γ-Secretase mediated proteolytic processing of the triggering receptor expressed on myeloid cells-2 – functional implications for intracellular signaling DISSERTATION zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen-Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Dipl. Biochem. Patrick Wunderlich aus Brühl Bonn, 21.12.2010 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: 21.12.2010 Tag der Promotion: 06.04.2011 Erscheinungsjahr: 2011 An Eides statt versichere ich, dass ich die Dissertation “γ-Secretase mediated proteolytic processing of the triggering receptor expressed on myeloid cells-2 – functional implications for intracellular signaling“ 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. Promotionsordnung vom 7. Januar 2004 _________________________ Patrick Wunderlich INDEX IV CONTENTS ABBREVIATIONS ...................................................................................................................IX 1 I NTRODUCTION .................................................................................................................14 1.1 Alzheimer's disease (AD) ............................................................................................................14 1.1.1 Neuropathological hallmarks of AD.................................................................................15 1.1.1.1 Neurofibrillary tangles.................................................................................................15 1.1.1.2 β-amyloid plaques.........................................................................................................17 1.1.2 Genetic factors involved in AD .........................................................................................18 1.1.2.1 Late onset or sporadic AD..........................................................................................18 1.1.2.2 Early onset or familiar AD..........................................................................................19 1.1.3 Characteristics of the β-amyloid precursor protein (APP).............................................20 1.1.4 APP processing.....................................................................................................................21 1.1.5 Structural and functional insights in the APP processing enzymes...............................22 1.1.5.1 The α-secretase: ADAM-10, ADAM-17 and ADAM-9..........................................22 1.1.5.2 The β-secretase: BACE-1............................................................................................23 1.1.5.3 The γ-secretase..............................................................................................................25 1.2 Aβ clearance in the brain..............................................................................................................28 1.3 Microglia.........................................................................................................................................30 1.4 The role of microglia & inflammation in AD...........................................................................31 1.4.1 Beneficial roles of microglia in Aβ clearance...................................................................31 1.4.2 Detrimental roles of microglia by chronic enhancement of inflammatory processes................................................................................................................................33 1.5 Triggering receptors expressed on myeloid cells (TREMs).....................................................34 1.5.1 TREM1...................................................................................................................................35 1.5.2 TREM2...................................................................................................................................35 1.6 DAP12 – structure and signaling................................................................................................37 1.7 Rationale.........................................................................................................................................39 2 M ATERIAL & M ETHODS ...................................................................................................40 2.1 Cell biological techniques.............................................................................................................41 2.1.1 Cell culture ............................................................................................................................41 2.1.2 Cell culture of ES cell derived microglia...........................................................................42 2.1.3 Transient transfection...........................................................................................................42 INDEX V 2.1.4 Immunocytochemistry (ICC)..............................................................................................43 2.2 Molecularbiological techniques...................................................................................................44 2.2.1 Polymerase chain reaction (PCR)........................................................................................44 2.2.2 Separation and visualization of DNA fragments.............................................................47 2.2.3 Isolation of DNA fragments from agarose gels..............................................................47 2.2.4 Restriction digestion.............................................................................................................47 2.2.5 Dephosphorylation of linearized DNA............................................................................47 2.2.6 Ligation...................................................................................................................................48 2.2.7 Generation of chemo competent E. coli Top10..............................................................48 2.2.8 Transformation of E. coli Top10.......................................................................................48 2.2.9 Cryo conservation of transformed E. coli........................................................................49 2.2.10 Extraction of plasmid DNA from E. coli.......................................................................49 2.2.11 Precipitation of DNA by isopropanol.............................................................................50 2.2.12 Precipitation of DNA by ethanol.....................................................................................50 2.2.13 DNA sequencing................................................................................................................50 2.2.14 Photometric determination of DNA concentration.....................................................50 2.3 Proteinbiochemical techniques....................................................................................................51 2.3.1 Extraction of membrane proteins out of eukaryotic cells.............................................51 2.3.2 Protein extraction out of eukaryotic cells.........................................................................51 2.3.3 Protein estimation.................................................................................................................52 2.3.4 Immunoprecipitation (IP)....................................................................................................52 2.3.5 Co-immunoprecipitation of DAP12 and TREM2...........................................................53 2.3.6 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)...................53 2.3.7 Western immunoblotting (WB)...........................................................................................54 2.3.8 Coomassie staining of proteins in polyacrylamide gels...................................................56 2.3.9 Precipitation of soluble proteins from cell culture supernatants by trichloroacetic acid (TCA)..............................................................................................................................56 2.3.10 Expression and isolation of glutathione S-transferase (GST) fusion proteins..........57 2.3.11 Biotinylation of cell surface proteins...............................................................................58 2.3.12 Radio-labeling with 32P-orthophosphate........................................................................58 2.3.13 TREM2 shedding assay......................................................................................................59 2.3.14 TREM2 activation assay.....................................................................................................59 2.3.15 Aβ-Phagocytosis assay.......................................................................................................60 2.4 Densitometric quantification of signals and statistical analysis..............................................60 INDEX VI 3 R ESULTS ............................................................................................................................61 3.1 Proteolytic processing of TREM2 by γ-secretase....................................................................61