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Optimisation and Use of Cell-Based Assays and in Vivo Assay for Screening Drugs for Alzheimer’S Disease

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Optimisation and Use of Cell-Based Assays and in Vivo Assay for Screening Drugs for Alzheimer’S Disease Optimisation and use of cell-based assays and in vivo assay for screening drugs for Alzheimer’s Disease A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science & Engineering. 2017 Maria Priscila del Castillo Frias School of Chemistry List of Contents Chapter 1. Introduction ............................................................................ 21 1.1. Alzheimer’s Disease (AD) ............................................................. 21 1.1.1 AD as a world health issue ......................................................... 21 1.1.2 AD Overview and pathological hallmarks ................................. 22 1.1.3 AD Genetics .............................................................................. 24 1.1.4 AD Enviromental factors ........................................................... 25 1.1.5 APP metabolism ......................................................................... 25 1.1.6 Aβ Clearance .............................................................................. 27 1.1.7 Tau protein ................................................................................. 28 1.1.8 Amyloid cascade hypothesis ...................................................... 28 1.1.9 Oligomers: refined cascade hypothesis ...................................... 30 1.1.10 Role of oxidative stress in AD.................................................. 31 1.2. Approved drugs to treat AD ........................................................... 32 1.3. Therapeutic strategies to treat AD.................................................. 33 1.3.1 Modifications in APP metabolism ............................................. 33 1.3.2 Immunotherapy .......................................................................... 36 1.3.3 Drugs targeting Aβ aggregation ................................................. 37 1.3.4 Reducing Aβ toxicity (oxidative stress, Calcium) ..................... 38 1.3.5 Drugs targeting Tau.................................................................... 39 1.4. Drug repositioning to find drugs for AD........................................ 40 1.4.1 Current candidates for drug repositioning based on rationale approaches 42 1.4.2 Drug repositioning with no therapeutic rationale. Drug library screening. 49 1.5. Cellular models for AD .................................................................. 50 1.6. Animal Models for AD .................................................................. 52 1.6.1 Natural/ Spontaneous models ..................................................... 52 1.6.2 Transgenic models ..................................................................... 53 3 1.7. Project Aims................................................................................... 56 Chapter 2. Optimisation of in vitro assays .............................................. 57 2.1. Methods.......................................................................................... 58 2.1.1 SH-SY5Y cell culturing and maintenance. ................................ 58 2.1.2 Aβ42 preparation using HFIP and DMSO ................................. 58 2.1.3 Aβ42 oligomers preparation using NaOH ................................. 59 2.1.4 MTT (Optimised method) .......................................................... 59 2.1.5 Evaluation of Aβ42 oligomers toxicity in SH-SY5Y by MTT .. 60 2.1.6 LDH (Cyto Tox-OneTM assay Promega).................................... 60 2.1.7 CytoTox-GloTM .......................................................................... 61 2.1.8 Meso scale Discovery system .................................................... 61 2.1.9 DCFH ......................................................................................... 63 2.1.10 ROS-GloTM ............................................................................... 64 2.1.11 GSH/GSSG- GloTM assay......................................................... 64 2.2. MTT ............................................................................................... 65 2.2.1 MTT principle ............................................................................ 65 2.2.2 Optimisation of MTT assay ....................................................... 66 2.2.3 Optimal Cell Density for MTT assay......................................... 68 2.2.4 Evaluation of Aβ42 toxicity using MTT assay .......................... 70 2.3. LDH principle ................................................................................ 71 2.3.1 LDH optimisation ...................................................................... 72 TM 2.4. CyToTox-Glo ............................................................................. 73 2.4.1 Optimal cell density ................................................................... 74 2.4.2 Experimental sensitivity............................................................. 75 TM 2.4.3 Evaluation of the toxicity of Aβ42 using CytoTox-Glo ........ 76 2.5. MSD immunoassay principle ......................................................... 77 2.5.1 MSD optimisation ...................................................................... 78 2.6. Oxidative Stress assays .................................................................. 80 2.6.1 DCFH principle .......................................................................... 80 TM 2.6.2 ROS-Glo H2O2 principle ........................................................ 83 2.6.3 GSH/GSSG principle ................................................................. 86 4 2.7. Discussion ...................................................................................... 88 Chapter 3. Primary Screening.................................................................. 95 3.1. Introduction .................................................................................... 95 3.2. Methods .......................................................................................... 95 3.2.1 Selection of drugs for primary screening ................................... 95 3.2.2 Preparation of Aβ42 and LOPAC compounds for MTT screening. 97 3.2.3 Drug setup for MTT screening ................................................... 98 3.2.4 MTT drug screening ................................................................... 99 3.2.5 MSD drug preparation and setup ............................................... 99 3.2.6 MSD immunoassay drug screening ......................................... 100 3.3. Results: MTT LOPAC sub-library screening .............................. 100 3.3.1 MTT first round ....................................................................... 101 3.3.2 MTT LOPAC second round ..................................................... 104 3.3.3 MTT LOPAC third round ........................................................ 107 3.3.4 Aβ42 Variation from batch to batch ........................................ 110 3.3.5 Hits confirmation ..................................................................... 111 3.3.6 Non-LOPAC compounds screening ......................................... 115 3.4. MSD immunoassays .................................................................... 117 3.4.1 MSD immunoassays primary screening LOPAC .................... 117 3.4.2 MSD immunoassays hits confirmation .................................... 127 3.5. Discussion .................................................................................... 131 Chapter 4. Secondary screening ............................................................. 137 4.1. Introduction .................................................................................. 137 4.2. Methods ........................................................................................ 138 4.2.1 Dose-response curve drug preparation ..................................... 138 4.2.2 Preparation of drug combinations ............................................ 138 4.2.3 Preparation of drugs for oxidative stress assays panel ............. 139 4.2.4 MTT and DCFH assay ............................................................. 139 4.2.5 GSH/GSSG- GloTM assay. ....................................................... 140 4.3. MTT dose-response curves .......................................................... 140 5 4.4. MTT Drug combination screening............................................... 143 4.5. Oxidative stress assay panel......................................................... 144 4.5.1 DCFH ....................................................................................... 144 4.5.2 GSH/GSSG ratio ...................................................................... 147 4.6. Discussion .................................................................................... 149 Chapter 5. Drosophila melanogaster assays ......................................... 151 5.1. Introduction .................................................................................. 151 5.2. Methods........................................................................................ 152 5.2.1 Drosophila melanogaster stocks .............................................. 152 5.2.2 Fly husbandry........................................................................... 153 5.2.3 Male/Female sorting ................................................................ 154 5.2.4 Virgin collection ...................................................................... 154 5.2.5 Quantification of soluble and insoluble Aβ42 ......................... 155 5.2.6 Lifespan assays
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