RESUMEN
Exogenous transplanted neural precursor cells (NPCs) exhibit miscellaneous immune-modulatory effects in models of autoimmune demyelination. However, the regional interactions of NPCs with the host brain tissue in remissive inflammatory events have not been adequately studied. In this study we used the chronic MOG-induced Experimental Autoimmune Encephalomyelitis (EAE) model in C57BL/six mice. Based on previous data, we focused on neuropathology at Day 50 post-induction (D50) and studied the expression of connexin43 (Cx43) and Cx47, two of the main glial gap junction (GJ) proteins, in relation to the intraventricular transplantation of GFP(+) NPCs and their integration with the host tissue. By D50, NPCs had migrated intraparenchymally and were found in the corpus callosum at the level of the lateral ventricles and hippocampus. The majority of GFP(+) cells differentiated with simple or ramified processes expressing mainly markers of mature GLIA (GFAP and NogoA) and significantly less of precursor glial cells. GFP(+) NPCs expressed connexins and formed GJs around the hippocampus more than lateral ventricles. The presence of NPCs did not alter the increase in Cx43 GJ plaques at D50 EAE, but prevented the reduction of oligodendrocytic Cx47, increased the number of oligodendrocytes, local Cx47 levels and Cx47 GJ plaques per cell. These findings suggest that transplanted NPCs may have multiple effects in demyelinating pathology, including differentiation and direct integration into the panglial syncytium, as well as amelioration of oligodendrocyte GJ loss, increasing the supply of potent myelinating cells to the demyelinated tissue.
Asunto(s)
Encéfalo/patología , Conexina 43/metabolismo , Conexinas/metabolismo , Encefalomielitis Autoinmune Experimental/cirugía , Regulación de la Expresión Génica/fisiología , Células-Madre Neurales/trasplante , Factores de Edad , Animales , Encéfalo/citología , Diferenciación Celular , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proteína Básica de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/fisiología , Células-Madre Neurales/ultraestructura , Neuroglía/metabolismo , Neuroglía/patología , Neuroglía/ultraestructura , Fragmentos de Péptidos/toxicidadRESUMEN
The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.
Asunto(s)
Espinas Dendríticas/metabolismo , Receptores AMPA/metabolismo , Simportadores/metabolismo , Sinapsis/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Membrana Celular/metabolismo , Difusión , Hipocampo/citología , Espacio Intracelular/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Cotransportadores de K ClRESUMEN
Dentate gyrus granule cells have been suggested to corelease GABA and glutamate both in juvenile animals and under pathological conditions in adults. Although mossy fiber terminals (MFTs) are known to express glutamic acid decarboxylase (GAD) in early postnatal development, the functional role of GABA synthesis in MFTs remains controversial, and direct evidence for synaptic GABA release from MFTs is missing. Here, using GAD67-GFP transgenic mice, we show that GAD67 is expressed only in a population of immature granule cells in juvenile animals. We demonstrate that GABA can be released from these cells and modulate mossy fiber excitability through activation of GABAB autoreceptors. However, unitary postsynaptic currents generated by individual, GAD67-expressing granule cells are purely glutamatergic in all postsynaptic cell types tested. Thus GAD67 expression does not endow dentate gyrus granule cells with a full GABAergic phenotype and GABA primarily instructs the pre- rather than the postsynaptic element.
