RESUMEN
Increase in aerobic glycolysis and mitochondrial dysfunction are important biochemical features observed in human cancers. Recent studies suggest oncogenic K-Ras can cause suppression of mitochondrial respiration and up-regulation of glycolytic activity through a yet unknown mechanism. Here we employed proteomic approach and used a K-RasG12V inducible cell system to investigate the impact of oncogenic K-Ras on mitochondria and cell metabolism. Mitochondria isolated from cells before and after K-Ras induction were subjected to protein analysis using stable isotope labeling with amino acids (SILAC) and liquid chromatography coupled with mass spectrometry (LC-MS). 70 mitochondrial proteins with significant expression alteration after K-Ras induction were identified. A majority of these proteins were involved in energy metabolism. Five proteins with significant decrease belong to mitochondrial respiratory chain complex I. NADH dehydrogenase 1 alpha subcomplex assembly factor 1 (NDUFAF1) showed most significant decrease by 50%. Such decrease was validated in primary human pancreatic cancer tissues. Knockdown of NDUFAF1 by siRNA caused mitochondrial respiration deficiency, accumulation of NADH and subsequent increase of glycolytic activity. Our study revealed that oncogenic K-Ras is able to induce significant alterations in mitochondrial protein expression, and identified NDUFAF1 as an important molecule whose low expression contributes to mitochondrial dysfunction induced by K-Ras.
Asunto(s)
Genes ras , Mitocondrias/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Proteínas ras/biosíntesis , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Proteómica , Activación Transcripcional , Transcriptoma , Proteínas ras/genéticaRESUMEN
CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti-CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive-like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.
Asunto(s)
Antígeno CD47/metabolismo , Diferenciación Celular/fisiología , Cerebelo/citología , Relaciones Interpersonales , Neuronas/metabolismo , Organogénesis/fisiología , Animales , Conducta Animal , Antígeno CD47/genética , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Ratones , Ratones NoqueadosRESUMEN
Nuclear respiratory factor-1 (NRF-1) is a major transcription factor in the human genome and functions in neurite outgrowth in neuroblastoma cells. Whether genes downstream from NRF-1 differentially regulate axonal and dendritic outgrowth in neurons remains largely unknown. Three hypothetical genes, C3orf10, FAM134C, and ENOX1, were investigated because their NRF-1 response elements are 100% conserved in humans and mice. We found that NRF-1 positively regulates FAM134C and ENOX1, but negatively regulates C3orf10 in human neuroblastoma IMR-32 cells and primary rat cortical neurons. In IMR-32 cells, FAM134C positively regulates and C3orf10 negatively regulates neurite outgrowth, but ENOX1 plays no role in neurite outgrowth regulation. FAM134C but not C3orf10 mediates NRF-1-enhanced neurite outgrowth. In primary rat hippocampal neurons, Fam134c is predominantly expressed in the axon hillock and C3orf10 is ubiquitously expressed in all neurites and cell bodies at different developmental stages, suggesting their roles in axonal and dendritic outgrowth. Knockdown of Fam134c decreased both axonal length and the number of axonal collaterals and dendrites, however, knockdown of C3orf10 only increased the number of axonal collaterals and dendrites. Overall, we annotated FAM134C, C3orf10, and ENOX1 as NRF-1-regulated genes, which have differential effects on neurite outgrowth in neuroblastoma cells as well as neurons. This study provided an effective platform for annotating hypothetical genes in the human genome and increasing our knowledge in the molecular network underlying neuronal differentiation.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Hipocampo/citología , Neuroblastoma/metabolismo , Neurogénesis/genética , Neuronas/citología , Factor Nuclear 1 de Respiración/metabolismo , Animales , Axones , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Hipocampo/metabolismo , Humanos , Lentivirus/genética , Neuritas/metabolismo , Células Neuroepiteliales/citología , Células Neuroepiteliales/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Factor Nuclear 1 de Respiración/genética , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Nuclear respiratory factor-1 (NRF-1) is a transcription factor that functions in neurite outgrowth; however, the genes downstream from NRF-1 that mediate this function remain largely unknown. This study employs a genome-wide analysis approach to identify NRF-1-targeted genes in human neuroblastoma IMR-32 cells. A total of 916 human genes containing the putative NRF-1 response element (NRE) in their promoter regions were identified using a cutoff score determined by results