Amyloid Precursor Protein - A Practical Approach - W. Xia, H. Xu (CRC, 2005) WW

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AmyloidPrecursor

ProteinA Practical Approach

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EDITED BY

Weiming Xia and Huaxi Xu

AmyloidPrecursor

ProteinA Practical Approach

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Library of Congress Cataloging-in-Publication Data

Xia, Weiming.Amyloid precursor protein : a practical approach / by Weiming Xia, Huaxi Xu.

p. cm.Includes bibliographical references and index.ISBN 0-8493-2245-6 (alk. paper)1. Amyloid beta-protein precursors — Laboratory manuals. I. Xu, Huaxi. II. Title.

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Preface

Amyloid precursor protein (APP) is an extensively studied single transmembraneprotein. Our vast knowledge of this protein is derived from thousands of reportspublished during the past decade. In fact, many scientists from different disciplineshave used their well-established experimental procedures to examine the character-istics of this molecule. Hence, their published reports display nearly all aspects ofbiological techniques used in genetics, molecular biology, cell biology, and biochem-istry. As a result, APP may be viewed as a unique model protein to illustrate a widearray of basic and advanced biological techniques used in many laboratories.

The major aim of this book is to demonstrate the critical techniques utilized inthe experiments selected from many significant findings that contribute to our under-standing of APP biology. Each technique will be presented in the format of a standardprotocol, providing step-by-step instructions for bench scientists carrying out similarstudies on APP and other proteins. Theoretical background and discussions willbe provided in the introduction section, and a brief description of the goal will also bepresented. These protocols will form the core of our approach in elucidating thefunction of a protein. The antibodies used in each experiment will be described andare cited on the list of antibodies to APP and Aβ proteins at the front of this bookIt is our intention to contrast basic and advanced methods and demonstrate howdevelopment of biological techniques significantly affects the way we examine ourmolecular targets.

An important feature of this book is the presentation of modifications appliedto standard procedures used in examining a membrane protein. These modificationswill likely help readers consider similar alterations in their own experimental pro-cedures. The fact that most experiments we perform every day do not lead toconclusive answers strongly indicates that actively modifying our approach is essen-tial to perform biological experiments and achieve definitive results. The descriptionsof the modifications will help justify similar alterations in readers’ own experimentalapproaches.

Another goal of this book is to include commonly used experimental proceduresand clearly present them in the format of a protocol to serve as a laboratory manualfor bench scientists working on different aspects of the biological functions of APPand other membrane proteins. Most experimental procedures can be carried out ina regularly equipped laboratory, but a few protocols require sophisticated corefacilities. In addition, multistep protocols will be broken down into several indepen-dent protocols, allowing an investigator to create parallel experiments to acceleratethe achievement of results. This book will also describe a set of previously publishedmilestone studies on APP. Results summarized here will not only provide a completepicture of our current understanding of APP, but also confer future direction forcontinued investigation of this protein in normal cellular function and in disease.

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Due to rapid expansion of our knowledge of APP biology, this book will cover themost up-to-date research activities. More importantly, we emphasize practical tech-niques used to address key questions related to APP and similar membrane proteins.Hence, this book will offer a framework for studying other membrane proteins andprovide detailed, step-wise procedures to achieve specific aims. It will be suitablefor students who are learning basic experimental approaches to address biologicalquestions and also for bench scientists who seek immediate assistance and practicalapproaches for studying proteins.

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Contributors

Karen Hsiao AsheDepartment of NeurologyUniversity of MinnesotaMinneapolis, Minnesota

Jorge BusciglioDepartment of Neurobiology and

BehaviorUniversity of CaliforniaIrvine, California

Dongming CaiFisher Center for Research on

Alzheimer’s DiseaseThe Rockefeller UniversityNew York, New York

William A. CampbellCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Roberto CappaiDepartment of PathologyThe University of MelbourneMelbourne, Victoria, Australia

Atul DeshpandeDepartment of Neurobiology and


Susanne C. FeilBiota Structural Biology LaboratorySt. Vincent’s Institute of Medical

ResearchFitzroy, Victoria, Australia

Erica A. FradingerCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Denise GalatisDepartment of PathologyThe University of MelbourneMelbourne, Victoria, Australia

Arun K. GhoshDepartment of ChemistryUniversity of Illinois at ChicagoChicago, Illinois

Gunnar GourasDepartment of Neurology and

NeuroscienceWeill Medical College of Cornell

UniversityNew York, New York

Heike S. GrimmCenter for Molecular BiologyUniversity of HeidelbergHeidelberg, Germany

Marcus O.W. GrimmCenter for Molecular BiologyUniversity of HeidelbergHeidelberg, Germany

Tobias HartmannCenter for Molecular BiologyUniversity of HeidelbergHeidelberg, Germany

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Pablo HelgueraDepartment of Neurobiology and


Stanley Jones Premkumar IyaduraiDepartment of NeurologyUniversity of MinnesotaMinneapolis, Minnesota

Gerald KoelschProtein Studies ProgramOklahoma Medical Research

FoundationOklahoma City, Oklahoma

Edward H. KooDepartment of NeurosciencesUniversity of California, San DiegoLa Jolla, California

Markus P. KummerDepartment of NeurosciencesUniversity of California, San DiegoLa Jolla, California

Noel D. LazoCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Cynthia A. LemereCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Feng LiCenter for Neuroscience and AgingThe Burnham InstituteLa Jolla, California

Samir Kumar MajiCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

William J. McKinstryBiota Structural Biology LaboratorySt. Vincent’s Institute of Medical


Chica MoriCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

William J. NetzerFisher Center for Research on

Alzheimer’s DiseaseThe Rockefeller UniversityNew York, New York

Andreas J. PaetzoldCenter for Molecular BiologyUniversity of HeidelbergHeidelberg, Germany

Michael W. ParkerBiota Structural Biology LaboratorySt. Vincent’s Institute of Medical


Alejandra PelsmanDepartment of Neurobiology and


Thomas RuppertCenter for Molecular BiologyUniversity of HeidelbergHeidelberg, Germany

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Dongwoo ShinDepartment of ChemistryUniversity of Illinois at ChicagoChicago, Illinois

Xiaoyan SunCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Reisuke H. TakahashiDepartment of Neurology and

NeuroscienceWeill Medical College of Cornell

UniversityNew York, New York

Akihiko TakashimaBrain Science InstituteInstitute of Physical and Chemical

Research Saitama, Japan

Jordan TangProtein Studies ProgramOklahoma Medical Research FoundationOklahoma City, Oklahoma

David B. TeplowCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Vajira WeerasenaProtein Studies ProgramOklahoma Medical Research FoundationOklahoma City, Oklahoma

Michael S. WolfeCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Weiming XiaCenter for Neurologic DiseasesBrigham and Women’s HospitalHarvard Medical SchoolBoston, Massachusetts

Huaxi XuCenter for Neuroscience and AgingThe Burnham InstituteLa Jolla, California

Tsuneo YamazakiDepartment of NeurologyGraduate School of MedicineGunma UniversityGunma, Japan

Hui ZhengDepartment of Molecular and Human

GeneticsBaylor College of MedicineHouston, Texas

Eva G. ZinserCenter for Molecular BiologyUniversity of HeidelbergHeidelberg, Germany

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Table of Contents

List of Antibodies to APP and Aβ Proteins

Chapter 1 Biochemical Characterization of Amyloid Precursor Protein

Weiming Xia

Chapter 2 Assays for Analysis of APP Secretion and Recycling

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

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

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

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

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

Chapter 5 Assays for Amyloid Precursor Protein γ-Secretase Activity

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

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

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

Chapter 7 Studying Amyloid β-Protein Assembly

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

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

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

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

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

Chapter 10 Regulation of Amyloid Precursor Protein Processing by Lithium

Xiaoyan Sun and Akihiko Takashima

Chapter 11 Immunocytochemical Analysis of Amyloid Precursor Protein and Its Derivatives

Gunnar Gouras and Reisuke H. Takahashi

Chapter 12 Pathological Detection of Aβ and APP in Brain

Chica Mori and Cynthia A. Lemere

Chapter 13 Creating APP Transgenic Lines in Mice

Stanley Jones Premkumar Iyadurai and Karen Hsiao Ashe

Chapter 14 Generation of Amyloid Precursor Protein Knockout Mice

Hui Zheng

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List of Antibodies to APP and Aβ Proteins

Name Source Immunogen (Specific)Host

(Formulation) MethodsDescription in Chapter

22C11 Chemicon Cat# MAB348 N terminal, purified recombinant APP A4 fusion protein

Monoclonal mouse (lyophilized, azide)

IH, WB 6

Alz-90 (1.D5)

Chemicon Cat# MAB349 Synthetic aa 511-608 of APP pre A4-695

Monoclonal mouse (purified, lyophilized, azide)

WB 8

369 S. [email protected]

C terminal APP Polyclonal IH, WB 6

APP 192 Elan Pharmaceuticals Specific to carboxyl terminus of β-secretase cleavage site of APP

Polyclonal IH, WB 1

1736 D. Selkoe [email protected]

Residues 595–611 of APP695 (α-sAPP)

Polyclonal WB 1

C7 D. Selkoe [email protected]

Against 20 C terminal residues of APP

Polyclonal IH, IP 1, 5, 12

C8 D. Selkoe [email protected]

Against 20 C terminal residues of APP

Polyconal IP, WB 8

R57 David [email protected]

Residues 672–695 of APP695

Polyclonal WB 5

8E5 Elan Pharmaceuticals Residues 444–591 of APP (β−APPs and APP)

Monoclonal WB 12

13G8 Elan Pharmaceuticals Residues 676–695 of APP695

Monoclonal IP, WB 1, 5

1G7 E.H. [email protected]

Residues 380–665 of APP Monoclonal IP, IF, WB

2

5A3 EH. [email protected]

Recognize nonoverlapping epitopes in extracellular region of APP

Monoclonal IP, IF, WB

2

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4G8 Signet Cat# 9200, 9220 Amino acid residues 17–24 of β-amyloid peptide (reactive to aa 17–24 Aβ and to APP)

Monoclonal, mouse (crude, ascites)

ELISA, IH, IP, WB

6, 12

6E10 Signet Cat# 9300, 9320 Amino acid residues 1–17 of β-amyloid peptide (reactive to aa 1–17 Aβ and to APP)

Monoclonal, mouse (crude, ascites)

ELISA, IH, IP, WB

6, 10, 12


Raised to Aβ 1–40 (reactive to Aβ and P3)

Polyclonal IP 1

0-2 Genetics CompanyCat# AB-10

aa 1–10 of N terminal of human Aβ (high affinity to amyloid peptides Aβ1-38, Aβ1-39, Aβ1-40, Aβ1-43, and Aβ1-44)

Monoclonal mouse

ELISA, WB

9


Amino acid residues 31–40 of human Aβ peptide at C terminal (Aβ-peptide, aa 31–40; not Aβ1-38, Aβ1-39, Aβ1-42, Aβ1-43, or Aβ1-44)

Monoclonal mouse

ELISA, WB

9


Amino acid residues 33–42 of human Aβ 42 peptide at C terminal (Aβ-peptide, aa 33–42; not Aβ1-38, Aβ1-39, Aβ1-40, Aβ1-43, or Aβ1-44)

Monoclonal mouse

ELISA, WB

9

BC40 H. Yamaguchi [email protected]

Amino acid residues 1–40 of β-amyloid peptide (reactive to Aβ 40)

Monoclonal IH, WB 10, 11, 12

BC42 H. Yamaguchi [email protected]

Amino acid residues 1–42 of β-amyloid peptide (reactive to Aβ 42)

Monoclonal IH, WB 10, 11, 12

-amyloid 1-42

Chemicon Cat# AB5078P

β-amyloid 1-42 (recognizes β-amyloid 1-42)

Polyclonal rabbit

WB, IH, IF, IP, ELISA

11

1F12 Elan Pharmaceuticals β-amyloid 33-42 Monoclonal mouse

IH, ELISA, WB

12


Aβ 1−40 Polyclonal rabbit

IH, IP 12

H = Immunohistochemistry; WB = Western blot; IP = Immunoprecipitation; IF = Immunofluorescence; ELISA =nzyme-linked immunosorbent assay.

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1
Biochemical Characterization of Amyloid Precursor Protein
Weiming Xia

CONTENTS

1.1 Introduction1.2 Main Scheme of Approaches1.3 Results1.4 Discussion1.5 Protocols

1.5.1 Overexpression of APP in Mammalian Cells by Transient Transfection

1.5.2 Selection of Stable Cell Line Overexpressing APP1.5.3 Determination of Protein Concentration by BCA1.5.4 Identification of APP and Its Derivatives by Western Blot1.5.5 Radiolabeling of Cells with [35S]-Met1.5.6 Identification of Full-Length APP and Its Derivatives by

Immunoprecipitation1.5.7 Determination of Half-Life of APP1.5.8 Co-Immunoprecipitation of APP-Interacting Protein

1.5.8.1 Preparation1.5.8.2 Pre-Absorption of Protein A-Sepharose1.5.8.3 Pre-Clearing of Cell Lysates1.5.8.4 Set Up Co-IP1.5.8.5 Wash Co-IP

1.5.9 Conjugation of Antibody to Protein A-SepharoseAcknowledgmentsReferences

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1.1 INTRODUCTION

Amyloid precursor protein (APP) is a single transmembrane protein that undergoessequential proteolysis to generate multiple peptides, including the amyloid β-peptide(Aβ) — the major component of the senile plaques that are diagnostic hallmarks ofAlzheimer’s disease (AD).1,2 AD accounts for more than 50% of cases of dementiain the elderly and has a prevalence estimated at 15 to 20 million patients worldwide.It is associated with progressive memory loss that leads to profound dementia andeventually death, although a patient can have the disease for as long as 10 to 15 yearsbefore death.