Asunto(s)
Lateralidad Funcional/fisiología , Hipocampo/citología , Fibras Musgosas del Hipocampo/metabolismo , Neuronas/citología , Terminales Presinápticos/fisiología , Transducción de Señal/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Animales Recién Nacidos , Venenos de Abeja/farmacología , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Antagonistas del GABA/farmacología , Glutamato Descarboxilasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas de Homeodominio/metabolismo , Imagenología Tridimensional , Técnicas In Vitro , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fibras Musgosas del Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Ácidos Fosfínicos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Terminales Presinápticos/efectos de los fármacos , Propanolaminas/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3ß both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3ß target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3ß induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Neuronas/metabolismo , Prolina/química , Serina/química , Estatmina/metabolismo , Animales , Diferenciación Celular , Glucógeno Sintasa Quinasa 3 beta , Células HeLa , Humanos , Microtúbulos/metabolismo , Modelos Biológicos , Neuritas/metabolismo , Fosforilación , Conejos , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Axon initial segments (AISs) and nodes of Ranvier (NRs) are essential regions for saltatory conduction of the action potential along the axon. These two domains are enriched in similar multimolecular complexes, which include voltage-gated sodium channels (Na(v)), NF186 (neurofascin 186), NrCAM (neuron glia-related cell adhesion molecule), and cytoskeleton linkers ankyrin G (AnkG) and betaIV-spectrin. Identification of novel members of these complexes is critical to better understand their formation, function, and maintenance. Here we report that IQCJ-SCHIP-1, a recently identified isoform of schwannomin-interacting protein-1 (SCHIP-1), is a novel component of both AISs and NRs in the central and peripheral nervous systems. We show that IQCJ-SCHIP-1 binds calmodulin in the absence of Ca(2+) and is highly enriched at AISs and NRs. IQCJ-SCHIP-1 accumulation at AISs and NRs is a late event, suggesting that IQCJ-SCHIP-1 is likely to play a role in mature AISs and NRs rather than during their formation. IQCJ-SCHIP-1 was not detected at AISs in the absence of AnkG and interacted in vitro with this protein. IQCJ-SCHIP-1 was also absent from central NRs and AISs of quivering mice, which have a mutation of betaIV-spectrin. We suggest that IQCJ-SCHIP-1 might participate, along with AnkG and betaIV-spectrin, in the stabilization or function of the multimolecular complexes of AISs and NRs, possibly by participating in Ca(2+)-mediated responses.
Asunto(s)
Axones/metabolismo , Proteínas Portadoras/metabolismo , Neuronas/citología , Nódulos de Ranvier/metabolismo , Animales , Ancirinas/deficiencia , Línea Celular Transformada , Chlorocebus aethiops , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/biosíntesis , Hipocampo/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Proteínas Asociadas a Microtúbulos/metabolismo , Espectrina/genética , Transfección/métodosRESUMEN
Schwannomin/merlin is the product of a tumor suppressor gene mutated in neurofibromatosis type 2 (NF2). Although the consequences of NF2 mutations on Schwann cell proliferation are well established, the physiological role of schwannomin in differentiated cells is not known. To unravel this role, we studied peripheral nerves in mice overexpressing in Schwann cells schwannomin with a deletion occurring in NF2 patients (P0-SCH-Delta39-121) or a C-terminal deletion. The myelin sheath and nodes of Ranvier were essentially preserved in both lines. In contrast, the ultrastructural and molecular organization of contacts between Schwann cells and axons in paranodal and juxtaparanodal regions were altered, with irregular juxtaposition of normal and abnormal areas of contact. Similar but more severe alterations were observed in mice with conditional deletion of the Nf2 gene in Schwann cells. The number of Schmidt-Lanterman incisures, which are cytoplasmic channels interrupting the compact myelin and characterized by distinct autotypic contacts, was increased in the three mutant lines. P0-SCH-Delta39-121 and conditionally deleted mice displayed exuberant wrapping of nonmyelinated fibers and short internodes, an abnormality possibly related to altered control of Schwann cell proliferation. In support of this hypothesis, Schwann cell number was increased along fibers before myelination in P0-SCH-Delta39-121 mice but not in those with C-terminal deletion. Schwann cell numbers were also more numerous in mice with conditional deletion. Thus, schwannomin plays an important role in the control of Schwann cell number and is necessary for the correct organization and regulation of axoglial heterotypic and glio-glial autotypic contacts.
Asunto(s)
Comunicación Celular/fisiología , Neurofibromina 2/fisiología , Células de Schwann/metabolismo , Células de Schwann/ultraestructura , Proteínas Supresoras de Tumor/fisiología , Animales , Proliferación Celular , Eliminación de Gen , Humanos , Ratones , Ratones Transgénicos , Neurofibromina 2/biosíntesis , Neurofibromina 2/deficiencia , Neurofibromina 2/genética , Nervios Periféricos/metabolismo , Nervios Periféricos/ultraestructura , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Polaridad Celular , Conos de Crecimiento/metabolismo , Proteínas SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Animales , Axones/metabolismo , Células COS , Adhesión Celular , Células Cultivadas , Chlorocebus aethiops , Exocitosis , Fusión de Membrana , Metaloendopeptidasas/metabolismo , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Toxina Tetánica/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
KCC2 regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in numerous neurological and psychiatric disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents is depolarized in KCC2 knockdown neurons, this shift is compensated by depolarization of the resting membrane potential. This reflects downregulation of leak potassium currents. We show KCC2 interacts with Task-3 (KCNK9) channels and is required for their membrane expression. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis, which could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that perturb network activity thus offering additional targets for therapeutic intervention.