from electrophoretic mobility shift assays (EMSA). Seventy-four NRF-1 target genes were listed according to the typical locations and high conservation of NREs. Fifteen genes, MAPRE3, NPDC1, RAB3IP, TRAPPC3, SMAD5, PIP5K1A, USP10, SPRY4, GTF2F2, NR1D1, SUV39H2, SKA3, RHOA, RAPGEF6, and SMAP1 were selected for biological confirmation. EMSA and chromatin immunoprecipitation confirmed that all NREs of these fifteen genes are critical for NRF-1 binding. Quantitative RT-PCR demonstrated that mRNA levels of 12 of these genes are regulated by NRF-1. Overexpression or knockdown of candidate genes demonstrated that MAPRE3, NPDC1, SMAD5, USP10, SPRY4, GTF2F2, SKA3, SMAP1 positively regulated, and RHOA and RAPGEF6 negatively regulated neurite outgrowth. Overall, our data showed that the combination of genome-wide bioinformatic analysis and biological experiments helps to identify the novel NRF-1-regulated genes, which play roles in differentiation of neuroblastoma cells.
Asunto(s)
Factor 1 Relacionado con NF-E2/metabolismo , Neuritas/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Línea Celular Tumoral , Biología Computacional/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Chronic exercise has been reported to improve cognitive function. However, whether and how different types of exercise affect various learning and memory tasks remain uncertain. To address this issue, male BALB/c mice were trained for 4 weeks under two different exercise protocols: moderate treadmill running or voluntary wheel running. After exercise training, their spatial memory and aversive memory were evaluated by a Morris water maze and by one-trial passive avoidance (PA), respectively. Levels of neural plasticity-related proteins, i.e. brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB) and synaptotagmin I (Syt I), in hippocampus and amygdala were determined by ELISA or immunoblotting. Finally, the functional roles of these proteins in the basolateral amygdala were verified by locally blocking them with K252a (a TrkB kinase inhibitor), or lentivirus expressing Syt I shRNA. We found that (1) although both moderate treadmill running and wheel running improved the Morris water maze performance, only the former improved PA performance; (2) likewise, both exercise protocols upregulated the BDNF-TrkB pathway and Syt I in the hippocampus, whereas only treadmill exercise upregulated their expression levels in the amygdala; (3) local injection of K252a abolished the treadmill exercise-facilitated PA performance and upregulation of amygdalar TrkB and Syt I; and (4) local administration of Syt I shRNA abolished the treadmill exercise-facilitated PA performance and upregulation of amygdalar Syt I. Therefore, our results support the notion that different forms of exercise induce neuroplasticity changes in different brain regions, and thus exert diverse effects on various forms of learning and memory.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Aprendizaje/fisiología , Memoria/fisiología , Actividad Motora/fisiología , Sinaptotagmina I/fisiología , Amígdala del Cerebelo/fisiología , Animales , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Secuencia de Bases , Carbazoles/farmacología , Corticosterona/sangre , Hipocampo/fisiología , Alcaloides Indólicos/farmacología , Aprendizaje/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Músculo Esquelético/fisiología , Plasticidad Neuronal/fisiología , ARN Interferente Pequeño/genética , Receptor trkB/antagonistas & inhibidores , Receptor trkB/fisiología , Carrera/fisiología , Sinaptotagmina I/antagonistas & inhibidores , Sinaptotagmina I/genéticaRESUMEN
Nuclear respiratory factor (NRF)-1 is a transcription factor with a novel function in neurite outgrowth. Synapsin I protein is a well-known phosphoprotein in neuronal terminals and has been implicated in neuronal differentiation. Human synapsin I gene promoter has a putative NRF-1 responsive element (NRE), but it is not known whether this NRE is functional. We hypothesized that synapsin I is downstream of NRF-1 and mediates its function in neurite outgrowth. Gel electrophoretic mobility shift assays, chromatin immunoprecipitation, site-directed mutagenesis, and promoter studies indicated that NRF-1 is a positive regulator of synapsin I promoter. Exogenous NRF-1 overexpression increased synapsin I protein levels in IMR-32 and HEK293T cells. Serum deprivation, which induces neurite outgrowth in IMR-32 cells, increased the binding activity of NRF-1 to synapsin I NRE and induced alternating synapsin I protein expression. Down-regulating synapsin I expression markedly decreased the percentage of neurite-bearing cells and the length of the longest neurite in IMR-32 cells that stably or transiently overexpressed NRF-1. We conclude that the human synapsin I gene is positively regulated by NRF-1 and mediates the function of NRF-1 in neurite outgrowth.