The pathology of AD is characterized by extracellular neuritic plaques consistingof Aβ and intracellular neurofibrillary tangles.3 A central role for Aβ in the patho-genesis of AD was first discovered by finding APP mutations in a subset of familialAD (FAD) cases that occurred as inherited autosomal dominant disease.4,5 The APPgene is located on chromosome 21, and mutations found in APP occur either withinthe Aβ peptide sequence (A692G6 and E693Q,7,8 APP770 numbering) or immediatelyflanking the Aβ peptide sequence including KM670/671NL (Swedish mutation),9

I716V,10 and V717I,G,F mutations.5,11,12

Mutations in APP are rare but they have been very informative. Patients carryingtrisomy 21 (Down’s syndrome) develop the histopathology of AD in early midlife,presumably because they have three copies of the APP gene and a documentedincrease in APP transcription13 that leads to augmented Aβ deposition. The doublemutation in APP at the Aβ N terminus (Swedish mutation) leads to a marked increasein Aβ production,9,14–16 as confirmed in primary skin fibroblasts and the plasma ofpresymptomatic and symptomatic carriers.17 The mutations at APP716 or 7175,11,12

lead to hypersecretion of the longer and more amyloidogenic Aβ42 peptide.10,18 Whenhuman APP containing the Val → Phe mutation19 or the Swedish mutation20 isoverexpressed in transgenic mice, a time-delayed accumulation of both diffuse andneuritic Aβ plaques develops. These various studies provide strong circumstantialevidence for the early mechanistic role of abnormal APP metabolism and Aβ dep-osition in AD neuropathology.

In this chapter, we will discuss several basic biochemical approaches routinelyused to study full-length APP and shorter peptides derived from proteolytic cleavagesof APP holoprotein (Figure 1.1); the same approaches can be used to characterizeany newly identified proteins.

1.2 MAIN SCHEME OF APPROACHES

To characterize a newly cloned gene product, transient transfection is a quick andsimple way to enrich the protein of interest for biochemical analysis. Here we presenta standard protocol to transiently transfect a mammalian expression vector contain-ing APP cDNA into Chinese hamster ovary (CHO) cells (Protocol 1.5.1). Duringthe 24 to 48 hr post-transfection, cells can either be harvested for immediate analysisor used for screening of clones to make a cell line stably expressing APP. In thelatter case, the selection drug (based on the selection marker in the expression vectorused for transient transfection) will be used for screening (Protocol 1.5.2).

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A large number of antibodies against different regions of APP are available frommany laboratories and commercial sources. Using these antibodies, cell lysates withequivalent amounts of protein concentrations (determined by Bicinchoninic AcidKit [BCA]; Protocol 1.5.3) can be separated by SDS-PAGE followed by Westernblotting analysis (Protocol 1.5.4). An alternative approach would be to metabolicallylabel cells with [35S]-methionine (Met) (Protocol 1.5.5), and lyse the radiolabeledcells for immunoprecipitation with specific antibodies (Protocol 1.5.6). Radiolabel-ing cells followed by immunoprecipitation usually enhances the signal-to-noiseratios of overexpressed proteins.

Proteins identified by Western blot and immunoprecipitation usually representmature species at steady state levels. To examine the process of protein maturation,cells can be pulse-labeled with [35S]-Met followed by chasing in nonradioactivemedia for various periods of time (Protocol 1.5.7). Cell lysates will then be immuno-precipitated with specific antibodies.

A simple co-immunoprecipitation method using an antibody against a candidateprotein (e.g., presenilin) can be used to determine whether this protein interacts withAPP (Protocol 1.5.8). By using free antibody or protein A sepharose-conjugated

FIGURE 1.1 Proteolytic cleavage of amyloid precursor protein by α-, β- and γ-secretases.APP is proteolytically processed by two alternative pathways. First, α-secretase cleavesslightly N terminal to the beginning of the APP transmembrane domain (at residues 16 and17 of the Aβ region) and generates a major secreted derivative (α-APPs) and a ~10-kDaC-terminal fragment (C83). C83 can be cleaved by a protease activity called γ-secretase toyield p3. Alternative cleavage of APP by the β-secretase (called BACE or memapsin 2)generates a soluble N-terminal fragment (β-APPs) and a 12-kDa C-terminal fragment of APP(C99) that can be further cleaved by γ-secretase to yield two major species of Aβ ending atresidue 40 (Aβ40) or 42 (Aβ42).

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antibody (Protocol 1.5.9), co-immunoprecipitation is widely used to confirm a can-didate protein that specifically interacts with APP.

1.3 RESULTS

APP occurs in three alternatively spliced forms of 695, 751, and 770 residues, andthese proteins undergo N- and N + O glycosylation as well as phosphorylation. InCHO cells stably expressing APP, both N- and N + O-glycosylated APP holoproteinscan be detected by immunoprecipitation of radiolabeled cells with antibody C7,which was raised against the last 21 amino acids of the APP C terminus(APP675–695, APP695 numbering; Figure 1.2a). APP is proteolytically processed byat least two broad alternative pathways (Figure 1.1).

First, cleavage of APP by β-secretase (called BACE or memapsin 2)21–24 gener-ates a soluble N-terminal fragment (β-APPs, Figure 1.2b), and a C-terminal stub ofAPP (C99, Figure 1.2d),25 which can be further cleaved by a protease activity calledγ-secretase to yield two major species of Aβ ending at residue 40 (Aβ40) or 42 (Aβ42).See Figure 1.2e.26,27 Since soluble β-APPs and Aβ generated in radiolabeled cellsare secreted into media, they can be detected by immunoprecipitation of media with

FIGURE 1.2 Detection of APP and its derivatives by immunoprecipitation. (a) Both N- andN+O-glycosylated APP holoproteins were immunoprecipitated from radiolabeled CHO cellsstably expressing APP with antibody C7, which was raised against the last 21 amino acidsof the APP C terminus. (b) Soluble β-APPs from radiolabeled cells were secreted into mediaand could be detected by immunoprecipitation of media with specific antibody 192 whichspecifically recognizes the C termini of β-APPs. (c) Soluble α-APPs can be detected byimmunoprecipitation of growth media with antibody 1736 which recognizes the C termini ofα-APPs. (d) APP-expressing cells were lysed and immunoprecipitated with antibody C7,followed by Western blotting with another C terminus antibody, 13G8, to detect C99 andC83. (e) Both Aβ and p3 were immunoprecipitated from growth media of radiolabeled APP-expressing CHO cells using antibody 1280, which was raised against the whole region of Aβ.

N-APP

N+O-APP

100

100 100β-APPs α-APPs

14

6

3C83C99

p3

Aβ

a.

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100 100β-APPs α-APPs

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p3

Aβ

a.

d.

b.

e.

c.

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specific antibodies. Antibody 192 can specifically recognize the C termini of β-APPs(Figure 1.2b). Antibody 1280 was raised against the whole region of Aβ, and canthus recognize both Aβ and p3 (Figure 1.2e).

Alternative cleavage slightly N terminal to the beginning of the APP transmem-brane domain (at residues 16–17 of the Aβ region) by a α-secretase protease28,29

generates the major secreted derivatives, α-APPs (Figure 1.2c), precluding Aβ for-mation (nonamyloidogenic).30,31 The ~10-kDa C-terminal stub (C83, Figure 1.2d)can be cleaved by γ-secretase to yield p340 and p342 (Figure 1.2e),32 which can berecognized by antibody 1280. Soluble α-APPs can be detected by immunoprecipi-tation of growth media with antibody 1736, which recognizes the C termini ofα-APPs (Figure 1.2c).

Another approach to detecting APP and its derivatives is to perform immunopre-cipitation followed by Western blotting; this approach does not require radiolabelingof cells. For example, APP-expressing cells can be lysed and immunoprecipitatedwith antibody C7. The immunoprecipitates, full length APP, C83, and C99, all carrythe antigen for C7, namely, the C terminus of APP. After immunoprecipitates areseparated by SDS-PAGE, Western blotting with another C-terminal antibody, 13G8,can be used to detect these peptides. This is a convenient approach to detect fulllength APP as well as C99 and C83 (Figure 1.2d).

The temporal events of APP glycosylation and proteolytic processing occurwithin a very short period. Within a half hour of protein translation, the majority ofAPP is glycosylated, as determined by pulse-chase labeling of APP expressing cellswith [35S]-Met (Figure 1.3). After APP-expressing cells were incubated with Met-free media for 45 min at 37˚C, cells were pulse-labeled with [35S]-Met for 5 min,and then the medium was changed to regular Dulbecco’s modified Eagle’s mediumand chased for 0.25 to 5 hr. Cells were lysed and immunoprecipitated with antibodyC7, followed by SDS-PAGE. The half life of APP was very short (~30 min) andmost of the holoprotein was either degraded or proteolytically cleaved by β- orα-secretase to generate C99/C83 within 1.5 hr (Figure 1.3). Like the APP holopro-tein, almost all of the C83/C99 was either degraded or cleaved by γ-secretase, andno C83/C99 was detectable after 5 hr (Figure 1.3).

To determine the spatial distribution of immature and mature APP, membranevesicles enriched in different subcellular compartments were separated by fraction-ation on discontinuous Iodixanol sucrose gradients (see Chapter 5). A total of 12fractions were collected, and each fraction was analyzed by Western blotting. Forendoplasmic reticulum (ER)-rich fractions, an antibody against the ER marker pro-tein, calnexin, was used (Figure 1.4a). The densest fractions (1 through 4) had thestrongest immunoreactivities for calnexin, indicating that these fractions containedER vesicles.

For Golgi/trans-Golgi network (TGN)-enriched fractions, β-1,4-galactosyltrans-ferase activity in each fraction was measured, i.e., the addition of [3H]-galactoseonto the oligosaccharides of an acceptor protein, ovomucoid, was measured.33 Sinceβ-1,4-galactosyltransferase is a marker for Golgi/TGN-type vesicles, fractions 4through 8 were enriched in Golgi/TGN vesicles (Figure 1.4b). Alternatively, an

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antibody against another Golgi/TGN marker protein, syntaxin 6, can be used tocharacterize these subcellular fractions. When these subcellular fractions wereprobed for APP distribution with APP monoclonal antibodies 5A3/1G7, which candetect both N- and N + O-glycosylated APP proteins (Figure 1.4c), fractions 1through 3 only had N-glycosylated APP proteins. The lack of further glycosylationof these immature APP proteins suggests that N-glycosylated APP resides primarilyin the ER (Figure 1.4c).34–36 Both N- and N + O-glycosylated APP proteins wereobserved from fraction 4 to fraction 8, indicating that post-translational modificationis completed during the passage of APP into the Golgi/TGN compartment.34

Post-translationally modified APP continues to transport through the centralvacuolar pathway and finally reaches the cell surface. A large portion of APPundergoes endocytosis and is re-internalized to endosomes (see Chapter 2).

To examine whether another AD-linked gene product, presenilin 1 (PS1), inter-acts with APP, co-immunoprecipitation of APP and PS1 was carried out (Protocol1.5.8). When cells were lysed and lysates were immunoprecipitated with PS1 anti-bodies X81, R22, or 4627 (against N-terminal, middle, and C-terminal regions ofPS1, respectively) followed by Western blotting with the APP C-terminal antibody13G8, full-length APP was clearly detected (Figure 1.5). The specificity of thisinteraction was demonstrated by the observation that only the N-glycosylated formof full-length APP co-precipitated with PS1 (Figure 1.5).

FIGURE 1.3 Maturation of APP and turnover of APP and C terminal fragments. CHO cellsstably expressing APP were pulse-labeled for 5 min and chased for 0 to 5 hr. Cell lysateswere immunoprecipitated with C7. The half life of APP was ~30 min, and a portion of APPholoprotein was cleaved by β- or α-secretase to generate C99/C83 within 1.5 hr. Most C83and C99 were either degraded or cleaved by γ-secretase, and no C83/C99 was detectable after5 hr.

14C83C99

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28

71

110

210

N-APPN+O-APP

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1.4 DISCUSSION

This chapter has presented several basic approaches to characterize the biochemicalproperties of APP. These experimental procedures are routinely utilized in manylaboratories, and the protocols listed in this chapter usually serve as starting methodsto study transmembrane proteins. Various modifications can be introduced to meetspecial needs for individual proteins.

