Amyloid Precursor Protein: A Practical Approach

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ISBN: 9780849322457 | 0849322456
Cover: Hardcover
Copyright: 12/16/2004

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Amyloid precursor protein (APP) is an extensively studied transmembrane protein, and its derivative, amyloid b-protein, has been implicated as the primary cause of Alzheimer's disease. Thousands of reports have been published on APP, and interest continues to grow in the research community. In the search for an effective treatment for Alzheimer's disease, APP is a unique model protein that illustrates the wide array of basic and sophisticated techniques available for characterization.Amyloid Precursor Protein: A Practical Approach presents a variety of biological techniques utilized in genetics, molecular biology, cell biology, and biochemistry to elucidate the structure and function of this transmembrane protein. Each technique is presented with detailed, step-by-step protocols to achieve reproducible results and provide a framework for studying other membrane proteins. These protocols also form the core of the authors' approaches to elucidating the function of APP.This timely book contrasts basic and advanced methods to explain how the development of biological techniques affects the way molecular targets are examined. Practical in scope, it provides useful lists of protocols and routinely used antibodies for easy reference and paints a complete picture of the current understanding of APP as well as direction for future research.

List of Antibodies to APP and Aβ Proteins

xiii

Chapter 1 Biochemical Characterization of Amyloid Precursor Protein

(18)

Weiming Xia

1.1 Introduction

(1)

1.2 Main Scheme of Approaches

(2)

1.3 Results

(3)

1.4 Discussion

(2)

1.5 Protocols

(6)

1.5.1 Overexpression of APP in Mammalian Cells by Transient Transfection

(1)

1.5.2 Selection of Stable Cell Line Overexpressing APP

(1)

1.5.3 Determination of Protein Concentration by BCA

(1)

1.5.4 Identification of APP and Its Derivatives by Western Blot

(1)

1.5.5 Radiolabeling of Cells with [35S]-Met

(1)

1.5.6 Identification of Full-Length APP and Its Derivatives by Immunoprecipitation

(1)

1.5.7 Determination of Half-Life of APP

(1)

1.5.8 Co-Immunoprecipitation of APP-Interacting Protein

(2)

1.5.8.1 Preparation

(1)

1.5.8.2 Pre-Absorption of Protein A-Sepharose

(1)

1.5.8.3 Pre-Clearing of Cell Lysates

(1)

1.5.8.4 Set Up Co-IP

(1)

1.5.8.5 Wash Co-IP

(1)

1.5.9 Conjugation of Antibody to Protein A-Sepharose

(1)

Acknowledgments

(1)

References

(4)

Chapter 2 Assays for Analysis of APP Secretion and Recycling

(10)

Markus P. Kummer, Tsuneo Yamazaki, and Edward H. Koo

Abstract

(1)

2.1 Introduction

(1)

2.2 Main Scheme of Approaches

(1)

2.3 Methods

(5)

2.3.1 Iodination of Antibody

(1)

2.3.2 Preparation of Cells

(1)

2.3.3 Kinetics of Secretion and Endocytosis of APP

(1)

2.3.4 Steady State Level of APP Endocytosis

(1)

2.3.5 Recycling of APP

(1)

2.3.6 Morphological Analysis

(1)

2.4 Discussion

(1)

Acknowledgments

(1)

References

(3)

Chapter 3 Strategies for Crystallizing the N-Terminal Growth Factor Domain of Amyloid Precursor Protein

(12)

William J. McKinstry, Susanne C. Feil, Denise Galatis, Roberto Cappai, and Michael W. Parker

Abstract

(1)

3.1 Introduction

(1)

3.2 Overview of Approach

(1)

3.3 Bioinformatics Analysis of APP

(1)

3.4 Biological Roles of GFD

(1)

3.5 Previous Crystallization Studies

(1)

3.6 Expression of Recombinant GFD in Pichia pastoris

(2)

3.6.1 Materials

(1)

3.6.2 Method for Cloning

(1)

3.6.3 Method for Expression

(1)

3.7 Purification of GFD

(1)

3.7.1 Method

(1)

3.8 Crystallization of GFD

(2)

3.8.1 Materials

(1)

3.8.2 Method

(1)

3.9 Discussion

(2)

Acknowledgments

(1)

References

(2)

