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1.
J Neurochem ; 120(5): 741-51, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22118475

RESUMO

Synaptic remodeling has been postulated as a mechanism underlying synaptic plasticity and cell adhesion molecules are thought to contribute to this process. We examined the role of nectin-1 ectodomain shedding on synaptogenesis in cultured rat hippocampal neurons. Nectins are Ca(2+) -independent immunoglobulin-like adhesion molecules, involved in cell-cell adherens junctions. Herein, we show that the processing of nectin-1 occurs by multiple endoproteolytic steps both in vivo and in vitro. We identified regions containing two distinct cleavage sites within the ectodomain of nectin-1. By alanine scanning mutagenesis, two point mutations that disrupt nectin-1 ectodomain cleavage events were identified. Expression of these mutants significantly alters the density of dendritic spines. These findings suggest that ectodomain shedding of nectin-1 regulates dendritic spine density and related synaptic functions.


Assuntos
Moléculas de Adesão Celular/metabolismo , Dendritos/ultraestrutura , Espinhas Dendríticas/fisiologia , Neurônios/citologia , Secretases da Proteína Precursora do Amiloide/farmacologia , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular/genética , Células Cultivadas , Espinhas Dendríticas/ultraestrutura , Embrião de Mamíferos , Feminino , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Nectinas , Neurônios/efeitos dos fármacos , Mutação Puntual/genética , Gravidez , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução Genética , Transfecção
2.
J Neurosci Methods ; 177(2): 348-54, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19014969

RESUMO

To facilitate genetic studies in primary neurons, we analyzed the efficiency of cationic lipid-mediated plasmid DNA transfection using adherent and acutely dissociated neuronal suspensions derived from embryonic mouse cortical tissue. Compared to transfections using adherent cultures, the in-tube procedure enhanced the delivery of a GFP reporter plasmid between four- to eightfold depending on the age of the harvested embryo. The procedure required relatively brief complex incubation times, and supported the transfection of cells expressing the neuronal markers NeuN and TuJ1 with improved uniformity in transfection events across the well surface. To demonstrate the utility of this approach in studying the genetic mechanisms controlling neuron development, we provide data regarding the role of the bZIP transcription factor c/EBP-beta in regulating neurite outgrowth. It is anticipated that this in vitro protocol will facilitate the identification of novel genes involved in both developmental and disease-relevant signaling pathways.


Assuntos
Técnicas de Transferência de Genes , Biologia Molecular/métodos , Neurônios/metabolismo , Transfecção/métodos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Humanos , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Biologia Molecular/instrumentação , Neuritos/metabolismo , Neuritos/ultraestrutura , Neurogênese/genética , Neurônios/ultraestrutura , Plasmídeos/genética , Polilisina/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Transfecção/instrumentação
3.
J Comp Neurol ; 507(2): 1228-44, 2008 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-18181141

RESUMO

Nectins are cell adhesion molecules that, together with the intracellular binding partner afadin, mediate adhesion and signaling at a variety of intercellular junctions. In this work we studied the distribution of nectin-1 and afadin during hippocampal synapse formation using cultured primary hippocampal neurons. Nectin-1 and afadin cluster at developing synapses between hippocampal neurons. These nectin-afadin clusters uniformly colocalize with N-cadherin-catenin pairs, suggesting that formation of developing synapses involves participation of both bimolecular systems. Nectin-1 is initially expressed at excitatory and inhibitory synapses but is progressively lost at inhibitory synapses during their maturation. Treatment of neurons with actin depolymerizing agents disrupts the synaptically localized nectin-1 and afadin cluster at an early stage and elicits nectin-1 ectodomain shedding. These data indicate that the synaptic localization of nectin-1 and l-afadin are F-actin-dependent and that the shedding of nectin-1 is a mechanism contributing to synaptic plasticity.


