Functional characterization of Cytoplasmic Polyadenylation Element Binding proteins in the developing and diseased brain Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Magdalena Skubal aus Tarnogrod, Polen Bonn, 2016 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. Christian Steinhäuser Institut für Zelluläre Neurowissenschaften Universität Bonn 2. Gutachter: Prof. Dr. Walter Witke Institut für Genetik Universität Bonn Tag der Promotion: 29.11.2016 Erscheinungsjahr: 2016 “One of the ultimate challenges for biology is to understand the brain's processing of unconscious and conscious perception, emotion, and empathy“ Eric R. Kandel ACKNOWLEDGEMENTS First of all, I would like to sincerely thank my supervisor Prof. Dr. Christian Steinhäuser for the scientific guidance, support and many constructive discussions. I am honestly grateful to PD Dr. Andreas Waha for co-supervising my project, giving valuable suggestions and encouraging me throughout my work. My special thanks go to my former supervisor Prof. Dr. Martin Theis for giving me the opportunity to work on this interesting project. I would like to thank Prof. Dr. Walter Witke, Prof. Dr. Volkmar Gieselmann and Prof. Dr. Dieter Meschede for accepting my request to be part of the examination committee and for their effort in reviewing this work. I am especially grateful to PD Dr. Gerald Seifert, Dr. med. Gerrit Gielen, Dr. Lech Kaczmarczyk and Dr. Peter Bedner for their help in multiple analyses and writing CPEB manuscripts. I want to thank PD. Dr. Ronald Jabs for introduction to the confocal microscopy. In addition, I would like to thank Prof. Dr. Christian Henneberger and Dr. Anne Boehlen for fruitful discussions and constructive suggestions. My special thanks go to the present and the former lab members: Aline, Björn, Camille, Daniel, Dimitri, Dilaware, Julia, Kirsten, Michel, Stefan, Steffi Anders, Steffi Griemsmann, and Tushar. Thank you for the great time together! I also would like to thank for administrative assistance to Dr. Silke Kunzel and Dr. Ines Heuer and for excellent technical support to Anja Matijevic, Thomas Erdmann, Dorota Denkhaus, Evelyn Dörfer, Verena Dreschmann, and Jennifer Hammes. Last but not least, I would like to express my deepest gratitude towards my family, especially my parents and Nicolas for their constant encouragement and incredible support. CONTENTS ABBREVIATIONS ......................................................................................................................... 11 1 INTRODUCTION .................................................................................................................. 14 1.1 Gliomas ............................................................................................................................. 14 1.1.1 Cellular origin of gliomas ......................................................................................... 14 1.1.2 Histopathological and molecular classification of gliomas ....................................... 15 1.1.3 Microenvironment of glioblastoma ........................................................................... 18 1.1.4 Implications for diagnosis and therapy ..................................................................... 19 1.2 Translational control by CPEBs ........................................................................................ 20 1.2.1 Principles of translation ............................................................................................. 20 1.2.2 CPEBs ....................................................................................................................... 21 1.2.3 Regulation of polyadenylation-induced translation ................................................... 22 1.3 Role of CPEBs in germline and somatic cells ................................................................... 24 1.3.1 CPEBs in cell cycle progression ............................................................................... 24 1.3.2 CPEBs in metabolism and senescence ...................................................................... 25 1.3.3 CPEBs in cancer ........................................................................................................ 26 1.3.4 CPEBs in the nervous system .................................................................................... 29 1.4 Alterations regulating expression and activity of CPEBs ................................................. 31 1.4.1 DNA methylation ...................................................................................................... 31 1.4.2 Alternative splicing ................................................................................................... 33 1.4.3 Phosphorylation ......................................................................................................... 35 2 AIM OF THE STUDY ............................................................................................................ 37 3 MATERIALS .......................................................................................................................... 39 3.1 Antibodies ......................................................................................................................... 39 3.2 Cell cultures ...................................................................................................................... 41 3.2.1 Reagents .................................................................................................................... 41 3.2.2 Media composition .................................................................................................... 41 3.2.3 Cell culture consumables .......................................................................................... 42 3.3 Chemicals .......................................................................................................................... 43 3.4 Extraction of nucleic acids ................................................................................................ 43 3.5 Fragment analysis .............................................................................................................. 44 3.5.1 Reagents .................................................................................................................... 44 3.5.2 Primers ...................................................................................................................... 44 3.6 Immunocytochemistry ....................................................................................................... 46 3.6.1 Reagents .................................................................................................................... 46 3.6.2 Buffers and solutions ................................................................................................. 46 3.7 Immunohistochemistry ...................................................................................................... 47 3.7.1 Reagents .................................................................................................................... 47 3.7.2 CSA II staining system for immunohistochemistry .................................................. 48 3.8 Laboratory equipment ....................................................................................................... 48 3.9 Methylation ....................................................................................................................... 49 3.9.1 Bisulfite conversion reagents .................................................................................... 49 3.9.2 Bisulfite-DNA amplification reagents ....................................................................... 50 3.9.3 Pyrosequencing reagents ........................................................................................... 50 3.9.4 Primers ...................................................................................................................... 51 3.10 Pathways activity assay ..................................................................................................... 51 3.11 Semi-quantitative real time PCR ....................................................................................... 52 3.11.1 Reverse transcription reagents................................................................................... 52 3.11.2 TaqMan semi-quantitative real time PCR reagents ................................................... 52 3.11.3 Primers and probes .................................................................................................... 52 3.12 Transfection ....................................................................................................................... 54 3.12.1 Reagents .................................................................................................................... 54 3.12.2 Expression vectors ..................................................................................................... 54 3.13 Western blotting ................................................................................................................ 54 3.13.1 Protein lysis buffer .................................................................................................... 54 3.13.2 BCA Protein Assay ..................................................................................................