While more than a dozen transfection reagents are available to introduce expres-sion vectors into mammalian cells, the toxicity of reagents usually counteracts thetransfection efficiency. Therefore, it is necessary to titrate the amount of DNA/trans-fection reagent to obtain the optimal transfection efficiency with minimum toxicity.A simple test is to co-transfect the gene of interest with another vector expressinggreen fluorescent protein (GFP), and the expression levels of GFP can be monitoredunder a fluorescent microscope 24 to 48 hr post-transfection. If cellular toxicity is

FIGURE 1.4 Characterization of immature and mature APP in subcellular fractions. (a)Distribution of the ER marker protein (calnexin) in discontinuous Iodixanol gradient fractionswas detected by Western blotting with anti-calnexin antibody (densest fraction, lane 1; lightestfraction, lane 12). (b) Distribution of the Golgi/TGN marker β-1,4-galactosyl transferaseactivity was mainly in fractions 4 through 8. (c) The same fractions were immunoblotted withAPP monoclonal antibodies 5A3/1G7. Fractions 1 through 3 were rich in ER vesicles andcontained solely N-glycosylated APP; fractions 4 through 8 were rich in Golgi/TGN vesicleswhich contained both N- and N + O-glycosylated APP. Fraction 4 represented a transitionfraction in the discontinuous gradient and contained both ER and Golgi/TGN proteins. (FromXia, W. et al. Proc. Natl. Acad. Sci. USA, 97, 9299–9304, 2000; Xia, W. et al. Biochemistry37, 16465–16471, 1998. With permission.)

N-APPN+O-APP

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68

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c.

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obvious even when a low amount of DNA/transfection reagent is used, then over-expressing the target gene may cause enough cellular damage that raising a stablecell line is not feasible.

Detection of proteins by immunoprecipitation and/or Western blot largelydepends on the specificity of an antibody. Nevertheless, the choice of detergent inthe lysis buffer is important to successfully lyse a membrane protein. This is espe-cially critical when co-immunoprecipitation is performed to search for any interact-ing proteins that form a complex with the transmembrane protein. Because manytransmembrane proteins tend to interact nonspecifically and form aggregates undernon-physiological conditions, i.e., overexpression of two transmembrane proteins intransiently transfected cells, stringent conditions should be tested to differentiate aspecific protein–protein interaction versus nonspecific hydrophobic aggregation. Insome cases, steric hindrance may interfere with an antibody binding to a specificregion during co-immunoprecipitation, Therefore, using multiple antibodies againstdifferent regions of the protein for co-immunoprecipitation is necessary to confirma specific interaction. Reverse co-immunoprecipitation should be carried out to provea direct interaction between two proteins. In addition, examining the occurrence andlocalization of the complex will also help explain the physiological significance ofthe interaction between two proteins.

In conclusion, the standard protocols listed in this chapter provide an outline ofexperiments that can be immediately performed to study a new protein. Understand-ing the basic biochemical properties of a protein is not only the basis for future invitro and in vivo studies, but also represents the first step to explore its biologicalfunction.

FIGURE 1.5 Co-immunoprecipitation of APP and PS1. Lysates from APP- and PS1-express-ing cells were co-immunoprecipitated with either PS1 antibodies (X81, 4627, or R22) orpreimmune serum (preimm), followed by Western blotting with APP antibody 13G8. TheAPP species that co-immunoprecipitated with PS1 co-migrated with the N-glycosylated formof APP detected on straight Western blots of the lysates. The lower band in lanes 2 through4 is nonspecific. (From Xia, W. et al. Proc. Natl. Acad. Sci. USA, 97, 9299–9304, 2000. Withpermission.)

N-APP

N+O-APP

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X81 4627 R22 Preimm C7

PS1 antibody APP antibody

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1.5 PROTOCOLS

1.5.1 OVEREXPRESSION OF APP IN MAMMALIAN CELLS BY TRANSIENT TRANSFECTION

1. Split CHO cells in a six-well plate the day before transfection so that theyare 90 to 95% confluent the following day. Plate cells in 1.5 ml of theirnormal growth medium.

2. For each well of cells, dilute 4 µg of DNA in 250 µl medium withoutserum (e.g., Opti-MEM I). Additionally, dilute 12 µl Lipofectamine 2000(Invitrogen, #11668-019) in 250 µl Opti-MEM I for each well of cellsand incubate for 5 min at room temperature.

3. Combine diluted Lipofectamine and the DNA within 30 min. Incubate atroom temperature for 20 min to allow DNA–Lipofectamine complexes toform.

4. Add 500 µl of the DNA–Lipofectamine mixture to each well of cells andmix gently.

5. Incubate cells at 37˚C in a CO2 incubator for 24 to 48 hr. Growth mediummay be replaced after 4 to 6 hr.

1.5.2 SELECTION OF STABLE CELL LINE OVEREXPRESSING APP

1. At 48 hr post-transfection, detach the cells by brief trypsin treatment,count the cells, and make serial dilutions to obtain a final concentrationof 1 cell/100 µl of media containing a selection drug (e.g., G418). Transfer100 µl of media to each well of a 96-well plate, resulting in a final cellcount of approximately one cell per well. A total of six to eight platesshould be prepared for selection of multiple clones of the stable cell line.

2. Formation of a single colony of cells in individual wells is monitoredunder a microscope after a growth period of 2 weeks. Only wells containingsingle colonies of cells will be selected. Transfer cells to a 24-well platefor growth.

3. Prepare duplicate wells of cells from the same clone and lyse one wellof cells for measuring expression levels of APP by Western blot.

4. Any candidate clones with satisfactory expression levels of APP will begrown in large quantities for long-term storage.

1.5.3 DETERMINATION OF PROTEIN CONCENTRATION BY BCA

1. Prepare BSA standards in lysis buffer [50 mM Tris, pH 7.6, 150 mMNaCl, 2 mM EDTA, 1% NP-40 and a protease inhibitor cocktail (5 µg/mlleupeptin, 5 µg/ml aprotinin, 2 µg/ml pepstatin A, and 0.25 mM phenyl-methylsulfonyl fluoride)] so that there is a serial dilution of BSA from1 mg/ml down to 15.5 µg/ml.

2. In a 96-well plate, add 25 µl of sample or standard to each well.

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3. Mix 50 parts of solution A to 1 part of solution B from the BCA ProteinAssay Reagent (Pierce, #23225). Add 200 µl of this working reagent toeach well.

4. Cover the plate with foil and incubate for 30 min at 37˚C.5. Read the plate at an absorbance of 562 nm.

1.5.4 IDENTIFICATION OF APP AND ITS DERIVATIVES BY WESTERN BLOT

1. Lyse samples with 3× sample buffer (10% SDS, 0.3 M Tris, 50% glycerol,10% β-mercaptoethanol and a trace amount of bromophenol blue).

2. Heat samples at 100˚C for 5 min.3. For BioRad Criterion gels, run at 200 V in running buffer (25 mM Tris,

192 mM glycine, 1% (w/v) SDS, pH 8.3) for 50 to 55 min.4. Transfer gel to a 0.2-µm supported nitrocellulose membrane (BioRad,

#162-0097) at 100 V for 1 hr at 4˚C in transfer buffer (20% methanol,25 mM Tris, 192 mM glycine, 1% (w/v) SDS, pH 8.3).

5. Block the membrane in 5% milk in PBS-T (0.05% Tween-20) with agi-tation for 30 min at room temperature.

6. Wash two times with PBS-T for 2 min.7. Incubate in primary antibody in PBS-T overnight at 4˚C or for 2 hr at

room temperature.8. Wash for 15 min, then wash for 5 min, three times in PBS-T.9. Incubate 1 hr at room temperature in secondary antibody diluted 1:10,000

in PBS-T. The type of secondary antibody (antimouse, antirabbit, etc.)will depend on the primary antibodies used (monoclonal/mouse vs. poly-clonal/rabbit antibodies).

10. Wash for 15 min, then wash for 5 min, three times in PBS-T.11. Place the membrane on a transparency and add ECL Plus mixture (1 ml

A plus 25 µl B; Amersham) to cover entire membrane for 1 min.12. Place second transparency over membrane and expose for various periods

(e.g., 15 sec, 30 sec, 1 min, and 5 min).

1.5.5 RADIOLABELING OF CELLS WITH [35S]-MET

1. Aspirate media from cells cultured in 35-, 60-, or 100-mm dishes. Incubatecells with methionine-free medium for 15 to 30 min at 37˚C.

2. Add appropriate volume of methionine-free medium (0.75 ml for 35-mmdish, 2 ml for 60-mm dish and 5 ml for 100-mm dish).

3. Add [35S]-methionine to each dish to reach a final specificity of 100 to200 µCi/ml. Incubate at 37˚C for the desired time; for abundant proteins,2 to 4 hr is generally sufficient. If labeling for >6 hr, it may be necessaryto supplement with fetal calf serum (10% or less) or 5 to 10% DMEM.

4. Collect cells for immunoprecipitation of target proteins. For secretedproteins, collect medium for immunoprecipitation.

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1.5.6 IDENTIFICATION OF FULL-LENGTH APP AND ITS DERIVATIVES BY IMMUNOPRECIPITATION

1. Culture cells to confluence in a 10-cm dish (~3 mg of protein), then brieflywash them twice in PBS. (For storage, 2 ml of 20 mM EDTA in PBS isadded and cells are collected and centrifuged at 3500 g for 5 min. Cellpellets can be frozen at –80˚C.)

2. Cells are lysed in an IP Lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl,2 mM EDTA, 1% NP-40 and a protease inhibitor cocktail [5 µg/mlleupeptin, 5 µg/ml aprotinin, 2 µg/ml pepstatin A, and 0.25 mM phenyl-methylsulfonyl fluoride (Sigma)]). Incubate lysates on ice for 20 min.Centrifuge lysates at 3500 g for 5 min. Transfer supernatant to a fresh tube.

3. Lysates are precleared with 20 µl of protein A-sepharose CL-4B (Sigma,#P-3391) at 100 mg/ml in STEN buffer (50 mM Tris, pH 7.6, 150 mMNaCl, 2 mM EDTA, 0.2% NP-40) for 0.5 hr at 4˚C. Supernatants aretransferred for immunoprecipitation with primary antibodies with 20 µlprotein A-sepharose or protein G-agarose (for a monoclonal antibody) at4˚C for 2 hr.

4. Immunoprecipitates are washed in a 0.5 M STEN buffer (50 mM Tris,pH 7.6, 500 mM NaCl, 2 mM EDTA, and the same protease inhibitorcocktail described above) for 15 min at 4˚C. Centrifuge the samples at3500 g for 5 min at 4˚C and discard the supernatant.

5. Immunoprecipitates are washed in a SDS-STEN buffer (50 mM Tris,pH 7.6, 150 mM NaCl, 0.1% SDS, 2 mM EDTA, and the protease inhibitorcocktail) for 15 min at 4˚C. Centrifuge the samples at 3500 g for 5 minat 4˚C and discard the supernatant.

6. Immunoprecipitates are washed in a STEN buffer (50 mM Tris, pH 7.6,150 mM NaCl, 0.2% NP-40, 2 mM EDTA, and the protease inhibitorcocktail) for 15 min at 4˚C. Centrifuge the samples at 3500 g for 5 minat 4˚C. Discard the supernatant, then elute the immunoprecipitates with3× sample buffer (10% SDS, 0.3 M Tris, 50% glycerol, 10% β-mercapto-ethanol and a trace amount of bromophenol blue), heat at 100˚C for 5 min,and separate the samples on a 4 to 20% tris-glycine gel by SDS-PAGE(BioRad, Criterion).

7. Gels are stained in Coomassie blue [0.2% Coomassie in destain solution(50% methanol, 18% acetic acid)] followed by destaining in destain solu-tion for 30 to 45 min to fix proteins. Dry gel after washing gel in Gel-Drysolution (Invitrogen, #LC4025-4) for 30 min. Expose gel in phosphor-imager for imaging or to film at –80˚C.

8. Alternatively, confluent cells can be directly lysed in lysis buffer withoutradiolabeling, and immunoprecipitates can be separated by electrophoresisfollowed by Western blot, using standard procedures provided by themanufacturer, e.g., ECL Plus detection kit from Amersham.

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1.5.7 DETERMINATION OF HALF-LIFE OF APP

The half-life of a target protein can be determined by pulse-chase labeling of cellsfollowed by immunoprecipitation with its specific antibody. In addition to determin-ing the rate of holoprotein turnover, the course of protein maturation can also beobserved.

1. Growth media of confluent CHO cells are replaced with methionine (Met)-free media, and cells are incubated at 37˚C for 45 min before the mediaare aspirated.

2. Cells are pulse labeled with prewarmed Met-free media containing100 µCi/ml of [35S]-Met for 5 to 15 min.

3. [35S]-Met-containing media are removed, and cells are washed twice withregular DMEM media.

4. Cells are chased in prewarmed DMEM media for an appropriate periodbefore they are collected for immunoprecipitation.

1.5.8 CO-IMMUNOPRECIPITATION OF APP-INTERACTING PROTEIN

1.5.8.1 Preparation

1. Thaw Co-IP lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 2 mMEDTA, 1% NP-40, 0.5% Triton X-100, 0.5% BSA, and a protease inhibitorcocktail) at room temperature.

2. Thaw cell pellets/membrane vesicles on ice, and leave protein A-sepharoseon ice.

1.5.8.2 Pre-Absorption of Protein A-Sepharose

1. Pipet 2 ml of Co-IP lysis buffer into an Eppendorf tube, then add up to200 µl of protein-A sepharose. Since each sample will be equally dividedand co-immunoprecipitated with an immune antibody or a control preim-mune serum, 40 µl of protein A-sepharose will be needed for each sampleof cell lysate.

2. Rotate the Eppendorf tube for at least 4 hr at 4˚C (in the cold room).

1.5.8.3 Pre-Clearing of Cell Lysates

1. Lyse the pellets/vesicles with 1 ml of Co-IP lysis buffer and incubate onice for 20 min.

2. Centrifuge at 3500 g for 5 min.3. Transfer supernatant into a new tube.4. Vortex protein A-sepharose briefly, and add 20 µl of protein A-sepharose

into each tube.5. Rotate samples for at least 4 hr at 4˚C (in the cold room).

