RESUMEN
BACKGROUND: Urokinase-type plasminogen activator (uPA) is a serine proteinase found in excitatory synapses located in the II/III and V cortical layers. The synaptic release of uPA promotes the formation of synaptic contacts and the repair of synapses damaged by various forms of injury, and its abundance is decreased in the synapse of Alzheimer's disease (AD) patients. Inactivation of the Wingless/Int1 (Wnt)-ß-catenin pathway plays a central role in the pathogenesis of AD. Soluble amyloid-ß (Aß) prevents the phosphorylation of the low-density lipoprotein receptor-related protein-6 (LRP6), and the resultant inactivation of the Wnt-ß-catenin pathway prompts the amyloidogenic processing of the amyloid-ß protein precursor (AßPP) and causes synaptic loss. OBJECTIVE: To study the role of neuronal uPA in the pathogenesis of AD. METHODS: We used in vitro cultures of murine cerebral cortical neurons, a murine neuroblastoma cell line transfected with the APP-695 Swedish mutation (N2asw), and mice deficient on either plasminogen, or uPA, or its receptor (uPAR). RESULTS: We show that uPA activates the Wnt-ß-catenin pathway in cerebral cortical neurons by triggering the phosphorylation of LRP6 via a plasmin-independent mechanism that does not require binding of Wnt ligands (Wnts). Our data indicate that uPA-induced activation of the Wnt-ß-catenin pathway protects the synapse from the harmful effects of soluble Aß and prevents the amyloidogenic processing of AßPP by inhibiting the expression of ß-secretase 1 (BACE1) and the ensuing generation of Aß40 and Aß42 peptides. CONCLUSION: uPA protects the synapse and antagonizes the inhibitory effect of soluble Aß on the Wnt-ß-catenin pathway by providing an alternative pathway for LRP6 phosphorylation and ß-catenin stabilization.
Asunto(s)
Enfermedad de Alzheimer , Activador de Plasminógeno de Tipo Uroquinasa , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Fibrinolisina/metabolismo , Lipoproteínas LDL , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Ratones , Neuronas/metabolismo , Fosforilación , Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , beta Catenina/metabolismoRESUMEN
Ischemic tolerance is a phenomenon whereby transient exposure to a non-injurious preconditioning stimulus triggers resistance to a subsequent lethal ischemic insult. Despite the fact that not only neurons but also astrocytes and endothelial cells have a unique response to preconditioning stimuli, current research has been focused mostly on the effect of preconditioning on neuronal death. Thus, it is unclear if the blood-brain barrier (BBB) can be preconditioned independently of an effect on neuronal survival. The release of tissue-type plasminogen activator (tPA) from perivascular astrocytes in response to an ischemic insult increases the permeability of the BBB. In line with these observations, treatment with recombinant tPA increases the permeability of the BBB and genetic deficiency of tPA attenuates the development of post-ischemic edema. Here we show that tPA induces ischemic tolerance in the BBB independently of an effect on neuronal survival. We found that tPA renders the BBB resistant to an ischemic injury by inducing TNF-α-mediated astrocytic activation and increasing the abundance of aquaporin-4-immunoreactive astrocytic end-feet processes in the neurovascular unit. This is a new role for tPA, that does not require plasmin generation, and with potential therapeutic implications for patients with cerebrovascular disease.
Asunto(s)
Isquemia Encefálica , Activador de Tejido Plasminógeno , Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Células Endoteliales/metabolismo , Humanos , Isquemia/metabolismo , Activador de Tejido Plasminógeno/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Urokinase-type plasminogen activator (uPA; encoded by Plau) is a serine proteinase that, in the central nervous system, induces astrocytic activation. ß-Catenin is a protein that links the cytoplasmic tail of cadherins to the actin cytoskeleton, thus securing the formation of cadherin-mediated cell adhesion complexes. Disruption of cell-cell contacts leads to the detachment of ß-catenin from cadherins, and ß-catenin is then degraded by the proteasome following its phosphorylation by GSK3ß. Here, we show that astrocytes release uPA following a scratch injury, and that this uPA promotes wound healing via a plasminogen-independent mechanism. We found that uPA induces the detachment of ß-catenin from the cytoplasmic tail of N-cadherin (NCAD; also known as CDH2) by triggering its phosphorylation at Tyr654. Surprisingly, this is not followed by degradation of ß-catenin because uPA also induces the phosphorylation of the low density lipoprotein receptor-related protein 6 (LRP6) at Ser1490, which then blocks the kinase activity of GSK3ß. Our work indicates that the ensuing cytoplasmic accumulation of ß-catenin is followed by its nuclear translocation and ß-catenin-triggered transcription of the receptor for uPA (Plaur), which in turn is required for uPA to induce astrocytic wound healing.
