The role of the metalloprotease ADAMTS4 in Aβ peptide generation and Alzheimer’s disease Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Susanne Walter aus Offenbach Düsseldorf, September 2017 aus dem Institut für Neuropathologie der Heinrich-Heine-Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Berichterstatter: 1. Prof. Dr. Sascha Weggen 2. Prof. Dr. Lutz Schmitt Tag der mündlichen Prüfung: Ich versichere an Eides Statt, dass die vorgelegte Dissertation The role of the metalloprotease ADAMTS4 in Aβ peptide generation and Alzheimer’s disease von mir selbständig und ohne unzulässige fremde Hilfe unter Beachtung der „Grundsätze zur Sicherung guter wissenschaftlicher Praxis an der Heinrich-Heine-Universität Düsseldorf“ erstellt worden ist. Des Weiteren versichere ich, dass die Dissertation zuvor keiner anderen Universität oder Fakultät vorgelegt wurde und ich bislang keinerlei andere Promotionsversuche getätigt habe. Düsseldorf, den 15.09.2017 _______________________ _____________________ Ort, Datum Susanne Walter Contents Summary .................................................................................................................................... 5 Zusammenfassung ...................................................................................................................... 6 Abbreviations ............................................................................................................................. 7 Amino acids .............................................................................................................................. 11 1. Introduction .......................................................................................................................... 12 1.1 Alzheimer’s disease ........................................................................................................ 12 1.1.1 Alzheimer’s disease pathology and clinical features ............................................... 12 1.1.2 The amyloid cascade hypothesis ............................................................................. 13 1.1.3 APP processing and functional APP metabolites ..................................................... 16 1.1.4 Properties of Aβ peptides and pathological mechanisms ....................................... 18 1.1.5 FAD mutations ......................................................................................................... 19 1.1.6 Murine animal models of AD ................................................................................... 20 1.1.7 N-terminal truncation and other Aβ peptide modifications ................................... 22 1.1.8 Other APP cleaving proteases .................................................................................. 23 1.1.9 The role of non-neuronal cells in Aβ generation and degradation ......................... 25 1.1.9.1 Astrocytes and microglia ...................................................................................... 25 1.1.9.2 Oligodendrocytes .................................................................................................. 26 1.1.10 Current AD therapeutic research ........................................................................... 28 1.2 The metalloprotease ADAMTS4 ..................................................................................... 29 1.2.1 The ADAMTS family and the extracellular matrix (ECM) ......................................... 29 1.2.2 ADAMTS proteases in arthritis, cancer, and coronary artery disease ..................... 32 1.2.3 ADAMTS proteases in the physiological CNS ........................................................... 34 1.2.4 ADAMTS proteases in CNS injuries and diseases ..................................................... 35 2. Objectives ............................................................................................................................. 37 3. Materials ............................................................................................................................... 38 3.1 Cell lines .......................................................................................................................... 38 3.2 Mouse strains ................................................................................................................. 38 3.3 Human brain samples ..................................................................................................... 39 3.4 Bacterial strain ................................................................................................................ 40 3.5 Adenoviral strain ............................................................................................................. 40 3.6 Antibodies ....................................................................................................................... 40 1 3.6.1 Primary antibodies ................................................................................................... 40 3.6.2 Secondary antibodies ............................................................................................... 42 3.7 Plasmids and Primers ...................................................................................................... 42 3.7.1 Plasmids ................................................................................................................... 42 3.7.2 Primers ..................................................................................................................... 43 3.8 Reagents ......................................................................................................................... 43 3.8.1 Chemicals ................................................................................................................. 43 3.8.2 Cell culture reagents ................................................................................................ 45 3.8.3 Antibiotics ................................................................................................................ 46 3.8.4 Synthetic Aβ peptides .............................................................................................. 47 3.8.5 BACE1 Inhibitor ........................................................................................................ 47 3.8.6 Transfection reagents .............................................................................................. 47 3.8.7 Beads ........................................................................................................................ 47 3.8.8 Size standards .......................................................................................................... 47 3.8.9 Enzymes and enzyme mixes .................................................................................... 48 3.8.10 Kits .......................................................................................................................... 48 3.9 Laboratory hardware and appliances ............................................................................. 48 3.10 Consumables ................................................................................................................. 50 3.11 Software ........................................................................................................................ 51 4. Methods ............................................................................................................................... 53 4.1 Molecular Cloning ........................................................................................................... 53 4.1.1 Polymerase chain reaction (PCR) ............................................................................. 53 4.1.2 Agarose gel electrophoresis and PCR product purification ..................................... 53 4.1.3 Gateway cloning ....................................................................................................... 55 4.1.4 Bacterial transformation and plasmid purification ................................................. 55 4.2 Cell culture ...................................................................................................................... 56 4.2.1 Immortalized cell lines ............................................................................................. 56 4.2.1.1 Transient transfection ........................................................................................... 58 4.2.1.2 Dose-response curve for antibiotic selection (Kill curve) ..................................... 58 4.2.1.3 Production of lentiviral particles .......................................................................... 58 4.2.1.4 Lentiviral transduction .......................................................................................... 59 4.2.1.5 Subcloning ............................................................................................................. 60 4.2.1.6 Induction with doxycycline (DOX) ......................................................................... 61 4.2.1.7 Immunocytochemistry .......................................................................................... 61 2 4.2.1.8 BACE1 inhibitor treatment .................................................................................... 63 4.2.2 Primary oligodendrocyte