Genetical analysis of neuronal glycine transporter GlyT1b function and its transcriptional regulation in vivo

神经元甘氨酸转运蛋白GlyT1b功能的遗传分析及其体内转录调控

• 批准号: 251864121
• 负责人: Professor Dr. Volker Eulenburg
• 金额: --
• 依托单位: Klinik für Anästhesiologie und Operative Intensivmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251864121?language=en
• 关键词: Genetical; analysis; neuronal; glycine; transporter

In the central nervous system, the amino-acid glycine functions as an important neurotransmitter at excitatory as well as inhibitory synapses. The extracellular concentration of glycine is controlled by high affinity glycine transporters, the GlyTs. Especially GlyT1, that is expressed by both glial cells and a yet not precisely defined subpopulation of neurons appears to have a key role in this process. Mice that are deficient in GlyT1 expression die due to over-activation of inhibitory synapses, whereas mice that carry a GlyT1 deficiency restricted to neurons display an increased resistance against pharmacological induced psychosis. Consistent with these findings, the treatment of human patients suffering from schizophrenia with GlyT1 inhibitors was shown to be beneficial. There is, however, up to now only limited information concerning the underlying mechanisms and involved cell populations that are responsible for these effects available. In this grant application, we now propose the characterization of mice that carry the insertion of a Luciferase-RFP fusion protein (LucR) within the exon 1b of the GlyT1 gene. This modification of the GlyT1 gene should result in the expression of LucR instead of the supposedly exclusively neuronal expressed GlyT1b, whereas the expression of GlyT1a that is encoded by the same gene is expected to be unaffected. Using these mice, we plan to analyse the function of GlyT1b on a cellular level but also its influence on the behaviour of the living animal, thereby obtaining a better understanding of how neuronal expressed GlyT1 affects glutamatergic neurotransmission. The expression of the LucR reporter protein under the control of the endogenous GlyT1b promoter will allow us to determine the expression pattern of GlyT1b/c under physiological and pathophysiological conditions. In addition, the labeling of these cells will allow the identification and subsequent isolation of this apparently special cell population and make them thereby accessible for an in depth characterization on basis of their transcriptome. Together with in silico predictions of transcription factor binding sites within the GlyT1b promoter region, this dataset will provide informations which transcription factors and/or signaling cascades are involved in the regulation of GlyT1b expression. These factors will be subsequently tested for their effect on GlyT1b expression in in vitro test systems. Following this approach we hope to obtain a better understanding of the transcriptional regulation of neuronal GlyT1. Furhtermore these experiments might provide the basis of a (pharamacological) manipulation of GlyT1b expression and thus for new treatment strategies for psychiatric diseases like schizophrenia.

在中枢神经系统中，氨基酸甘氨酸在兴奋性和抑制性突触中充当重要的神经递质。甘氨酸的细胞外浓度由高亲和力甘氨酸转运蛋白 GlyT 控制。尤其是 GlyT1，它由神经胶质细胞和尚未精确定义的神经元亚群表达，似乎在这一过程中发挥着关键作用。 GlyT1 表达缺陷的小鼠会因抑制性突触的过度激活而死亡，而仅限于神经元的 GlyT1 缺陷的小鼠表现出对药理学诱发的精神病的抵抗力增强。与这些发现一致的是，用 GlyT1 抑制剂治疗精神分裂症人类患者是有益的。然而，到目前为止，关于造成这些效应的潜在机制和所涉及的细胞群的信息有限。在此拨款申请中，我们现在提出了对在 GlyT1 基因的外显子 1b 内插入荧光素酶-RFP 融合蛋白 (LucR) 的小鼠进行表征。 GlyT1 基因的这种修饰应该导致 LucR 的表达，而不是假定的仅神经元表达的 GlyT1b，而由同一基因编码的 GlyT1a 的表达预计不会受到影响。利用这些小鼠，我们计划在细胞水平上分析 GlyT1b 的功能及其对活体动物行为的影响，从而更好地了解神经元表达的 GlyT1 如何影响谷氨酸能神经传递。 LucR 报告蛋白在内源性 GlyT1b 启动子控制下的表达将使我们能够确定 GlyT1b/c 在生理和病理生理条件下的表达模式。此外，这些细胞的标记将允许识别和随后分离这种明显特殊的细胞群，从而使它们能够根据其转录组进行深入表征。 与 GlyT1b 启动子区域内转录因子结合位点的计算机预测一起，该数据集将提供哪些转录因子和/或信号级联参与 GlyT1b 表达调节的信息。随后将在体外测试系统中测试这些因素对 GlyT1b 表达的影响。 通过这种方法，我们希望更好地了解神经元 GlyT1 的转录调控。此外，这些实验可能为 GlyT1b 表达的（药理学）操纵提供基础，从而为精神分裂症等精神疾病的新治疗策略提供基础。