Asunto(s)
Activador de Plasminógeno de Tipo Uroquinasa , beta Catenina , Cadherinas/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Cicatrización de Heridas , beta Catenina/genéticaRESUMEN
The synapse is a complex structure where the transmission of information takes place. Synaptic dysfunction is one of the earliest pathophysiological events in several diseases, such as traumatic brain injury, cerebral ischemia, and neurodegenerative diseases. Thus, a methodology to study synaptic structure and function is crucial for the development of potential strategies for the treatment of many neurological diseases. Synaptoneurosomes (SNs) are structures assembled by the sealed presynaptic bouton and the attached post-synaptic density. Despite the fact that for a long time it has been recognized that SNs are a powerful tool to study synaptic function, composition, and structure, its use has been limited by the requirement of relatively large amounts of material to successfully isolate them. Here we describe a three-step centrifugation procedure performed under hypotonic conditions to isolate SNs from small volumes of the cerebral cortex. Graphic abstract: Schematic flowchart for the preparation of synaptoneurosomes.
RESUMEN
Urokinase-type plasminogen activator (uPA) is a serine proteinase that catalyzes the generation of plasmin on the cell surface and activates cell signaling pathways that promote remodeling and repair. Neuronal cadherin (NCAD) is a transmembrane protein that in the mature brain mediates the formation of synaptic contacts in the II/III and V cortical layers. Our studies show that uPA is preferentially found in the II/III and V cortical laminae of the gyrencephalic cortex of the non-human primate. Furthermore, we found that in murine cerebral cortical neurons and induced pluripotent stem cell (iPSC)-derived neurons prepared from healthy human donors, most of this uPA is associated with pre-synaptic vesicles. Our in vivo experiments revealed that in both, the gyrencephalic cortex of the non-human primate and the lissecephalic murine brain, cerebral ischemia decreases the number of intact synaptic contacts and the expression of uPA and NCAD in a band of tissue surrounding the necrotic core. Additionally, our in vitro data show that uPA induces the synthesis of NCAD in cerebral cortical neurons, and in line with these observations, intravenous treatment with recombinant uPA three hours after the onset of cerebral ischemia induces NCAD-mediated repair of synaptic contacts in the area surrounding the necrotic core.