Asunto(s)
Giro Dentado/metabolismo , Neuronas/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio/metabolismo , Convulsiones/metabolismo , Simportadores/metabolismo , Animales , Giro Dentado/efectos de los fármacos , Potenciales Evocados/genética , Potenciales Evocados/fisiología , Antagonistas del GABA/farmacología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Convulsiones/genética , Simportadores/genética , Ácido gamma-Aminobutírico/metabolismo , Cotransportadores de K ClRESUMEN
Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at approximately 21 d after birth and control the complex spike waveform.
Asunto(s)
Fibras Nerviosas/fisiología , Células de Purkinje/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Cerebelo/citología , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , N-Metilaspartato/farmacología , Técnicas de Placa-Clamp/métodos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Nanodiamonds that were prepared by high pressure/high temperature were functionalized with biomolecules for biological applications. Nanodiamonds (NDs, < or =35 nm) that were coated by silanization or with polyelectrolyte layers were grafted with a fluorescent thiolated peptide via a maleimido function; this led to an aqueous colloidal suspension that was stable for months. These substituted NDs were not cytotoxic for CHO cells. Their capacity to enter mammalian cells, and their localisation inside were ascertained after labelling the nucleus and actin, by examining the cells by confocal, reflected light and fluorescence microscopy.
Asunto(s)
Células/efectos de los fármacos , Células/metabolismo , Diamante/metabolismo , Diamante/toxicidad , Nanopartículas/química , Oligopéptidos/química , Animales , Células CHO , Muerte Celular/efectos de los fármacos , Células/citología , Cricetinae , Cricetulus , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/enzimología , Oxidorreductasas/metabolismoRESUMEN
The membrane-trafficking pathway mediated by tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) in neurons is still unknown. We show herein that TI-VAMP expression is necessary for neurite outgrowth in PC12 cells and hippocampal neurons in culture. TI-VAMP interacts with plasma membrane and endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptors, suggesting that TI-VAMP mediates a recycling pathway. L1, a cell-cell adhesion molecule involved in axonal outgrowth, colocalized with TI-VAMP in the developing brain, neurons in culture, and PC12 cells. Plasma membrane L1 was internalized into the TI-VAMP-containing compartment. Silencing of TI-VAMP resulted in reduced expression of L1 at the plasma membrane. Finally, using the extracellular domain of L1 and N-cadherin immobilized on beads, we found that the silencing of TI-VAMP led to impaired L1- but not N-cadherin-mediated adhesion. Furthermore, TI-VAMP- but not synaptobrevin 2-containing vesicles accumulated at the site of the L1 bead-cell junction. We conclude that TI-VAMP mediates the intracellular transport of L1 and that L1-mediated adhesion controls this membrane trafficking, thereby suggesting an important cross talk between membrane trafficking and cell-cell adhesion.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuritas/metabolismo , Neuronas/metabolismo , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Encéfalo/embriología , Encéfalo/metabolismo , Cadherinas/metabolismo , Cadherinas/fisiología , Compartimento Celular , Membrana Celular/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Gránulos Citoplasmáticos/fisiología , Embrión de Mamíferos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Microscopía por Video , Molécula L1 de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología , Neuronas/fisiología , Células PC12 , Unión Proteica , Estructura Terciaria de Proteína , Proteínas R-SNARE , ARN Interferente Pequeño/farmacología , Ratas , Proteínas SNARE , Transducción de Señal , Fracciones SubcelularesRESUMEN
Brain development involves extensive migration of neurons. Microtubules (MTs) are key cellular effectors of neuronal displacement that are assembled from α/ß-tubulin heterodimers. Mutation of the α-tubulin isotype TUBA1A is associated with cortical malformations in humans. In this study, we provide detailed in vivo and in vitro analyses of Tuba1a mutants. In mice carrying a Tuba1a missense mutation (S140G), neurons accumulate, and glial cells are dispersed along the rostral migratory stream in postnatal and adult brains. Live imaging of Tuba1a-mutant neurons revealed slowed migration and increased neuronal branching, which correlated with directionality alterations and perturbed nucleus-centrosome (N-C) coupling. Tuba1a mutation led to increased straightness of newly polymerized MTs, and structural modeling data suggest a conformational change in the α/ß-tubulin heterodimer. We show that Tuba8, another α-tubulin isotype previously associated with cortical malformations, has altered function compared with Tuba1a. Our work shows that Tuba1a plays an essential, noncompensated role in neuronal saltatory migration in vivo and highlights the importance of MT flexibility in N-C coupling and neuronal-branching regulation during neuronal migration.
