The role of amylin in Alzheimer’s disease A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy (PhD) In the faculty of Biology, Medicine and Health School of Biological Sciences Ben James Allsop 2017 TABLE OF CONTENTS List of figures .............................................................................................................6 List of tables ..............................................................................................................7 List of appendices .......................................................................................................8 List of abbreviations ...................................................................................................9 Abstract....................................................................................................................11 Declaration and copyright statement...........................................................................12 Acknowledgements....................................................................................................13 CHAPTER 1: INTRODUCTION ........................................................................ 14 1.1 Protein misfolding disorders .........................................................................14 1.1.1 Amyloid aggregation and oligomerisation ...............................................14 1.1.2 Relationship between PMDs and ageing .................................................16 1.2 Type II diabetes mellitus ..............................................................................18 1.2.1 Pathological hallmarks of T2D ...............................................................19 1.2.2 Physiological role of amylin ...................................................................23 1.2.3 Amylin aggregation is a central event in T2D .........................................24 1.2.4 Proposed toxic species of amylin ...........................................................25 1.3 Alzheimer’s disease ......................................................................................26 1.3.1 Symptoms and pathological hallmarks of AD ..........................................28 1.3.2 Generation of Aβ ..................................................................................29 1.3.3 Amyloid cascade hypothesis ..................................................................31 1.3.4 Cell surface receptors of Aβ oligomers ...................................................31 1.3.5 Tau .....................................................................................................34 1.3.6 Other pathological features of AD ..........................................................35 1.4 Connections between T2D and AD ................................................................36 1.4.1 Etiological links between T2D and AD ....................................................36 1.4.2 “Type 3 diabetes” .................................................................................36 1.4.3 Toxic oligomer species ..........................................................................37 1.4.4 Pathology in apparently non-diseased individuals ...................................38 1.4.5 Non-canonical amyloid deposition in AD and T2D ...................................38 1.4.6 Amylin deposition in the brain ...............................................................39 1.4.7 Repurposing antidiabetic drugs for AD ...................................................40 1.5 Thesis aims .................................................................................................42 1.5.1 Investigating the role of amylin in the brain in AD and T2D .....................43 2 1.5.2 Investigating whether amylin and Aβ share downstream signalling ..........43 1.5.3 The therapeutic potential of quercetin in AD ..........................................43 CHAPTER 2: MATERIALS AND METHODS ...................................................... 44 2.1 Cell Culture .................................................................................................44 2.1.1 Cell Lines .............................................................................................44 2.1.2 iPSC differentiation protocol ..................................................................44 2.2 Animal tissue ...............................................................................................45 2.3 Human Brain samples ..................................................................................46 2.3.1 Ethics and permissions .........................................................................46 2.3.2 Homogenisation protocol ......................................................................48 2.4 Sample Preparation ......................................................................................49 2.4.1 Preparation of cell lysates and media .....................................................49 2.4.2 Lysis buffer and loading sample preparation...........................................49 2.4.3 Bicinchoninic acid (BCA) assay ..............................................................50 2.5 Oligomer preparation ...................................................................................50 