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Zelluläre Modulatoren Der Ektodomänenspaltung Von App

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Zelluläre Modulatoren Der Ektodomänenspaltung Von App Aus dem Adolf-Butenandt-Institut der Ludwig-Maximilians-Universität München Lehrstuhl: Stoffwechselbiochemie Vorstand: Prof. Dr. rer. nat. Dr. h.c. Christian Haass ZELLULÄRE MODULATOREN DER EKTODOMÄNENSPALTUNG VON APP Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Anna Münch aus Altötting 2012 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig- Maximilians-Universität München Betreuer: Priv.-Doz. Dr. Stefan Lichtenthaler Zweitgutachter: Priv.-Doz. Dr. Christian Ries Dekan: Prof. Dr. med. Dr. h.c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 31.07.2012 Ehrenwörtliche Erklärung Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation „Zelluläre Modulatoren der Ektodomänenspaltung von APP“ von mir selbständig und ohne unerlaubte Hilfe angefertigt wurde. Ich habe mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Hilfen und Quellen bedient. Die Dissertation wurde in der jetzigen oder ähnlichen Form bei keiner anderen Hochschule eingereicht und hat auch noch keinen anderen Prüfungszwecken gedient. Anna Münch Inhaltsverzeichnis 5 ZELLULÄRE MODULATOREN DER EKTODOMÄNENSPALTUNG VON APP 1 Einleitung ...................................................................................................................... 8 1.1 Die Alzheimer-Krankheit ..................................................................................................... 8 1.1.1 Beschreibung der Alzheimer-Krankheit ............................................................................. 8 1.1.2 Die Amyloid-Kaskaden-Hypothese .................................................................................. 10 1.1.3 Therapeutische Ansätze zur Behandlung der Alzheimer-Krankheit ................................ 12 1.2 Molekulare Grundlagen der Alzheimer-Krankheit ......................................................... 13 1.2.1 Das Amyloid-Vorläuferprotein („Amyloid Precursor Protein“, APP) ............................. 13 1.2.1.1 Allgemeine Beschreibung von APP .......................................................................... 13 1.2.1.2 Funktion von APP ..................................................................................................... 14 1.2.1.3 Proteolytische Prozessierung von APP ..................................................................... 15 1.2.2 Die Sekretasen .................................................................................................................. 17 1.2.2.1 Die α-Sekretase ......................................................................................................... 17 1.2.2.2 Die β-Sekretase ......................................................................................................... 18 1.2.2.3 Die γ-Sekretase .......................................................................................................... 18 1.2.3 Regulation der Prozessierung von APP ............................................................................ 19 1.2.3.1 Regulation der Aktivität der APP-Sekretasen ........................................................... 19 1.2.3.2 Der intrazelluläre Transport von APP ....................................................................... 21 1.3 Das RNA-Interferenz Prinzip ............................................................................................ 24 2 Ziele der Arbeit .......................................................................................................... 29 3 Ergebnisse ................................................................................................................... 32 3.1 TMEM59 ist ein neuartiger Modulator der Ektodomänenspaltung von APP .............. 32 3.1.1 Identifizierung von TMEM59 .......................................................................................... 32 3.1.2 TMEM59 und TMEM59L modulieren die Ektodomänenspaltung und die Maturierung von APP ................................................................................................. 32 3.1.3 TMEM59 moduliert die Maturierung und die Ektodomänenspaltung von endogenem APP ........................................................................................................ 34 3.1.4 TMEM59 moduliert die Maturierung von endogenem Nicastrin ..................................... 36 3.1.5 TMEM59 ist in allen Golgi-Subkompartimenten vorhanden ........................................... 36 3.1.6 TMEM59 hat keinen Einfluss auf die Aktivität der Sekretasen ....................................... 39 3.1.7 TMEM59 verändert den APP-Transport .......................................................................... 