Asunto(s)
Neuritas/fisiología , Neuroblastoma/patología , Factor Nuclear 1 de Respiración/metabolismo , Sinapsinas/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/métodos , Ensayo de Cambio de Movilidad Electroforética/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Mutagénesis/fisiología , Neuritas/efectos de los fármacos , Factor Nuclear 1 de Respiración/genética , ARN Mensajero , ARN Interferente Pequeño/farmacología , Suero/metabolismo , Sinapsinas/genética , Transfección/métodosRESUMEN
While serotonin (5-HT) may impair learning and memory, exercise has been reported to improve them. Whether chronic exercise can facilitate fear memory via regulating the serotonin system is unknown. We examined the effects of 4-week treadmill exercise training on levels of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), the protein expression of its receptor 5-HT(1A) and transporter in the amygdala, hippocampus and prefrontal cortex of male Sprague-Dawley rats. Our results demonstrated that treadmill exercise (1) improved the passive avoidance learning performance; (2) decreased the 5-HT level in the hippocampus; (3) decreased the expression of 5-HT(1A) receptor in the amygdala without altering the transporter expression. Moreover, pretreatment with 0.1 mg/kg 8-hydroxy-di-n-propylamino tetralin, a selective 5-HT(1A) receptor agonist, impaired the passive avoidance performance and completely abolished the exercise-enhanced fear memory. Our results suggest that down-regulation of the 5-HT system in the limbic system, i.e., the reduction of the hippocampus 5-HT content and the amygdala 5-HT(1A) receptor expression, may be involved in the exercise-enhanced fear memory.
Asunto(s)
Reacción de Prevención/fisiología , Sistema Límbico/fisiología , Condicionamiento Físico Animal/fisiología , Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Amígdala del Cerebelo/fisiología , Animales , Reacción de Prevención/efectos de los fármacos , Regulación hacia Abajo/fisiología , Prueba de Esfuerzo , Miedo/fisiología , Ácido Hidroxiindolacético/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
Although exercise has been known to regulate brain plasticity, its impact on psychostimulant reward and the associated mesolimbic dopamine system remained scarcely explored. A psychostimulant, 3,4-methylenedioxymethamphetamine (MDMA), is currently a worldwide abused drug of choice. We decided to examine the modulating effects of long-term, compulsive treadmill exercise on the hedonic value of MDMA in male C57BL/6J mice. MDMA-induced conditioned place preference (CPP) was used as a behavioral paradigm to indicate the reward efficacy of MDMA. We observed that sedentary control mice all demonstrated reliable MDMA-induced CPP with our conditioning protocol. Interestingly, pre-exposure to a treadmill exercise decreased the later MDMA-induced CPP in a running period-dependent manner. Specifically, mice undergoing a 12-week treadmill running exercise did not exhibit any approaching bias toward the MDMA-associated compartment in this CPP paradigm. Twelve weeks of treadmill running did not alter peripheral metabolism of MDMA 30min following single intraperitoneal injection of MDMA (3mg/kg). We further used microdialysis technique to study the underlying mechanisms for the impaired MDMA reward produced by the12-week exercise pre-exposure. We found that acute MDMA-stimulated dopamine release in nucleus accumbens was abolished in the exercised mice, whereas an obvious elevation of accumbal dopamine release was observed in sedentary control mice. Finally, the 12-week exercise program did not alter the protein levels of primary dopamine receptors, vesicular or membrane transporters in this area. We conclude that the long-term, compulsive exercise is effective in curbing the reward efficacy of MDMA possibly via its direct effect on reversing the MDMA-stimulated dopamine release in nucleus accumbens.