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1.5.8.4 Set Up Co-IP

1. Label new Eppendorf tubes: two per cell lysate, one for the sample to beimmunoprecipitated with preimmune serum (Pre) and the other for theimmune serum.

2. Pipet 2 µl of preimmune serum (1:200 dilution) into each preimmunesample. For easier pipetting, create a larger volume by combining the totalamount of preimmune serum for all Pre samples with about 100 µl oflysis buffer. Add equal amounts to each tube.

3. For a co-IP of presenilin, for example, put 1 µl of antibody X81 and 1 µlof antibody 4627 (1:200 dilution; dilution ratio should be the same as thatof preimmune; adjust if necessary) to each sample. For easier pipetting,add the total amounts of X81 and 4627 for all immune samples into atube with about 100 µl of lysis buffer. Add equal amounts to each tube.

4. Spin down the cell samples and preabsorbed protein A-sepharose at 3500 gfor 5 min.

5. Equally divide the supernatant from each sample and aliquot it into twotubes that contain preimmune serum or immune serum.

6. Remove most of the supernatant of preabsorbed protein A-sepharose, andleave a sufficient amount of buffer equivalent to two times the volume ofprotein A-sepharose.

7. Vortex preabsorbed protein A-sepharose and transfer 20 µl into each tube.8. Rotate samples for at least 4 hr (in the cold room).

1.5.8.5 Wash Co-IP

Two solutions will be used: 0.5 M STEN (50 mM Tris, pH 7.6, 500 mM NaCl, 2 mMEDTA, and the protease inhibitor cocktail) and STEN (50 mM Tris, pH 7.6, 150 mMNaCl, 0.2% NP-40, 2 mM EDTA, and the protease inhibitor cocktail).

1. Spin samples at 3500 g for 5 min.2. Aspirate the supernatant without touching the immunoprecipitates.3. Pipet 750 µl of 0.5 M STEN into each tube.4. Rotate samples for 15 min (in the cold room).5. Spin samples at 3500 g for 5 min.6. Aspirate the supernatant.7. Pipet 750 µl STEN into each tube.8. Rotate samples for 15 min in the cold room.9. Spin samples at 3500 g for 5 min.

10. Aspirate most, but not all, of the supernatant. Use a P20 Pipetman toremove the last of the supernatant. Do not take up any beads.

11. Add 20 µl of 3× sample buffer (10% SDS, 0.3 M Tris, 50% glycerol, 10%β-mercaptoethanol, and a trace amount of bromophenol blue) into eachsample.

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12. Vortex the samples, and heat at 100˚C for 5 min.13. Spin at 18,000 g for 5 min.14. Load samples into the gel. Do not take up any beads.15. Proceed to Western blot for detection of co-immunoprecipitates.

Many elements must be monitored carefully for successful detection of proteincomplexes. Several key components are:

1. Proper preabsorption of protein A-sepharose and preclearing of cell lysatewill reduce nonspecific binding of proteins to protein A-sepharose. With-out these two steps, the complex will be immunoprecipitated in samplesincubated with preimmune serum due to nonspecific binding to proteinA-sepharose.

2. The choice of detergents affects the stringency of co-immunoprecipitationas well as the maintenance of the intact complex. Another detergent thatcan be used to replace 0.5% Triton X-100 and 1% NP-40 is 1% CHAPSO.A battery of detergents must be tested to detect a specific interactionbetween two proteins.

1.5.9 CONJUGATION OF ANTIBODY TO PROTEIN A-SEPHAROSE

Most co-immunoprecipitation experiments are carried out using unconjugated anti-body (e.g., serum) and protein A-sepharose (or protein G-agarose), and the immuno-precipitates are detected by Western blot. The best method is to use polyclonalantibodies for immunoprecipitation and monoclonal antibodies for Western blot.However, if the choice of antibodies is limited and the same type of antibody mustbe used for both immunoprecipitation and Western blot, the cross-reactivity of IgGheavy and light chains eluted from the immunoprecipitates will lead to a much higherbackground at molecular weights of ~25 kDa and above ~55 kDa. Although it doesnot completely eliminate free heavy and light chains, conjugation of primary anti-body for immunoprecipitation to protein A-sepharose will significantly reduce thenumber of IgG heavy and light chains eluted from immunoprecipitates, thus reducingthe background of the Western blot.

1. Suspend 2 g of protein A-sepharose beads in 16 ml of conjugation buffer(50 mM Tris, pH 7.6, 150 mM NaCl, 2% BSA) to make a final concen-tration of 125 mg/ml protein A-sepharose. This will give a final yield ofeight tubes of 2-ml conjugated beads.

2. Combine each 1 ml of resuspended beads with 125 µl antiserum. Incubate1 hr at room temperature with rocking, then place 2 ml of beads plusantiserum per 15 ml conical tube.

3. Centrifuge for 5 min at 3000 g. Save the supernatant in case of a couplingproblem.

4. Resuspend the beads in 10 ml of 0.2 M sodium borate buffer, pH 9.0 (heatto dissolve the precipitates prior to usage). Centrifuge the beads and repeatthe wash as before. Bring up in 10 ml of sodium borate buffer. Remove100 µl of supernatant to check later (A).

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5. Add 50 mg dimethyl pimelimidate (DMP; Sigma, #D8388, store at –20˚C)to each tube. Ensure that pH >8.3. Incubate for 30 min at room temperaturewith rocking. Remove 100 µl of supernatant to check later (B).

6. To stop the reaction, spin the beads at 3000 g for 5 min. Add 10 ml 0.2 Methanolamine (pH 8.0) per tube. Wash one more time with 10 ml 0.2 Methanolamine, then incubate in 10 ml ethanolamine for 2 hr at roomtemperature with rocking.

7. Centrifuge and transfer each pellet to a 2-ml Eppendorf tube.8. Add 1 ml 100 mM glycine, pH 3.0; mix and then spin at 10,000 g for

30 sec. Remove the supernatant and wash one time with 1 ml 100 mMTris pH 8.0; mix and spin. Expose beads to glycine buffer for as short atime as possible.

9. Resuspend beads in 1 ml PBS with 0.01% thimerosal for each tube.Remove 100 µl of supernatant to check later (C).

To check the efficiency of conjugation, the amount of remaining IgG can bedetected in the supernatant (A, B, and C) by Western blot. With the above protocol,the conjugated antibodies with protein A-sepharose are usually stable for >1 yearat 4˚C.

ACKNOWLEDGMENTS

This work was supported in part by National Institutes of Health (AG 17593) andthe Foundation for Neurologic Diseases. I would like to thank many of my colleaguesat the Center for Neurologic Diseases, especially Dr. Dennis Selkoe, who fosters asuperb research environment.

REFERENCES

1. Selkoe, D.J. The genetics and molecular pathology of Alzheimer’s disease, in Neu-rologic Clinics: Dementia, DeKosky, S.T. et al., Eds., W.B. Saunders, Philadelphia,2000, 18, 903–921.

2. Selkoe, D.J. and Podlisny, M.B. Deciphering the genetic basis of Alzheimer’s disease.Annu. Rev. Genomics Hum. Genet. 3, 67–99, 2002.

3. Selkoe, D.J. Cell biology of the amyloid β-protein precursor and the mechanism ofAlzheimer’s disease. Annu. Rev. Cell Biol. 10, 373–403, 1994.

4. Kang, J. et al. The precursor of Alzheimer’s disease amyloid A4 protein resemblesa cell-surface receptor. Nature 325, 733–736, 1987.

5. Goate, A. et al. Segregation of a missense mutation in the amyloid precursor proteingene with familial Alzheimer’s disease. Nature 349, 704–706, 1991.

6. Hendriks, L. et al. Presenile dementia and cerebral haemorrhage linked to a mutationat codon 692 of the β-amyloid precursor protein gene. Nature Genet. 1, 218–221, 1992.

7. Levy, E. et al. Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebralhemorrhage, Dutch-type. Science 248, 1124–1126, 1990.

8. van Broeckhoven, C. et al. Amyloid β-protein precursor gene and hereditary cerebralhemorrhage with amyloidosis (Dutch). Science 248, 1120–1122, 1990.

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9. Mullan, M. et al. A pathogenic mutation for probable Alzheimer’s disease in the APPgene at the N-terminus of β-amyloid. Nature Genet. 1, 345–347, 1992.

10. Eckman, C. et al. A new pathogenic mutation in the APP gene (I716V) increases therelative proportion of Aβ 42(43). Human Mol. Genet. 6, 2087–2089, 1997.

11. Chartier-Harlin, M.C., Crawford, F., and Houlden, H. Early-onset Alzheimer’s diseasecaused by mutations at codon 717 of the β-amyloid precursor protein gene. Nature353, 844–846, 1991.

12. Murrell, J., Farlow, M., Ghetti, B., and Benson, M.D. A mutation in the amyloidprecursor protein associated with hereditary Alzheimer’s disease. Science 254, 97–99,1991.

13. Neve, R.L., Finch, E.A., and Dawes, L.R. Expression of the Alzheimer amyloidprecursor gene transcripts in the human brain. Neuron 1, 669–677, 1988.

14. Citron, M. et al. Mutation of the β-amyloid precursor protein in familial Alzheimer’sdisease increases β-protein production. Nature 360, 672–674, 1992.

15. Cai, X.D., Golde, T.E., and Younkin, G.S. Release of excess amyloid β protein froma mutant amyloid β protein precursor. Science 259, 514–516, 1993.

16. Citron, M. et al. Excessive production of amyloid β-protein by peripheral cells ofsymptomatic and presymptomatic patients carrying the Swedish familial Alzheimer’sdisease mutation. Proc. Natl. Acad. Sci. USA 91, 11993–11997, 1994.

17. Scheuner, D. et al. Secreted amyloid β-protein similar to that in the senile plaquesof Alzheimer’s disease is increased in vivo by the presenilin 1 and 2 and APPmutations linked to familial Alzheimer’s disease. Nature Med. 2, 864–870, 1996.

18. Suzuki, N. et al. An increased percentage of long amyloid β protein secreted byfamilial amyloid β protein precursor (βAPP717) mutants. Science 264, 1336–1340,1994.

19. Games, D. et al. Alzheimer-type neuropathology in transgenic mice overexpressingV717F β-amyloid precursor protein. Nature 373, 523–527, 1995.

20. Hsiao, K. et al. Correlative memory deficits, Aβ elevation, and amyloid plaques intransgenic mice. Science 274, 99–102, 1996.

21. Vassar, R. et al. β-secretase cleavage of Alzheimer’s amyloid precursor protein bythe transmembrane aspartic protease BACE. Science 286, 735–741, 1999.

22. Sinha, S. et al. Purification and cloning of amyloid precursor protein β-secretase fromhuman brain. Nature 402, 537–540, 1999.

23. Yan, R. et al. Membrane-anchored aspartyl protease with Alzheimer’s disease β-secre-tase activity. Nature 402, 533–537, 1999.

24. Lin, X. et al. Human aspartic protease memapsin 2 cleaves the β-secretase site ofβ-amyloid precursor protein. Proc. Natl. Acad. Sci. USA 97, 1456–1460, 2000.

25. Seubert, P. et al. Secretion of β-amyloid precursor protein cleaved at the amino-terminus of the β-amyloid peptide. Nature 361, 260–263, 1993.

26. Haass, C. et al. Amyloid β-peptide is produced by cultured cells during normalmetabolism. Nature 359, 322–325, 1992.

27. Shoji, M. et al. Production of the Alzheimer amyloid β protein by normal proteolyticprocessing. Science 258, 126-129, 1992.

28. Lammich, S. et al. Constitutive and regulated α-secretase cleavage of Alzheimer’samyloid precursor protein by a disintegrin metalloprotease. Proc. Natl. Acad. Sci.USA 96, 3922–3927, 1999.

29. Buxbaum, J.D. et al. Evidence that tumor necrosis factor alpha converting enzymeis involved in regulated α-secretase cleavage of the Alzheimer amyloid protein pre-cursor. J. Biol. Chem. 273, 27765–27767, 1998.

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30. Esch, F.S. et al. Cleavage of amyloid β-peptide during constitutive processing of itsprecursor. Science 248, 1122–1124, 1990.

31. Sisodia, S.S., Koo, E.H., Beyreuther, K., Unterbeck, A., and Price, D.L. Evidencethat β-amyloid protein in Alzheimer’s disease is not derived by normal processing.Science 248, 492–495, 1990.

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2
Assays for Analysis of APP Secretion and Recycling
Markus P. Kummer, Tsuneo Yamazaki, and Edward H. Koo

CONTENTS

Abstract2.1 Introduction2.2 Main Scheme of Approaches2.3 Methods

2.3.1 Iodination of Antibody2.3.2 Preparation of Cells2.3.3 Kinetics of Secretion and Endocytosis of APP2.3.4 Steady State Level of APP Endocytosis2.3.5 Recycling of APP2.3.6 Morphological Analysis

2.4 DiscussionAcknowledgmentsReferences

ABSTRACT

The trafficking of the amyloid precursor protein (APP) involves the concomitantsecretion and endocytosis of APP from the cell surface. In addition, APP recyclesbetween the endocytic compartment and the cell surface. This complex sequence ofevents can be studied by a reliable and reproducible assay based on the binding of aradiolabeled APP antibody. Using this technique, the secretion and internalizationof APP can be measured simultaneously under normal and perturbated conditionsin APP transfected cells. Furthermore, this method is readily adaptable to morpho-logically examine the pathways of APP trafficking from the cell surface.