2.5.1 Peptide film preparation ........................................................................50 2.5.2 Amyloid-β preparation ..........................................................................50 2.5.3 Amylin preparation ...............................................................................51 2.5.4 Small molecule inhibitors ......................................................................51 2.6 Western Blotting and Dot blotting .................................................................51 2.6.1 Western Blotting ..................................................................................51 2.6.2 Dot blotting ..........................................................................................52 2.7 Glutaraldehyde cross-linking assay ................................................................54 2.8 Atomic Force Microscopy ..............................................................................54 2.8.1 Protocol ...............................................................................................54 2.8.2 Data acquisition ...................................................................................54 2.8.3 Data analysis .......................................................................................54 2.9 Thioflavin-T Assay .......................................................................................55 2.10 Immunofluorescence microscopy ..................................................................55 2.10.1 Protocol ...............................................................................................55 2.10.2 Antibodies ............................................................................................56 2.10.3 Microscopes .........................................................................................56 2.10.4 Data Analysis .......................................................................................56 2.11 Immunohistochemistry .................................................................................57 2.11.1 Fixing and slicing ..................................................................................57 2.11.2 DAB immunohistochemistry ...................................................................57 2.11.3 Slide scanner and quantification ............................................................58 2.11.4 Congo red staining ...............................................................................58 3 2.12 Multiplex immunoassays and ELISAs .............................................................58 2.12.1 Multiplex electro-chemiluminescence immunoassays ...............................59 2.12.2 Sandwich ELISA ...................................................................................59 2.13 PCR ............................................................................................................59 2.13.1 RNA isolation and generation of cDNA ...................................................59 2.13.2 RT-PCR ................................................................................................60 2.13.3 Resolving PCR product by agarose gel ...................................................60 2.13.4 RT-PCR data analysis ............................................................................61 2.14 Mass Spectrometry ......................................................................................61 2.14.1 Ion-mobility mass spectrometry ............................................................61 2.14.2 Tandem mass spectrometry ..................................................................63 2.15 Statistical Analysis .......................................................................................66 2.16 Nomenclature ..............................................................................................66 CHAPTER 3: INVESTIGATING THE ROLE OF AMYLIN IN THE BRAIN IN AD AND T2D 67 3.1 Introduction ................................................................................................67 3.1.1 Similarities between AD and T2D ...........................................................67 3.1.2 Complications associated with T2D correlate with amylin deposition in peripheral organs ................................................................................................68 3.1.3 Amylin deposition in the brain ...............................................................69 3.1.4 Chapter Aims .......................................................................................70 3.2 Results ........................................................................................................71 3.2.1 Amylin is deposited in the brain .............................................................71 3.2.2 Amylin deposits co-localise with Aβ CAA and form heterocomplexes ........81 3.2.3 Quantification of amylin in human brain fractions by multiple methods ....87 3.2.4 The IAPP gene is expressed in the occipital lobe and up-regulated in AD…. ……………………………………………………………………………………………………..100 3.2.5 Amylin upregulates Aβ secretion in OX1-19 neurons ............................. 