Assuntos
Moléculas de Adesão Celular/metabolismo , Hipocampo/embriologia , Hipocampo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Hipocampo/ultraestrutura , Camundongos , Nectinas , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/ultraestrutura , Estrutura Terciária de Proteína/fisiologia , Ratos , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Fatores de Tempo , beta Catenina/metabolismo
4.
Neurobiol Aging ; 29(11): 1690-701, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17537546

RESUMO

Parkinson's disease (PD) is the most common neurodegenerative movement disorder afflicting >500,000 patients in the United States alone. This age-related progressive disorder is typified by invariant loss of dopaminergic substantia nigra neurons (DAN), dystrophic neurites, the presence of alpha-synuclein (SYN) positive intracytoplasmic inclusions (Lewy bodies) in the remaining DAN, and activated microglia. As such, microglial activation and resultant increase in proinflammatory molecules have moved to the forefront of PD research as a potential pathobiologic mechanism of disease. Herein, we present data demonstrating early microglial activation in mice that over-express wild-type SYN, the release of SYN from a SYN overexpressing MN9D cell line, and dose-dependent SYN-mediated activation of primary microglial cultures with consequent increases in proinflammatory molecules. Furthermore, we provide evidence that the CD36 scavenger receptor and downstream kinases are involved in SYN-mediated microglial activation. Together, our data suggest an early role for SYN and inflammation in PD pathogenesis.


Assuntos
Citocinas/metabolismo , Modelos Animais de Doenças , Microglia/efeitos dos fármacos , Microglia/metabolismo , Transtornos Parkinsonianos/metabolismo , Sinucleínas/efeitos dos fármacos , Sinucleínas/metabolismo , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
5.
Biochem Biophys Res Commun ; 361(3): 599-604, 2007 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-17673176

RESUMO

Nerve growth factor mediates neuronal survival, synaptogenesis, and synaptic remodeling. We utilized primary hippocampal cultures to investigate the intrinsic motifs of proNGF that might contribute to its processing and subsequent allocation to a regulated versus constitutive secretory pathway. The addition of a carboxypeptidase E motif to proNGF did not alter the secretion of NGF. However, mutagenesis of proNGF proteolytic processing sites had significant effects on the final NGF product and its secretion. The furin recognition site (R118-S-K-R121) is essential for the proper processing of proNGF to its 13.5kDa mature product and mutating the furin site exposed an alternative processing site resulting in an intermediate NGF product of approximately 22kDa. Finally, inhibiting the processing of proNGF abolished regulated secretion of the resulting NGF product. These experiments demonstrate that hippocampal neurons harbor multiple pathways to process proNGF of which the furin consensus sequence is the preferred processing site.


Assuntos
Fator de Crescimento Neural/metabolismo , Neurônios/metabolismo , Precursores de Proteínas/metabolismo , Motivos de Aminoácidos , Diamino Aminoácidos/genética , Diamino Aminoácidos/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Carboxipeptidase H/metabolismo , Furina/genética , Furina/metabolismo , Hipocampo/metabolismo , Modelos Genéticos , Mutação , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/genética , Ratos , Ratos Sprague-Dawley
6.
Exp Neurol ; 203(1): 221-32, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16999955

RESUMO

The orphan nuclear receptor Nurr1 is required for the development of the ventral mesencephalic dopaminergic neurons. These are the same neurons that are invariantly lost in patients with Parkinson's disease. Nurr1 mRNA expression is not confined to the developing midbrain, and yet Nurr1 appears to be essential for either the maturation of progenitors into fully post-mitotic dopaminergic neurons and/or once formed, their survival. The function of Nurr1 in the transactivation of gene(s) important for neuronal development and/or maintenance is uncharacterized. To characterize potential downstream target genes of Nurr1, we sought to identify mRNAs that are differentially affected by Nurr1 expression. Using a dopaminergic cell line in which Nurr1 content was tightly regulated, differential display analysis identified transcripts altered by Nurr1 expression, including the mRNA encoding vasoactive intestinal peptide (VIP). Herein, we demonstrate that Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP promoter through Nurr1-responsive cis elements. In addition, dopaminergic cells release and utilize VIP to mediate survival when challenged with paraquat. Nurr1 regulation of VIP is also demonstrated in vivo as loss of Nurr1 function results in diminished VIP mRNA levels within the developing midbrain.


