(Ebook) Amyloid Precursor Protein A Practical Approach 1st Edition by Weiming Xia, Huaxi Xu ISBN 0849322456 9780849322457

(Ebook) Amyloid Precursor Protein A Practical Approach 1st Edition by Weiming Xia, Huaxi Xu ISBN 0849322456 9780849322457

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ISBN 10: 0849322456 
ISBN 13: 9780849322457
Author: Weiming Xia, Huaxi Xu

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 characterization techniques available. Exploring a variety of biological techniques to clarify the structure and function of this transmembrane protein, this text presents each method with detail

(Ebook) Amyloid Precursor Protein A Practical Approach 1st Table of contents:

1 Biochemical Characterization of Amyloid Precursor Protein
1.1 Introduction
1.2 Main Scheme of Approaches
1.3 Results
1.4 Discussion
1.5 Protocols
1.5.1 Overexpression of APP in Mammalian Cells by Transient Transfection
1.5.2 Selection of Stable Cell Line Overexpressing APP
1.5.3 Determination of Protein Concentration by BCA
1.5.4 Identification of APP and Its Derivatives by Western Blot
1.5.5 Radiolabeling of Cells with [35S]-Met
1.5.6 Identification of Full-Length APP and Its Derivatives by Immunoprecipitation
1.5.7 Determination of Half-Life of APP
1.5.8 Co-Immunoprecipitation of APP-Interacting Protein
1.5.8.1 Preparation
1.5.8.2 Pre-Absorption of Protein A-Sepharose
1.5.8.3 Pre-Clearing of Cell Lysates
1.5.8.4 Set Up Co-IP
1.5.8.5 Wash Co-IP
1.5.9 Conjugation of Antibody to Protein A-Sepharose
Acknowledgments
References
2 Assays for Analysis of APP Secretion and Recycling
Abstract
2.1 Introduction
2.2 Main Scheme of Approaches
2.3 Methods
2.3.1 Iodination of Antibody
2.3.2 Preparation of Cells
2.3.3 Kinetics of Secretion and Endocytosis of APP
2.3.4 Steady State Level of APP Endocytosis
2.3.5 Recycling of APP
2.3.6 Morphological Analysis
2.4 Discussion
Acknowledgments
References
3 Strategies for Crystallizing the N-Terminal Growth Factor Domain of Amyloid Precursor Protein
Abstract
3.1 Introduction
3.2 Overview of Approach
3.3 Bioinformatics Analysis of APP
3.4 Biological Roles of GFD
3.5 Previous Crystallization Studies
3.6 Expression of Recombinant GFD in
3.6.1 Materials
3.6.2 Method for Cloning
3.6.3 Method for Expression
3.7 Purification of GFD
3.7.1 Method
3.8 Crystallization of GFD
3.8.1 Materials
3.8.2 Method
3.9 Discussion
Acknowledgments
References
4 Analysis of Amyloid Precursor Protein Processing Protease β-Secretase: Tools for Memapsin 2 (β-Secretase) Inhibition Studies
