RESUMEN
Excitatory neurons undergo dendritic spine remodeling in response to different stimuli. However, there is scarce information about this type of plasticity in interneurons. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate to mediate this plasticity as it participates in neuronal remodeling and is expressed by some mature cortical interneurons, which have reduced dendritic arborization, spine density, and synaptic input. To study the connectivity of the dendritic spines of interneurons and the influence of PSA-NCAM on their dynamics, we have analyzed these structures in a subpopulation of fluorescent spiny interneurons in the hippocampus of glutamic acid decarboxylase-enhanced green fluorescent protein transgenic mice. Our results show that these spines receive excitatory synapses. The depletion of PSA in vivo using the enzyme Endo-Neuraminidase-N (Endo-N) increases spine density when analyzed 2 days after, but decreases it 7 days after. The dendritic spine turnover was also analyzed in real time using organotypic hippocampal cultures: 24 h after the addition of EndoN, we observed an increase in the apparition rate of spines. These results indicate that dendritic spines are important structures in the control of the synaptic input of hippocampal interneurons and suggest that PSA-NCAM is relevant in the regulation of their morphology and connectivity.
Asunto(s)
Espinas Dendríticas/metabolismo , Regulación de la Expresión Génica/fisiología , Interneuronas/ultraestructura , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/fisiología , Ácidos Siálicos/metabolismo , Ácidos Siálicos/fisiología , Animales , Animales Recién Nacidos , Calbindina 2/metabolismo , Colecistoquinina/metabolismo , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato Descarboxilasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/efectos de los fármacos , Neuraminidasa/farmacología , Técnicas de Cultivo de Órganos , Somatostatina/metabolismo , Factores de Tiempo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
The present study investigated the involvement of 5-HT(1A) receptors in the inhibitory effect of single administration of cocaine (COC, 15 mg/kg i.p.) on the induction of long-term potentiation (LTP) in slices of rat dentate gyrus (DG), prepared 30 min and 2 days after COC administration. These effects of COC were blocked by an antagonist of 5-HT(1A) receptors, WAY 100635 (0.4 mg/kg i.p.), which had been administered 20 min before COC. The detrimental effect of COC on LTP in slices prepared 30 min after COC administration could be prevented by blocking glucocorticoid receptors (GRs) using mifepristone (RU 38486, 10 mg/kg s.c. given 1 h before COC), similar as in slices obtained 2 days after COC as reported previously [Mackowiak et al. (2008) Eur J Neurosci 27:2928-2937]. After a single administration of an agonist of 5-HT(1A) receptors, 8-OH-DPAT, (0.5 mg/kg i.p.), the level of LTP in slices prepared 2 days later was significantly decreased resembling the effect of COC. This effect of 8-OH-DPAT was antagonized by WAY 100635 (0.4 mg/kg i.p.), administered 20 min before 8-OH-DPAT and by RU 38486, given 1 h before 8-OH-DPAT. COC-induced inhibition of LTP could be blocked by the inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK1/2), SL 327 (50 mg/kg i.p.), administered 1 h before COC, but not by the inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), LY 294002 (80 mg/kg i.p.). COC-induced reduction in the number of polysialylated neural cell adhesion molecule (PSA-NCAM)-positive neurons in rat dentate gyrus could also be prevented by WAY 100635, given 20 min before COC. These data indicate that the indirect 5-HT(1A) receptor activation by a single COC administration and subsequent stimulation of extracellular signal-regulated kinases (ERK 1/2) signaling pathway result in a decrease of the potential for long-term increase in synaptic efficacy in rat DG lasting at least two but less than 7 days, most likely via activation of the hypothalamic-pituitary-adrenal (HPA) axis.