Asunto(s)
Inhibidores de Captación Adrenérgica/farmacología , N-Metil-3,4-metilenodioxianfetamina/farmacología , Condicionamiento Físico Animal/fisiología , Recompensa , Inhibidores de Captación Adrenérgica/sangre , Animales , Western Blotting , Condicionamiento Operante/efectos de los fármacos , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Masculino , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microdiálisis , Actividad Motora/fisiología , N-Metil-3,4-metilenodioxianfetamina/sangre , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/efectos de los fármacos , Receptores de Dopamina D2/metabolismoRESUMEN
New neurons are continuously generated in hippocampal subgranular zone throughout life, and the amount of neurogenesis is suggested to be correlated with the hippocampus-dependent function. Several extrinsic stimuli are known to modulate the neurogenesis process. Among them, physical exercise has advantageous effects on neurogenesis and brain function, while inflammation shows the opposite. Herein we showed that a moderate running exercise successfully restored the peripheral lipopolysaccharide (LPS)-impaired neurogenesis in the dentate area. LPS treatment obstructed neuronal differentiation, but not proliferation. Exercise training facilitated both the proliferation of the neural stem cells and their differentiation into neurons. Interestingly, exercise replenished the LPS-reduced levels of brain-derived neurotrophic factor and its receptor, TrkB, and rescued the LPS-disturbed performance in water maze; while the LPS-elicited up-regulation of tumor necrosis factor-alpha and interleukin-1beta remained unaltered. In conclusion, our findings suggest that running exercise effectively ameliorates the LPS-disturbed hippocampal neurogenesis and learning and memory performance. Such advantageous effects of running exercise are not due to the alteration of inflammatory response, but possibly by the restoring the LPS-lessened brain-derived neurotrophic factor signaling pathway.
Asunto(s)
Proliferación Celular , Encefalitis/terapia , Terapia por Ejercicio/métodos , Hipocampo/fisiopatología , Plasticidad Neuronal/fisiología , Condicionamiento Físico Animal/fisiología , Animales , Diferenciación Celular/fisiología , Encefalitis/inducido químicamente , Encefalitis/metabolismo , Prueba de Esfuerzo , Hipocampo/citología , Mediadores de Inflamación/farmacología , Aprendizaje/fisiología , Lipopolisacáridos/farmacología , Masculino , Memoria/fisiología , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/fisiopatología , Trastornos de la Memoria/terapia , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Células Madre/fisiologíaRESUMEN
Context-induced drug craving and continuous drug use manifest the critical roles of specific memory episodes associated with the drug use experiences. Drug-induced conditioned place preference (CPP) in C57BL/6J mouse model, in this regard, is an appropriate behavioral paradigm to study such drug use-associated memories. Requirement of protein synthesis in various forms of long-term memory formation and storage has been phylogenetically demonstrated. This study was undertaken to study the requirement of protein synthesis in the learning and memory aspect of the conditioned place preference induced by cocaine and methamphetamine, two abused drugs of choice in local area. Since pCREB has been documented as a candidate substrate for mediating the drug-induced neuroadaptation, the pCREB level in hippocampus, nucleus accumbens, and prefrontal cortex was examined for its potential participation in the formation of CPP caused by these psychostimulants. We found that cocaine (2.5 and 5.0 mg/kg/dose)-induced CPP was abolished by the pretreatment of anisomycin (50 mg/kg/dose), a protein synthesis inhibitor, whereas methamphetamine (0.5 or 1.0 mg/kg/dose)-induced CPP was not affected by the anisomycin pretreatment. Likewise, cocaine-induced CPP was mitigated by another protein synthesis inhibitor, cycloheximide (15 mg/kg/injection) pretreatment, whereas methamphetamine-induced CPP remained intact by such pretreatment. Moreover, anisomycin treatment 2h after each drug-place pairing disrupted the cocaine-induced CPP, whereas the same treatment did not affect methamphetamine-induced CPP. An increase of accumbal pCREB level was found to associate with the learning phase of cocaine, but not with the learning phase of methamphetamine. We further found that intraaccumbal CREB antisense oligodeoxynucleotide infusion diminished cocaine-induced CPP, whereas did not affect the methamphetamine-induced CPP. Taken together, these data suggest that protein synthesis and accumbal CREB phosphorylation are essential for the learning and consolidation of the cocaine-induced CPP, whereas methamphetamine-induced CPP may be unrelated to the synthesis of new proteins.