4.1 Introduction
4.2 Assay of Memapsin 2 Activity
4.3 Synthesis of Memapsin 2 Inhibitors Based on APP Sequence
4.3.1 N-(tert-butoxycarbonyl)-L-leucine-N′-methoxy-N′-methylamide (3)
4.3.2 N-(tert-butoxycarbonyl)-L-leucinal (4)
4.3.3 Ethyl (4S,5S)- and (4R,5S)-5-[(tert-butoxycarbonyl)amino]-4hydroxy-7-methyloct-2-ynoate (5)
4.3.4 (5S,1′S)-5-[1′-[(tert-Butoxycarbonyl)amino]-3′-methylbutyl]-dihydrofuran-2(3H)-one (7)
4.3.5 (3R,5S,1′S)-5-[1′-[(tert-butoxycarbonyl)amino)]-3′-methylbutyl]-3 methyl dihydrofuran-2(3H)-one (8)
4.3.6 (2R,4S,5S)-5-[(tert-Butoxycarbonyl)amino]-4-[(tertbutyldimethylsilyl)oxy ]-2,7-dimethyloctanoicacid (9)
4.3.7 (2R,4S,5S)-5-[(fluorenylmethyloxycarbonyl)amino]-4-[(tertbutyldimethyl silyl)oxy]-2,7-dimethyloctanoic acid (10)
4.3.8 Coupling of Di-Isostere in Solid-Phase Peptide Synthesis
4.4 Determination of Inhibition Constants
4.5 Summary
References
5 Assays for Amyloid Precursor Protein γ-Secretase Activity
Abstract
5.1 Introduction
5.2 Main Scheme of Approaches
5.3 Method
5.3.1 Assaying γ-Secretase Activity in Living Cells
5.3.2 Subcellular Fractionation of Membrane Vesicles
5.3.3
5.3.4 Determining pH Dependence of γ-Secretase Activity Using Vesicles
5.3.5 Protease Inhibitor Profiling of γ-Secretase Activity Using Fractions
5.3.6 Cell Membrane Preparation for Exogenous Substrate Assay
5.3.6.1 Buffers
5.3.7 M2 Flag Purification of
5.3.7.1 Buffers
5.3.8
5.4 Discussion
References
6 Cell-Free Reconstitution of β-Amyloid Production and Trafficking
Abstract
6.1 Introduction
6.2 Methods
6.2.1 Pulse-Labeling of N2a Cells and Temperature Blocking of Nascent Protein Transport
6.2.2 Preparation of Permeabilized N2a Cells through Osmotic Shock and Mechanical Shear
6.2.3 Preparation of Cytosol for Cell-Free Reconstitution System
6.2.4 Preparation of Energy-Regenerating System for Cell-Free Reconstitution System
6.2.5 Aβ Generation in Cell-Free System Utilizing ERS in the Absenc of Cytosol
6.2.6 Detection of Aβ Production from Cell-Free System
6.2.7 Formation of Nascent Secretory Vesicles in Cell-Free System Supplemented with ERS and Cytosol
6.2.8 Detection of βAPP in Different Fractions through Immunoprecipitation
6.3 Discussion
6.3.1 ATP-Dependent Aβ Generation in Cell-Free Reconstitution System
6.3.2 Cytosol-Dependent βAPP Trafficking from TGN/ER in the Cell-Free Reconstitution System
6.3.3 Utilizing the Cell-Free Reconstitution System to Demonstrate PS1-Regulated Intracellular Trafficking and Surface Delivery of βAPP