Asunto(s)
Cocaína/farmacología , Giro Dentado/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Molécula L1 de Adhesión de Célula Nerviosa/efectos de los fármacos , Receptor de Serotonina 5-HT1A/efectos de los fármacos , Animales , Recuento de Células , Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/fisiopatología , Giro Dentado/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Interacciones Farmacológicas/fisiología , Inhibidores Enzimáticos/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Potenciación a Largo Plazo/fisiología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Mifepristona/farmacología , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/metabolismo , Ratas , Ratas Wistar , Receptor de Serotonina 5-HT1A/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismo , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Ácidos Siálicos/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. We recently reported that induction of a chemoresistant phenotype in the PDAC cell line PT45-P1 by long-term chemotherapy involves an increased interleukin 1 beta (IL1beta)-dependent secretion of nitric oxide (NO) accounting for efficient caspase inhibition. In the present study, we elucidated the involvement of L1CAM, an adhesion molecule previously found in other malignancies, in this NO-dependent chemoresistance. Chemoresistant PT45-P1res cells, but not chemosensitive parental PT45-P1 cells, express high levels of L1CAM in an ILbeta-dependent fashion. PT45-P1res cells subjected to short interfering RNA (siRNA)-mediated L1CAM knock-down exhibited reduced inducible nitric oxide synthase expression and NO secretion, as well as a significant increase of anti-cancer drug-induced caspase activation, an effect reversed by the NO donor S-nitroso-N-acetyl-D,L-penicillamine. Conversely, overexpression of L1CAM in PT45-P1 cells conferred anti-apoptotic protection to anti-cancer drug treatment. Interestingly, L1CAM ectodomain shedding, in example, by ADAM10, as reported for other L1CAM-related activities, seemed to be dispensable for anti-apoptotic protection by L1CAM. Neither the shedded L1CAM ectodomain was detected in chemoresistant L1CAM-expressing PT45-P1 cells nor did the administration of various metalloproteinase inhibitors affect L1CAM-dependent chemoresistance. Immunohistochemical analysis revealed L1CAM expression in 80% of pancreatic cancer specimens, supporting a potential role of L1CAM in the malignancy of this tumor. These findings substantiate our understanding of the molecular mechanisms leading to chemoresistance in PDAC cells and indicate the importance of L1CAM in this scenario.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Apoptosis/fisiología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Resistencia a Antineoplásicos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Inhibidores de Caspasas , Caspasas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Etopósido/farmacología , Humanos , Interleucina-1beta/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Células Tumorales CultivadasRESUMEN
Transforming growth factor (TGF) beta1 and ethanol retard the migration of young, post-mitotic neurons to the developing cerebral cortex. The coordination of this migration depends upon cell adhesion proteins (CAPs). We examined the effects of TGFbeta1 and ethanol on genes related to both TGF and CAPs. Rat B104 neuroblastoma cells were treated with TGFbeta1 (0 or 10 ng/mL) and ethanol (0 or 400 mg/dL) for 6-48 h. Total RNA was purified from each sample and analyzed using the Rat U34A GeneChip (Affymetrix). Candidate genes were those up- or down-regulated by either TGFbeta1 or ethanol. Twenty transcripts of CAPs were identified as being expressed by B104 cells and as being affected by treatment with TGFbeta1 or ethanol. The expression was verified for five representative genes (neural cell adhesion molecule, L1, and integrins alpha1, alpha7, and beta1) using assays with real-time reverse transcriptase-polymerase chain reactions. Each of these genes showed time-dependent changes. The changes were reflected in increases in protein expression that appeared within 24 or 48 h. Thus, the effects of TGFbeta1 and ethanol on CAPs parallel changes described in vivo and likely underlie changes associated with ethanol-induced alterations in neuronal migration.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Etanol/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Trastornos del Sistema Nervioso Inducidos por Alcohol/genética , Trastornos del Sistema Nervioso Inducidos por Alcohol/metabolismo , Trastornos del Sistema Nervioso Inducidos por Alcohol/fisiopatología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Integrinas/efectos de los fármacos , Integrinas/genética , Integrinas/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/efectos de los fármacos , Molécula L1 de Adhesión de Célula Nerviosa/genética , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuroblastoma , Neuronas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/fisiología , Ratas , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1 , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Hippocampus dentate gyrus (DG) is characterized by neuronal plasticity processes in adulthood, and polysialylation of NCAM promotes neuronal plasticity. In previous investigations we found that alpha-tocopherol increased the PSA-NCAM-positive granule cell number in adult rat DG, suggesting that alpha-tocopherol may enhance neuronal plasticity. To verify this hypothesis, in the present study, structural remodeling in adult rat DG was investigated under alpha-tocopherol supplementation conditions. PSA-NCAM expression was evaluated by Western blotting, evaluation of PSA-NCAM-positive granule cell density, and morphometric analysis of PSA-NCAM-positive processes. In addition, the optical density of synaptophysin immunoreactivity and the synaptic profile density, examined by electron microscopy, were evaluated. Moreover, considering that PSA-NCAM expression has been found to be related to PKCdelta activity and alpha-tocopherol has been shown to inhibit PKC activity in vitro, Western blotting and immunohistochemistry followed by densitometry were used to analyze PKC. Our results demonstrated that an increase in PSA-NCAM expression and optical density of DG molecular layer synaptophysin immunoreactivity occurred in alpha-tocopherol-treated rats. Electron microscopy analysis showed that the increase in synaptophysin expression was related to an increase in synaptic profile density. In addition, Western blotting revealed a decrease in phospho-PKC Pan and phospho-PKCdelta, demonstrating that alpha-tocopherol is also able to inhibit PKC activity in vivo. Likewise, immunoreactivity for the active form of PKCdelta was lower in alpha-tocopherol-treated rats than in controls, while no changes were found in PKCdelta expression. These results demonstrate that alpha-tocopherol is an exogenous factor affecting neuronal plasticity in adult rat DG, possibly through PKCdelta inhibition.