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press

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2.1 INTRODUCTION

APP is a type I membrane protein that undergoes constitutive shedding both intra-cellularly and at the cell surface to release a large secreted ectodomain derivative(sAPP). APP is transported in the secretory pathway from the golgi to the plasmamembrane where it can be cleaved by α-secretase to release the N terminal APPectodomain (sAPPα) or internalized and transported to the endosomal–lysosomalpathway. APP trafficking has been actively investigated in order to determine thecellular sites where Aβ is generated. From these studies, a major pathway of Aβgeneration is from APP that is processed following internalization from the cellsurface in the endocytic pathway, although the precise organelles where β- andγ-secretase cleavages take place remain to be defined.1

APP internalization is mediated via clathrin-coated pits2,3 in the canonical recep-tor-mediated endocytic pathway. Recently, however, it has been shown that a fractionof APP is associated with detergent-resistant membranes and that lipid rafts areapparently sites of Aβ production as well.4–7 Whether raft-associated APP is internal-ized and, if so, whether this pool subsequently merges with the nonraft pool is unclear.

Following internalization, a fraction of APP returns to the cell surface foradditional rounds of endocytosis or secretion. The internalization signal for APPresides within its cytoplasmic domain and belongs to the NPXY-type signal, one ofseveral well-recognized motifs. This signal is present in APP, β-integrin, and lowdensity lipoprotein receptor (LDLR) protein family members among others, andcontrols the rapid internalization of these integral membrane proteins.8 Althoughthis signal represents the minimal amino acid sequence shared by all these proteins,it may be not sufficient for efficient internalization. In LDLR, an additional aromaticamino acid residue upstream of the NPXY motive has become an accepted signalfor internalization. In the case of APP, the signal is mediated by the longerGYENPTY sequence. Mutagenesis studies of this motif revealed that the predomi-nant signal lies in the YENP tetrapeptide.9

Several cytosolic adaptor proteins such as Fe65, X11, and the mammalian Dab1bind to the cytoplasmic YENPTY motif via their phosphotyrosine interactiondomains.10–12 They affect the subcellular trafficking and proteolytic processing ofAPP in different ways. Fe65, for example, increases the secretion of APP and pro-motes A secretion, whereas X11 retards APP catabolism and inhibits Aβ secretion.13,14


The analysis of APP processing in the endocytic pathway has been difficult becauseof the concurrent secretion and internalization of APP molecules from the cellsurface. The assay described presents a reliable and reproducible method to measurethe secretion of sAPP and APP internalization from the cell surface simultaneously.Radiolabeled antibodies have been successfully used as surrogates to natural ligandsto investigate the internalization of other transmembrane receptors like LDLR,transferrin, CD4, or macrophage Fc receptors.15–18

This protocol contains the procedure for radioiodination of the monoclonal APPantibody, 1G7, and four variations for analyzing the trafficking of APP (Figure 2.1).

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The first method describes a pulse/chase experiment to analyze the kinetics of APPsecretion and internalization. In the second method, APP internalization is investi-gated under steady state conditions. The third method addresses the recycling ofAPP to the cell surface. The fourth and final is a morphological approach to examineAPP internalization. These methods using the monoclonal antibody 1G7 have beensuccessfully applied to determine the trafficking of APP in a variety of cells includingprimary neurons and transfected CHO, B103, and N2a cell lines.9,19–21

2.3 METHODS

2.3.1 IODINATION OF ANTIBODY

The monoclonal antibody 1G7 was raised against human APPs purified from APP-transfected CHO cells and recognizes an epitope in the extracellular domain of APPbetween residues 380 and 665, as defined by its reactivity against a bacterial fusionprotein with this sequence, thereby excluding both the KPI and Aβ domains.19 Thespecificity of this antibody for APP was demonstrated previously by immunopre-cipitation and immunofluoresence studies.1,22

The 1G7 antibody is radioiodinated using IODO-GEN precoated iodinationtubes (Pierce, Rockford, IL; #28601). This method results in indirect labeling withoutcontact of the antibody to the iodination reagent, thereby reducing oxidative damage

FIGURE 2.1 Schematical overview about the different methods to characterize APP traffick-ing using a radiolabeled antibody.

Washand

incubateat 37°C

at 37°C at 4°C

Steady-statelevel of

internalization(method 2.3.4)

Labelingof cell

surface pool

Cool to 4°C,acid wash,

warm to 37°C

Recycledmolecules

(method 2.3.5)

Kinetics ofsecretion andinternalization(method 2.3.3)

Incubation withradiolabeled

antibody

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to the antibody. Excessive iodine is finally removed by gel filtration, typically witha disposable NAP-5 (Sephadex G-25) column (Amersham Biosciences, Piscataway,NJ; #17-0853-01). The entire procedure must be performed in an approved protectivehood to avoid uptake of free radioactive iodine.

1. Pre-equilibrate a NAP-5 column with 10 ml of Dulbecco’s phosphate-buffered saline (DPBS).

2. Rinse an IODO-GEN-coated iodination tube with 0.5 ml DPBS.3. Add 300 µl DPBS directly to the bottom of the tube. Add 1 mCi Na125I

in 10 mM NaOH. Incubate for 6 min, gently swirling the tube every 30 sec.This step results in the generation of iodous ions (I+) by oxidation ofiodide (I–) by the iodination reagent.

4. Remove and add the activated iodide to 200 µl DPBS containing 100 µgof 1G7 antibody in a new screw cap tube. The final volume with theactivated iodide solution is 500 µl.

5. Incubate the mixture for 6 min, gently flicking the tube every 30 sec.During this incubation, iodous ions undergo electrophilic attack at theortho ring positions of tyrosine residues.

6. Remove the labeling solution and add it to a pre-equilibrated NAP-5column. Drain the column and discard the flow-through.

7. Elute the column with 980 µl DPBS. Add 20 µl of 100 mg/ml crystallineBSA as a carrier protein to a final concentration of 2 mg/ml.

8. Determine radiospecific activity of the labeled antibody in a gammacounter.

Typically, the radiospecific activity will be 7,500 to 15,000 cpm/fmol (3 to6 µCi/µg) when 66 nm (100 µg) of antibody is labeled by this procedure. In ourexperience, the antibody is stable for up to 4 weeks when stored at 4˚C.

2.3.2 PREPARATION OF CELLS

APP-transfected cells are seeded in 12 well plates 48 hr before the assay and grownto confluence. Nonspecific binding by the antibody and the endogenous APP signalare subtracted by analyzing untransfected cells grown in parallel. Alternatively,radiolabeled antibody binding can be competed with excess cold antibody.

2.3.3 KINETICS OF SECRETION AND ENDOCYTOSIS OF APP

This protocol examines the trafficking of APP by pulse/chase analysis. A populationof surface APP molecules is initially bound to radiolabeled antibody at 4˚C and isthen, after removal of unbound antibody, allowed to transit when rewarmed to 37˚C.At various time points, the supernatant containing the secreted APP is collected andany remaining antibody is removed from the cell surface by acid washes. Finally,the cell pellet is lysed (acid resistant fraction) to obtain the internalized APP fractionand all samples are measured in a gamma counter.

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1. Chill the cells on ice to stop membrane trafficking.2. Wash the cells twice with 2 ml of ice cold binding medium consisting of

RPMI containing 20 mM HEPES (pH 7.5) supplemented with 0.2%bovine serum albumin.

3. Add approximately 0.4 µg 125I-IG7 antibody in 400 µl binding mediumper well (~7 nM final) and allow binding of the antibody for 1 hr on ice.The Kd for this reaction is 1.23 nM for a CHO cell line stably overexpressingAPP.19 Therefore, this concentration is at least fivefold the concentrationneeded for half-maximal saturation.

4. Wash the cells twice with binding medium and twice with ice cold DPBS.5. Place the cells in 2 ml of prewarmed medium and incubate at 37˚C for

various periods.6. Collect the medium and chill cells rapidly by adding 2 ml of ice cold

DPBS at pH 2.5. Incubate the cells for another 5 min. Collect the super-natant, repeat the acidic wash to remove residual surface-bound antibodyand pool both supernatants. The acid wash detaches 90 to 95% of cellsurface-bound antibody

7. Lyse the cells by adding 2 ml of 0.2 N NaOH.8. Measure the radioactivity of all fractions in a gamma counter.

To calculate the specific binding, the radioactivity from untransfected controlcells is subtracted from the counts obtained from the transfected cells in each con-dition. The results obtained at each time point are expressed as a percentage of totalradioactivity from the three fractions (medium, acid wash and cell lysate).

The anticipated result should show a rapid release of sAPP into the mediumwith a half life of 10 min. Consequently, cell surface APP recovered by the two acidwashes should rapidly decline and remain at low levels. The remaining cell surfaceAPP is internalized within 10 min of rewarming to 37˚C. The internalized pooldeclines concurrently with an increase in the secreted pool, because of recycling ofAPP to the cell surface and subsequent secretion into the medium. After 30 min,the secreted pool remains stable whereas the internalized fraction declines, presum-ably because of degradation.

If the experiment is prolonged to more than 30 min, one should take into accountthat some of the radiolabeled antibody may be degraded and therefore free radio-activity-liberated. Under these circumstances, the medium and cell lysate shouldfirst be precipitated with trichloroacetic acid to recover the antibody-bound radio-activity only. As a consequence of the precipitation, the total radioactivity will beless than 100%.

2.3.4 STEADY STATE LEVEL OF APP ENDOCYTOSIS

To measure the rate of APP endocytosis under steady state conditions, the radiolabeledantibody is allowed to bind at 37˚C, resulting in a concomitant uptake of radioiodinatedantibody by APP internalization. This follows the method established for assessing

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the internalization of transferrin receptor under steady state conditions. After incubation,the cell surface-bound pool is removed by acid washes whereas the internalized fractionis recovered by cell lysis. This straightforward method is a facile approach for com-paring the efficiency of APP endocytosis in cell lines transfected with different APPmutants or constructs of other proteins that may affect APP internalization.

1. Wash the cells twice with prewarmed binding medium.2. Add approximately 7 nmol 125I-IG7 antibody in 400 µl binding medium

per well and allow binding and internalization of the antibody for 30 to60 min at 37˚C.

3. Chill the cells on ice and wash the cells four times with ice cold DPBS.4. Remove the cell surface-bound antibody by two consecutive washes with

2 ml of ice cold DPBS at pH 2.5 for 5 min each on a shaker and poolboth supernatants.

5. Lyse the cells by adding 2 ml of 0.2 N NaOH.6. Measure the radioactivity of both fractions in a gamma counter.

The radioactivity from untransfected control cells is subtracted from the countsresulting from the transfected cells to obtain specific binding values. The results areexpressed as percents of the cell lysate (acid-resistant) and cell surface (acid-labile)fractions to reflect the percent of APP internalized from the cell surface.

2.3.5 RECYCLING OF APP

Internalized APP recycles and is secreted 10 to 30 min after endocytosis.19 Theamounts of APP and secreted APP released from the endocytosed pool are analyzedby incubation of the cells with radioiodinated antibody at 37˚C and subsequentremoval of any cell surface-bound antibody by acid washes. After rewarming themedium containing secreted APP is collected and cell surface APP is detached bytwo consecutive acid washes. The medium is precipitated with trichloroacetic acidto recover the antibody-bound radioactivity only.

1. Wash the cells twice with prewarmed binding medium.2. Add approximately 7 nmol 125I-IG7 antibody in 400 µl binding medium

per well and allow binding and internalization of the antibody for 15 minat 37˚C.

3. Wash the cells four times with ice cold DPBS.4. Remove the cell surface-bound antibody by two consecutive washes with

2 ml of ice cold DPBS at pH 2.5 for 3 min each and discard the supernatants.5. Wash the cells twice with ice cold binding medium, then add prewarmed

medium and return cells to the 37˚C incubator.6. Collect the medium after 5 to 30 min. Precipitate the medium with TCA.7. Lyse the cells by adding 2 ml of 0.2 N NaOH.8. Measure the radioactivity of both fractions in a gamma counter.

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To obtain the specific binding, the radioactivity from untransfected control cellsis subtracted from the counts resulting from the transfected cells. The radioactivityfrom the medium is computed after precipitation with trichloroacetic acid to measurethe antibody-bound radioactivity. The results are expressed as radioactivity from theTCA precipitation as a percentage of the radioactivity from both fractions (lysateand TCA precipitate). The expected result should be a gradual increase of APP inthe medium over 30 min, suggesting that internalized APP and therefore the boundradiolabeled antibody are recycled to the cell surface and secreted into the medium.In contrast to that, the counts in the pellets should decrease.

2.3.6 MORPHOLOGICAL ANALYSIS

To visualize directly the trafficking routes of cell surface APP, immunofluorescencedetection of APP antibody added to living cells combined with suitable organellemarkers is a straightforward method to morphologically evaluate the internalizationpathways of APP localization sites. However, it is important to be aware that theantibody may induce perturbations to the normal internalization pathways. To avoidcross-linking of cell surface APP, the results from whole antibody should be con-firmed with Fab fragments or with other antibodies recognizing different epitopes.The choice of cell types is also an important consideration.

