NEW HORIZON OF NEURON-ASTROCYTE INTERACTION : NEUROTROPHICACTIVITY OF L-SERINE

该项目的总体目的是确定星形胶质细胞来源的L-Ser对体内中枢神经系统神经元存活和发育的生理意义。需要验证的假设是，在动物细胞中，星形胶质细胞特异性表达3PGDH（一种催化l -丝氨酸生物合成初始步骤的酶）是l -丝氨酸对中枢神经系统神经元的神经营养作用的分子机制。在这个项目中，我们致力于了解星形胶质细胞特异性表达3PGDH基因的分子基础。我们首先分离并测序了含有小鼠3PGDH基因的BAC克隆。基于这些结构信息，我们随后检测了该基因的启动子活性。我们使用报告基因检测系统发现，在星形胶质细胞中，起始密码子上游的1 .8 kb区域在5'-侧翼序列中指导报告基因的高水平表达。我们现在正试图通过删除这个区域来确定负责星形胶质细胞特异性转录激活的序列。在分析启动子的同时，我们还在CNS神经元中建立了3PGDH cDNA异位表达的转基因小鼠。我们构建了在神经元特异性启动子控制下表达大鼠3pGDH cDNA的转基因，最近获得了方正小鼠，我们正在选择高水平神经元表达cDNA的细胞系。我们相信这些转基因小鼠将是检验我们假设的有用工具。

The overall aim of the project was to determine the physiological significance of astrocyte-derived L-Ser on the surival and development of CNS neurons in vivo. The hypothesis to be tested is that astrocyte-specific expression of 3PGDH, an enzyme catalyzing the initial step, of L-Ser biosynthesis in animal cells, is the molecular mechanism underlying neurotrophic action of L-Ser on CNS neurons. In the project, we set our effort to understand the molecular basis of astrocyte-specific expression of 3PGDH gene. We first isolated and sequenced a BAC clone which contains the mouse 3PGDH gene. Based on this structural information, we then examined promoter activity of the gene. We found using a reporter gene assay system that I.8 kb region upstream from the initiation codon in the 5'-flanking sequence directs high-level expression of reporter in astrocytes. We are now trying to identify sequence responsible for astrocyte-specific transcriptional activation by deletion of this region. In parallel with this promoter analysis, our effort was also directed to establish transgenic mice with ectopic expression of 3PGDH cDNA in CNS neurons. We constructed the transgene in which rat 3pGDH cDNA could be expressed under the control of neuron-specific promoter, Founder mice were recently obtained and we are now selecting lines with high-level neuronal expression of the cDNA. We believe that these transgenic mice will be a useful tool for testing our hypothesis.