Asunto(s)
Isquemia Encefálica/fisiopatología , Cadherinas/metabolismo , Sinapsis/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , RatonesRESUMEN
Soluble amyloid ß (Aß)-induced synaptic dysfunction is an early event in the pathogenesis of Alzheimer's disease (AD) that precedes the deposition of insoluble Aß and correlates with the development of cognitive deficits better than the number of plaques. The mammalian plasminogen activation (PA) system catalyzes the generation of plasmin via two activators: tissue-type (tPA) and urokinase-type (uPA). A dysfunctional tPA-plasmin system causes defective proteolytic degradation of Aß plaques in advanced stages of AD. In contrast, it is unknown whether uPA and its receptor (uPAR) contribute to the pathogenesis of this disease. Neuronal cadherin (NCAD) plays a pivotal role in the formation of synapses and dendritic branches, and Aß decreases its expression in cerebral cortical neurons. Here we show that neuronal uPA protects the synapse from the harmful effects of soluble Aß. However, Aß-induced inactivation of the eukaryotic initiation factor 2α halts the transcription of uPA mRNA, leaving unopposed the deleterious effects of Aß on the synapse. In line with these observations, the synaptic abundance of uPA, but not uPAR, is decreased in the frontal cortex of AD patients and 5xFAD mice, and in cerebral cortical neurons incubated with soluble Aß. We found that uPA treatment increases the synaptic expression of NCAD by a uPAR-mediated plasmin-independent mechanism, and that uPA-induced formation of NCAD dimers protects the synapse from the harmful effects of soluble Aß oligomers. These data indicate that Aß-induced decrease in the synaptic abundance of uPA contributes to the development of synaptic damage in the early stages of AD.SIGNIFICANCE STATEMENT Soluble amyloid ß (Aß)-induced synaptic dysfunction is an early event in the pathogenesis of cognitive deficits in Alzheimer's disease (AD). We found that neuronal urokinase-type (uPA) protects the synapse from the deleterious effects of soluble Aß. However, Aß-induced inactivation of the eukaryotic initiation factor 2α decreases the synaptic abundance of uPA, leaving unopposed the harmful effects of Aß on the synapse. In line with these observations, the synaptic expression of uPA is decreased in the frontal cortex of AD brains and 5xFAD mice, and uPA treatment abrogates the deleterious effects of Aß on the synapse. These results unveil a novel mechanism of Aß-induced synaptic dysfunction in AD patients, and indicate that recombinant uPA is a potential therapeutic strategy to protect the synapse before the development of irreversible brain damage.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Corteza Cerebral/efectos de los fármacos , Neuronas/efectos de los fármacos , Sinapsis/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.
Asunto(s)
Proteína GAP-43/metabolismo , Sinapsis/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Proteína GAP-43/análisis , Ratones , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Receptores de N-Metil-D-Aspartato/análisis , Receptores de N-Metil-D-Aspartato/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/análisisRESUMEN
Cerebral ischemia causes the presynaptic release of tissue-type plasminogen activator (tPA). The postsynaptic density (PSD) is a postsynaptic structure that provides a matrix where signaling transduction of excitatory synapses takes place. The postsynaptic density protein-95 (PSD-95) is the most abundant scaffolding protein in the postsynaptic density (PSD), where it modulates the postsynaptic response to the presynaptic release of glutamate by regulating the anchoring of glutamate receptors to the PSD. We found that tPA induces the local translation of PSD-95 mRNA and the subsequent recruitment of PSD-95 protein to the PSD, via plasminogen-independent activation of TrkB receptors. Our data show that PSD-95 is removed from the PSD during the early stages of cerebral ischemia, and that this effect is abrogated by either the release of neuronal tPA, or intravenous administration of recombinant tPA (rtPA). We report that the effect of tPA on PSD-95 is associated with inhibition of the phosphorylation and recruitment of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to the PSD, known to amplify the effect of the excitotoxic injury, and that this is followed by TrkB-mediated protection of dendritic spines from the harmful effects of the hypoxic insult. These data reveal that tPA is a synaptic protector in the ischemic brain.
Asunto(s)
Isquemia Encefálica/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Densidad Postsináptica/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Densidad Postsináptica/efectos de los fármacos , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Axonal injury is a common cause of neurological dysfunction. Unfortunately, in contrast to axons from the peripheral nervous system, the limited capacity of regeneration of central nervous system (CNS) axons is a major obstacle for functional recovery in patients suffering neurological diseases that involve the subcortical white matter. Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to the urokinase-type plasminogen activator receptor (uPAR) catalyzes the conversion of plasminogen into plasmin on the cell surface. uPAR expression increases after an injury, and signaling through uPAR promotes tissue remodeling. However, it is yet unknown whether uPA binding to uPAR has an effect on axonal recovery in the CNS. Here, we used in vitro and in vivo models of CNS axonal injury to test the hypothesis that uPA binding to uPAR promotes axonal regeneration in the CNS. We found that newly formed growth cones from axons re-emerging from an axonal injury express uPAR and that binding of uPA to this uPAR promotes axonal recovery by a mechanism that does not require the generation of plasmin. Our data indicate that the binding of recombinant uPA or endogenous uPA to uPAR induces membrane recruitment and activation of ß1 integrin via the low density lipoprotein receptor-related protein-1 (LRP1), which leads to activation of the Rho family small GTPase Rac1 and Rac1-induced axonal regeneration. Our results show that the uPA/uPAR/LRP1 system is a potential target for the development of therapeutic strategies to promote axonal recovery following a CNS injury.
