German Title: TrkB und Genexpression

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Abstract

The neurotrophins are a family of secreted proteins that potently regulate diverse neuronal responses. Family members include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4/5(NT4/5). Neurotrophins bind the Trks receptor family (TrkA, B, C). NGF is the preferred ligand for TrkA, BDNF and NT4/5 are preferred for TrkB, and NT3 for TrkC, although NT3 also binds with less affinity to TrkA and TrkB. During my PhD I have focused my interest in understanding how neurotrophins regulate gene expression and, in particular, how BDNF does this through its high affinity receptor TrkB during neurogenesis. In order to answer this question, I planned to adopt the following approaches. First, to analyze global changes in gene expression after TrkB/BDNF activation, using microarray technology. Second, once a set of regulated genes was identified, to characterize the regulation of these genes at the promoter level, in order to understand which common elements are important for their regulation. The high-density oligonucleotide array of Affymetrix was performed using mRNAs that were obtained from cortical neurons of wild type mouse embryos (E15.5), and of mouse embryos possessing TrkB receptors mutated at either tyrosine 515 (trkB/shc point signaling mutants), or tyrosine 816 (trkB/plc-g point signaling mutants), or at both sites. In all cases the primary neurons were either unstimulated or stimulated with BDNF. The sensitivity of the Affymetrix system allowed me to identify a set of transcription factors that showed a higher fold induction compared to the others class of genes. This group consisted of: egr1, egr2, c-fos and mGIF/Tieg1. These genes were found to be differentially regulated in the signaling point mutant mice. Although the promoter of mGif/TIEG1 is not yet characterized and also the function of this gene is not completely clear, egr1, egr2 and c-fos are well characterized, and, several data suggest that these genes share cis acting 5’ regulatory elements. To better understand which elements and transcription factors are important for BDNF-dependent gene expression I choose the c-fos promoter as a model. Using luciferase reporter gene constructs transfected in E15.5 cortical neurons isolated from wild-type and signaling point mutant mice, I discovered that the pathways activated through the shc site promoted higher activation of c-fos promoter than pathways activated through the plc-g_site, and the two sites are both required for BDNF-dependent activation of c-fos promoter. Additionally experiments using c-fos promoter constructs, mutated at single or multiple elements, revealed that the c/ebp binding site together with the E-box are fundamental for the activation of c-fos downstream BDNF/TrkB. This result suggested that C/EBPs and bHLH transcription factors might collaborate to induce the activation of the promoter downstream of BDNF. I have demonstrated, both in vivo and in vitro, that Mash1 and NeuroD are the members of bHLH family that bind C/EBP transcription factors at different domains. That interaction is BDNF independent, and the complex is constitutively present on the c-fos promoter. The BDNF regulation of gene expression is through the post-translation modification of that complex. In fact BDNF stimulation induces an increase in C/EBPb phosphorylation on Thr188 (ERK1/2-dependent). The phosphorylation by ERK1/2 could explain the transcriptional activation of the C/EBP-Mash1-NeuroD complex downstream BDNF/TrkB. These studies identify a novel neurotrophins-regulated signaling cascade that mediates the gene expression during neurogenesis.