Asunto(s)
Encéfalo/metabolismo , Movimiento Celular , Microtúbulos/metabolismo , Neurogénesis , Neuronas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Encéfalo/patología , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Ratones Endogámicos C3H , Ratones Mutantes , Microscopía Fluorescente , Microtúbulos/patología , Simulación de Dinámica Molecular , Mutación Missense , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/patología , Fenotipo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transducción de Señal , Relación Estructura-Actividad , Factores de Tiempo , Imagen de Lapso de Tiempo , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Grabación en VideoRESUMEN
Maturation of functional neuronal circuits during central nervous system development relies on sophisticated mechanisms. First, axonal and dendritic growth should reach appropriate targets for correct synapse elaboration. Second, pruning and neuronal death are required to eliminate redundant or inappropriate neuronal connections. Serotonin, in addition to its role as a neurotransmitter, actively participates in postnatal establishment and refinement of brain wiring in mammals. Brain resident macrophages, that is, microglia, also play an important role in developmentally regulated neuronal death as well as in synaptic maturation and elimination. Here, we tested the hypothesis of cross-regulation between microglia and serotonin during postnatal brain development in a mouse model of synaptic refinement. We found expression of the serotonin 5-HT2B receptor on postnatal microglia, suggesting that serotonin could participate in temporal and spatial synchronization of microglial functions. Using two-photon microscopy, acute brain slices, and local delivery of serotonin, we observed that microglial processes moved rapidly toward the source of serotonin in Htr2B(+/+) mice, but not in Htr2B(-/-) mice lacking the 5-HT2B receptor. We then investigated whether some developmental steps known to be controlled by serotonin could potentially result from microglia sensitivity to serotonin. Using an in vivo model of synaptic refinement during early brain development, we investigated the maturation of the retinal projections to the thalamus and observed that Htr2B(-/-) mice present anatomical alterations of the ipsilateral projecting area of retinal axons into the thalamus. In addition, activation markers were upregulated in microglia from Htr2B(-/-) compared to control neonates, in the absence of apparent morphological modifications. These results support the hypothesis that serotonin interacts with microglial cells and these interactions participate in brain maturation.
Asunto(s)
Cuerpos Geniculados/crecimiento & desarrollo , Microglía/fisiología , Receptor de Serotonina 5-HT2A/metabolismo , Retina/crecimiento & desarrollo , Serotonina/metabolismo , Sinapsis/fisiología , Animales , Receptor 1 de Quimiocinas CX3C , Células Cultivadas , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Cuerpos Geniculados/fisiología , Hipocampo/crecimiento & desarrollo , Hipocampo/fisiología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptor de Serotonina 5-HT2A/genética , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Retina/fisiología , Técnicas de Cultivo de Tejidos , Vías Visuales/crecimiento & desarrollo , Vías Visuales/fisiologíaRESUMEN
Thyroid hormones (TH) and receptors (TRs) may play an important role in the pathophysiology of acute cerebral ischemia. In the present study, we sought to determine whether serum triodothyronine (T3)/thyroxine (T4) and brain TRs (TRα1, TRß1) might change after experimental stroke. Male adult Wistar rats were subjected to permanent middle cerebral artery occlusion (group P) and compared to sham-operated controls (group S). Animals were followed clinically for 14 days until brain collection for Western blot (WB) or neuropathological analysis of TRs in three different brain areas (infarcted tissue, E1; noninfarcted ipsilateral hemisphere, E2; and contralateral hemisphere, E3). Analysis of serum TH levels showed a reduction of T4 in group P (p = 0.002) at days 2 to 14, while half of the animals also displayed "low T3" values (p = 0.012) on day 14. This T4 reduction was inversely correlated to the clinical severity of stroke and the concomitant body weight loss (p < 0.005). WB analysis of TRα1 and TRß1 protein expression showed heterogenic responses at day 14: total and nuclear TRα1 were similar between the two groups, while total TRß1 decreased 7.5-fold within E1 (p ≤ 0.001) with a concomitant 1.8-fold increase of nuclear TRß1 in E2 area (p = 0.03); TRß1 expression did not differ in E3. Neuropathological analysis revealed that activated macrophages/microglia exclusively expressed nuclear TRα1 within the infarct core. Astrocytes mildly expressed nuclear TRα1 in and around the infarct, along with a prominent TRß nuclear signal restricted in the astrocytic scar. Neurons around the infarct expressed mainly TRα1 and, to a milder degree, TRß. Surprisingly enough, we detected for the first time a TRß expression in the paranodal region of Ranvier nodes, of unknown significance so far. Our data support that cerebral ischemia induces a low TH response, associated with significant and heterogenic changes in brain TR expression. These findings could imply an important role of TH signaling in cerebral ischemia.