Asunto(s)
Axones/fisiología , Sistema Nervioso Central/metabolismo , Regeneración Nerviosa , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Humanos , Integrina beta1/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Unión ProteicaRESUMEN
Tissue-type plasminogen activator (tPA) is a serine proteinase released by the presynaptic terminal of cerebral cortical neurons following membrane depolarization (Echeverry et al., 2010). Recent studies indicate that the release of tPA triggers the synaptic vesicle cycle and promotes the exocytosis (Wu et al., 2015) and endocytic retrieval (Yepes et al., 2016) of glutamate-containing synaptic vesicles. Here we used electron microscopy, proteomics, quantitative phosphoproteomics, biochemical analyses with extracts of the postsynaptic density (PSD), and an animal model of cerebral ischemia with mice overexpressing neuronal tPA to study whether the presynaptic release of tPA also has an effect on the postsynaptic terminal. We found that tPA has a bidirectional effect on the composition of the PSD of cerebral cortical neurons that is independent of the generation of plasmin and the presynaptic release of glutamate, but depends on the baseline level of neuronal activity and the extracellular concentrations of calcium (Ca2+). Accordingly, in neurons that are either inactive or incubated with low Ca2+ concentrations tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-mediated phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in neurons with previously increased baseline levels of pCaMKIIα in the PSD due to neuronal depolarization in vivo or incubation with high concentrations of either Ca2+ or glutamate in vitro, tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. We found that these effects of tPA are mediated by synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. Our data indicate that by regulating the pCaMKIIα/PP1 balance in the PSD tPA acts as a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate.
RESUMEN
The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca(+2) elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein ßII-spectrin to the AZ and promotes the binding of SVs to ßII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca(+2)-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission.
Asunto(s)
Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Neuronas/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Activador de Tejido Plasminógeno/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Isquemia Encefálica/patología , Canales de Calcio/efectos de los fármacos , Células Cultivadas , Exocitosis/efectos de los fármacos , Fibrinolisina/biosíntesis , Inmunohistoquímica , Masculino , Fosforilación/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Proteómica , Ratas , Ratas Sprague-Dawley , Espectrina/biosíntesis , Sinapsinas/metabolismoRESUMEN
Spines are dendritic protrusions that receive most of the excitatory input in the brain. Early after the onset of cerebral ischemia dendritic spines in the peri-infarct cortex are replaced by areas of focal swelling, and their re-emergence from these varicosities is associated with neurological recovery after acute ischemic stroke (AIS). Urokinase-type plasminogen activator (uPA) is a serine proteinase that plays a central role in tissue remodeling via binding to the urokinase plasminogen activator receptor (uPAR). We report that cerebral cortical neurons release uPA during the recovery phase from ischemic stroke in vivo or hypoxia in vitro. Although uPA does not have an effect on ischemia- or hypoxia-induced neuronal death, genetic deficiency of uPA (uPA(-/-)) or uPAR (uPAR(-/-)) abrogates functional recovery after AIS. Treatment with recombinant uPA after ischemic stroke induces neurological recovery in wild-type and uPA(-/-) but not in uPAR(-/-) mice. Diffusion tensor imaging studies indicate that uPA(-/-) mice have increased water diffusivity and decreased anisotropy associated with impaired dendritic spine recovery and decreased length of distal neurites in the peri-infarct cortex. We found that the excitotoxic injury induces the clustering of uPAR in dendritic varicosities, and that the binding of uPA to uPAR promotes the reorganization of the actin cytoskeleton and re-emergence of dendritic filopodia from uPAR-enriched varicosities. This effect is independent of uPA's proteolytic properties and instead is mediated by Rac-regulated profilin expression and cofilin phosphorylation. Our data indicate that binding of uPA to uPAR promotes dendritic spine recovery and improves functional outcome following AIS.
