenchyma and in the cerebrospinal fluid. By using an in vitro model of BBB, we have confirmed that tPA can cross the intact BBB. Its passage was blocked at 4 degrees C, was saturable, and was independent of its proteolytic activity. We have shown that tPA crosses the BBB by transcytosis, mediated by a member of the LDL receptor-related protein family. CONCLUSIONS: We demonstrate that blood-derived tPA can reach the brain parenchyma without alteration of the BBB. The molecular mechanism of the passage of tPA from blood to brain described here could represent an interesting target to improve thrombolysis in stroke.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Activador de Tejido Plasminógeno/efectos adversos , Activador de Tejido Plasminógeno/farmacocinética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Frío , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , N-Metilaspartato/administración & dosificación , Síndromes de Neurotoxicidad/etiología , Inhibidor 1 de Activador Plasminogénico/administración & dosificación , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
Stroke represents a major health problem in the ever-ageing population of industrialized nations. Each year, over three million people in the USA alone suffer from this affliction. Stroke, which results from the obstruction of an intra- or extra-cerebral artery, induces irreversible neuronal damage. The clot-busting drug tissue-type plasminogen activator (tPA) is the only FDA-approved therapy for acute stroke. Although tPA has been successfully used to treat myocardial infarction due to clot formation, its use in the treatment of occlusive cerebrovascular diseases remains controversial. Indeed, tPA is clearly beneficial as a thrombolytic agent. However, increasing evidence suggests that tPA could have direct and deleterious effects on neurons and glial cells.
Asunto(s)
Fibrinolíticos/uso terapéutico , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Fibrinolíticos/efectos adversos , Ácido Glutámico/metabolismo , Humanos , Fármacos Neuroprotectores/efectos adversos , Fármacos Neuroprotectores/uso terapéutico , Transducción de Señal/efectos de los fármacos , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/efectos adversos , Factor de Crecimiento Transformador beta/uso terapéutico , Factor de Crecimiento Transformador beta1RESUMEN
BACKGROUND AND PURPOSE: Despite uncontroversial benefit from its thrombolytic activity, the documented neurotoxic effect of tissue plasminogen activator (tPA) raises an important issue: the current emergency stroke treatment might not be optimum if exogenous tPA can enter the brain and thus add to the deleterious effects of endogenous tPA within the cerebral parenchyma. Here, we aimed at determining whether vascular tPA crosses the blood-brain barrier (BBB) during cerebral ischemia, and if so, by which mechanism. METHODS: First, BBB permeability was assessed in vivo by measuring Evans Blue extravasation following intravenous injection at 0 or 3 hours after middle cerebral artery electrocoagulation in mice. Second, the passage of vascular tPA was investigated in an in vitro model of BBB, subjected or not to oxygen and glucose deprivation (OGD). RESULTS: We first demonstrated that after focal permanent ischemia in mice, the BBB remains impermeable to Evans Blue in the early phase (relative to the therapeutic window of tPA), whereas at later time points massive Evans Blue extravasation occurs. Then, the passage of tPA during these 2 phases, was investigated in vitro and we show that in control conditions, tPA crosses the intact BBB by a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent transcytosis, whereas OGD leads to an exacerbation of tPA passage, which switches to a LRP-independent process. CONCLUSIONS: We evidence 2 different mechanisms through which vascular tPA can reach the brain parenchyma, depending on the state of the BBB. As discussed, these data show the importance of taking the side effects of blood-derived tPA into account and offer a basis to improve the current thrombolytic strategy.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Proteínas Relacionadas con Receptor de LDL/fisiología , Activador de Tejido Plasminógeno/metabolismo , Animales , Isquemia Encefálica/etiología , Hipoxia de la Célula , Vesículas Citoplasmáticas/química , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Glucosa/fisiología , Infarto de la Arteria Cerebral Media/complicaciones , Proteínas Relacionadas con Receptor de LDL/antagonistas & inhibidores , Masculino , Ratones , Transporte de Proteínas , Activador de Tejido Plasminógeno/análisis , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Although transforming growth factor (TGF)-alpha, a member of the epidermal growth factor (EGF) family, has been shown to protect neurons against excitotoxic and ischemic brain injuries, its mechanism of action remains unknown. In the present study, we used in vitro models of apoptotic or necrotic paradigms demonstrating that TGF-alpha rescues neurons from N-methyl-D-aspartate (NMDA)-induced excitotoxic death, with the obligatory presence of astrocytes. Because neuronal tissue-type plasminogen activator (t-PA) release was shown to potentiate NMDA-induced excitotoxicity, we observed that TGF-alpha treatment reduced NMDA-induced increase of t-PA activity in mixed cultures of neurons and astrocytes. In addition, we showed that although TGF-alpha induces activation of the extracellular signal-regulated kinases (ERKs) in astrocytes, it failed to activate p42/p44 in neurons. Finally, we showed that TGF-alpha, by an ERK-dependent mechanism, stimulates the astrocytic expression of PAI-1, a t-PA inhibitor, which mediates the neuroprotective activity of TGF-alpha against NMDA-mediated excitotoxic neuronal death. Taken together, we indicate that TGF-alpha rescues neurons from NMDA-induced excitotoxicity in mixed cultures through inhibition of t-PA activity, involving PAI-1 overexpression by an ERK-dependent pathway in astrocytes.