Assuntos
Proteínas de Ligação a DNA/genética , Dopamina/metabolismo , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/genética , Peptídeo Intestinal Vasoativo/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Herbicidas/toxicidade , Mesencéfalo/citologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Paraquat/toxicidade , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Elementos Reguladores de Transcrição/genética , Substância Negra/citologia , Substância Negra/embriologia , Substância Negra/metabolismo , Ativação Transcricional/genética , Peptídeo Intestinal Vasoativo/metabolismo
7.
Cancer Res ; 62(22): 6545-51, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12438249

RESUMO

Development of effective antitumor immune responses depends on timely interactions of effector cells. A bimodal approach that involves coexpression of chemokines and costimulatory molecules within the tumor bed may elaborate a more optimal antitumor response. One candidate includes secondary lymphoid tissue chemokine (SLC), which promotes the colocalization of naïve, nonpolarized memory T cells and dendritic cells (DCs) within lymph nodes and Peyer's patches. CD40L-mediated DC activation could induce maturation, enhance antigen presentation, and facilitate priming of the recruited naïve T cells. To this end, the antitumor activity of SLC and CD40L expressed singly or in combination using the herpes simplex virus (HSV)-derived amplicon was examined in two murine models: A20, a B-cell lymphoma, and CT-26, an adenocarcinoma. Administration of amplicons encoding SLC (HSV-SLC) into s.c. tumors established previously resulted in heavy infiltration of CD4+ and CD8+ T cells, and DCs, and the generation of cytolytic T-cell activity. Combined transduction of either tumor with HSV-SLC and HSV-CD40L resulted in a more enhanced antitumor activity that was CD8+ T cell-dependent than observed with either vector alone. mRNA expression of the Th1 markers IFN-gamma, perforin, and interleukin 12 was detectable only in transduced regressing tumors. In addition to identifying a potent antitumor immune strategy, we show that amplicon-mediated SLC and CD40L delivery may mimic lymph node conditions necessary for priming naïve T cells within the tumor bed, and demonstrate the importance of DC activation status on antigen presentation and cytokine expression for priming of newly recruited T cells.


Assuntos
Ligante de CD40/imunologia , Quimiocinas CC/imunologia , Imunoterapia/métodos , Simplexvirus/genética , Adenocarcinoma/imunologia , Adenocarcinoma/terapia , Animais , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/genética , Quimiocina CCL21 , Quimiocinas CC/genética , Células Dendríticas/imunologia , Terapia Genética/métodos , Vetores Genéticos/genética , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Linfócitos T Citotóxicos/imunologia
8.
Mol Ther ; 5(6): 723-30, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12027556

RESUMO

Modern cell biologists typically use reporter genes either alone or co-expressed with a protein of interest to facilitate the localization or quantification of protein expression. Our work demonstrates that reporter genes should be used cautiously, as several common reporter gene products are toxic to primary cortical neuronal cultures. We used the herpes simplex virus-based viral amplicon vector to transduce cortical neurons with three different reporter genes and assessed whether any reporter gene products were toxic over time, by monitoring neurite disintegration and apoptosis. Toxicity varied as a function of the reporter gene, the gene product localization, and the level of reporter gene expression. Transduction of enhanced green fluorescent protein or nuclear-localized beta-galactosidase was more toxic than non-nuclear localized beta-galactosidase. This work underscores the need for careful design of gene expression constructs. Moreover, in studies where cell injury or toxicity is being evaluated, their use should be carefully considered.


Assuntos
Apoptose , Genes Reporter , Proteínas Luminescentes/toxicidade , Neurônios/patologia , beta-Galactosidase/toxicidade , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Camundongos , Neurônios/citologia , Simplexvirus/metabolismo , Transgenes , beta-Galactosidase/genética
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