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Selection and Characterization of Amyloid-Β1-42 Binding D-Enantiomeric Peptides for Potential Therapeutic Intervention of Alzheimer´S Disease

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Selection and Characterization of Amyloid-Β1-42 Binding D-Enantiomeric Peptides for Potential Therapeutic Intervention of Alzheimer´S Disease Selection and characterization of Amyloid-β1-42 binding D-enantiomeric peptides for potential therapeutic intervention of Alzheimer´s disease Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Stephan Rudolph aus Dessau Düsseldorf, April 2015 Die vorliegende Arbeit wurde in der Zeit von April 2011 bis Februar 2015 am Institut für Physikalische Biologie der Heinrich-Heine-Universität Düsseldorf unter der Leitung von Prof. Dr. Dieter Willbold angefertigt. Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. Dieter Willbold Koreferent: Prof. Dr. Georg Groth Tag der mündlichen Prüfung: „Die Dummheiten wechseln, und die Dummheit bleibt.“ (Erich Kästner) „Die dunkelste Stunde ist die vor Sonnenaufgang.“ (chinesisches Sprichwort) Für Tante Elli Index Index Index I - IV 1. Introduction 1 1.1 Alzheimer´s disease (AD) – an overview .................................................... 1 1.1.1 General information ............................................................................. 1 1.1.2 History and pathology of AD ................................................................ 3 1.1.3 The amyloid precursor protein (APP) ................................................... 4 1.1.4 From APP to Aβ ................................................................................... 7 1.1.5 The physiological role of Aβ and Tau .................................................. 9 1.1.6 Pathological aspects of Aβ and Tau .................................................. 10 1.1.6.1 The role of Tau ............................................................................ 10 1.1.6.2 The amyloid cascade hypothesis ................................................ 11 1.1.6.3 Contribution of different Aβ species to AD .................................. 15 1.2 Diagnosis of AD ....................................................................................... 16 1.3 Therapy of AD .......................................................................................... 17 1.3.1 Therapies targeting the pathogenesis of AD ...................................... 18 1.3.1.1 Immunotherapy ........................................................................... 18 1.3.1.2 Intervention of APP processing ................................................... 19 1.3.1.3 Modulation of Aβ aggregation and degradation ......................... 21 1.4 Drug design by phage display .................................................................. 22 1.4.1 Mirror image phage display ............................................................... 24 1.5 Objective of my thesis .............................................................................. 25 2. Materials 27 2.1 Peptides ................................................................................................... 27 2.2 Buffers, media, kits ................................................................................... 28 2.3 Antibodies ................................................................................................ 30 2.4 Bacterial strains ........................................................................................ 30 2.5 Molecular weight markers ........................................................................ 31 2.6 Devices, laboratory equipment and software ........................................... 32 2.7 Cell culture equipment .............................................................................. 34 I Index 2.8 Chemicals ................................................................................................ 35 3. Methods 37 3.1 Competitive mirror image phage display .................................................. 37 3.1.1 Selection rounds ................................................................................ 37 3.1.2 Enrichment ELISA ............................................................................. 41 3.1.3 Single phage amplification ................................................................. 43 3.1.4 Single phage ELISA ........................................................................... 43 3.1.5 Phage DNA preparation ..................................................................... 44 3.2 Preparation of Aβ1-42 monomers, - oligomers and -fibrils ......................... 45 3.2.1 General pretreatment of Aβ1-42 samples ............................................ 45 3.2.2 Separation of Aβ1-42 species by size exclusion chromatography ....... 45 3.2.3 Preparation of Aβ1-42 samples with a broad range of differently sized species .................................................................................... 46 3.2.4 Preparation of Aβ1-42 fibrils and high molecular weight aggregates ... 46 3.3 Concentration determination of Aβ1-42 samples ........................................ 47 3.3.1 BCA assay ......................................................................................... 47 3.4 Density gradient centrifugation ................................................................. 47 3.5 Determination of iodixanol solution concentrations .................................. 48 3.6 Tris-tricine-SDS-PAGE ............................................................................. 49 3.6.1 Casting gels ....................................................................................... 49 3.6.2 Running SDS gels ............................................................................. 50 3.7 Silver staining of SDS gels ....................................................................... 50 3.8 Thioflavin T assay .................................................................................... 51 3.9 Transmission electron microscopy ........................................................... 52 3.10 Reversed phase high performance liquid chromatography ................... 53 3.11 Turbidity assay ...................................................................................... 53 3.12 Cell culture ............................................................................................ 54 3.12.1 Cultivation of PC-12 cells ............................................................... 54 3.12.2 Microscopy of PC-12 cells .............................................................. 54 3.12.3 Coating of tissue culture flasks ....................................................... 54 3.12.4 Medium change and passaging of cells .......................................... 55 3.12.5 Cell count determination with a Neubauer counting chamber ........ 56 3.12.6 Cell viability assay (MTT reduction assay) ..................................... 56 3.12.7 Western blot for γ-secretase inhibition ............................................ 57 II Index 4. Results 59 4.1 Development of an Aβ1-42 monomer specific mirror image phage display 59 4.2 Competitive mirror image phage display for monomeric Aβ1-42 with alternating plastic surfaces ...................................................................... 68 4.2.1 Preparation of SEC-derived Aβ1-42 monomers and oligomers and DGC-derived aggregates and fibrils .................................................. 69 4.2.2 Enrichment ELISA ............................................................................. 75 4.2.3 Single phage ELISA ........................................................................... 77 4.2.4 Sequencing of single phage clones ................................................... 81 4.2.5 Amino acid composition and chemical properties of MoRu peptides . 83 4.3 Characterization of MoRu peptides .......................................................... 84 4.3.1 Solubility of MoRu peptides ............................................................... 84 4.3.2 ThT assay with MoRu peptides .......................................................... 84 4.3.3 Modulation of Aβ1-42 distribution by MoRu peptides ........................... 90 4.3.4 Turbidity assay ................................................................................... 93 4.4 Characterization of MoRu3 ....................................................................... 98 4.4.1 Modulation of Aβ1-42 distribution by MoRu3 ....................................... 98 4.4.2 Impact of MoRu3 on Aβ1-42 aggregation shown by TEM .................. 103 4.4.3 Influence of MoRu peptides on Aβ1-42 mediated cell toxicity ............ 103 4.4.4 Influence of MoRu3 on the phenotype of murine neuroblastoma cells ................................................................................................. 107 4.4.5 Influence of MoRu3 on γ-secretase activity in APP-transfected Neuro-2a cells ................................................................................. 110 4.5 Repetition of mirror image phage display #5 with an optimized elution procedure (mipd#6) ............................................................................... 111 4.5.1 Enrichment ELISA ........................................................................... 111 4.5.2 Sequencing of single phage clones ................................................. 113 4.5.3 Single phage ELISA ......................................................................... 114 5. Discussion 118 5.1 A mirror image phage display for identification of monomer specific Aβ1-42 binding peptides ........................................................................... 118 5.2 Chemical
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