The ideal cells should be adherent and have large cytoplasms for easy visual-ization. Importantly, the cells must remain adherent during the many washes andincubations on ice. Rat hippocampal neurons cultured at low density are also suitable,but the neuritic processes are easily damaged during the procedure. The following isour standard protocol for visualizing internalization of cell surface APP in CHO cells.

1. CHO cells stably transfected with APP 751 are grown on coverslips andcultured in standard medium.

2. The cells are chilled on ice for 15 min and washed with ice cold DPBSto stop membrane trafficking.

3. Coverslips are then incubated with anti-APP monoclonal antibodies (1G7alone or combined with 5A3) in cold DPBS for 1 hr on ice.

4. The cells are washed 5 times with cold DPBS and then incubated withprewarmed regular medium for various times (0 to 60 min) at 37°C. Thecells are then fixed with cold 4% formaldehyde (freshly prepared fromparaformaldehyde) in PBS for 15 min.

5. Following fixation, the cells are permeabilized for 5 min with 0.3% TritonX-100 in PBS, washed 3 times with PBS, and incubated with fluoresceinisothionate (FITC)-conjugated anti-mouse secondary antibody for 1 hr atroom temperature.

6. If desired, double labeling with another antibody, such as to organellemarker, can be used at this point.

7. The cover slips are mounted on slide glasses and visualized with conven-tional epifluorescence or confocal microscopy.

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2.4 DISCUSSION

In this chapter, we have described assays based on the binding of a radioiodinatedor unlabeled monoclonal antibody to investigate the aspects of APP trafficking incultured cells. The use of an antibody for kinetic analysis of APP trafficking fromthe cell surface may be the only possible approach because of the lack of a physi-ological soluble ligand for APP at the cell surface. Biotinylation of cell surface APPis possible but this would be very cumbersome for obtaining kinetic information.

The 1G7 antibody used in this protocol has been shown to bind to cell surfaceAPP in a saturable and competitive manner. This antibody is also not labile at 37°C,i.e., not readily detached from APP. Other APP antibodies recognizing the extracel-lular region of APP can certainly be used, but these same parameters should betested first. Additionally, the concentration for half-maximal saturation should bemeasured by Scatchard plot analysis to determine the antibody concentration nec-essary for the assay.

To verify that the experimental conditions do not result in a general perturbationof endocytosis, the internalization of another transmembrane protein should bemeasured. In this case, the uptake of transferrin by the transferrin receptor is fre-quently used as a control.21 For this purpose bovine holo-transferrin (Sigma) isiodinated as described and added to the cells exactly as described by Zuk et al.23

In summary, the approach described in this chapter provides rapid and accurateestimates for APP secretion and endocytosis as well as morphological assessmentof the internalization pathways. In this way, the complex APP trafficking pathwaysin neurons and non-neural cells can be analyzed. In particular, the influence ofmutations within the cytoplasmic domains of APP or the impacts of cytosolic APPbinding proteins on the processing of APP can be determined.

ACKNOWLEDGMENTS

This work was supported in part by NIH Grant AG 12376. We thank Drs. ChristianHaass, Claus Pietrzik, Ruth Perez, and Dennis Selkoe for helpful discussions.

REFERENCES

1. Koo, E.H. and Squazzo, S.L. Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 269, 17386, 1994.

2. Nordstedt, C. et al. Identification of the Alzheimer beta/A4 amyloid precursor proteinin clathrin-coated vesicles purified from PC12 cells. J. Biol. Chem. 268, 608, 1993.

3. Yamazaki, T., Koo, E.H., and Selkoe, D.J. Trafficking of cell-surface amyloid beta-protein precursor. II. Endocytosis, recycling and lysosomal targeting detected byimmunolocalization. J. Cell Sci. 109, 999, 1996.

4. Simons, M. et al. Cholesterol depletion inhibits the generation of beta-amyloid inhippocampal neurons. Proc. Natl. Acad. Sci. USA 95, 6460, 1998.

5. Aplin, A.E. et al. Effect of increased glycogen synthase kinase-3 activity upon thematuration of the amyloid precursor protein in transfected cells. Neuroreport 8, 639,1997.

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6. Bouillot, C. et al. Axonal amyloid precursor protein expressed by neurons in vitro ispresent in a membrane fraction with caveolae-like properties. J. Biol. Chem. 271,7640, 1996.

7. Ehehalt, R. et al. Amyloidogenic processing of the Alzheimer beta-amyloid precursorprotein depends on lipid rafts. J. Cell Biol. 160, 113, 2003.

8. Bonifacino, J.S. and Traub, L.M. Signals for sorting of transmembrane proteins toendosomes and lysosomes. Annu. Rev. Biochem. 72, 395, 2003.

9. Perez, R.G. et al. Mutagenesis identifies new signals for beta-amyloid precursorprotein endocytosis, turnover, and the generation of secreted fragments, includingAbeta42. J. Biol. Chem. 274, 18851, 1999.

10. Borg, J.P. et al. The phosphotyrosine interaction domains of X11 and FE65 bind todistinct sites on the YENPTY motif of amyloid precursor protein. Mol. Cell. Biol.16, 6229, 1996.

11. Fiore, F. et al. The regions of the Fe65 protein homologous to the phosphotyrosineinteraction/phosphotyrosine binding domain of Shc bind the intracellular domain ofthe Alzheimer’s amyloid precursor protein. J. Biol. Chem. 270, 30853, 1995.

12. Howell, B.W. et al. The disabled 1 phosphotyrosine-binding domain binds to theinternalization signals of transmembrane glycoproteins and to phospholipids. Mol.Cell. Biol. 19, 5179, 1999.

13. Borg, J.P. et al. The X11alpha protein slows cellular amyloid precursor proteinprocessing and reduces Abeta40 and Abeta42 secretion. J. Biol. Chem. 273, 14761,1998.

14. Sabo, S.L. et al. Regulation of beta-amyloid secretion by FE65, an amyloid proteinprecursor-binding protein. J. Biol. Chem. 274, 7952, 1999.

15. Pelchen-Matthews, A., Armes, J.E., and Marsh, M. Internalization and recycling ofCD4 transfected into HeLa and NIH3T3 cells. EMBO J. 8, 3641, 1989.

16. Mellman, I.S. et al. Internalization and degradation of macrophage Fc receptors duringreceptor-mediated phagocytosis. J. Cell Biol. 96, 887, 1983.

17. Hopkins, C.R. and Trowbridge, I.S. Internalization and processing of transferrin andthe transferrin receptor in human carcinoma A431 cells. J. Cell Biol. 97, 508, 1983.

18. Beisiegel, U. et al. Monoclonal antibodies to the low density lipoprotein receptor asprobes for study of receptor-mediated endocytosis and the genetics of familial hyper-cholesterolemia. J. Biol. Chem. 256, 11923, 1981.

19. Koo, E.H. et al. Trafficking of cell-surface amyloid beta-protein precursor. I. Secre-tion, endocytosis and recycling as detected by labeled monoclonal antibody. J. CellSci. 109, 991, 1996.

20. Soriano, S. et al. Expression of beta-amyloid precursor protein-CD3gamma chimerasto demonstrate the selective generation of amyloid beta(1-40) and amyloid beta(1-42) peptides within secretory and endocytic compartments. J. Biol. Chem. 274, 32295,1999.

21. Pietrzik, C.U. et al. The cytoplasmic domain of the LDL receptor-related proteinregulates multiple steps in APP processing. EMBO J. 21, 5691, 2002.

22. Yamazaki, T., Selkoe, D.J., and Koo, E.H. Trafficking of cell surface beta-amyloidprecursor protein: retrograde and transcytotic transport in cultured neurons. J. CellBiol. 129, 431, 1995.

23. Zuk, P.A. and Elferink, L.A. Rab15 mediates an early endocytic event in Chinesehamster ovary cells. J. Biol. Chem. 274, 22303, 1999.

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3
Strategies for Crystallizing the N-Terminal Growth Factor Domain of Amyloid Precursor Protein
William J. McKinstry, Susanne C. Feil, Denise Galatis, Roberto Cappai and Michael W. Parker

CONTENTS

Abstract3.1 Introduction3.2 Overview of Approach3.3 Bioinformatics Analysis of APP3.4 Biological Roles of GFD3.5 Previous Crystallization Studies3.6 Expression of Recombinant GFD in Pichia pastoris.

3.6.1 Materials3.6.2 Method for Cloning3.6.3 Method for Expression

3.7 Purification of GFD3.7.1 Method

3.8 Crystallization of GFD3.8.1 Materials3.8.2 Method


ABSTRACT

The normal physiological roles of amyloid precursor protein (APP) remain largelyunknown despite much research. A knowledge of APP function will not only provideinsights into the genesis of Alzheimer’s disease, but may also prove vital in thedevelopment of an effective therapy. Here we describe our strategies for determiningthe three-dimensional atomic structure of APP, highlighting our work on the N-terminalgrowth factor domain.

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press

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3.1 INTRODUCTION

The normal physiological function of amyloid precursor protein (APP) remainslargely unknown although a number of studies suggest that it acts as a cell surfacereceptor. APP shares a similar architecture,1 cellular orientation, and localization2,3

to known (type I) cell surface receptors. APP mutations associated with familialAlzheimer’s disease (FAD) cause constitutive activation of Go, a member of theheteromeric G protein family whose members serve as signal tranducers of cellsurface receptors.4 It has been suggested that the FAD mutations may interfere withthe possible dimerization of APP that leads to signal transduction, as may be thecase with other cell surface receptors.

The APP cytoplasmic domain binds to a number of proteins consistent with thepossibility that APP functions as a receptor involved in signal transduction. Forexample, this domain binds to Fe65 protein, a protein related to oncogenic signaltransducers.5 Other binding partners have been discovered including APP-BP1,6

X11,7 UV-DDB,8 Tip60,9 Numb,10 and ShcA/Grb2.11 Another series of studies haveshown that an antibody directed toward the APP N-terminal domain stimulatesG protein and MAP kinase activity.12,13

The antibody is presumably mimicking the action of a still-to-be-identifiedphysiological ligand. The APP gene has been knocked out in mice, resulting inreduced body mass, reduced locomotor activity, and in some cases gliosis, indicatingimpaired neuronal function.14 In summary, current knowledge suggests APP is apotential Go-coupled receptor with ligand-regulated function, although the physio-logical roles of APP remain to be established.

The uncertainty about the normal physiological roles of APP led us to embarkon a structural investigation of the molecule. The availability of an atomic structureof APP might greatly aid studies directed toward understanding the normal functionsof APP and might also prove useful for the design of novel therapeutics to combatAlzheimer’s disease.

3.2 OVERVIEW OF APPROACH

APP represents a difficult target for crystallization. It is a heterogeneous membraneprotein with multiple glycosylation, phosphorylation, and sulfation sites. Membraneproteins are very difficult to crystallize and only a small fraction of all protein crystalstructures are of this type. In the case of APP, this problem can be circumvented byexpressing APP fragments missing the transmembrane anchor. Highly heterogeneousproteins are difficult to crystallize and thus it is a common practice to minimizeheterogeneity wherever possible. The heterogeneity of the protein bought about byglycosylation can be minimized or eliminated by glycosylases or mutating outpotential glycosylation sites. Excessive phosphorylation can be overcome with thejudicious use of phosphatases.

APP is likely to be a highly mobile protein since it consists of numerous domains(see below). Multidomain proteins can be difficult to crystallize as protein flexibilitycan interfere with the crystallization process. A common strategy to overcome thisproblem is to target smaller fragments that might be more amenable to crystallization.

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The structure of the entire molecule can then be constructed by assembling theindividual structures together.

3.3 BIOINFORMATICS ANALYSIS OF APP

Before we embarked on crystallization studies, an in-depth analysis of the APPprimary structure was carried out. Such an analysis can be very useful in decidingwhat fragments should be expressed and purified for crystallization trials. APP is a90- to 130-kDa protein that is conserved among animal species and is expressedand secreted from a variety of tissues (see References 15 and 16 for reviews). Thereare at least 10 isoforms of APP due to alternative splicing of a single gene. Thepredominant isoform in neuronal tissues is a 695-amino acid protein. Its amino acidsequence reveals it is an integral membrane protein with a single transmembranedomain near the C-terminal end of the molecule (Figure 3.1).

APP molecules from a variety of organisms have been sequenced. The locationsof putative domains in the primary structure of APP have been determined based onextensive sequence alignments, secondary structure predictions, and databasesearches for similar sequences (Figure 3.1). The most highly conserved region ofthe molecule occurs at the N-terminal end, a cysteine-rich region that includes aheparin-binding domain (D1 or growth factor domain, GFD) and a metal-bindingdomain (D2 or copper binding domain, CuBD). All APP isoforms contain highlyacidic domains; 45% of their residues are either Asp or Glu (D3). Two largerisoforms, APP751 and APP770, include an additional exon that encodes a domain withsequence similarity to a Kunitz protease inhibitor domain (KPI).17,18 The APP770

isoform also possesses a domain (D5) with similarity to the MRC OX-2 antigen, aneuronal membrane glycoprotein belonging to the immunoglobulin superfamily.18

Next is a 275-amino acid stretch that secondary structure predictions suggest consistsof two domains: a highly helical domain (D6a) and a domain of little regular structure

FIGURE 3.1 Domain structure of APP. Domains are labeled D1 to D8 and the number ofresidues in each domain is indicated. The three-dimensional structure of GFD is shown as analpha-carbon trace at the N-terminal end of the molecule. Known locations of carbohydrateattachment are denoted by “CHO.” Sites of proteolytic degradation are marked by Greekletters. The transmembrane (TM) is highlighted by dark shading. The location of the pro-teolytic breakdown product, Aβ, is also indicated.