Chapter 4 Analysis of Amyloid Precursor Protein Processing Protease Β-Secretase: Tools for Memapsin 2(β-Secretase) Inhibition Studies

(10)

Gerald Koelsch, Vajira Weerasena, Dongwoo Shin, Arun K. Ghosh, and Jordan Tang

4.1 Introduction

(1)

4.2 Assay of Memapsin 2 Activity

(1)

4.3 Synthesis of Memapsin 2 Inhibitors Based on APP Sequence

(4)

4.3.1 N-(tert-butoxycarbonyl)-L-leucine-N'-methoxy-N'-methylamide (3)

(2)

4.3.2 N-(tert-butoxycarbonyl)-L-leucinal (4)

(1)

4.3.3 Ethyl (4S,5S)- and (4R,5S)-5-[(tert-butoxycarbonyl)amino]-4-hydroxy-7-methyloct-2-ynoate (5)

(1)

4.3.4 (5S,1'S)-5-[1'-[(tert-Butoxycarbonyl)amino]-3'-methylbutyl]-dihydrofuran-2(3H)-one (7)

(1)

4.3.5 (3R,5S,I'S)-5-[1'-[(tert-butoxycarbonyl)amino)]-3'-methylbutyl]-3-methyl dihydrofuran-2(3H)-one (8)

(1)

4.3.6 (2R,4S,5S)-5-[(tert-Butoxycarbonyl)amino]-4-[(tert-butyldimethylsilyl)oxy ]-2,7-dimethyloctanoicacid (9)

(1)

4.3.7 (2R,4S,5S)-5-[(fluorenylmethyloxycarbony])amino]-4-[(tert-butyldimethyl silyl)oxy]-2,7-dimethyloctanoic acid (10)

(1)

4.3.8 Coupling of Di-Isostere in Solid-Phase Peptide Synthesis

(1)

4.4 Determination of Inhibition Constants

(1)

4.5 Summary

(1)

References

(1)

Chapter 5 Assays for Amyloid Precursor Protein γ-Secretase Activity

(18)

William A. Campbell, Michael S. Wolfe, and Weiming Xia

Abstract

(1)

5.1 Introduction

(1)

5.2 Main Scheme of Approaches

(2)

5.3 Method

(9)

5.3.1 Assaying γ-Secretase Activity in Living Cells

(1)

5.3.2 Subcellular Fractionation of Membrane Vesicles

(1)

5.3.3 In Vitro γ-Secretase Activity Assay Using Endogenous Substrate

(2)

5.3.4 Determining pH Dependence of γ-Secretase Activity Using Vesicles

(1)

5.3.5 Protease Inhibitor Profiling of γ-Secretase Activity Using Fractions

(1)

5.3.6 Cell Membrane Preparation for Exogenous Substrate Assay

(1)

5.3.6.1 Buffers

(1)

5.3.7 M2 Flag Purification of E. coli-Generated γ-Secretase Substrates

(1)

5.3.7.1 Buffers

(1)

5.3.8 In Vitro γ-Secretase Activity Assay Using Exogenous Substrate

(1)

5.4 Discussion

(1)

References

(5)

Chapter 6 Cell-Free Reconstitution of β-Amyloid Production and Trafficking

(14)

Dongming Cai, William J. Netzer, Feng Li, and Huaxi Xu

Abstract

(1)

6.1 Introduction

(2)

6.2 Methods

(4)

6.2.1 Pulse-Labeling of N2a Cells and Temperature Blocking of Nascent Protein Transport

(1)

6.2.2 Preparation of Permeabilized N2a Cells through Osmotic Shock and Mechanical Shear

(1)

6.2.3 Preparation of Cytosol for Cell-Free Reconstitution System

(1)

6.2.4 Preparation of Energy-Regenerating System for Cell-Free Reconstitution System

(1)

6.2.5 Aβ Generation in Cell-Free System Utilizing ERS in the Absence of Cytosol

(1)

6.2.6 Detection of Aβ Production from Cell-Free System

(1)

6.2.7 Formation of Nascent Secretory Vesicles in Cell-Free System Supplemented with ERS and Cytosol

(1)

6.2.8 Detection of βAPP in Different Fractions through Immunoprecipitation

(1)

6.3 Discussion

(3)