Asunto(s)
Aprendizaje por Asociación/efectos de los fármacos , Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Cocaína/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Metanfetamina/farmacología , Animales , Conducta Adictiva/metabolismo , Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Fosforilación , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismoRESUMEN
Neuroplasticity after perinatal programming may allow for neuroprotection against hypoxic-ischemia (HI) at birth. The cAMP response element-binding protein (CREB) is a key mediator of stimulus-induced nuclear responses that underlie survival, memory and plasticity of nervous system. Chronic treatment of fluoxetine, a selective serotonin reuptake inhibitor, can upregulate CREB activation in the hippocampus. We examined whether fluoxetine administration before HI may protect against neonatal HI brain injury through CREB-mediated mechanisms. We found that low-dose fluoxetine pretreatment in a neonatal HI brain injury model significantly reduced functional deficits at adulthood. The neuroprotective mechanisms were associated with increased CREB phosphorylation and increased brain-derived neurotrophic factor and synapsin I mRNA expression in the hippocampus. Neurogenesis also increased because of greater precursor cell survival in the hippocampal dentate gyrus. These findings suggest that functional deficits after HI in the developing brain can be reduced by agents that enhance neural plasticity and neurogenesis through CREB activation.
Asunto(s)
Fluoxetina/farmacología , Hipoxia-Isquemia Encefálica/psicología , Fármacos Neuroprotectores , Desempeño Psicomotor/fisiología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Antimetabolitos/toxicidad , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factor Neurotrófico Derivado del Encéfalo/genética , Bromodesoxiuridina/toxicidad , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Giro Dentado/crecimiento & desarrollo , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Hipoxia-Isquemia Encefálica/fisiopatología , Locomoción/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Microscopía Confocal , Neuronas/efectos de los fármacos , Tamaño de los Órganos/fisiología , Fosforilación , Desempeño Psicomotor/efectos de los fármacos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsinas/biosíntesis , Sinapsinas/genéticaRESUMEN
Alpha-Pal/NRF-1 is a critical regulator of the promoter of human IAP/CD47 gene, a gene related to memory formation in rodents. However, its function in neurons was unknown. We found that stable or transient expression of full-length alpha-Pal/NRF-1 in human neuroblastoma IMR-32 cells significantly induced neurite outgrowth and increased the length of neurites both in medium containing 10% fetal bovine serum and in serum-free medium. In contrast, the dominant-negative mutant of alpha-Pal/NRF-1 inhibited the induction and extension of neurites. Ectopic expression of full-length alpha-Pal/NRF-1 also increased the induction of neurite outgrowth in primary mouse cortical neurons. The IAP antisense cDNA significantly inhibited the increase of neurite outgrowth by alpha-Pal/NRF-1. These findings indicate that a novel function of alpha-Pal/NRF-1 is to regulate neuronal differentiation, and that this function is mediated partly via its downstream IAP gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias/metabolismo , Neuritas/metabolismo , Neuritas/patología , Neuroblastoma/metabolismo , Neuroblastoma/patología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/genética , Humanos , Factor Nuclear 1 de Respiración , Factores Nucleares de Respiración , Proteínas Recombinantes/metabolismo , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Adult rats with early-life frequently repetitive febrile seizures (FRFS), but not single febrile seizure (SFS), exhibited impaired performance in inhibitory avoidance tasks but without significant hippocampal neuronal loss. The mechanisms of long-term memory impairment in the hippocampus of adult rats with early-life FRFS remain