6.3.4 Characterization of Vesicle and Membrane Fractions by Sucrose Gradient and Electron Microscopy
Acknowledgments
References
7 Studying Amyloid β-Protein Assembly
7.1 Introduction
7.2 Background
7.3 Peptide Production and Preparation
7.3.1 Production of Aβ Peptides
7.3.2 Preparation of Starting Peptide Stocks
7.3.2.1 HFIP Pretreatment
7.3.2.2 NaOH Pretreatment
7.3.2.3 Sample Clarification and Fibril Isolation
7.3.2.4 Preparation of LMW Aβ by Size Exclusion Chromatography
7.3.2.5 Preparation of LMW Aβ by Filtration
7.3.2.6 Preparation of Aggregate-Free Aβ by Ultracentrifugation
7.3.2.7 Preparation of Oligomeric Aβ
7.3.2.8 Preparation of Aβ Protofibrils
7.3.2.9 Preparation of Aβ Fibrils
7.4 Monitoring Aβ Assembly.
7.4.1 Oligomerization
7.4.1.1 Determination of Oligomer Size Distributions Using PICUP
7.4.1.2 Determination of Oligomer Size Using SEC
7.4.1.3 Determination of Particle Diffusion Coefficients Using QLS
7.4.2 Fibril Formation
7.4.2.1 Thioflavin T (ThT) Binding
7.4.2.2 Congo Red Binding
7.4.2.2.1 Turbidity
7.4.2.2.2 Secondary Structure Determination
7.4.2.3 Turbidity
7.4.3 Secondary Structure Determination
7.4.3.1 CD Spectroscopy
7.4.3.2 Fourier Transform Infrared Spectroscopy (FTIR)
7.4.4 Topographical Analysis
7.4.4.1 8-Anilino-1-Naphthalenesulfonic Acid (ANS) Binding
7.4.4.2 Intrinsic Fluorescence
7.4.4.3 Electron Paramagnetic Resonance (EPR)
7.4.4.4 Hydrogen–Deuterium Exchange
7.4.5 NMR Spectroscopy
7.4.6 Morphological Analysis
7.4.6.1 Electron Microscopy
7.4.6.2 Atomic Force Microscopy
7.5 Discussion
Acknowledgments
References
8 Intracellular Accumulation of Amyloid β and Mitochondrial Dysfunction in Down’s Syndrome
Abstract
8.1 Introduction
8.2 Experimental Procedures
8.2.1 Cell Culture
8.2.2 Antibodies
8.2.3 Immunocytochemical Procedures
8.2.3.1 Extraction
8.2.3.2 Fixation
8.2.3.3 Second Labeling Protocol
8.2.4 Western Blot.
8.2.5 Inhibition of Energy Metabolism
8.2.6 Assessment of Mitochondrial Function
8.2.6.1 MTS Assay
8.2.6.2 JC1 Protocol
8.2.7 Cell Viability Assays
8.2.7.1 Trypan Blue
8.2.7.2 Propidium Iodide
8.2.8 Neuroprotection Assays
8.3 Results
8.3.1 Detection of Intracellular Aβ in DS Astrocytes
8.3.2 DS-Like Alterations in APP Processing Induced in Normal Astrocytes by Energy Depletion
8.3.3 Mitochondrial Dysfunction in DS Astrocytes
8.3.4 APPs Rescues DS Cortical Neurons from Apoptosis
8.4 Discussion Acknowledgments
Acknowledgments
References
9 Linking Alzheimer’s Disease, β-Amyloid, and Lipids: A Technical Approach
9.1 Introduction
9.2 Cholesterol and Aβ.
9.2.1 Cell Culture and Cholesterol Depletion
9.2.2 Protein Analysis
9.2.3 Results
9.3 Sphingolipids and AD
9.3.1 Cell Culture
9.3.2 Enzymatic Assay
9.3.3 Lipid Extraction
9.3.4 Phosphorus Determination
9.3.5 Scintillation Count
9.4 Analysis of Lipids by Mass Spectrometry
9.4.1 Theoretical Background
9.4.2 Sample Preparation
9.4.3 Sample Application
9.4.4 Measurement
9.4.4.1 Instrumentation
9.4.4.2 TOF pos Measurement.
9.4.4.3 Precursor Ion Scans on m/z = 184.1, 188.1, and 264.3
9.4.5 Data Interpretation
9.4.6 Key Steps and Modifications
9.4.6.1 Sample Preparation
9.4.6.2 Sample Application
9.4.6.3 TOF pos Measurement
9.4.6.4 Precursor Ion Scan
9.4.7 Results
Acknowledgments
References
10 Regulation of Amyloid Precursor Protein Processing by Lithium
Abstract
10.1 Introduction
10.2 Experimental Procedures
10.2.1 Lithium Treatment in Transfected Cultured Cells
10.2.2 Aβ ELISA