6.3.1 ATP-Dependent Aβ Generation in Cell-Free Reconstitution System

(1)

6.3.2 Cytosol-Dependent βAPP Trafficking from TGN/ER in the Cell-Free Reconstitution System

(1)

6.3.3 Utilizing the Cell-Free Reconstitution System to Demonstrate PSI-Regulated Intracellular Trafficking and Surface Delivery of βAPP

(1)

6.3.4 Characterization of Vesicle and Membrane Fractions by Sucrose Gradient and Electron Microscopy

(2)

Acknowledgments

(1)

References

(3)

Chapter 7 Studying Amyloid β-Protein Assembly

(28)

Erica A. Fradinger, Samir Kumar Maji, Noel D. Law, and David B. Teplow

7.1 Introduction

(1)

7.2 Background

(1)

7.3 Peptide Production and Preparation

(7)

7.3.1 Production of AβPeptides

(1)

7.3.2 Preparation of Starting Peptide Stocks

(6)

7.3.2.1 HFIP Pretreatment

(1)

7.3.2.2 NaOH Pretreatment

(1)

7.3.2.3 Sample Clarification and Fibril Isolation

(1)

7.3.2.4 Preparation of LMW Aβ by Size Exclusion Chromatography

(1)

7.3.2.5 Preparation of LMW Aβ by Filtration

(1)

7.3.2.6 Preparation of Aggregate-Free Aβ by Ultracentrifugation

(1)

7.3.2.7 Preparation of Oligomeric Aβ42

(1)

7.3.2.8 Preparation of Aβ Protofibrils

(1)

7.3.2.9 Preparation of Aβ Fibrils

(1)

7.4 Monitoring Aβ Assembly

(12)

7.4.1 Oligomerization

(2)

7.4.1.1 Determination of Oligomer Size Distributions Using PICUP

(1)

7.4.1.2 Determination of Oligomer Size Using SEC

(1)

7.4.1.3 Determination of Particle Diffusion Coefficients Using QLS

(1)

7.4.2 Fibril Formation

(3)

7.4.2.1 Thioflavin T (ThT) Binding

(1)

7.4.2.2 Congo Red Binding

(2)

7.4.2.3 Turbidity

(1)

7.4.3 Secondary Structure Determination

(2)

7.4.3.1 CD Spectroscopy

(2)

7.4.3.2 Fourier Transform Infrared Spectroscopy (FTIR)

(1)

7.4.4 Topographical Analysis

(3)

7.4.4.1 8-Anilino-l-Naphthalenesulfonic Acid (ANS) Binding

(1)

7.4.4.2 Intrinsic Fluorescence

100

(1)

7.4.4.3 Electron Paramagnetic Resonance (EPR)

100

(1)

7.4.4.4 Hydrogen-Deuterium Exchange

101

(1)

7.4.5 NMR Spectroscopy

102

(1)

7.4.6 Morphological Analysis

103

(10)

7.4.6.1 Electron Microscopy

103

(1)

7.4.6.2 Atomic Force Microscopy

103

(1)

7.5 Discussion

104

(1)

Acknowledgments

104

(1)

References

104

(7)

Chapter 8 Intracellular Accumulation of Amyloid β and Mitochondrial Dysfunction in Down's Syndrome

111

(18)

Jorge Busciglio, Alejandra Pelsman, Pablo Helguera, and Atul Deshpande

Abstract

112

(1)

8.1 Introduction

112

(1)

8.2 Experimental Procedures

113

(5)

8.2.1 Cell Culture

113

(1)

8.2.2 Antibodies

113

(1)

8.2.3 Immunocytochemical Procedures

113

(2)

8.2.3.1 Extraction

114

(1)

8.2.3.2 Fixation

114

(1)

8.2.3.3 Second Labeling Protocol

115

(1)

8.2.4 Western Blot

115

(1)

8.2.5 Inhibition of Energy Metabolism

115

(1)

8.2.6 Assessment of Mitochondrial Function

115

(2)

8.2.6.1 MTS Assay

116

(1)

8.2.6.2 JC1 Protocol

116

(1)

8.2.7 Cell Viability Assays

117

(1)

8.2.7.1 Trypan Blue

117

(1)

8.2.7.2 Propidium Iodide

117

(1)

8.2.8 Neuroprotection Assays

118

(1)