unknown. Using a heated-air febrile seizures (FS) paradigm, male rat pups were subjected to single or nine episodes of brief FS at days 10 to 12 postpartum. We found that early-life FRFS led to long-term bidirectional modulation in hippocampal synaptic plasticity, i.e., impaired long-term potentiation and facilitated long-term depression. Three hours after inhibitory avoidance training, phosphorylation of hippocampal extracellular signal-regulated kinase (ERK) 1/2 was significantly less in the FRFS group than in controls. Furthermore, there was a selective alteration in NMDA receptor-mediated ERK1/2 phosphorylation in the hippocampus of the FRFS group. Although the expression levels of NMDA receptor subunits and interaction of NMDA receptor and postsynaptic density 95 did not alter quantitatively, there was a specific alteration in NR2A, but not NR2B, subunit tyrosine phosphorylation after NMDA stimulation in the FRFS group. These data offer a potential molecular explanation for the hippocampus-dependent memory deficits observed in the rats with early-life FRFS.
Asunto(s)
Trastornos de la Memoria/metabolismo , Plasticidad Neuronal/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones Febriles/metabolismo , Convulsiones Febriles/fisiopatología , Animales , Animales Recién Nacidos , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas/fisiología , Trastornos de la Memoria/enzimología , Trastornos de la Memoria/fisiopatología , Fosforilación , Ratas , Convulsiones Febriles/enzimología , Sinapsis/enzimología , Sinapsis/metabolismoRESUMEN
Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5'-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions -232 and -12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a alpha-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the alpha-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the alpha-Pal/NRF-1 site. Overexpression of the DNA binding domain of alpha-Pal/NRF-1 in cells enhanced DNA-alpha-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length alpha-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that alpha-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regiones Promotoras Genéticas , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Clonación Molecular , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Genes Dominantes , Genes Reporteros , Humanos , Integrinas/metabolismo , Luciferasas/metabolismo , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factor Nuclear 1 de Respiración , Factores Nucleares de Respiración , Plásmidos/metabolismo , Unión Proteica , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , TransfecciónRESUMEN
The long-term effects of brief but repetitive febrile seizures (FS) on memory have not been as thoroughly investigated as the impact of single and prolonged seizure in the developing brain. Using a heated-air FS paradigm, we subjected male rat pups to one, three, or nine episodes of brief FS on days 10 to 12 postpartum. Neither hippocampal neuronal damage nor apoptosis was noted within 72 hours after FS, nor was there significant hippocampal neuronal loss, aberrant mossy fiber sprouting, or altered seizure threshold to pentylenetetrazol in any FS group at adulthood. The adult rats subjected to nine episodes of early-life FS, however, showed long-term memory deficits as assessed by the Morris water maze. They also exhibited impaired intermediate and long-term memory but spared short-term memory in the inhibitory avoidance task. Three hours after inhibitory avoidance training, phosphorylation of cAMP response-element binding (CREB) protein in the hippocampus was significantly lower in nine-FS-group rats than in controls. Furthermore, rolipram administration, which activated the cAMP-CREB signaling pathway by inhibiting phosphodiesterase type IV, reversed the long-term memory deficits in nine-FS-group rats by enhancing hippocampal CREB phosphorylation. These results raise concerns about the long-term cognitive consequences of even brief frequently repetitive FS during early brain development.