ΑΑΑΑββββ

CHO

100 56 19 275 24 46

_ββββ γγγγNH2- -COOH

secretasesites

membraneanchor

Go binding

clathrinbinding

115

D1growthfactor

D2CuBD

D3acidic

D4KPI

D5OX-2

D6heparinbinding TM

D8cytoplasmic

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(D6b). The transmembrane (TM) region and cytoplasmic tail (D8) are located at theC-terminal end of the molecule.

3.4 BIOLOGICAL ROLES OF GFD

The N-terminal region of APP, including GFD, has previously been shown to stim-ulate neurite outgrowth19 and regulate synaptogenesis.20 Furthermore, an antibodydirected toward GFD stimulates G protein and MAP kinase activity.12,13 GFD has aknown heparin-binding site.19 Heparan sulfate proteoglycans may play a key role inAlzheimer’s disease pathogenesis: exogenous heparin induces APP production andamyloidogenic secretion21 and a number of heparin-binding growth factors areexpressed at increased levels in the brains of affected patients.22 Small heparinfragments have been proposed as possible drugs in preventing or retarding thedisease.23 To learn more about this critical domain, we determined its three-dimen-sional structure by x-ray crystallography.

3.5 PREVIOUS CRYSTALLIZATION STUDIES

We presented our original crystallization of GFD in 1999.24 We expressed a fragmentof APP consisting of residues 18 to 350 and hence encompassing the putative Nterminal domain, copper-binding domain and acidic-rich region of the molecule(Figure 3.1). Our intention was to crystallize the intact fragment but trace proteasesgenerated a smaller fragment in the crystallization trials. Nevertheless, the smallerfragment yielded well diffracting crystals that led to the structural determination ofGFD.

N-terminal sequencing and mass spectrometry revealed the crystals consisted ofa proteolytic breakdown product, residues 23 to 128. The final atomic model con-sisted of residues 28 to 123 indicating that residues 23 to 27 and 124 to 128 weretoo mobile to be seen in the electron density maps calculated from x-ray diffractionpatterns generated from the crystals. To obtain better crystals of GFD, we decidedto express GFD alone. We chose domain boundaries based on the crystal structureand omitted the most flexible regions to enhance the chances of obtaining highquality crystals (see below). We have now shown that expression of a fragmentconsisting of residues 28 to 123 crystallizes identically to the longer length fragmentand the details are presented below.

3.6 EXPRESSION OF RECOMBINANT GFD IN PICHIA PASTORIS

Recombinant secreted GFD (APP residues 28 to 123) was produced in the methylo-trophic yeast Pichia pastoris using standard molecular biology protocols andP. pastoris protocols (Invitrogen, Carlsbad, CA). We chose the P. pastoris systemas it offered high-level expression in a eukaryotic cell. In the first step, GFD wascloned into the P. pastoris expression plasmid pIC9 and then introduced into theP. pastoris cells as described below.

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3.6.1 MATERIALS

The following media were used:

1. Yeast extract peptone dextrose (YPD) medium consisting of 1% (w/v)bacto yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose.

2. Minimal methanol (MM) medium consisting of 1.34% (yeast nitrogenbase without amino acids, 4 × 10-5% w/v) biotin and 2.0% (v/v) methanol.

3. Buffered methanol complex (BMMY) consisting of MM plus 1% (w/v)bacto yeast extract, 2% w/v peptone in 0.1 M phosphate buffer, pH 6.0.

4. Minimal dextrose (MD) plates, consisting of 1.34% (yeast nitrogen basewithout amino acids, 4 × 10-5% w/v) biotin, 1% dextrose, and 15 g agarfor 1 liter.

5. Yeast extract peptone methanol (YPM) consisting of 1% (w/v) bacto yeastextract, 2% (w/v) peptone, and 3% (v/v) methanol.

3.6.2 METHOD FOR CLONING

1. The DNA encoding GFD is generated by polymerase chain reactin (PCR)using primers GGTCGACAAAAGAGAGGCTCTGCTGGCTGAACCC-CAGATTG and GAATTCTTATACAAACTCACCAACTAAG.

2. The PCR product is cloned as a Xho1-EcoR1 fragment into the P. pastorisvector pIC9 (Invitrogen).

3. The construct is linearized with BglII prior to transformation into P. pastorisstrain GS115 by electroporation.

4. A 5-ml quantity of GS115 is grown overnight in YPD in a 50-ml conicalflask at 30oC.

5. On the following day, inoculate the overnight culture into 500 ml YPDin a 2-liter flask. Grow to OD600 of 1.3 to 1.5.

6. Centrifuge (1500 × g for 5 min) and resuspend cells in 500 ml ice coldwater.

7. Centrifuge and resuspend in 250 ml ice-cold water.8. Centrifuge and resuspend in 20 ml ice-cold 1 M sorbitol.9. Centrifuge and resuspend in 1 ml ice-cold 1 M sorbitol.

10. Mix 80 µl of cells with 10 µg linearized pIC9-GFD DNA in a 0.2-cmelectroporation cuvette on ice. Electroporate according to manufacturer’srecommended conditions for yeast.

11. Cells are spread onto MD plates and grown at 30oC for 2 to 5 days. 12. Colonies are grown in 5 ml YPD for 2 days, centrifuged and then grown

in 1 ml BMMY for 2 days.13. Expressing clones are identified by silver stain sodium dodecyl sulfate–

polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the culturesupernatants.

The expressing clones are then grown in culture to produce large amounts ofGFD for crystallography and biological assays as described below.

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3.6.3 METHOD FOR EXPRESSION

1. Shaker flask cultures are grown by inoculating a suitable high-level expres-sion clone into 500 ml YPD in a 2-liter baffled flask.

2. Cultures are grown for 48 hr at 3oC on an orbital shaker (250 rpm) to acell density between 45 and 60 × 107 cells/ml (optical density of 15 to20 at 600 nm).

3. Cells are harvested by centrifugation (2000 × g for 5 min), resuspendedin 500 ml YPM, and grown for 48 hr at 30ºC on an orbital shaker (250 rpm).

4. Condition media containing the expressed GFD are harvested by centrif-ugation (14000 × g for 30 min) and filtered through a 0.45-µm filter.

3.7 PURIFICATION OF GFD

GFD is known to bind heparin,19 and this property was used to purify the proteinfrom the cell supernatants. Purification was performed using a Beckman 510 ProteinPurification Workstation (Beckman Instruments, Fullerton, CA) fitted with a single-channel wavelength detector tuned to 280 nm and a 1-cm path length analytical flowcell.

3.7.1 METHOD

1. A heparin–hyperD column (1.6 × 12 cm, Biosepra S.A., Cergy SaintChristophe, France) is equilibrated in 10 mM sodium phosphate buffer,pH 7.0, at a flow rate of 2.5 ml/min. The column is washed with equili-bration buffer until the baseline returns to zero.

2. The supernatant is loaded directly onto the heparin–hyperD column.3. Bound proteins are eluted with a 250-ml linear 0 to 2.0 M NaCl gradient

in column equilibration buffer.4. Next, 5-ml fractions are collected and analyzed by both SDS-PAGE and

immunoblotting using a monoclonal antibody that recognizes thisdomain.12,13,25

5. Fractions containing GFD are pooled and buffer-exchanged into 20 mMTris HCl, pH 8.0.

6. A QHyperD anion exchange column (4.6 × 100 mm, Biosepra S.A.) isequilibrated with 20 mM Tris HCl, pH 8.0.

7. The pooled fractions from the heparin column are loaded onto the QHyperDcolumn and bound proteins eluted with a 50-ml linear gradient of NaCl(0 to 500 mM) in column equilibration buffer.

8. GFD elutes at a concentration of 50 mM NaCl.9. The purified GFD will be >99% pure as judged by Coomassie blue

staining of an overloaded SDS–polyacrylamide gel (Figure 3.2).10. The purified GFD is concentrated between 4 and 5 mg/ml for crystalli-

zation trials.

Care must be taken to maintain the purified GFD at 4oC because the proteinreadily forms microcrystals if allowed to warm up.

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3.8 CRYSTALLIZATION OF GFD

Proteins can be made to crystallize by the addition of certain precipitants such assalts and organic solvents, most commonly ammonium sulfate or polyethyleneglycol, under unusually precise conditions of pH, temperature and protein concen-tration. Many factors can influence successful crystallization including protein andprecipitant concentrations, ionic strength, vibration, protein flexibility, protein purity,small molecule additives, temperature, and so on.

The detailed physics behind crystallization are not well understood. The processis usually considered in terms of phase diagrams where the vertical axis correspondsto the protein solubility and the horizontal axis refers to some experimental parametersuch as pH or precipitant concentration. Consider the behavior of a typical proteinsolution. At low protein and precipitant concentrations, the protein stays in solution(i.e., it is undersaturated). As the concentration of protein or precipitant increases,the protein becomes less soluble until supersaturation occurs whereby the proteincomes out of solution as either an amorphous precipitate or as ordered crystals.

All crystallization experiments for APP were conducted using the hanging dropvapor diffusion method.26 The wells of a tissue culture tray were filled with aprecipitant solution and a mixture of protein and precipitant solution was applied tothe surface of a coverslip that was then placed over the well of a culture dish, thedrop facing down (Figure 3.3).

The protein and precipitant in the drop slowly become more concentrated anda point is reached when the protein reaches supersaturation and will form an amor-phous precipitate or crystal nuclei. When the precipitate or crystals form, the proteinconcentration decreases. Crystals may begin to form at any time, from the start ofthe equilibration process until long after equilibrium has been reached, and mayform after precipitation of the protein has occurred.

FIGURE 3.2 GFD purity as assessed by SDS-PAGE and Coomassie blue staining. Molecularweight markers are shown in the left column, with their masses in kDa.

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The point of supersaturation is governed by the protein and the type of precipitantused. The rate of equilibration is governed by temperature, drop size, and the typeof precipitant used. The protein in the drop only becomes more concentrated if theprecipitant or salt concentration is higher in the well. It is not possible to determinea priori what conditions will be required for the crystallization of a new protein.Many possible crystallization conditions could have been tested and trials werecarried out using large screens under many different conditions. The crystallizationprotocol for GFD is explained below.

3.8.1 MATERIALS

1, Tissue culture plates are available from ICN Biochemicals, Inc. (Aurora,OH).

2. Chemicals for crystallization may be obtained from Fluka (Buchs, Switzer-land) or Sigma-Aldrich (Sydney, Australia).

3. Amicon protein microconcentrators may be obtained from Amicon, Inc.(Beverly, MA).

3.8.2 METHOD

1. The purified protein is dialyzed into 5 mM Tris HCl buffer, pH 7.5.2. The protein is concentrated to 10 mg/ml in Amicon concentrators.3. Take a tissue culture plate and fill the reservoirs with 1 ml of solutions

containing from 18 to 26% (w/w) PEG 10K (steps of 2%) and 100 mMHEPES buffer, ranging from pH 7.0 to 8.0 (steps of 0.5 pH units).

4. Grease the rims of the wells with petroleum jelly.5. Mix 2 µl of protein with 2 µl of reservoir solution on a cover slip and

hang the cover slip over 1 ml of reservoir solution.6. Store the trays in a constant temperature room set to 22°C.7. Crystals should appear in a number of the drops after 4 days.8. The crystals take 2 weeks to grow to maximal dimensions of approxi-

mately 0.2 × 0.2 × 0.4 mm (Figure 3.4).

FIGURE 3.3 Crystallization by the vapor diffusion hanging drop method. (Courtesy of GeoffreyKong.)

Hanging Drop Cover Slip

Reservoir

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3.9 DISCUSSION

The availability of well-diffracting crystals was the vital first step in determiningthe three-dimensional atomic structure of GFD by x-ray crystallography.26 GFD wasfound to adopt a compact, globular fold consisting of nine β-strands and one α-helixtethered together by three disulfide bridges (Cys 38 to Cys 62; Cys 73 to Cys 117;Cys 98 to Cys 105; see Figure 3.5). Sequence alignments of APP orthologues andparalogues show their GFD regions are well conserved with sequence identitiesranging from 36 to 84%; all the cysteine residues are strictly conserved. This suggeststhat the fold described here is maintained across the APP family.

An electrostatic calculation based on the three-dimensional structure demon-strated a highly positively charged surface on one side of the domain including apeptide region, residues 96 to 110, that was previously identified as part of a heparin-binding site.19 Maintenance of the disulfide bridge in this region is critical for neuriteoutgrowth19 and activation of MAP kinase,27 suggesting that the conformation of theloop is important. The surface is dominated by the β-hairpin loop (residues 96 to110) representing the most mobile region of the structure. The overall fold of theGFD did not resemble any protein of known three-dimensional structure. However,like APP, a number of growth factors also possess disulfide-bonded β-hairpin loopsimplicated in proteoglycan binding. These include midkine,28 hepatocyte growthfactor29 and vascular endothelial growth factor.30 In all cases, the loop is long, flexible,and highly charged with basic residues. These properties appear ideal for bindingheparin oligosaccharides where the flexibility would allow induced fit binding viathe positively charged residues around the sulfate moieties of the carbohydrate.

FIGURE 3.4 Crystals of GFD. The largest crystal is 0.3 mm in its longest dimension.

