Amyloid Precursor Protein A Practical Approach

Amyloid Precursor Protein: A Practical Approach......Page 1
Preface......Page 4
Contributors......Page 6
Table of Contents......Page 9
List of Antibodies to APP and Aβ Proteins......Page 11
Contents......Page 0
CONTENTS......Page 13
1.2 MAIN SCHEME OF APPROACHES......Page 14
1.3 RESULTS......Page 16
1.4 DISCUSSION......Page 19
1.5.3 DETERMINATION OF PROTEIN CONCENTRATION BY BCA......Page 21
1.5.5 RADIOLABELING OF CELLS WITH [35S]-MET......Page 22
1.5.6 IDENTIFICATION OF FULL-LENGTH APP AND ITS DERIVATIVES BY IMMUNOPRECIPITATION......Page 23
1.5.8.3 Pre-Clearing of Cell Lysates......Page 24
1.5.8.5 Wash Co-IP......Page 25
1.5.9 CONJUGATION OF ANTIBODY TO PROTEIN A-SEPHAROSE......Page 26
REFERENCES......Page 27
ABSTRACT......Page 30
2.2 MAIN SCHEME OF APPROACHES......Page 31
2.3.1 IODINATION OF ANTIBODY......Page 32
2.3.3 KINETICS OF SECRETION AND ENDOCYTOSIS OF APP......Page 33
2.3.4 STEADY STATE LEVEL OF APP ENDOCYTOSIS......Page 34
2.3.5 RECYCLING OF APP......Page 35
2.3.6 MORPHOLOGICAL ANALYSIS......Page 36
REFERENCES......Page 37
ABSTRACT......Page 39
3.2 OVERVIEW OF APPROACH......Page 40
3.3 BIOINFORMATICS ANALYSIS OF APP......Page 41
3.6 EXPRESSION OF RECOMBINANT GFD......Page 42
3.6.2 METHOD FOR CLONING......Page 43
3.7.1 METHOD......Page 44
3.8 CRYSTALLIZATION OF GFD......Page 45
3.8.2 METHOD......Page 46
3.9 DISCUSSION......Page 47
REFERENCES......Page 49
CONTENTS......Page 51
4.1 INTRODUCTION......Page 52
4.2 ASSAY OF MEMAPSIN 2 ACTIVITY......Page 53
4.3.1 N-(TERT-BUTOXYCARBONYL)-L-LEUCINE-N′-METHOXY-N′-METHYLAMIDE (3)......Page 54
4.3.4 (5S,1′S)-5-[1′-[(TERT-BUTOXYCARBONYL)AMINO]-3′-METHYLBUTYL]-DIHYDROFURAN-2(3H)-ONE (7)......Page 56
4.3.7 (2R,4S,5S)-5-[(FLUORENYLMETHYLOXYCARBONYL)AMINO]- 4-[(TERT-BUTYLDIMETHYL SILYL)OXY]-2,7-DIMETHYLOCTANOIC ACID (10)......Page 57
4.4 DETERMINATION OF INHIBITION CONSTANTS......Page 58
4.5 SUMMARY......Page 59
REFERENCES......Page 60
ABSTRACT......Page 61
5.1 INTRODUCTION......Page 62
5.2 MAIN SCHEME OF APPROACHES......Page 63
5.3.1 ASSAYING y-SECRETASE ACTIVITY IN LIVING CELLS......Page 65
5.3.2 SUBCELLULAR FRACTIONATION OF MEMBRANE VESICLES......Page 66
5.3.3 IN VITRO γ-SECRETASE ACTIVITY ASSAY USING ENDOGENOUS SUBSTRATE......Page 67
5.3.4 DETERMINING PH DEPENDENCE OF y-SECRETASE ACTIVITY USING VESICLES......Page 69
5.3.5 PROTEASE INHIBITOR PROFILING OF y-SECRETASE ACTIVITY USING FRACTIONS......Page 70
5.3.6 CELL MEMBRANE PREPARATION FOR EXOGENOUS SUBSTRATE ASSAY......Page 71
5.3.7 M2 FLAG PURIFICATION OF E. COLI-GENERATED y-SECRETASE SUBSTRATES......Page 72
5.3.8 IN VITRO γ-SECRETASE ACTIVITY ASSAY USING EXOGENOUS SUBSTRATE......Page 73
REFERENCES......Page 74
