RESUMO
The PC12 is the widely used cell line to study neuronal differentiation. We had extensively investigated the details of protein expression in differentiated PC12 cells by proteomic analysis. The cells were incubated at the presence of nerve growth factor. We had analyzed the expression changes in the differentiating PC12 cells by 2-dimensional electrophoresis and the identification of the proteins using MALDI-TOF MS. By comparing expression pattern in the time course, we identified the candidate genes which are associated with neuronal differentiation. Among these genes, we performed real-time PCR analysis to validate Idh3alpha expression by the time course. To identify the function of Idh3alpha in neuronal differentiation stage, the transfection of Idh3alpha to PC12 cells was performed. As a result, we proved that up-regulation of Idh3alpha causes reduction in neural differentiation of PC12 cells. Based on these data, we suggest that Idh3alpha plays a role to the neuronal differentiation.
Assuntos
Diferenciação Celular , Isocitrato Desidrogenase/metabolismo , Isoenzimas/metabolismo , Neurônios/fisiologia , Células PC12/fisiologia , Proteoma/análise , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Isocitrato Desidrogenase/genética , Isoenzimas/genética , Neurônios/citologia , Ratos , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para CimaRESUMO
The study of ES cell-mediated neuronal differentiation allows elucidating the mechanism of neuronal development in spite of the complexity and the difficult accessibility. During the differentiation of embryonic stem cells into neuronal cell, the expression profiles in the level of protein were extensively investigated by proteomic analysis. These cells were analyzed for charges in proteome during the differentiation of ES cells by 2-dimensional electrophoresis (2-DE) and MALDI-TOF MS. Seven unique proteins were identified, some of which were differentially expressed at each stage. A complex system of neuronal differentiation can be activated in cultured embryonic stem cells and our two dimensional electrophoresis data should be useful for investigating some of the mechanism that regulates neuronal differentiation.
Assuntos
Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Neurônios/citologia , Proteômica , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Eletroforese em Gel Bidimensional , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Concentração de Íons de Hidrogênio , Camundongos , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Tretinoína/farmacologiaRESUMO
ABSTRACT Methamphetamine (MAP) is a commonly used, addictive drug, and a powerful stimulant that dramatically affects the central nervous system. In this study, we used the conditioned place preference (CPP) paradigm in order to study the reinforcing properties of MAP and the herewith associated changes in proteins within the mesolimbic dopamine system. A CPP was induced by MAP after three intermittent intraperitoneal injections (1 mg/kg) in rats and protein profiles in the nucleus accumbens, striatum, prefrontal cortex, cingulate cortex and hippocampus were compared with a saline-treated control group. In addition, a group of animals was run through extinction and protein profiles were compared with a non-extinguished group. Protein screening was conducted using two-dimensional electrophoresis analysis which identified 27 proteins in the group that showed MAP-induced CPP. Some of the proteins were confirmed by Western lot analysis. Identified proteins had functions related to the cytoskeleton, transport/endocytosis or exocytosis (e.g. profilin-2 and syntaxin-binding protein), and signal transduction, among others.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Dopamina/metabolismo , Sistema Límbico/efeitos dos fármacos , Metanfetamina/farmacologia , Proteômica/métodos , Reforço Psicológico , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Eletroforese em Gel Bidimensional , Masculino , Metanfetamina/administração & dosagem , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Methamphetamine is an illicit drug that is often abused and can cause neuropsychiatric and neurotoxic damage. Repeated administration of psychostimulants such as methamphetamine induces a behavioral sensitization. According to a previous study, Bax was involved in neurotoxicity by methamphetamine, but the function of Bax in rewarding effect has not yet been elucidated. Therefore, we have studied the function of Bax in a rewarding effect model. In the present study, we treated chronic methamphetamine exposure in a Bax-deficient mouse model and examined behavioral change using a conditioned place preference (CPP) test. The CPP score in Bax knockout mice was decreased compared to that of wild-type mice. Therefore, we screened for Bax-related genes that are involved in rewarding effect using microarray technology. In order to confirm microarray data, we applied the RT-PCR method to observe relative changes of Bcl2, a pro-apoptotic family gene. As a result, using our experiment microarray, we selected genes that were associated with Bax in microarray data, and eventually selected the Tgfbr2 gene. Expression of the Tgfbr2 gene was decreased by methamphetamine in Bax knockout mice, and the gene was overexpressed in Bax wild-type mice. Additionally, we confirmed that Creb, FosB, and c-Fos were related to rewarding effect and Bax using immunohistochemistry.