8.3 Results

118

(5)

8.3.1 Detection of Intracellular Aβ in DS Astrocytes

118

(2)

8.3.2 DS-Like Alterations in APP Processing Induced in Normal Astrocytes by Energy Depletion

120

(1)

8.3.3 Mitochondrial Dysfunction in DS Astrocytes

120

(2)

8.3.4 APPs Rescues DS Cortical Neurons from Apoptosis

122

(1)

8.4 Discussion

123

(3)

Acknowledgments

126

(1)

References

126

(3)

Chapter 9 Linking Alzheimer's Disease, β-Amyloid, and Lipids: A Technical Approach

129

(16)

Marcus O.W. Grimm, Andreas J. Paetzold, Heike S. Grimm, Eva G. Zinser, Thomas Ruppert, and Tobias Hartmann

9.1 Introduction

130

(1)

9.2 Cholesterol and Aβ

131

(4)

9.2.1 Cell Culture and Cholesterol Depletion

134

(1)

9.2.2 Protein Analysis

134

(1)

9.2.3 Results

135

(1)

9.3 Sphingolipids and AD

135

(3)

9.3.1 Cell Culture

135

(1)

9.3.2 Enzymatic Assay

135

(1)

9.3.3 Lipid Extraction

136

(1)

9.3.4 Phosphorus Determination

136

(1)

9.3.5 Scintillation Count

137

(1)

9.4 Analysis of Lipids by Mass Spectrometry

138

(3)

9.4.1 Theoretical Background

138

(2)

9.4.2 Sample Preparation

140

(1)

9.4.3 Sample Application

140

(1)

9.4.4 Measurement

140

(1)

9.4.4.1 Instrumentation

140

(1)

9.4.4.2 TOF pos Measurement

140

(1)

9.4.4.3 Precursor Ion Scans on m/z = 184.1, 188.1, and 264.3

140

(1)

9.4.5 Data Interpretation

141

(1)

9.4.6 Key Steps and Modifications

141

(1)

9.4.6.1 Sample Preparation

141

(1)

9.4.6.2 Sample Application

141

(1)

9.4.6.3 TOF pos Measurement

141

(1)

9.4.6.4 Precursor Ion Scan

141

(1)

9.4.7 Results

141

(1)

Acknowledgments

141

(2)

References

143

(2)

Chapter 10 Regulation of Amyloid Precursor Protein Processing by Lithium

145

(10)

Xiaoyan Sun and Akihiko Takashima

Abstract

145

(1)

10.1 Introduction

146

(1)

10.2 Experimental Procedures

146

(2)

10.2.1 Lithium Treatment in Transfected Cultured Cells

146

(1)

10.2.2 Aβ ELISA

147

(1)

10.2.3 Preparation of fibrillar Aβ (fAβ)

147

(1)

10.2.4 Microinjection of fAβ into Tau Transgenic Mice

147

(1)

10.2.5 Lithium Administration

148

(1)

10.3 Results

148

(3)

10.3.1 Establishment of Selective Sandwich Aβ ELISA

148

(1)

10.3.2 Effect of LiCI on Aβ Secretion in Association with GSK-3β Activity from APP C100-Transfected COS7 Cells

149

(1)

10.3.3 Effect of GSK-3β on fAβ-Induced Tau Pathology In Vivo

150

(1)

10.4 Discussion

151

(2)

References

153

(2)

Chapter 11 Immunocytochemical Analysis of Amyloid Precursor Protein and Its Derivatives

155

(10)

Gunnar Gouras and Reisuke H. Takahashi

11.1 Introduction

155

(1)

11.2 Experimental Approaches

156

(4)

11.2.1 Immunocytochemistry: Fixation

156

(1)

11.2.2 Immunocytochemistry: Controls

157

(1)

11.2.3 Immunocytochemistry for APP and Aβ

157

(1)

11.2.4 Immuno-Electron Microscopy for APP and Aβ

158

(2)

11.3 Conclusion

160

(1)

11.4 Experimental Procedures

160

(4)

11.4.1 Protocol I: Immunostaining Protocol for Paraffin-Embedded Sections

160

(1)

11.4.2 Protocol II: Immunoperoxidase Staining Protocol for Floating Sections

161

(1)

11.4.3 Protocol III: Immunoperoxidase Electron Microscopy

162

(1)