41 3.1.8 Auch der Doppel-„Knockdown” von TMEM59 und TMEM59L verändert die Ektodomänenspaltung von APP ................................................................. 42 3.2 Genomweites RNAi-„Screening“ zur Identifizierung neuer zellulärer Modulatoren der Ektodomänenspaltung von APP .......................................................... 46 3.2.1 Etablierung eines Drosophila-„Screenings“ ..................................................................... 46 3.2.1.1 Etablierung einer Drosophila-Reporterzelllinie......................................................... 46 3.2.1.2 Etablierung eines „Screening“-Verfahrens in Drosophila-S2-Zellen ........................ 48 3.2.2 Auswertung und Resultate des genomweiten Drosophila RNAi-„Screenings“ ............... 50 3.2.3 Identifizierung orthologer humaner Gene zu den Kandidatengenen aus dem Drosophila RNAi-„Screening“ ......................................................................................... 54 3.2.4 Etablierung eines „Screening“-Verfahrens in humanen SH-SY5Y-Zellen ...................... 55 3.2.5 Untersuchung der humanen orthologen Gene mit Dharmacon siRNA-Pools .................. 58 3.2.6 Untersuchung der humanen orthologen Gene mit Qiagen siRNA-Pools ......................... 60 Inhaltsverzeichnis 6 3.2.7 Untersuchung der humanen orthologen Gene mit einzelnen siRNA-Sequenzen ............ 62 3.2.8 Untersuchung der ESCRT-Maschinerie in RNAi-Studien zur Validierung von VPS24 als Modulator der APP-Ektodomänenspaltung.................................................... 65 3.2.9 Weitere Validierung von VPS24 als Modulator der APP-Ektodomänenspaltung durch stabilen „Knockdown” mittels shRNAs ................................................................. 68 4 Diskussion ................................................................................................................... 71 4.1 TMEM59 ist ein neuartiger Modulator der Ektodomänenspaltung von APP ............... 71 4.1.1 TMEM59 inhibiert die Ektodomänenspaltung von APP ................................................. 71 4.1.2 Mechanismen von TMEM59 in der Ektodomänenspaltung von APP ............................. 73 4.1.2.1 TMEM59 hat keinen negativen Einfluss auf die Aktivität der APP-Sekretasen ....... 73 4.1.2.2 TMEM59 inhibiert den intrazellulären Transport von APP ...................................... 74 4.2 Erfolg und Limitationen des genomweiten RNAi-„Screenings“ zur Identifizierung neuartiger Modulatoren der Ektodomänenspaltung von APP ............. 76 4.2.1 Identifizierung neuer zellulärer Modulatoren der Ektodomänenspaltung von APP ........ 76 4.2.2 Genomweite RNAi-„Screenings“ in Drosophila-Zellen zur Untersuchung humaner Genfunktionen ................................................................................................... 77 4.2.3 Strategie zur Validierung von potentiellen Kandidatengenen in humanen Zellen ........... 80 4.3 Die ESCRT-Maschinerie moduliert die Ektodomänenspaltung von APP ..................... 85 4.3.1 Aufbau und zelluläre Funktionen der ESCRT-Maschinerie ............................................ 85 4.3.2 Potentielle Mechanismen der ESCRT-Maschinerie in der Ektodomänenspaltung von APP ........................................................................................................................... 89 4.3.2.1 Die ESCRT-Maschinerie könnte die Endozytose von BACE1 modulieren .............. 89 4.3.2.2 Die ESCRT-Maschinerie könnte die Sekretion von APPsβ und Aβ regulieren ........ 91 4.3.2.3 Die ESCRT-Maschinerie könnte indirekt die β-Sekretase-Spaltung von APP modulieren .................................................................................................. 93 5 Ausblick ...................................................................................................................... 95 6 Zusammenfassung ..................................................................................................... 97 7 Summary .................................................................................................................. 100 8 Material und Methoden .......................................................................................... 103 8.1 Material .............................................................................................................................. 103 8.1.1 Geräte und Verbrauchsmaterialien................................................................................. 103 8.1.1.1 Allgemein ................................................................................................................ 103 8.1.1.2 Molekularbiologische Methoden ............................................................................. 103 8.1.1.3 Zellkultur ................................................................................................................
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