Molecular roles of the Prion Protein in zebrafish embryos and cultured cells


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SEMPOU, Aimilia, 2014. Molecular roles of the Prion Protein in zebrafish embryos and cultured cells

@phdthesis{Sempou2014Molec-26739, title={Molecular roles of the Prion Protein in zebrafish embryos and cultured cells}, year={2014}, author={Sempou, Aimilia}, address={Konstanz}, school={Universität Konstanz} }

eng 2014-03-03T13:25:58Z 2014 Sempou, Aimilia Molecular roles of the Prion Protein in zebrafish embryos and cultured cells deposit-license Sempou, Aimilia The prion protein (PrP) is a cell surface glycoprotein, best known for its key role in infectious brain disorders in which it misfolds, forms aggregates and triggers neurodegeneration. Although the misfolding and aggregation of PrP in the brain constitute hallmarks of these disorders, it has become clear that normally folded PrP molecules contribute to neuronal death via an activity carried out on neuronal surfaces. Thus, aggregates of misfolded PrP trigger neurodegeneration possibly by subverting the function of normal PrP molecules. A physiological role of PrP in signal transduction is consistent with this thesis, but the exact nature of PrP’s function and the underlying molecular mechanisms have remained elusive. Our laboratory identified the zebrafish orthologue PrP-1 as a positive regulator of E-cadherin based cell-cell adhesion during early embryonic development. Due to their impaired tissue cohesiveness, PrP-1 knockdown embryos failed to undergo morphogenesis and became arrested at gastrulation. Interestingly, mammalian (mouse) PrP could compensate for PrP-1 loss of function, indicating that the control of cell adhesion is a basic, conserved function of PrPs. The goal of my thesis was to elucidate the conserved cellular and molecular mechanisms by which PrP exerts its regulation over E-cadherin-based adhesion in the zebrafish gastrula. Our morpholino knockdown experiments revealed that the Src tyrosine kinases Fyn and Yes act downstream of PrP-1 to prevent the endocytosis of cell surface adhesion complexes, composed of transmembrane E-cadherin and its intracellular binding partner β-catenin. Accordingly, downregulation of Fyn/Yes or PrP-1 produce similar gastrulation phenotypes and defects in cell adhesion, whereas the exogenous expression of these kinases in PrP-1 knockdown embryos leads to developmental recovery.<br /><br />We further show that zebrafish and mouse PrPs positively regulate the activity of Src kinases and that these have an unexpected, positive effect on E-cadherin-mediated cell adhesion in vivo. In addition, while PrP knockdown disrupts the cell surface localization of E-cadherin, PrP mRNA overexpression enhances it, thereby sequestering β-catenin at the plasma membrane and antagonizing its parallel role as a transcription activator in the nucleus. This, in turn, leads to impaired development of embryonic dorsal structures, which largely depends on β-catenin nuclear signaling. Through mutational analysis, we found that the ability of PrP to localize at cell-cell contacts and influence zebrafish gastrulation phenotypes depends on its anchorage at the plasma membrane as well as on its repetitive and globular domains. In addition, we show that a small polybasic stretch within PrP’s N-terminus, reportedly encoding neuroprotective properties, is essential to the role of PrP during zebrafish gastrulation. Conversely, PrP activity was not affected by deletion of a central stretch that controls neurotoxicity in transgenic mice, although the corresponding zebrafish mutant PrPs were toxic to mammalian cells. Finally, in light of the recent identification of PrPC as a receptor for neurotoxic Aβ oligomers in Alzheimer’s disease, we assessed the effects of these species on PrP-mediated signaling in zebrafish embryonic cells. These experiments revealed that exposure to Aβ oligomers leads to the PrP-1-dependent activation of Src kinases and a simultaneous increase in the levels of E-cadherin, thus mimicking a PrP gain-of-function scenario. Taken together, these findings highlight the remarkable functional conservation of vertebrate PrPs in the regulation of complex intracellular signals relevant to the fields of development, neurodegeneration and cell adhesion. Molekulare Rollen des Prion-Proteins in Zebrafischembryonen und Zellen

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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