γ-secretase [gamma-secretase] modulators and inhibitors in Alzheimer's disease [Elektronische Ressource] : influence of genetic background on efficacy and mechanism of action / Eva Czirr

Eva Lecomte

Sujets

• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2008
• Nombre de lectures: 25
• Poids de l'ouvrage: 1 Mo

Extrait

γ-secretase modulators and inhibitors in
Alzheimer’s disease:
Influence of genetic background
on efficacy and mechanism of action.





Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften


Am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz


Eva Czirr
geb. am 27. Juni 1979 in Kreuztal

Mainz, 2008










































Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 17. Juni 2008
Contents

Abbreviations .........................................................................................................................4
Amino acids............................................................................................................................5
1 Introduction .........................................................................................................................1
1.1 Alzheimer’s disease .....................................................................................................1
1.2 APP processing and function .....................................................................................3
1.3 γ-secretase7
1.4 Treatment strategies for Alzheimer’s disease.........................................................12
1.5 γ-secretase modulation .............................................................................................15
1.6 Objectives ...................................................................................................................20
2 Material...............................................................................................................................21
2.1 Mouse strains .............................................................................................................21
2.2 Cell lines.....21
2.3 Bacterial strains..........................................................................................................22
2.4 Plasmids and Primer..................................................................................................22
2.4.1 Plasmids...............................................................................................................22
2.4.2 Primer ...................................................................................................................23
2.5 Antibodies...................................................................................................................24
2.5.1 Primary antibodies ..............................................................................................24
2.5.2 Secondary antibodies .........................................................................................24
2.6 Reagents..25
2.6.1 Chemicals.............................................................................................................25
2.6.2 Antibiotics............................................................................................................26
2.6.3 γ-secretase modulators and inhibitors .............................................................27
2.6.4 Size standards .....................................................................................................27
2.6.5 Enzymes...............................................................................................................27
2.6.5.1 General enzymes ..........................................................................................27
2.6.5.2 Restriction endonucleases..........................................................................27
2.6.6 Kits........................................................................................................................28
2.7 Laboratory hardware and appliances.......................................................................28
2.8 Consumables..............................................................................................................29
2.9 Software ......................................................................................................................30
3 Methods..............................................................................................................................31
3.1 Treatment of mice with γ-secretase inhibitors ........................................................31
3.2 Tissue Culture.............................................................................................................31
3.1.1 Passaging of adherent cell lines........................................................................32
- 1 -
3.1.2 Cryopreservation of cells ...................................................................................32
3.1.3 Thawing of frozen cell stocks ............................................................................33
3.1.4 Compound treatment ..........................................................................................33
3.1.5 Generation of retroviral particles.......................................................................34
3.1.6 Infection with retroviral particles35
3.1.7 Killing curve.........................................................................................................36
3.1.8 Subcloning of cell lines ......................................................................................37
3.2 Protein Biochemistry .................................................................................................37
3.2.1 Harvesting of secreted proteins.........................................................................37
3.2.2 Harvesting of cellular lysates.............................................................................38
3.2.3 Bicinchonic acid protein assay (BCA)...............................................................38
3.2.5 Western-Blot ........................................................................................................41
3.2.6 High-resolution urea gelelectrophoresis ..........................................................42
3.2.7 Immunostaining of membranes46
3.2.8 PonceauS staining of membranes.....................................................................47
3.2.9 Coomassie staining.............................................................................................48
3.2.10 HRP-coupling of antibodies .............................................................................48
3.2.11 Enzyme-linked immunosorbent assay (ELISA) ..............................................49
3.2.12 Liquid-phase electrochemiluminescence assay (LPECL) .............................51
3.2.13 Matrix-assisted desorption/ionization time of flight (MALDI-TOF) ...............53
3.3 Molecular Biology.......................................................................................................53
3.3.1 DNA preparation..................................................................................................53
3.3.2 Agarose gelelectrophoresis ...............................................................................53
3.3.3 Restriction-enzyme digest..................................................................................54
3.3.4 Gelelution.............................................................................................................55
3.3.5 Dephosphorylation of DNA.................................................................................55
3.3.7 Transformation ....................................................................................................56
3.3.8 Polymerase chain reaction (PCR)......................................................................57
3.3.9 Colony-PCR..........................................................................................................58
3.3.10 Site directed mutagenesis................................................................................59
3.3.11 DNA-Sequencing ...............................................................................................60
4 Results ...............................................................................................................................61
4.1 Presenilin-1 mutations strongly affect the cellular response to γ-secretase
modulators and inhibitors in vitro and in vivo ..............................................................61
4.1.1 Genetic dissection of the PS1- ∆Exon9 mutation .............................................61
- 2 -
4.1.2 The pathogenic increase in the A β42/40 ratio induced by the PS1- ∆Exon9
mutation is caused by a synergistic effect of the S290C point mutation and the
lack of endoproteolytic cleavage................................................................................63
4.1.3 The attenuated response of PS1- ∆Exon9 to γ-secretase modulators is
mainly caused by its lack of endoproteolytic cleavage............................................65
4.1.4 Insensitivity to γ-secretase modulators and inhibitors is common among
aggressive FAD-associated PS1 mutations...............................................................68
4.1.5 The higly potent γ-se