Asunto(s)
Astrocitos/efectos de los fármacos , Neuronas/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/genética , Factor de Crecimiento Transformador alfa/farmacología , Animales , Apoptosis/efectos de los fármacos , Astrocitos/citología , Astrocitos/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , N-Metilaspartato/farmacología , Neuronas/citología , Fosforilación/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activador de Tejido Plasminógeno/efectos de los fármacos , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Tissue-type plasminogen activator (tPA) is available for the treatment of thromboembolic stroke in humans. However, adverse effects of tPA have been observed in animal models of ischemic brain injuries. In the present study, we have used a synthetic tPA inhibitor, named 2,7-bis-(4-amidino-benzylidene)-cycloheptan-1-one dihydrochloride (tPA stop), to investigate the role of endogenous tPA in the cerebral parenchyma. In mouse cortical cell cultures, we observed that although tPA stop reduced N-methyl-D-aspartic acid (NMDA)-mediated excitotoxic neuronal death, it failed to modulate alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate-mediated necrosis. In addition, we found that tPA stop could prevent the deleterious effects of both endogenous and exogenous tPA during NMDA exposure. At the functional level, tPA stop was found to prevent tPA-dependent potentiation of NMDA receptor-evoked calcium influx. The relevance of those findings was strengthened by the observation of a massive reduction of NMDA-induced excitotoxic lesion in rats when tPA stop was co-injected. Altogether, these data demonstrate that the blockade of the endogenous proteolytic activity of tPA in the cerebral parenchyma could be a powerful neuroprotective strategy raised against brain pathologies associated with excitotoxicity.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Activador de Tejido Plasminógeno/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Cicloheptanos , Agonistas de Aminoácidos Excitadores/toxicidad , Técnicas In Vitro , Masculino , Ratones , N-Metilaspartato/toxicidad , Neuronas/citología , Neurotoxinas/toxicidad , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
To correlate brain metabolic status with the molecular events during cerebral ischemia, a cDNA array was performed after positron emission tomography scanning in a model of focal cerebral ischemia in baboons. Cluster analysis for the expression of 74 genes allowed the identification of 4 groups of genes. In each of the distinct groups, the authors observed a marked inflection in the pattern of gene expression when the CMRo was reduced by 48% to 66%. These patterns of coordinated modifications in gene expression could define molecular checkpoints for the development of an ischemic infarct and a molecular definition of the penumbra.
Asunto(s)
Isquemia Encefálica/genética , Encéfalo/metabolismo , Regulación de la Expresión Génica , Animales , Encéfalo/irrigación sanguínea , Isquemia Encefálica/metabolismo , Arterias Cerebrales/metabolismo , Arterias Cerebrales/patología , Clonación Molecular , Análisis por Conglomerados , ADN Complementario , Modelos Animales de Enfermedad , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Consumo de Oxígeno , PapioRESUMEN
Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Hipocampo/citología , Microscopía/métodos , Terminales Presinápticos/metabolismo , Animales , Supervivencia Celular , Espinas Dendríticas/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Despite their low abundance, phosphoinositides are critical regulators of intracellular signaling and membrane compartmentalization. However, little is known of phosphoinositide function at the postsynaptic membrane. Here we show that continuous synthesis and availability of phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) at the postsynaptic terminal is necessary for sustaining synaptic function in rat hippocampal neurons. This requirement was specific for synaptic, but not extrasynaptic, AMPA receptors, nor for NMDA receptors. PIP(3) downregulation impaired PSD-95 accumulation in spines. Concomitantly, AMPA receptors became more mobile and migrated from the postsynaptic density toward the perisynaptic membrane within the spine, leading to synaptic depression. Notably, these effects were only revealed after prolonged inhibition of PIP(3) synthesis or by direct quenching of this phosphoinositide at the postsynaptic cell. Therefore, we conclude that a slow, but constant, turnover of PIP(3) at synapses is required for maintaining AMPA receptor clustering and synaptic strength under basal conditions.