11.4.4 Protocol IV: Immuno-Electron Microscopy with Gold

163

(1)

Acknowledgments

164

(1)

References

164

(1)

Chapter 12 Pathological Detection of Aβ and APP in Brain

165

(20)

Chica Mori and Cynthia A. Lemere

12.1 Introduction

165

(2)

12.2 Methods

167

(6)

12.2.1 Tissue Processing Protocols

167

(1)

12.2.1.1 Paraffin Tissue

167

(1)

12.2.1.2 Frozen Tissue

168

(1)

12.2.2 Immunohi stochemi stry

168

(2)

12.2.2.1 Paraffin Sections

168

(1)

12.2.2.2 Frozen Sections

169

(1)

12.2.3 Congo Red

170

(1)

12.2.4 Thioflavin S Staining Protocol

170

(1)

12.2.5 Double Immunofluorescent Labeling

170

(1)

12.2.6 Double Labeling with DAB and Other Colored Markers

171

(2)

12.3 Results and Discussion

173

(9)

12.3.1 Aβ40 vs. Aβ42 in Down's Syndrome

173

(1)

12.3.2 Aβ40 vs. Aβ42 in Familial Alzheimer's Disease (FAD)

173

(2)

12.3.3 Intraneuronal Aβ in Down's Syndrome

175

(3)

12.3.4 Intraneuronal Aβ in APP Transgenic Mice

178

(1)

12.3.5 APP Immunoreactivity

178

(1)

12.3.6 Colocalization of Aβ with Glia

178

(4)

References

182

(3)

Chapter 13 Creating APP Transgenic Lines in Mice

185

(16)

Stanley Jones Premkumar Iyadurai and Karen Hsiao Ashe

13.1 Introduction

185

(6)

13.1.1 Origins of Gene Manipulation and Gene Transfer into Mouse Genome

186

(2)

13.1.2 Basic Principles for Consideration in Generating Transgenic Mice

188

(1)

13.1.3 APP Gene Structure: Its Isoforms and Promoters Used in Creating APP Transgenics

188

(3)

13.2 Experimental Procedures

191

(5)

13.2.1 Steps in Generating APP Transgenic Mice by Microinjection

191

(12)

13.2.1.1 Strain Selection

191

(1)

13.2.1.2 Isolation of Target DNA from Bacterial Host

192

(1)

13.2.1.3 Purification of DNA for Microinjection

193

(1)

13.2.1.4 Preparation of DNA for Microinjection

194

(1)

13.2.1.5 Microinjection with Admixed DNAs

194

(1)

13.2.1.6 Indentification of Founders

194

(1)

13.2.1.7 Maintenance and Analysis of Founders

195

(1)

13.3 Concluding Remarks

196

(1)

References

196

(5)

Chapter 14 Generation of Amyloid Precursor Protein Knockout Mice

201

(18)

Hui Zheng

14.1 Introduction

201

(2)

14.2 Experimental Procedures

203

(11)

14.2.1 Construction of APP Gene Targeting Vector

204

(3)

14.2.2 Embryonic Stem Cells: Culturing and Maintenance

207

(2)

14.2.2.1 Growing SNL Cells and Preparation of Feeders

207

(1)

14.2.2.2 Culturing and Electroporating Embryonic Stem Cells

208

(1)

14.2.3 Selection of Homologous Recombinant Clones

209

(3)

14.2.3.1 Selection of Recombinant Colonies

209

(1)

14.2.3.2 Expansion and Duplication of Recombinant Clones

210

(1)

14.2.3.3 Freezing ES Cells in 96-Well Plates

210

(1)

14.2.3.4 Mini-Southern Analysis of ES Clones to Identify Gene Targeting Events

211

(1)

14.2.3.5 Preparation of Gene-Targeted Clones for Blastocyst Injection

211

(1)

14.2.4 Generation of Gene (APP) Knockout Mice

212

(2)

14.2.4.1 Microinjection, Assessment of Chimerism, and Test for Germline Transmission

212

(1)

14.2.4.2 Breeding and Generation of Homozygous APP Knockout Mice

213

(1)

Acknowledgments

214

(5)

References

Index

219

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Amyloid Precursor Protein: A Practical Approach

Summary

Table of Contents

Supplemental Materials