Extracellular phosphorylation of the Amyloid β-peptide [beta-peptide] promotes aggregation [Elektronische Ressource] / vorgelegt von Sathish Kumar H. S.

Sathish Kumar H.S.

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2009
• Nombre de lectures: 37
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

Extracellular Phosphorylation of the
Amyloid β-Peptide Promotes
Aggregation



Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn



vorgelegt von

Sathish Kumar H.S.
aus
Chamarajanagar, Indien



– Bonn, 2009 –
Angefertigt mit Genehmigung der Mathematisch-
Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-
Universität Bonn













Gutachter

1. Prof. Dr. rer. nat. Jochen Walter
2. Prof. Dr. rer. nat. Michael Hoch


Eingereicht am: 08. April 2009

Tag der Promotion: 01. July 2009



Diese Dissertation ist auf dem Hochschulschriftenserver der ULB Bonn
unter, http://hss.ulb.uni-bonn.de/diss_online“ elektronisch publiziert.
Die vorliegende Arbeit wurde in der Zeit von Februar 2005 bis April 2009
in der Klinik und Poliklinik für Neurologie, Molekulare Zellbiologie, Uni-
versitätsklinikum Bonn, Sigmund-Freud-Str. 25, Bonn unter Leitung von
Prof. Dr. Jochen Walter durchgeführt. Index

Contents .....................................................................................................................................................i

List of Figures..........................................................................................................................................iv

List of tables ............................................................................................................................................. v

Abbreviations............................................................................................................................................ v


SUMMARY / ABSTRACT .................................................................................................................viii


1. INTRODUCTION ............................................................................................................................. 1

1.1. Protein misfolding, aggregation and age-related neurodegenerative diseases........................... 1

1.2. Alzheimer’s disease (AD) ................................................................................................................ 7

1.2.1. Neuropathological hallmarks of AD......................................................................................... 8
1.2.2. The Amyloid Precursor Protein (APP) and generation of Aβ ................................................ 11
1.2.3. Genetic factors of AD ............................................................................................................ 15
1.2.4. Amyloid β toxicity: The importance of structure .................................................................. 17
1.2.5. The “Amyloid hypothesis” or “Aβ hypothesis” .................................................................... 21

1.3. Protein phosphorylation ............................................................................................................... 23

1.3.1. Protein phosphorylation in the human brain .......................................................................... 25
1.3.2. Phosphorylation of proteins by extracellular protein kinases ................................................ 25
1.3.3. Altered protein phosphorylation in AD ................................................................................. 26
1.3.4. Phosphorylation of AD related proteins................................................................................. 27


2. AIM OF THE STUDY...................................................................................................................... 30


3. MATERIALS AND METHODS .................................................................................................... 32

3.1. MATERIALS........................................................................................................................ 32
3.1.1. Chemicals used............................................................................................................. 32
3.1.2. Ready to use solutions/reagents ................................................................................... 32
3.1.3. Kits .............................................................................................................................. 32
3.1.4. Buffers and Solutions for Protein Biochemistry .......................................................... 33
3.1.5. Solutions for Histochemistry and Immunofluorescence ............................................. 36
3.1.6. Solutions for eukaryotic cell culture and primary mouse neuronal cell culture .......... 36
3.1.7. Antibodies .................................................................................................................... 37
3.1.7.1. Primary antibodies .................................................................................................... 37
3.1.7.2. Secondary antibodies ................................................................................................ 38
3.1.8. Mouse lines .................................................................................................................. 38
3.1.9. General Lab Materials ................................................................................................. 38
3.1.10. Laboratory Devices .................................................................................................... 39

i Index
3.2. APPLIED METHODS ......................................................................................................... 40

3.2.1. In silico analysis of putative phospho-sites of Aβ and the responsible kinases........... 40
3.2.2. In vitro Aβ phosphorylation assay .............................................................................. 41
3.2.3. Kinetic and Stoichiometry of Aβ phosphorylation ...................................................... 41
3.2.4. Phosphoamino acid analysis ....................................................................................... 41
3.2.5. In vivo phosphorylation of Aβ by cultured cells ......................................................... 42
3.2.6. Primary culture of mouse cortical neurons and phosphorylation of
Aβ in vivo ............................................................................................................. 42
3.2.7. Stimulation and induced release of ecto-PKA from intact cells ................................. 43
3.2.8. Cell surface biotinylation of ecto-PKA ....................................................................... 43
3.2.9. Human CSF (huCSF) handling, and Ex vivo phosphorylation .................................... 44
3.2.10. Preparation of Aβ stock solutions .............................................................................. 45
3.2.11. Quantifying Aβ Aggregation by CR and ThT dye binding studies............................ 45
3.2.12. Circular Dichroism (CD) Spectroscopy .................................................................... 46
3.2.13. Aggregation kinetics analysis .................................................................................... 47
3.2.14. Nuclear magnetic resonance (NMR) ......................................................................... 47
3.2.15. Analysis of size of the Aβ aggregates by Dynamic Light Scattering......................... 48
3.2.16. Analysis of Aβ oligomers by Dot blot assay .............................................................. 48
3.2.17. Transmission Electron Microscopy (TEM)................................................................ 49
3.2.18. Generation of phosphorylation-site specific Aβ antibody.......................................... 49
3.2.19. Transgenic mice, protein extraction and immunohistochemistry............................... 49
3.2.20. Dephosphorylation of mouse brain lysates and synthetic pAβ samples .................... 50
3.2.21.Immunohistochemistry and double-label confocal microscopy of human AD brain . 51
3.2.22. SDS-PAGE and Western blotting .............................................................................. 51

4. RESULTS …….............................................................................................................................. 53

4.1. Phosphorylation of Aβ ............................................................................................................ 53
4.1.1. In silico analysis of putative phosphorylation sites of Aβ ..................................................... 53
4.1.2. Identification of kinase-specific consensus sequences in Aβ and
responsible kinases ............................................................................................................... 55
4.1.3. In vitro phosphorylation of Aβ............................................................................................... 56
4.1.3.1. Phosphoamino acid analysis of in vitro phosphorylated Aβ ..................................... 57
4.1.