Fakultät für Medizin der Technischen Universität München Function and substrates of the Alzheimer´s disease protease BACE1 Jasenka Rudan Njavro Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Thomas Misgeld Prüfende/-r der Dissertation: 1. Prof. Dr. Stefan Lichtenthaler 2. Prof. Dr. Wolfgang Wurst Die Dissertation wurde am 12.04.2019 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 08.10.2019 angenommen. TABLE OF CONTENT ABSTRACT ........................................................................................................................................................... 3 ZUSAMMENFASSUNG ...................................................................................................................................... 4 LIST OF ABBREVIATIONS ............................................................................................................................... 5 1. INTRODUCTION ........................................................................................................................................ 7 1.1. History of Alzheimer´s disease .............................................................................................................. 7 1.2. Epidemiology of Alzheimer´s disease ................................................................................................... 7 1.3. Pathology of the disease ........................................................................................................................ 7 1.4. Familial and sporadic Alzheimer´s disease............................................................................................ 8 1.5. The amyloid hypothesis of Alzheimer´s disease ................................................................................... 9 1.6. Amyloid precursor protein (APP) and its processing .......................................................................... 11 1.7. The β-site amyloid precursor protein cleaving enzyme 1 (BACE1) .................................................... 12 1.8. BACE1 cellular localization and trafficking........................................................................................ 14 1.9. BACE1 deficient mice ......................................................................................................................... 14 1.10. BACE1 substrates and functions ......................................................................................................... 14 1.11. BACE1 as a drug target in Alzheimer´s disease .................................................................................. 16 1.12. The Seizure protein family .................................................................................................................. 19 1.13. Seizure protein family as the main BACE1 substrates ........................................................................ 20 2. AIM OF THE WORK ................................................................................................................................ 22 3. MATERIAL AND METHODS ................................................................................................................. 23 3.1. Material................................................................................................................................................ 23 3.1.1. General reagents and chemicals .................................................................................................. 23 3.1.2. Primers ........................................................................................................................................ 25 3.1.3. Buffers ........................................................................................................................................ 26 3.1.4. Kits .............................................................................................................................................. 27 3.1.5. Equipment ................................................................................................................................... 27 3.2. DNA methods ...................................................................................................................................... 28 3.2.1. Polymerase Chain Reaction (PCR) ............................................................................................. 28 3.2.2. Cloning of DNA constructs ........................................................................................................ 28 3.2.3. Gibson assembly ......................................................................................................................... 30 3.2.4. Transformation of competent E. coli bacteria ............................................................................. 30 3.2.5. Mini preps – preparation of DNA for construct screening .......................................................... 30 3.2.6. Midi preps – preparation of DNA for transfection ...................................................................... 30 3.2.7. Sequencing of cloned DNA constucts ........................................................................................ 31 3.2.8. DNA isolation ............................................................................................................................. 31 3.2.9. Genotyping ................................................................................................................................. 31 3.3. Cell culture methods ............................................................................................................................ 33 3.3.1. Cell culture .................................................................................................................................. 33 3.3.2. Cell lines ..................................................................................................................................... 33 3.3.3. Cryopreservation ......................................................................................................................... 33 3.3.4. Transient transfection.................................................................................................................. 33 3.3.5. Lentivirus production .................................................................................................................. 33 3.3.6. Stable cell line production .......................................................................................................... 34 3.3.7. Primary cortical neurons isolation and culture ............................................................................ 35 3.3.8. Compound treatment ................................................................................................................... 35 3.4. Protein analysis methods ..................................................................................................................... 35 3.4.1. Cell lysis ..................................................................................................................................... 35 3.4.2. Protein quantification .................................................................................................................. 36 3.4.3. Concanavalin A enrichment ........................................................................................................ 36 3.4.4. Co-immunoprecipitation ............................................................................................................. 36 1 3.4.5. Mouse brain fractionation: membrane and soluble fraction ........................................................ 37 3.4.6. Crude membrane preparation ...................................................................................................... 37 3.4.7. Surface Spanning Protein Enrichment with Click Sugars – SUSPECS ...................................... 37 3.4.8. Western blot analysis .................................................................................................................. 38 3.4.9. Quantification and statistical analysis ......................................................................................... 40 3.4.10. Brain slice preparation ................................................................................................................ 40 3.4.11. Immunostaining of brain sections ............................................................................................... 40 3.4.12. Sample preparation for mass spectrometric measurement .......................................................... 40 3.4.13. LC-MS/MS analysis ................................................................................................................... 41 3.4.14. LC-MS/MS data analysis and statistical evaluation .................................................................... 42 3.5. Animal work ........................................................................................................................................ 42 3.5.1. Mouse lines ................................................................................................................................. 42 3.5.2. Behavioural testing ....................................................................................................................