ALCOHOL AND NMDA GENE EXPRESSION IN A NEURONAL CELL LINE

描述：N-甲基-D-天冬氨酸（NMDA）受体，一种兴奋性 脑中的神经递质受体，对神经元发育至关重要， 突触可塑性 最近的研究表明，NMDA受体 是乙醇作用的重要部位，并与 酒精依赖和戒断综合征的发展。 急性暴露 乙醇导致抑制NMDA受体功能，而慢性 乙醇处理上调NMDA受体功能和结合。 结果 来自PI实验室的研究表明， 处理增加NMDA R1和R2 B亚基多肽的合成 合成，伴随着仅NMDA R2 B亚基mRNA水平的增加。 细胞质稳定性和基因转录测定结果表明， NMDA R1亚基在翻译水平的调节和NMDA R2 B亚基在翻译水平的调节 在基因转录水平上。 为了进一步阐明 作为这种差异调节的基础的分子机制， 需要研究NMDA受体基因表达的调节， 转录水平和翻译水平。 为承接 在这些研究中，必须有一种神经元细胞系， 天然表达功能性NMDA受体。 在这份提案中，PI的 目的是通过永生化小鼠胎儿来建立这样的细胞系 皮质细胞与猿猴病毒40大肿瘤抗原，一个永生化的 癌基因 PI将产生克隆细胞系，其将被广泛地 其特征在于1）通过免疫细胞化学来检查细胞来源， 克隆细胞系;只有那些对神经元特异性 将进一步检查烯醇化酶2）NMDA受体的存在 亚基多肽和mRNA，3）NMDA受体的存在和4） 功能性NMDA受体的存在。 表达功能性 然后将NMDA受体暴露于乙醇以确定NMDA受体是否 受体以与体内相当的方式被调节。 细胞系 表达功能性NMDA受体的技术将在未来用于 定义乙醇如何影响NMDA受体的确切机制 亚基基因表达

DESCRIPTION: The N-methyl-D-aspartate (NMDA) receptor, an excitatory neurotransmitter receptor in brain, is critical for neuronal development and synaptic plasticity. Recent studies have suggested that the NMDA receptor is an important site of action of ethanol, and has been implicated in the development of alcohol dependence and withdrawal syndrome. Acute exposure to ethanol results in inhibition of NMDA receptor function whereas chronic ethanol treatment upregulates NMDA receptor function and binding. Results from the PI's laboratory demonstrated that exposure to chronic ethanol treatment augments the synthesis of NMDA Rl and R2B subunit polypeptide synthesis with a concomitant increase in only NMDA R2B subunit MRNA levels. Cytoplasmic stability and gene transcription assay results point to a regulation of NMDA RI subunit at the translation level and NMDA R2B subunit at the gene transcription level. To further elucidate the potential molecular mechanisms that underlie this differential regulation, it is necessary to study regulation of the NMDA receptor gene expression at the transcriptional level and at the translation level. In order to undertake such investigations, it is essential to have a neuronal cell line that naturally expresses functional NMDA receptors. In this proposal, the PI's objective is to develop such a cell line by immortalizing murine fetal cortical cells with Simian virus 40 large tumor antigen, an immortalizing oncogene. The PI will generate clonal cell lines which will be extensively characterized 1) by immunocytochemistry to examine the cellular origin of the clonal cell lines; only those that are positive for neuron specific enolase will be further examined for 2) the presence of NMDA receptor subunit polypeptides and mRNAs, 3)the presence of NMDA receptors and 4) the presence of functional NMDA receptors. Cell lines expressing functional NMDA receptors will then be exposed to ethanol to determine if NMDA receptors are regulated in a manner comparable to in vivo. Cell lines expressing functional NMDA receptors will then be used in the future to define the precise mechanism(s) as to how ethanol influences NMDA receptor subunit gene expression.