Intramembrane proteolysis of ephrin-B2 by γ-secretase regulates podosome dynamics and migration of microglia Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen-Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Nadja Kemmerling, MSc aus Düsseldorf Bonn, 31.08.2015 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. rer. nat. Jochen Walter 2. Gutachter: Prof. Dr. rer. nat. Sven Burgdorf Tag der Abgabe: 31.08.2015 Tag der Promotion: 30.11.2015 Erscheinungsjahr: 2016 2 An Eides statt versichere ich, dass ich die Dissertation “Intramembrane proteolysis of ephrin-B2 by γ-secretase regulates podosome dynamics and migration of microglia“ selbst und ohne jede unerlaubte Hilfe angefertigt habe und dass diese oder eine ähnliche Arbeit noch an keiner anderen Stelle als Dissertation eingereicht worden ist. Auszüge der ausgewiesenen Arbeit wurden beim Journal Glia eingereicht und die Möglichkeit einer Veröffentlichung dieser wird momentan vom genannten Journal geprüft. Promotionsordnung vom 17. Juni 2011 ________________________________________ Nadja Kemmerling 3 Dedicated to my mother and my grandparents Carl & Marianne 4 Table of Contents Index ......................................................................................................................... 8 List of figures ..................................................................................................................... 8 List of tables ...................................................................................................................... 9 Abbreviations ................................................................................................................. 10 1. Introduction ........................................................................................................ 15 1.1 The Eph-ephrin system ............................................................................................ 15 1.1.1 The Eph-ephrin system during CNS development ............................................................ 17 1.1.2 The Eph-ephrin system in the adult brain ........................................................................... 20 1.1.3 Signaling of the Eph-ephrin system ...................................................................................... 20 1.1.4 The Eph-ephrin system in pathological conditions ........................................................... 25 1.2 The γ-secretase complex ....................................................................................... 27 1.2.1 Assembly of the y-secretase complex ................................................................................ 29 1.2.2 Regulation of y-secretase activity ....................................................................................... 30 1.2.3 γ-Secretase substrates in cell adhesion and the function of RIP ................................... 31 1.2.4 The γ-secretase in AD ............................................................................................................. 34 1.3 Microglia .................................................................................................................. 35 1.3.1 Microglial migration ................................................................................................................ 38 1.4 Rationale and aim of the study .............................................................................. 41 2. Materials and Methods ...................................................................................... 42 2.1 Molecular biological techniques ........................................................................... 43 2.1.1 Polymerase chain reaction (PCR) ........................................................................................ 43 2.1.2 Separation and purification of DNA fragments ................................................................ 44 2.1.3 DNA restriction and dephosphorylation ............................................................................. 45 2.1.4 DNA ligation ............................................................................................................................. 45 2.1.5 Generation of chemically competent E.coli (Top 10) ..................................................... 45 2.1.6 Transformation of E.coli (Top10) and E.coli colony screen ............................................. 46 2.1.7 Cryo conservation of transformed E.coli ............................................................................ 46 2.1.8 Purification of plasmid DNA from E.coli .............................................................................. 47 2.1.9 RNA isolation from eukaryotic cells ...................................................................................... 47 2.1.10 DNA digestion and reverse transcription (RT)-PCR ......................................................... 47 5 2.1.11 Photometric determination of DNA concentration ....................................................... 47 2.1.12 Analysis of gene expression by qPCR ............................................................................... 48 2.2 Cell biological techniques ...................................................................................... 49 2.2.1 Cell Culture ............................................................................................................................... 49 2.2.2 Generation of lentiviral particles .......................................................................................... 49 2.2.3 Transduction of cells with lentiviral particles and selection ............................................ 50 2.2.4 Transfection of cells with siRNA ............................................................................................. 50 2.2.5 In-Cell Western Assay ............................................................................................................. 50 2.2.6 Immunocytochemistry and Total Internal Reflection Fluorescence (TIRF) microscopy .............................................................................................................................................................. 51 2.3 Protein biochemical techniques ............................................................................ 52 2.3.1 Preparation of cell lysates ..................................................................................................... 52 2.3.2 Cell fractionation .................................................................................................................... 52 2.3.3 Protein estimation.................................................................................................................... 53 2.3.4 Deglycosylation of proteins ................................................................................................... 53 2.3.5 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)/NuPAGE .............................................................................................................................................................. 54 2.3.6 Western Immunoblotting (WB) and ECL imaging ............................................................. 55 2.3.7 Co-Immunoprecipitation (Co-IP) ......................................................................................... 57 2.3.8 Protein precipitation with trichloroacetic acid (TCA) ...................................................... 57 2.4 Analysis of cell migration ........................................................................................ 58 2.5 Statistical analysis .................................................................................................... 58 3. Results.................................................................................................................. 59 3.1 Ephrin-B expression and processing in microglial cells ....................................... 59 3.1.1 Endogenous expression of ephrin-B in primary microglia and ESdM ............................ 59 3.1.2 Generation of ephrin-B2 overexpressing ESdM ................................................................. 60 3.1.3 Proteolytic processing of ephrin-B2 ..................................................................................... 61 3.1.4 Maturation of ephrin-B2 FL overexpressed in BV-2 ........................................................... 64 3.2 γ-Secretase mediates reverse signaling of ephrin-B2 .......................................... 67 3.2.1 EphB1 stimulates phosphorylation of Src and FAK ............................................................ 69 3.2.2 EphB1 induced phosphorylation of Src is dependent on γ-secretase ......................... 70 3.2.3 Eph receptor stimulated phosphorylation of Src and FAK is dependent on ephrin-B2 ICD generation.................................................................................................................................. 71 3.2.4 Effects of the ephrin-B2 ICD on Akt phosphorylation ....................................................... 74 6 3.3 Functional effects of γ-secretase/ephrin-B2 signaling ......................................... 75 3.3.1 Inhibition of γ-secretase affects podosomal surface....................................................... 75 3.3.2 Involvement of γ-secretase activity in motility of microglia ..........................................