Asunto(s)
Isquemia Encefálica/enzimología , Espinas Dendríticas/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Recuperación de la Función/fisiología , Accidente Cerebrovascular/enzimología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Células Cultivadas , Espinas Dendríticas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/enzimología , Enfermedades del Sistema Nervioso/patología , Unión Proteica/fisiología , Recuperación de la Función/efectos de los fármacos , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Resultado del Tratamiento , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/uso terapéuticoRESUMEN
Adenosine monophosphate-activated protein kinase (AMPK) is an energy sensor that regulates cellular adaptation to metabolic stress. Tissue-type plasminogen activator (tPA) is a serine proteinase found in the intravascular space, where its main role is as thrombolytic enzyme, and in neurons, where its function is less well understood. Here, we report that glucose deprivation induces the mobilization and package of neuronal tPA into presynaptic vesicles. Mass spectrometry and immunohistochemical studies show that the release of this tPA in the synaptic space induces AMPK activation in the postsynaptic terminal, and an AMPK-mediated increase in neuronal uptake of glucose and neuronal adenosine 5'(tetrahydrogen triphosphate; ATP) synthesis. This effect is independent of tPA's proteolytic properties, and instead requires the presence of functional N-methyl-D-aspartate receptors (NMDARs). In agreement with these observations, positron emission tomography (PET) studies and biochemical analysis with synaptoneurosomes indicate that the intravenous administration of recombinant tPA (rtPA) after transient middle cerebral artery occlusion (tMCAO) induces AMPK activation in the synaptic space and NMDAR-mediated glucose uptake in the ischemic brain. These data indicate that the release of neuronal tPA or treatment with rtPA activate a cell signaling pathway in the synaptic space that promotes the detection and adaptation to metabolic stress.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Oxígeno/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Sinaptosomas/patología , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/uso terapéuticoRESUMEN
Here we explore the role of semaphorin 3A and 3F (Sema3A, Sema3F) in the formation of the mesotelencephalic pathway. We show that Sema3A and 3F are expressed in the ventral mesencephalon (VM) of E13.5 rat embryos; the receptors Neuropilin 1 and Neuropilin 2, and co-receptors L1CAM, NrCAM, and Plexins A1 and A3 but not A4 are expressed by VM dopaminergic neurons; these neurons bind Sema3A and 3F in vitro which induces collapse of their growth cones and elicits, with different potencies, a repulsive response; and this response is absent in axons from Nrp1 and Nrp2 null embryos. Despite these in vitro effects, only very mild anatomical defects were detected in the organization of the mesotelencephalic pathway in embryonic and adult Nrp1 or Nrp2 null mice. However, the dopaminergic meso-habenular pathway and catecholaminergic neurons in the parafascicular and paraventricular nuclei of the thalamus were significantly affected in Nrp2 null mice. These data are consistent with a model whereby Sema3A and 3F, in combination with other guidance molecules, contributes to the navigation of DA axons to their final synaptic targets.
Asunto(s)
Dopamina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Semaforina-3A/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Quimiotaxis/genética , Diencéfalo/citología , Diencéfalo/embriología , Diencéfalo/metabolismo , Femenino , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Péptidos y Proteínas de Señalización Intracelular/genética , Mesencéfalo/citología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Vías Nerviosas/citología , Vías Nerviosas/embriología , Vías Nerviosas/metabolismo , Neuronas/citología , Ratas , Ratas Wistar , Semaforina-3A/genética , Telencéfalo/citología , Telencéfalo/embriología , Telencéfalo/metabolismoRESUMEN
The brain produces two brain-derived neurotrophic factor (BDNF) transcripts, with either short or long 3' untranslated regions (3' UTRs). The physiological significance of the two forms of mRNAs encoding the same protein is unknown. Here, we show that the short and long 3' UTR BDNF mRNAs are involved in different cellular functions. The short 3' UTR mRNAs are restricted to somata, whereas the long 3' UTR mRNAs are also localized in dendrites. In a mouse mutant where the long 3' UTR is truncated, dendritic targeting of BDNF mRNAs is impaired. There is little BDNF in hippocampal dendrites despite normal levels of total BDNF protein. This mutant exhibits deficits in pruning and enlargement of dendritic spines, as well as selective impairment in long-term potentiation in dendrites, but not somata, of hippocampal neurons. These results provide insights into local and dendritic actions of BDNF and reveal a mechanism for differential regulation of subcellular functions of proteins.
