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

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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.