101 3.3 Discussion ................................................................................................. 103 3.3.1 Amylin is deposited in the human brain ............................................... 103 3.3.2 Effect of amylin on pericyte viability .................................................... 107 3.3.3 Expression of IAPP mRNA in the brain and up regulation in AD ............. 108 3.3.4 Mechanism of amylin induced increase of Aβ secretion ......................... 110 3.4 Chapter Summary ...................................................................................... 112 CHAPTER 4: INVESTIGATING WHETHER AMYLIN AND AΒ SHARE DOWNSTREAM SIGNALLING ........................................................................... 114 4.1 Introduction .............................................................................................. 114 4 4.1.1 Complex world of Aβ oligomers ........................................................... 114 4.1.2 Aβ oligomers bind cell surface receptors .............................................. 115 4.1.3 Tau phosphorylation in AD .................................................................. 116 4.1.4 Similarities between Aβ and amylin...................................................... 117 4.1.5 Aims .................................................................................................. 118 4.2 Results ...................................................................................................... 119 4.2.1 Characterisation of Aβ and amylin oligomers ........................................ 119 4.2.2 Amylin causes activation of Fyn kinase ................................................ 126 4.2.3 Fyn kinase activation in iPSC derived neurons ...................................... 130 4.2.4 Tau phosphorylation in iPSC-derived neurons ....................................... 135 4.3 Discussion ................................................................................................. 139 4.3.1 Characterising oligomers ..................................................................... 139 4.3.2 Amylin activates Fyn kinase, potentially through PrPC ........................... 141 4.3.3 Downstream signalling in iPSC derived neuronal cultures ...................... 144 4.4 Chapter summary ...................................................................................... 146 CHAPTER 5: THERAPEUTIC POTENTIAL OF QUERCETIN IN ALZHEIMER’S DISEASE………………………………………………………………………………………..148 5.1 Introduction .............................................................................................. 148 5.1.1 Therapeutic targets in AD ................................................................... 148 5.1.2 Comparing therapeutic strategies in AD ............................................... 148 5.1.3 Small molecule inhibitors of Aβ aggregation ......................................... 149 5.1.4 Aims .................................................................................................. 152 5.2 Results ...................................................................................................... 153 5.2.1 Quercetin prevents the oligomerisation of Aβ ....................................... 153 5.2.2 Quercetin does not alter the structure of preformed oligomers .............. 160 5.2.3 Quercetin prevents Aβ oligomer binding to SH-SY5Y-PrPC cells .............. 165 5.2.4 Quercetin reduces Aβ and Aβ production ..................................... 167 1-40 1-42 5.3 Discussion ................................................................................................. 168 5.3.1 A critical appraisal of quercetin as an aggregation inhibitor ................... 168 5.3.2 How does quercetin affect preformed Aβ oligomers? ............................ 171 5.3.3 How may quercetin mediate a reduction of APP processing? ................. 172 5.4 Chapter summary ...................................................................................... 174 CHAPTER 6: FINAL DISCUSSION ................................................................ 176 6.1 Amylin deposition: a link between AD and T2D or indicative of other problems? 176 6.2 Does amylin contribute to AD pathology? .................................................... 177 6.3 Further discussion of IAPP expression in the brain ....................................... 180 5 6.4 Amylin induced Fyn activation: Pathological or physiological signalling? ......... 181 6.5 Therapeutic viability of quercetin in AD and T2D .......................................... 182 6.6 Concluding remarks ................................................................................... 