CONTENTS......Page 79
6.1 INTRODUCTION......Page 80
6.2.2 PREPARATION OF PERMEABILIZED N2A CELLS THROUGH OSMOTIC SHOCK AND MECHANICAL SHEAR......Page 82
6.2.5 Aß GENERATION IN CELL-FREE SYSTEM UTILIZING ERS IN THE ABSENCE OF CYTOSOL......Page 83
6.2.7 FORMATION OF NASCENT SECRETORY VESICLES IN CELL-FREE SYSTEM SUPPLEMENTED WITH ERS AND CYTOSOL......Page 84
6.2.8 DETECTION OF bAPP IN DIFFERENT FRACTIONS THROUGH IMMUNOPRECIPITATION......Page 85
6.3.1 ATP-DEPENDENT Aß GENERATION IN CELL-FREE RECONSTITUTION SYSTEM......Page 86
6.3.4 CHARACTERIZATION OF VESICLE AND MEMBRANE FRACTIONS BY SUCROSE GRADIENT AND ELECTRON MICROSCOPY......Page 87
ACKNOWLEDGMENTS......Page 89
REFERENCES......Page 90
CONTENTS......Page 92
7.2 BACKGROUND......Page 93
7.3.1 PRODUCTION OF Ab- Peptides......Page 94
7.3.2 PREPARATION OF STARTING PEPTIDE STOCKS......Page 95
7.3.2.4 Preparation of LMW Aβ by Size Exclusion Chromatography......Page 97
7.3.2.5 Preparation of LMW Aβ by Filtration......Page 98
7.3.2.7 Preparation of Oligomeric Aβ42......Page 99
7.3.2.9 Preparation of Aβ Fibrils......Page 100
7.4.1.1 Determination of Oligomer Size Distributions Using PICUP......Page 101
7.4.1.3 Determination of Particle Diffusion Coefficients Using QLS......Page 102
7.4.2.2 Congo Red Binding......Page 103
7.4.2.3 Turbidity......Page 105
7.4.3.1 CD Spectroscopy......Page 106
7.4.4.1 8-Anilino-1-Naphthalenesulfonic Acid (ANS) Binding......Page 108
7.4.4.3 Electron Paramagnetic Resonance (EPR)......Page 109
7.4.4.4 Hydrogen-Deuterium Exchange......Page 110
7.4.5 NMR SPECTROSCOPY......Page 111
7.4.6.2 Atomic Force Microscopy......Page 112
REFERENCES......Page 113
CONTENTS......Page 120
8.1 INTRODUCTION......Page 121
8.2.3 IMMUNOCYTOCHEMICAL PROCEDURES......Page 122
8.2.3.2 Fixation......Page 123
8.2.6 ASSESSMENT OF MITOCHONDRIAL FUNCTION......Page 124
8.2.6.2 JC1 Protocol......Page 125
8.2.7.2 Propidium Iodide......Page 126
8.3.1 DETECTION OF INTRACELLULAR AB in ADS ASTROCYTES......Page 127
8.3.3 MITOCHONDRIAL DYSFUNCTION IN DS ASTROCYTES......Page 129
8.3.4 APPS RESCUES DS CORTICAL NEURONS FROM APOPTOSIS......Page 131
8.4 DISCUSSION......Page 132
REFERENCES......Page 135
CONTENTS......Page 138
9.1 INTRODUCTION......Page 139
9.2 CHOLESTEROL AND AND AB......Page 140
9.2.2 PROTEIN ANALYSIS......Page 143
9.3.2 ENZYMATIC ASSAY......Page 144
9.3.4 PHOSPHORUS DETERMINATION......Page 145
9.3.5 SCINTILLATION COUNT......Page 146
9.4.1 THEORETICAL BACKGROUND......Page 147
9.4.4.3 Precursor Ion Scans on m/z = 184.1, 188.1, and 264.3......Page 149
ACKNOWLEDGMENTS......Page 150
REFERENCES......Page 152
ABSTRACT......Page 154
10.2.1 LITHIUM TREATMENT IN TRANSFECTED CULTURED CELLS......Page 155