Asunto(s)
Regiones no Traducidas 3'/análisis , Regiones no Traducidas 3'/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Animales , Dendritas/química , Ratones , Ratones Endogámicos C57BL , Neuronas/química , Neuronas/citología , Poliadenilación , Biosíntesis de Proteínas , Receptor trkB/análisisRESUMEN
BACKGROUND & AIMS: The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal and postnatal enteric neuronal cell lines using H-2K(b)-tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 large tumor antigen gene under the control of an interferon gamma-inducible H-2K(b) promoter element. METHODS: Enteric neuronal precursors were isolated from the intestines of E13-mouse fetuses and second day postnatal mice using magnetic immunoselection with a p75NTR antibody. The cells were maintained at the permissive temperature, 33 degrees C, and interferon-gamma for 24 or 48 hours, and then transferred to 39 degrees C in the presence of glial cell line-derived neurotrophic factor for 7 days for further differentiation. Neuronal markers were assessed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Neuronal function was assessed by transplanting these cells into the colons of Piebald or nNOS(-/-) mice. RESULTS: Expression analysis of cells showed the presence of neuronal markers peripherin, PGP9.5, HuD, tau, synaptic marker synaptophysin, characteristic receptors of enteric neurons, Ret, and 5-hydroxytryptamine-receptor subtypes at 33 degrees C and 39 degrees C. Nestin, S-100beta, and alpha-smooth muscle actin were expressed minimally at 39 degrees C. Glial cell line-derived neurotrophic factor resulted in increased phosphorylation of Akt in these cells, similar to primary enteric neurons. Transplantation of cells into the piebald or nNOS(-/-) mice colon improved colonic motility. CONCLUSIONS: We have developed novel enteric neuronal cell lines that have neuronal characteristics similar to primary enteric neurons. These cells can help us in understanding newer therapeutic options for Hirschsprung's disease.
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Colon/inervación , Sistema Nervioso Entérico/embriología , Motilidad Gastrointestinal/fisiología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , ARN/genética , Actinas/biosíntesis , Actinas/genética , Animales , Western Blotting , Línea Celular , Colon/embriología , Colon/cirugía , Proteínas ELAV/biosíntesis , Proteínas ELAV/genética , Proteína 4 Similar a ELAV , Sistema Nervioso Entérico/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas de Filamentos Intermediarios/genética , Contracción Isométrica/fisiología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Liso/inervación , Músculo Liso/fisiología , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/biosíntesis , Nestina , Neuroglía/citología , Neuroglía/metabolismo , Neuroglía/trasplante , Neuronas/citología , Periferinas , Embarazo , Proteínas Proto-Oncogénicas c-ret/biosíntesis , Proteínas Proto-Oncogénicas c-ret/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/biosíntesis , Proteínas S100/genética , Serotonina/biosíntesis , Serotonina/genética , Sinaptofisina/biosíntesis , Sinaptofisina/genética , Ubiquitina Tiolesterasa/biosíntesis , Ubiquitina Tiolesterasa/genética , Proteínas de Xenopus , Proteínas tau/biosíntesisRESUMEN
The dogma that the adult central nervous system (CNS) is nonpermissive to axonal regeneration is beginning to fall in the face of increased understanding of the molecular and cellular biology of axon outgrowth. It is now appreciated that axon growth is regulated by a combination of extracellular factors related to the milieu of the developing or adult CNS and the presence of injury, and intracellular factors related to the "growth state" of the developing or regenerating neuron. Several critical points of convergence within the developing or regenerating neuron for mediating intracellular cell signaling effects on the growth cone cytoskeleton have been identified, and their modulation has produced marked increases in axon outgrowth within the "nonpermissive" milieu of the adult injured CNS. One such critical convergence point is the small GTPase RhoA, which integrates signaling events produced by both myelin-associated inhibitors (e.g., NogoA) and astroglial-derived inhibitors (chondroitin sulfate proteoglycans) and regulates the activity of downstream effectors that modulate cytoskeletal dynamics within the growth cone mediating axon outgrowth or retraction. Inhibition of RhoA has been associated with increased outgrowth on nonpermissive substrates in vitro and increased axon regeneration in vivo. We are developing lentiviral vectors that modulate RhoA activity, allowing more long-term expression than is possible with current approaches. These vectors may be useful in regenerative strategies for spinal cord injury, brain injury, and neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, and Huntington's disease.