184 References............................................................................................................187 List of figures Figure 1.1 Schematic of amyloid aggregation ...............................................................15 Figure 1.2 Pancreas anatomy and the islets of Langerhans. ..........................................20 Figure 1.3 Islet amyloidosis in diabetic human pancreas ...............................................22 Figure 1.4 Demonstration of the brain atrophy in AD ....................................................28 Figure 1.5 Pathological hallmarks of AD .......................................................................29 Figure 1.6 Pathways of APP processing ........................................................................30 Figure 1.7 Sequence analysis of amylin and Aβ ............................................................40 Figure 2.1 Age at death comparison between human samples .......................................46 Figure 2.2 Human brain tissue homogenisation protocol ...............................................48 Figure 3.1 Amylin antibody testing in hIAPP+/- mouse pancreas .....................................72 Figure 3.2 Immunohistochemistry of Aβ in human samples ...........................................73 Figure 3.3 Immunohistochemistry of amylin in human occipital lobe ..............................75 Figure 3.4 Immunohistochemistry of amylin in human temporal lobe .............................78 Figure 3.5 Serial staining of Aβ and amylin shows co-deposition in vasculature ..............81 Figure 3.6 Quantification of pericyte viability ................................................................83 Figure 3.7 Ion mobility mass spectrometry demonstrates that amylin and Aβ interact .....84 Figure 3.8 Amylin and Aβ form heterocomplexes ......................................................86 1-42 Figure 3.9 Soluble and Insoluble Aβ levels from fractionated human brain tissue ............88 Figure 3.10 Amylin in human brain fractions ................................................................90 Figure 3.11 Selective reaction monitoring method for Aβ ..............................................92 Figure 3.12 Selective reaction monitoring method for amylin .........................................93 Figure 3.13 Separation of amylin and Aβ standards by HPLC .........................................94 Figure 3.14 Retention time and chromatogram of Aβ in human brain fractions ...............95 Figure 3.15 Retention time and chromatogram of amylin in human brain fractions .........96 Figure 3.16 IAPP is expressed in the brain and is up-regulated in AD .............................98 Figure 3.17 Intra-neuronal amylin staining ...................................................................99 Figure 3.18 Amylin increases secreted Aβ isoforms in OX1-19 neurons ........................ 101 Figure 3.19 Amylin impairs autophagy to increase Aβ ................................................. 102 Figure 3.20 Potential role of amylin in AD .................................................................. 113 Figure 4.1 Characterisation of Aβ oligomers and aggregation ...................................... 120 Figure 4.2 Characterisation of Aβ oligomers by atomic force microscopy (AFM) ............ 121 6 Figure 4.3 Characterisation of amylin aggregation ...................................................... 123 Figure 4.4 Characterisation of amylin oligomers by AFM .............................................. 124 Figure 4.5 Amylin monomer and oligomer species phosphorylate Fyn .......................... 125 Figure 4.6 Effect of Phospholipase-C pretreatment on amylin induced Fyn phosphorylation ............................................................................................................................... 127 Figure 4.7 Effect of 6D11 pretreatment on amylin induced Fyn phosphorylation. .......... 129 Figure 4.8 PrPC expression in iPSC derived cortical neurons ......................................... 132 Figure 4.9 Aβ induces Fyn phosphorylation in OX1-19 neurons. ................................. 133 O Figure 4.10 Amylin induces Fyn phosphorylation in OX1-19 neurons. ........................... 134 Figure 4.11 Tau phosphorylation in OX1-19 neurons. .................................................. 137 Figure 5.1 Structures of quercetin and rutin ............................................................... 151 Figure 5.2 Characterisation of Aβ oligomers and aggregation ...................................... 151 Figure 5.3 Rutin prevents Aβ fibril but not oligomer formation ..................................... 154 Figure 5.4 Quercetin prevents Aβ aggregation ............................................................ 155 Figure 5.5 AFM demonstrates quercetin prevents Aβ aggregation and produces smaller oligomers ................................................................................................................. 156 Figure 5.6 Modelling AFM data shows Q preparation significantly smaller than Aβ .... 157 OT O Figure 5.7 Rutin binds Aβ ..................................................................................... 159 1-42 Figure 5.8 Biochemistry characterisation of the effect of quercetin on preformed Aβ oligomers ................................................................................................................. 162 Figure 5.9 AFM characterisation of the effect of quercetin on preformed Aβ oligomers .. 163 Figure 5.10 Characterisation of Q preparation by AFM .............................................. 164 OP Figure 5.11 Quercetin prevents Aβ binding to cells ..................................................... 166 Figure 5.12 Quercetin reduces production of Aβ isoforms ............................................ 