Asunto(s)
Infarto de la Arteria Cerebral Media/metabolismo , Receptores alfa de Hormona Tiroidea/metabolismo , Receptores beta de Hormona Tiroidea/metabolismo , Animales , Astrocitos/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Microglía/citología , Microglía/metabolismo , Neuronas/metabolismo , Especificidad de Órganos , Ratas , Ratas Wistar , Receptores alfa de Hormona Tiroidea/genética , Receptores beta de Hormona Tiroidea/genética , Hormonas Tiroideas/sangreRESUMEN
Over the course of cortical neurogenesis, the transition of progenitors from proliferation to differentiation requires a precise regulation of involved gene networks under varying environmental conditions. In order to identify such regulatory mechanisms, we analyzed microRNA (miRNA) target networks in progenitors during early and late stages of neurogenesis. We found that cyclin D1 is a network hub whose expression is miRNA-dosage sensitive. Experimental validation revealed a feedback regulation between cyclin D1 and its regulating miRNAs miR-20a, miR-20b, and miR-23a. Cyclin D1 induces expression of miR-20a and miR-20b, whereas it represses miR-23a. Inhibition of any of these miRNAs increases the developmental stage-specific mean and dynamic expression range (variance) of cyclin D1 protein in progenitors, leading to reduced neuronal differentiation. Thus, miRNAs establish robustness and stage-specific adaptability to a critical dosage-sensitive gene network during cortical neurogenesis. Understanding such network regulatory mechanisms for key developmental events can provide insights into individual susceptibilities for genetically complex neuropsychiatric disorders.
Asunto(s)
Redes Reguladoras de Genes , MicroARNs/genética , Neurogénesis/genética , Células Madre/citología , Animales , Diferenciación Celular/genética , Humanos , Ratones , Ratones TransgénicosRESUMEN
Dentate gyrus granule cells (GCs) have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2-4 weeks post-mitotic GCs. These rather immature cells are able to fire action potentials (APs) and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus GCs is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage.
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Giro Dentado/enzimología , Regulación Enzimológica de la Expresión Génica , Glutamato Descarboxilasa/biosíntesis , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Giro Dentado/citología , Hipocampo/citología , Hipocampo/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de ÓrganosRESUMEN
Heterotopic or aberrantly positioned cortical neurons are associated with epilepsy and intellectual disability. Various mouse models exist with forms of heterotopia, but the composition and state of cells developing in heterotopic bands has been little studied. Dcx knockout (KO) mice show hippocampal CA3 pyramidal cell lamination abnormalities, appearing from the age of E17.5, and mice suffer from spontaneous epilepsy. The Dcx KO CA3 region is organized in two distinct pyramidal cell layers, resembling a heterotopic situation, and exhibits hyperexcitability. Here, we characterized the abnormally organized cells in postnatal mouse brains. Electron microscopy confirmed that the Dcx KO CA3 layers at postnatal day (P) 0 are distinct and separated by an intermediate layer devoid of neuronal somata. We found that organization and cytoplasm content of pyramidal neurons in each layer were altered compared to wild type (WT) cells. Less regular nuclei and differences in mitochondria and Golgi apparatuses were identified. Each Dcx KO CA3 layer at P0 contained pyramidal neurons but also other closely apposed cells, displaying different morphologies. Quantitative PCR and immunodetections revealed increased numbers of oligodendrocyte precursor cells (OPCs) and interneurons in close proximity to Dcx KO pyramidal cells. Immunohistochemistry experiments also showed that caspase-3 dependent cell death was increased in the CA1 and CA3 regions of Dcx KO hippocampi at P2. Thus, unsuspected ultrastructural abnormalities and cellular heterogeneity may lead to abnormal neuronal function and survival in this model, which together may contribute to the development of hyperexcitability.