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Axones/fisiología , Sistema Nervioso Central/lesiones , Regeneración Nerviosa/fisiología , Transducción de Señal/fisiología , Traumatismos del Sistema Nervioso/terapia , Proteína de Unión al GTP rhoA/metabolismo , Animales , Astrocitos/metabolismo , Vectores Genéticos , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Glicoproteína Asociada a Mielina/metabolismo , Neuronas/citología , Neuronas/fisiología , Traumatismos del Sistema Nervioso/patología , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1(-/-)). HIP1(-/-) mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1(-/-) mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis.
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Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Endocitosis , Receptores AMPA/metabolismo , Animales , Encéfalo/metabolismo , Clatrina/metabolismo , Colorimetría , Femenino , Técnica del Anticuerpo Fluorescente , Ratones , Fenotipo , Embarazo , Transporte de Proteínas , Médula Espinal/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Coat proteins cycle between soluble and membrane-bound locations at the time of vesicle biogenesis and act to regulate the assembly of the vesicle coat that determines the specificity in cargo selection and the destination of the vesicle. A transmembrane cargo protein, an Arf GTPase, and a coat protein (e.g. COPs, APs, or GGAs) are minimal components required for budding of vesicles. Munc18 interacting proteins (MINTs) are a family of three proteins implicated in the localization of receptors to the plasma membrane. We show that MINTs bind Arfs directly, co-localize with Arf and the Alzheimer's precursor protein (beta-APP) to regions of the Golgi/trans-Golgi network, and can co-immunoprecipitate clathrin. We demonstrate that MINTs bind Arfs through a region of the PTB domain and the PDZ2 domain, and Arf-MINT interaction is necessary for the increased cellular levels of beta-APP produced by MINT overexpression. Knockdown (small interference RNA) experiments implicate beta-APP as a transmembrane cargo protein that works together with MINTs. We propose that MINTs are a family of Arf-dependent, vesicle-coat proteins that can regulate the traffic of beta-APP.
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Precursor de Proteína beta-Amiloide/química , Cadherinas , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Brefeldino A/farmacología , Células COS , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Modelos Genéticos , Proteínas Munc18 , Mutación , Proteínas del Tejido Nervioso/química , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte Vesicular/químicaRESUMEN
Stigmoid bodies (SBs) are structures present in the cytoplasm of neurons. Many brain regions including hypothalamus, thalamus, amygdala, septum, hippocampus, colliculi, and brainstem contain neurons with at least one SB. Despite this widespread distribution their function remains unknown. SBs contain a brain protein called huntingtin-associated protein 1 (HAP1) and have more recently been found to contain the apolipoprotein E receptor LR11 (Lipoprotein Receptor containing 11 LDL binding domains, also called SorLA for sorting protein-related receptor containing LDLR class A repeats) and sortilin. To provide a first step towards further identification of their components and perhaps shed some light on their neurobiological role, we have developed a method for isolating SBs from rat brain. The protocol relies on a combination of centrifugational forces, sucrose gradient, and immunoisolation. Samples enriched in SBs were incubated with antibodies to HAP1B or to LR11 followed by incubation with FITC conjugated secondary antibodies. Anti-FITC coated beads were incubated with samples and SB-bead complexes formed were separated by magnetic sorting without pelleting the complexes during the isolation procedure. Immunopurified SBs, visualized by light and electron microscopy, show similar ultrastructure to those present in neurons.