167 Figure 5.13 Potential therapeutic actions of quercetin in AD ........................................ 175 Figure 6.1 Possible contributions of amylin to AD ....................................................... 187 List of tables Table 1.1 Native proteins associated with PMDs ...........................................................16 Table 1.2 Table of reported Aβ and amylin receptors ....................................................33 Table 2.1 Media used in neuronal induction and differentiation of iPSCs .........................45 Table 2.2 Identification and demographic data of human samples .................................47 Table 2.3 Primary antibodies used in western blotting and dot blotting. .........................53 Table 2.4 Table of antibodies used in immunofluorescence microscopy ..........................56 Table 2.5 Table of antibodies used in immunohistochemistry ........................................58 Table 2.6 Table of primer designs used in RT-PCR experiments .....................................61 Table 2.7 IMMS instrument parameters .......................................................................62 7 Table 2.8 HPLC gradient. ............................................................................................63 Table 2.9 LC-MS/MS Quatpump gradient. ....................................................................64 Table 2.10 Triple-Quad source conditions. ...................................................................64 Table 2.11 SRM methods for amylin and Aβ. ................................................................65 List of appendices Appendix 1 Intra-neuronal amylin staining (T-4150) ................................................... 224 Appendix 2 Intra-neuronal amylin staining (H-017-03) ................................................ 225 Appendix 3 Ion-mobility mass spectrometry instrument parameters ............................ 226 Appendix 4 Evidence of Aβ aggregation in tip during IMMS ......................................... 229 Word count: 52,907 8 List of Abbreviations AD Alzheimer's disease ADAM10 A Disintegrin and metalloproteinase domain-containing protein 10 ADDL Aβ-derived diffusible ligand AFM Atomic force microscopy AICD Amyloid intracellular domain AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AMPK 5'-AMP activated Kinase AMY Calcitonin receptor + RAMP isoform 1, 2 or 3 1/2/ 3 AMYm Amylin monomer preparation AMYo Amylin oligomer preparation APOE Apolipoprotein-E APP Amyloid precursor protein ASC Apoptosis-associated speck-like protein containing a CARD Aβ Amyloid-β Aβm Aβ monomer preparation AβO Aβ oligomer preparation BACE1 Beta-site APP cleaving enzyme 1 BBB Blood-brain barrier BCA Bicinchoninic acid BSA Bovine serum albumin CAA Cerebral amyloid angiopathy CaMKII Ca2+/calmodulin-dependent protein kinase II CGRP calcitonin gene related peptide CQ Chloroquine DAPI Diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide EphA4 Ephrin type A receptor 4 EphB2 Ephrin type B receptor 2 fAD Familial AD FGF2 Fetal growth factor-2 FOXG1 forkhead box protein G1 FTD frontotemporal lobar dementia GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase GFAP Glial fibrillary acidic protein GPCR G-protein coupled receptor GSK3 Glycogen synthase kinase-3 HFIP 1,1,1,3,3,3-hexafluoropropanol-2-ol HPLC High performance liquid chromatography IL-1β /-6 Interleukin-1β /-6 IMMS Ion-mobility mass spectrometry iPSC Induced pluripotent stem cells JNK c-Jun N-terminal kinase LC3 Microtubule-associated protein 1A/1B-light chain 3 LC-MS/MS liquid chromatography linked tandem mass spectrometry 9 LRP1 Low density lipoprotein (LDL) receptor-related protein 1 LTP Long term potentiation MAP2 Microtubule associated protein-2 MAPK Mitogen-activated protein kinase MAPT Microtubule associated protein tau mGluR5 Metabotropic glutamate receptor 5 NLRP3 Nod-like receptor protease-3 NMDAR N-methyl-D-asapartate receptor NSAID Non-steroidal anti-inflammatory drug OC Structural antibody designed against fibrillar oligomers OG Occipital lobe grey matter OW Occipital lobe white matter PAX6 Paired box protein-6 PBS Phosphate buffered saline PBST Phosphate buffered saline with Tween-20 PDGFRβ Platelet derived growth factor receptor-β PFA Paraformaldehyde PI3K phosphatidylinositol 3-kinase PKA Protein kinase A PKC Protein kinase C PMD Protein misfolding disorder PrPC Cellular prion protein PSD-95 Post-synaptic density-95 pTau Phosphorylated tau Pyk2 Protein tyrosine kinase 2 RAMP1/2/3 Receptor activity-modifying protein isoform 1, 2 or 3 ROS Reactive oxygen species RPMI Roswell park memorial institute 1640 medium RT-PCR Real time polymerase chain reaction sAPPα / β Soluble APPα / β SATB2 Special AT-rich sequence-binding protein 2 SDS-PAGE Sodium dodecyl sulphate polyacrylamide electrophoresis SH-SY5Y-APP SH-SY5Y neuroblastoma cells stably expressing APP695 695 SH-SY5Y-PrPC SH-SY5Y neuroblastoma cells stably expressing PrPC Sox-2 Sex determining region-Y-box-2 STZ Streptozotocin T2D Type II diabetes mellitus TBR1 T-box brain 1 TDP-43 TAR DNA-binding protein 43 TEAB Triethylammonium bicarbonate ThT Thioflavin-T TLR2 Toll-like receptor 2 TNFα Tumor necrosis factor-α TW Temporal lobe white matter α-syn α-synuclein 10