10.2.4 MICROINJECTION OF FAβ INTO TAU TRANSGENIC MICE......Page 156
10.3.1 ESTABLISHMENT OF SELECTIVE SANDWICH Ab ELISA......Page 157
10.3.2 EFFECT OF LICL ON Ab SECRETION IN ASSOCIATION WITH GSK-3b ACTIVITY FROM APP C100-TRANSFECTED COS7 CELLS......Page 158
10.3.3 EFFECT OF GSK-3b ON FAb-INDUCED TAU PATHOLOGY IN VIVO......Page 159
10.4 DISCUSSION......Page 160
REFERENCES......Page 162
11.1 INTRODUCTION......Page 163
11.2.1 IMMUNOCYTOCHEMISTRY: FIXATION......Page 164
11.2.3 IMMUNOCYTOCHEMISTRY FOR APP AND AB......Page 165
11.2.4 IMMUNO-ELECTRON MICROSCOPY FOR APP and AB......Page 166
11.4.1 PROTOCOLI: IMMUNOSTAINING PROTOCOL FOR PARAFFIN-EMBEDDED SECTIONS......Page 168
11.4.2 PROTOCOL II: IMMUNOPEROXIDASE STAINING PROTOCOL......Page 169
11.4.3 PROTOCOL III: IMMUNOPEROXIDASE ELECTRON MICROSCOPY......Page 170
11.4.4 PROTOCOL IV: IMMUNO-ELECTRON MICROSCOPY......Page 171
REFERENCES......Page 172
12.1 INTRODUCTION......Page 173
12.2.1.1 Paraffin Tissue......Page 175
12.2.2.1 Paraffin Sections......Page 176
12.2.2.2 Frozen Sections......Page 177
12.2.5 DOUBLE IMMUNOFLUORESCENT LABELING......Page 178
12.2.6 DOUBLE LABELING WITH DAB AND OTHER COLORED MARKERS......Page 179
12.3.2 Aβ40 VS. Aβ42 IN FAMILIAL ALZHEIMER’S DISEASE (FAD)......Page 181
12.3.3 INTRANEURONAL Aβ IN DOWN’S SYNDROME......Page 183
12.3.6 COLOCALIZATION OF Aβ WITH GLIA......Page 186
REFERENCES......Page 190
13.1 INTRODUCTION......Page 192
13.1.1 ORIGINS OF GENE MANIPULATION AND GENE TRANSFER INTO MOUSE GENOME......Page 193
13.1.3 APP GENE STRUCTURE: ITS ISOFORMS AND PROMOTERS USED IN CREATING APP TRANSGENICS......Page 195
13.2.1.1 Strain Selection......Page 198
13.2.1.2 Isolation of Target DNA from Bacterial Host......Page 199
13.2.1.3 Purification of DNA for Microinjection......Page 200
13.2.1.6 Indentification of Founders......Page 201
13.2.1.7 Maintenance and Analysis of Founders......Page 202
REFERENCES......Page 203
14.1 INTRODUCTION......Page 208
14.2 EXPERIMENTAL PROCEDURES......Page 210
14.2.1 CONSTRUCTION OF APP GENE TARGETING VECTOR......Page 211
14.2.2.1 Growing SNL Cells and Preparation of Feeders......Page 214
14.2.2.2 Culturing and Electroporating Embryonic Stem Cells......Page 215
14.2.3.1 Selection of Recombinant Colonies......Page 216
14.2.3.3 Freezing ES Cells in 96-Well Plates......Page 217
14.2.3.5 Preparation of Gene-Targeted Clones for Blastocyst Injection......Page 218
14.2.4.1 Microinjection, Assessment of Chimerism, and Test......Page 219
14.2.4.2 Breeding and Generation of Homozygous APP......Page 220
ACKNOWLEDGMENTS......Page 221
REFERENCES......Page 222
COLOR FIGURE 12.8......Page 229
COLOR FIGURE 8.3......Page 226
COLOR FIGURE 8.5......Page 227
COLOR FIGURE 10.3......Page 228