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Encéfalo/metabolismo , Encéfalo/patología , Hipocampo/metabolismo , Hipocampo/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Animales , Encéfalo/ultraestructura , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Región CA1 Hipocampal/ultraestructura , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Región CA3 Hipocampal/ultraestructura , Caspasa 3/metabolismo , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Femenino , Aparato de Golgi/metabolismo , Aparato de Golgi/patología , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Mitocondrias/ultraestructura , Neuropéptidos/genéticaRESUMEN
Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.
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Anticuerpos Monoclonales/farmacología , Regulación hacia Abajo , Neuroblastoma/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasa de Linfoma Anaplásico , Secuencia de Bases , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Activación Enzimática/efectos de los fármacos , Humanos , Mutación , Neuroblastoma/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/agonistas , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Transcripción GenéticaRESUMEN
In migrating neurons, the centrosome nucleates and anchors a polarized network of microtubules that directs organelle movements. We report here that the mother centriole of neurons migrating tangentially from the medial ganglionic eminence (MGE) assembles a short primary cilium and exposes this cilium to the cell surface by docking to the plasma membrane in the leading process. Primary cilia are built by intraflagellar transport (IFT), which is also required for Sonic hedgehog (Shh) signal transduction in vertebrates. We show that Shh pathway perturbations influenced the leading process morphology and dynamics of MGE cells. Whereas Shh favored the exit of MGE cells away from their tangential migratory paths in the developing cortex, cyclopamine or invalidation of IFT genes maintained MGE cells in the tangential paths. Our findings show that signals transmitted through the primary cilium promote the escape of future GABAergic interneurons from their tangential routes to colonize the cortical plate.
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Movimiento Celular/fisiología , Centrosoma/fisiología , Corteza Cerebral/embriología , Cilios/fisiología , Proteínas Hedgehog/fisiología , Neuronas/fisiología , Animales , Ganglios Basales/citología , Ganglios Basales/embriología , Polaridad Celular/fisiología , Centriolos/fisiología , Corteza Cerebral/citología , Ratones , Ratones Mutantes Neurológicos , Ratones Transgénicos , Neurogénesis/fisiología , Neuronas/citología , Transducción de Señal/fisiologíaRESUMEN
Chronic acquired neuropathies of unknown origin are classified as chronic inflammatory demyelinating polyneuropathies (CIDP) and chronic idiopathic axonal polyneuropathies (CIAP). The diagnosis can be very difficult, although it has important therapeutic implications since CIDP can be improved by immunomodulating treatment. The aim of this study was to examine the possible abnormalities of nodal and paranodal regions in these two types of neuropathies. Longitudinal sections of superficial peroneal nerves were obtained from biopsy material from 12 patients with CIDP and 10 patients with CIAP and studied by immunofluorescence and in some cases electron microscopy. Electron microscopy revealed multiple alterations in the nodal and paranodal regions which predominated in Schwann cells in CIDP and in axons in CIAP. In CIDP paranodin/Caspr immunofluorescence was more widespread than in control nerves, extending along the axon in internodes where it appeared intense. Nodal channels Nav and KCNQ2 were less altered but were also detected in the internodes. In CIAP paranodes, paranodin labeling was irregular and/or decreased. To test the consequences of acquired primary Schwann cells alteration on axonal proteins, we used a mouse model based on induced deletion of the transcription factor Krox-20 gene. In the demyelinated sciatic nerves of these mice we observed alterations similar to those found in CIDP by immunofluorescence, and immunoblotting demonstrated increased levels of paranodin. Finally we examined whether the alterations in paranodin immunoreactivity could have a diagnosis value. In a sample of 16 biopsies, the study of paranodin immunofluorescence by blind evaluators led to correct diagnosis in 70 ± 4% of the cases. This study characterizes for the first time the abnormalities of nodes of Ranvier in CIAP and CIDP, and the altered expression and distribution of nodal and paranodal proteins. Marked differences were observed between CIDP and CIAP and the alterations in paranodin immunofluorescence may be an interesting tool for their differential diagnosis.