Asunto(s)
Giro Dentado/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Proteína Quinasa C-delta/antagonistas & inhibidores , alfa-Tocoferol/farmacología , Animales , Antioxidantes/farmacología , Dendritas/metabolismo , Dendritas/ultraestructura , Giro Dentado/enzimología , Giro Dentado/ultraestructura , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Molécula L1 de Adhesión de Célula Nerviosa/efectos de los fármacos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Plasticidad Neuronal/fisiología , Terminales Presinápticos/enzimología , Terminales Presinápticos/ultraestructura , Proteína Quinasa C-delta/metabolismo , Ratas , Ratas Sprague-Dawley , Ácidos Siálicos/metabolismo , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/enzimología , Membranas Sinápticas/ultraestructura , Sinaptofisina/efectos de los fármacos , Sinaptofisina/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
To study nicotine's effects on neurogenesis in the dentate gyrus of the hippocampus, nicotine was injected intraperitoneally into adult rats. After sacrificing, the hippocampal formation was processed for immunohistochemical staining of PSA-NCAM, NeuN and GFAP. Nicotine decreased numbers of PSA-NCAM(+) and NeuN(+) cells dose-dependently.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Hipocampo/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Nicotina/farmacología , Animales , Biomarcadores , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Proteínas de Unión al ADN , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Inmunohistoquímica , Inyecciones Intraperitoneales , Masculino , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/efectos de los fármacos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Agonistas Nicotínicos/farmacología , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/metabolismo , Ratas , Ratas Endogámicas BB , Ácidos Siálicos/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
The NMDA class of glutamate receptors plays a critical role in CNS, such as synaptic plasticity, axonal sprouting, growth, and migration. NMDA receptor stimulation has been shown to regulate polysialylated neural cell adhesion molecule (PSA-NCAM) expression in glial cell cultures and in hippocampal slice cultures. There is also growing evidence that molecular chaperons and ROS are related to the synaptic plasticity phenomena. We have examined the neuroprotective effect of subtoxic dose of NMDA in retinoic acid differentiated SH-SY5Y neuroblastoma cells. SH-SY5Y cell line differentiated with retinoic acid (10 muM) was exposed to NMDA (100 microM) or to antagonist MK-801 (200 nM) + NMDA and cells harvested after 24 h of treatment for PSA-NCAM, NCAM, and HSP70 expression study and for biochemical analysis. A significant increase was observed in PSA-NCAM, NCAM-180, NCAM-140, and HSP70 expression as seen by Western blotting and immunocytofluorescent studies in NMDA-treated cultures. Biochemical analysis revealed a significant increase in the activities of glutathione peroxidase (GPx) and copper zinc-superoxide dismutase (CuZnSOD) upon exposure to NMDA. No significant change was observed in the level of lipid peroxidation. All the changes observed reverted back to the control values upon pretreatment of cultures with MK-801, a non-competitive NMDA receptor antagonist, prior to NMDA exposure indicating the involvement of NMDA receptor in these changes. These results illustrate the neuroprotective role of subtoxic dose of NMDA in SH-SY5Y neuroblastoma cells.