ALCOHOL AND NMDA GENE EXPRESSION IN A NEURONAL CELL LINE

描述：N-甲基-D-天冬氨酸(NMDA)受体，一种兴奋性物质 大脑中的神经递质受体，对神经元发育和 突触可塑性。最近的研究表明，NMDA受体 是乙醇的一个重要作用部位，并与 酒精依赖和戒断综合征的发展。急性暴露 酒精导致NMDA受体功能抑制，而慢性 乙醇处理上调NMDA受体的功能和结合。结果 来自PI实验室的证据表明，长期接触乙醇 治疗促进NMDARL和R2b亚基多肽的合成 合成只伴随着NMDAR2b亚单位mRNA水平的增加。 细胞质稳定性和基因转录检测结果表明 NMDARI亚基和NMDAR2b亚基在翻译水平的调节 在基因转录水平上。以进一步阐明潜在的 这种不同调控的分子机制，是 有必要研究NMDA受体基因表达的调控 在转录层面和翻译层面。为了承担 在这样的研究中，拥有一种神经细胞系是必不可少的 自然地表达功能性的NMDA受体。在这份提案中，私家侦探的 目的是通过使小鼠胚胎永生化来建立这样的细胞系 皮质细胞携带猿猴病毒40大肿瘤抗原，永生 致癌基因。PI将产生克隆细胞系，将广泛应用于 特征1)通过免疫细胞化学检查细胞起源 克隆细胞系；只有那些神经元特异性阳性的细胞系 将进一步检查烯醇化酶2)NMDA受体的存在 亚基多肽和mRNAs，3)NMDA受体的存在和4) 功能正常的NMDA受体的存在。表达功能性的细胞系 然后将NMDA受体暴露在乙醇中以确定NMDA是否 受体的调节方式与体内相当。细胞系 表达功能性NMDA受体将在未来用于 确定酒精影响N-甲基-D-天冬氨酸受体的精确机制(S) 亚单位基因表达。

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.