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

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作为重要的神经递质发挥作用。甘氨酸的细胞外浓度由高亲和力甘氨酸转运蛋白GlyTs控制。特别是GlyT 1，它由神经胶质细胞和尚未精确定义的神经元亚群表达，似乎在这一过程中起着关键作用。缺乏GlyT 1表达的小鼠由于抑制性突触的过度激活而死亡，而携带仅限于神经元的GlyT 1缺乏的小鼠显示出对药物诱导的精神病的抵抗力增加。与这些发现一致，用GlyT 1抑制剂治疗患有精神分裂症的人类患者显示出有益。然而，到目前为止，只有有限的信息有关的基本机制和涉及的细胞群负责这些影响。在本授权申请中，我们现在提出了携带GlyT 1基因外显子1b内插入荧光素酶-RFP融合蛋白（LucR）的小鼠的表征。GlyT 1基因的这种修饰应该导致LucR的表达，而不是推测的神经元特异性表达的GlyT 1b，而由相同基因编码的GlyT 1a的表达预计不受影响。使用这些小鼠，我们计划在细胞水平上分析GlyT 1b的功能，以及它对活体动物行为的影响，从而更好地了解神经元表达的GlyT 1如何影响神经元能神经传递。 在内源GlyT 1b启动子的控制下LucR报告蛋白的表达将使我们能够确定在生理和病理生理条件下GlyT 1b/c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，从而使它们可用于基于其转录组的深入表征。 连同GlyT 1b启动子区域内转录因子结合位点的计算机预测，该数据集将提供哪些转录因子和/或信号级联参与GlyT 1b表达调控的信息。随后将在体外试验系统中检测这些因子对GlyT 1b表达的影响。 通过这种方法，我们希望获得更好的理解神经元GlyT 1的转录调控。此外，这些实验可能为GlyT 1b表达的（药理学）操纵提供基础，从而为精神疾病如精神分裂症的新治疗策略提供基础。

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.