10.2.3 Preparation of fibrillar Aβ (fAβ)
10.2.4 Microinjection of fAβ into Tau Transgenic Mice
10.2.5 Lithium Administration
10.3 Results
10.3.1 Establishment of Selective Sandwich Aβ ELISA
10.3.2 Effect of LiCl on Aβ Secretion in Association with GSK-3β Activity from APP C100-Transfected COS7 Cells
10.3.3 Effect of GSK-3β on fAβ-Induced Tau Pathology
10.4 Discussion References
References
11 Immunocytochemical Analysis of Amyloid Precursor Protein and Its Derivatives
11.1 Introduction
11.2 Experimental Approaches
11.2.1 Immunocytochemistry: Fixation
11.2.2 Immunocytochemistry: Controls
11.2.3 Immunocytochemistry for APP and Aβ
11.2.4 Immuno-Electron Microscopy for APP and Aβ
11.3 Conclusion
11.4 Experimental Procedures
11.4.1 Protocol I: Immunostaining Protocol for Paraffin-Embedded Sections
11.4.2 Protocol II: Immunoperoxidase Staining Protocol for Floating Sections
11.4.3 Protocol III: Immunoperoxidase Electron Microscopy
11.4.4 Protocol IV: Immuno-Electron Microscopy with Gold
Acknowledgments
References
12 Pathological Detection of Aβ and APP in Brain
12.1 Introduction
12.2 Methods
12.2.1 Tissue Processing Protocols
12.2.1.1 Paraffin Tissue
12.2.1.1.1 Humans
12.2.1.1.2 Mice
12.2.1.2 Frozen Tissue
12.2.2 Immunohistochemistry
12.2.2.1 Paraffin Sections
12.2.2.2 Frozen Sections
12.2.3 Congo Red
12.2.4 Thioflavin S Staining Protocol
12.2.5 Double Immunofluorescent Labeling
12.2.6 Double Labeling with DAB and Other Colored Markers
12.3 Results and Discussion
12.3.1 Aβ40 vs. Aβ42 in Down’s Syndrome
12.3.2 Aβ40 vs. Aβ42 in Familial Alzheimer’s Disease (FAD)
12.3.3 Intraneuronal Aβ in Down’s Syndrome
12.3.4 Intraneuronal Aβ in APP Transgenic Mice
12.3.5 APP Immunoreactivity
12.3.6 Colocalization of Aβ with Glia
References
13 Creating APP Transgenic Lines in Mice
13.1 Introduction
13.1.1 Origins of Gene Manipulation and Gene Transfer into Mouse Genome
13.1.2 Basic Principles for Consideration in Generating Transgenic Mice
13.1.3 APP Gene Structure: Its Isoforms and Promoters Used in Creating APP Transgenics
13.2 Experimental Procedures
13.2.1 Steps in Generating APP Transgenic Mice by Microinjection
13.2.1.1 Strain Selection
13.2.1.2 Isolation of Target DNA from Bacterial Host
13.2.1.3 Purification of DNA for Microinjection
13.2.1.4 Preparation of DNA for Microinjection
13.2.1.5 Microinjection with Admixed DNAs
13.2.1.6 Indentification of Founders
13.2.1.7 Maintenance and Analysis of Founders
13.3 Concluding Remarks
References
14 Generation of Amyloid Precursor Protein Knockout Mice
14.1 Introduction
14.2 Experimental Procedures
14.2.1 Construction of APP Gene Targeting Vector
14.2.2 Embryonic Stem Cells: Culturing and Maintenance
14.2.2.1 Growing SNL Cells and Preparation of Feeders
14.2.2.2 Culturing and Electroporating Embryonic Stem Cells
14.2.3 Selection of Homologous Recombinant Clones
14.2.3.1 Selection of Recombinant Colonies
14.2.3.2 Expansion and Duplication of Recombinant Clones
14.2.3.3 Freezing ES Cells in 96-Well Plates
14.2.3.4 Mini-Southern Analysis of ES Clones to Identify Gene Targeting Events
14.2.3.5 Preparation of Gene-Targeted Clones for Blastocyst Injection
14.2.4 Generation of Gene (APP) Knockout Mice
14.2.4.1 Microinjection, Assessment of Chimerism, and Test for Germline Transmission
14.2.4.2 Breeding and Generation of Homozygous APP Knockout Mice

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Tags: Weiming Xia, Huaxi Xu, Amyloid, Precursor