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The extracellular domain of APP has been shown to be a potent mediator ofthyrocyte proliferation31 and can potentiate the phosphorylation of the tyrosine kinase(trkA) receptor caused by nerve growth factor binding.32 The N-terminal cysteine-rich region can inhibit platelet activation33 emphasizing the role of this region inmodulating cellular pathways and function. Presumably, the growth-promoting activ-ity of APP is expressed after it is released from membranes through the action ofsecretases. In conclusion, the structural similarities to some growth factors in concertwith the known growth promoting properties of APP and its N-terminal domain ledto the conclusion that GFD can be classified as a new member of the cysteine-richgrowth factor superfamily.

FIGURE 3.5 Structure of GFD. A ribbon diagram indicating the location of secondarystructure with helices as coils and β-strands as arrows. The disulfide bridges are shown inball-and-stick form and the tip of the β-hairpin loop is indicated. This figure was drawn withMOLSCRIPT.34

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ACKNOWLEDGMENTS

This work was supported by grants from the National Health and Medical ResearchCouncil of Australia to R.C. and M.W.P. W.J.M. is a NHMRC Industry Fellow andM.W.P. is a NHMRC Senior Principal Research Fellow.

REFERENCES

1. Kang, J. et al. The precursor of Alzheimer’s disease amyloid A4 protein resemblesa cell-surface receptor. Nature 325, 733, 1987.

2. Weidemann, A. et al. Identification, biogenesis, and localization of precursors ofAlzheimer’s disease A4 amyloid protein. Cell 57, 115, 1989.

3. Schubert, W. et al. Localization of Alzheimer beta A4 amyloid precursor protein atcentral and peripheral synaptic sites. Brain Res. 563, 184, 1991.

4. Okamoto, T. et al. Intrinsic signaling function of APP as a novel target of three V642mutations linked to familial Alzheimer’s disease. EMBO J. 15, 3769, 1996.

5. Zambrano, N. et al. Interaction of the phosphotyrosine interaction/phosphotyrosinebinding-related domains of Fe65 with wild-type and mutant Alzheimer’s beta-amyloidprecursor proteins. J. Biol. Chem. 272, 6399, 1997.

6. Chow, N. et al. APP-BP1, a novel protein that binds to the carboxyl-terminal regionof the amyloid precursor protein. J. Biol. Chem. 271, 11339, 1996.

7. Borg, J.P. et al. The phosphotyrosine interaction domains of X11 and FE65 bind todistinct sites on the YENPTY motif of amyloid precursor protein. Mol. Cell. Biol.16, 6229, 1996.

8. Watanabe, T. et al. A 127-kDa protein (UV-DDB) binds to the cytoplasmic domainof the Alzheimer’s amyloid precursor protein. J. Neurochem. 72, 549, 1999.

9. Cao, X. and Südhof., T.C. A transcriptionally active complex of APP with Fe65 andhistone acetyltransferase Tip60. Science 293, 115, 2001.

10. Roncarati, R. et al. The gamma-secretase-generated intracellular domain of beta-amyloid precursor protein binds Numb and inhibits Notch signaling. Proc. Natl. Acad.Sci. USA 99, 7102, 2002.

11. Russo, C. et al. Signal transduction through tyrosine-phosphorylated C-terminal frag-ments of amyloid precursor protein via an enhanced interaction with Shc/Grb2 adaptorproteins in reactive astrocytes of Alzheimer’s disease brain. J. Biol. Chem. 277, 35282,2002.

12. Okamoto, T. et al. Ligand-dependent G protein coupling function of amyloid trans-membrane precursor. J. Biol. Chem. 270, 4205, 1995.

13. Murayama, Y. et al. Cell surface receptor function of amyloid precursor protein thatactivates Ser/Thr kinases. Gerontology 42, 2, 1996.

14. Zheng, H. et al. beta-Amyloid precursor protein-deficient mice show reactive gliosisand decreased locomotor activity. Cell 81, 525, 1995.

15. Hendriks, L. and Van Broeckhoven, C. A beta A4 amyloid precursor protein geneand Alzheimer’s disease. Eur. J. Biochem. 237, 6, 1996.

16. Mattson, M.P. Cellular actions of beta-amyloid precursor protein and its soluble andfibrillogenic derivatives. Physiol. Revs. 77, 1081, 1997.

17. Kitaguchi, N. et al. Novel precursor of Alzheimer’s disease amyloid protein showsprotease inhibitory activity. Nature 331, 530, 1988.

18. Ponte, P. et al. A new A4 amyloid mRNA contains a domain homologous to serineproteinase inhibitors. Nature 331, 525, 1988.

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19. Small, D.H et al. A heparin-binding domain in the amyloid protein precursor ofAlzheimer’s disease is involved in the regulation of neurite outgrowth. J. Neurosci.14, 2117, 1994.

20. Morimoto, T. et al. Involvement of amyloid precursor protein in functional synapseformation in cultured hippocampal neurons. J. Neurosci. Res. 51, 185, 1998.

21, Leveugle, B. et al. Heparin promotes beta-secretase cleavage of the Alzheimer’samyloid precursor protein. Neurochem. Int. 30, 543, 1997.

22. Fenton, H. et al. Hepatocyte growth factor (HGF/SF) in Alzheimer’s disease. BrainRes. 779, 262, 1998.

23. Leveugle, B. et al. Heparin oligosaccharides that pass the blood–brain barrier inhibitbeta-amyloid precursor protein secretion and heparin binding to beta-amyloid peptide.J. Neurochem. 70, 736, 1998.

24. Rossjohn, J. et al. Crystal structure of the N-terminal, growth factor-like domain ofAlzheimer amyloid precursor protein. Nature Struct. Biol. 6, 327, 1999.

25. Hilbich, C. et al. Amyloid-like properties of peptides flanking the epitope of amyloidprecursor protein-specific monoclonal antibody 22C11. J. Biol. Chem. 268, 26571,1993.

26. McPherson, A., Crystallization of Biological Macromolecules, Cold Spring HarborLaboratory Press, New York, 1999.

27. Greenberg, S.M. et al. Amino-terminal region of the beta-amyloid precursor proteinactivates mitogen-activated protein kinase. Neurosci. Lett. 198, 52, 1995.

28. Iwasaki, W. et al. Solution structure of midkine, a new heparin-binding growth factor.EMBO J. 16, 6936, 1997.

29. Zhou, H. et al. The solution structure of the N-terminal domain of hepatocyte growthfactor reveals a potential heparin-binding site. Structure 6, 109, 1998.

30. Fairbrother, W.J. et al. Solution structure of the heparin-binding domain of vascularendothelial growth factor. Structure 6, 637, 1998.

31. Pietrzik, C.U. et al. From differentiation to proliferation: the secretory amyloidprecursor protein as a local mediator of growth in thyroid epithelial cells. Proc. Natl.Acad. Sci. USA 95, 1770, 1998.

32. Akar, C.A. and Wallace, W.C. Amyloid precursor protein modulates the interactionof nerve growth factor with p75 receptor and potentiates its activation of trkA phos-phorylation. Mol. Brain. Res. 56, 125, 1998.

33. Henry A. et al. Inhibition of platelet activation by the Alzheimer’s disease amyloidprecursor protein. Br. J. Haematol. 103, 402, 1998.

34. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plotsof proteins. J. Appl. Crystallogr. 24, 946, 1991.

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4
Analysis of Amyloid Precursor Protein Processing Protease β-Secretase: Tools for Memapsin 2 (β-Secretase) Inhibition Studies
Gerald Koelsch, Vajira Weerasena, Dongwoo Shin, Arun K. Ghosh, and Jordan Tang

CONTENTS

4.1 Introduction4.2 Assay of Memapsin 2 Activity4.3 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]-4-

hydroxy-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-[(tert-

butyldimethylsilyl)oxy ]-2,7-dimethyloctanoicacid (9)4.3.7 (2R,4S,5S)-5-[(fluorenylmethyloxycarbonyl)amino]-4-[(tert-

butyldimethyl silyl)oxy]-2,7-dimethyloctanoic acid (10)4.3.8 Coupling of Di-Isostere in Solid-Phase Peptide Synthesis

4.4 Determination of Inhibition Constants4.5 SummaryReferences

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press

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4.1 INTRODUCTION

Memapsin 2 (BACE, ASP-2) is the membrane-anchored aspartic protease that pro-cesses APP at the β-secretase site.1–5 The resulting C terminal fragment, C99, isfurther processed by γ-secretase to produce amyloid-β (Aβ) peptide.6 Since Aβ isintimately related to the pathogenesis of Alzheimer’s disease, a great deal of interestsurrounds the design and testing of memapsin 2 inhibitors. The first potent transitionstate inhibitor of memapsin 2, OM99-2 (Figure 4.1), was designed based on a slightlymodified sequence around the β-secretase processing site of APP Swedish mutant.7

This inhibitor had a Ki of 1.6 nM. When the complete subsite specificity of memapsin 2was determined,8 the most preferred residues were used to design OM00-3(Figure 4.2) which had a Ki of 0.3 nM.9 The crystal structures of these inhibitorsbound to the catalytic unit of memapsin 2 have been reported.10,11 These structuresdefine the interactions of the protease active sites with the inhibitors and also predictthe locations of the APP substrate binding positions during the hydrolysis.

Memapsin 2 is a type I transmembrane protein with the catalytic domain locatedon the lumenal face of the plasma membrane.1–5 Likewise, its most notorious sub-strate, APP, has the same topology as the β-secretase cleavage site located within28 amino acids of the plasma membrane surface. In vivo memapsin 2 may beregulated by factors including expression, post-translational modifications and mem-brane component compositions such as “lipid rafts” and endocytotic and vesiculartrafficking to acidic compartments such as endosomes. Nonetheless, small peptidesubstrates representing the β-secretase cleavage site in APP are capably cleaved bymemapsin 2,5,12 demonstrating the independence of in vivo biochemical regulationfrom its fundamental proteolytic function.

Similarly, retroviral proteases demonstrate complex in vivo characteristics,requiring dimerization in the form of gag-pol precursor proteins with subsequentactivation from these precursor multiproteins. These proteases are still able to cleavepeptide substrates in vitro and autoprocess a mini-precursor form of the protease.13,14

Despite the complexity of the in vivo environment of the retroviral proteases, clearly

FIGURE 4.1 Continuous fluorescence assay of memapsin 2 activity using an Mca/Dnp inter-nally quenched fluorogenic substrate. Memapsin 2 was incubated with substrate (3 µM) atpH 4.0, 37˚C and emission at 393 nm was monitored continuously with excitation at 328 nm.Increased fluorescence intensity over time indicates proteolysis of the substrate. Linear regres-sion is used to determine the initial velocity (V0) of the uninhibited enzyme.

0.20

0.25

0.30

0.35

0 50 100 150 200

Time (sec)

Flu

ore

scen

ce In

ten

sity

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the development of potent HIV inhibitors has been successful using peptide-basedin vitro enzyme assays. Thus it is likely that memapsin 2 inhibitors with pharma-ceutical application for modulation of Aβ production may be developed from theassay of small peptide substrates that model the fundamental proteolytic function,superseding the complexity of the memapsin 2 function in vivo. The assay ofmemapsin 2 activity, syntheses of transition-state memapsin 2 inhibitors, and mea-surements of their potencies are described in the following sections.

4.2 ASSAY OF MEMAPSIN 2 ACTIVITY

Continuous assay of memapsin 2 activity is a necessary prelude to the measure ofits inhibition and permits rapid analysis of inhibition potency and determination ofinhibition constants. An internally quenched fluorogenic substrate for memapsin 2was designed to mimic the β-secretase cleavage site of APP (substrate FS-2 inReference 12) with the sequence Mca–Ser–Glu–Val–Asn–Leu–Asp–Ala–Glu–Phe–Lys(Dnp)–NH2 [Mca–(Asn670, Leu671)–APP770 (667–675)–Lys(Dnp) amide] and isavailable from Bachem (Torrance, CA, #2485). It contains the (7-methoxycoumarin-4-yl)acetyl fluorophore (Mca) at the amino terminus (effectively at the P6 position)and the quenching chromophore group N-2,4-dinitrophenyl (Dnp) attached to theε-amino group of Lys in the P ′5 position. Upon excitation of the Mca group at328 nm, energy is transferred to the Dnp group with limited photon emissiondetectible at 393 nm (fluorescence resonance energy transfer or FRET).

Cleavage of the intervening peptide results in diffusion of the two products, eachcontaining a respective fluorophore and quenching chromophore group. This permitsthe excitation of the Mca group of the N-terminal product, resulting in unquenchedemission at a wavelength of 393 nm (λex = 328 nm, λem = 393 nm). Increasedfluorescence intensity at this excitation–emission wavelength pair allows continuousmonitoring of proteolytic activity (Figure 4.1). Cleavage at the β-secretase site aloneby memapsin 2 was confirmed by mass spectrometry.12

The assay of β-secretase activity using the Mca–Dnp substrate is accomplishedby the addition of 1.75 ml 0.1 M sodium acetate, pH 4.0, to an aliquot of dimethylsulfoxide (DMSO) in a 1.0 × 1.0-cm quartz cuvette thermostatted cell holder pre-equilibrated to 37˚C. The DMSO is added to the aliquot of sodium acetate such thatthe final DMSO concentration is 10% (including the amounts of substrate andinhibitor to be added, which are dissolved in DMSO). Memapsin 2 activity versussubstrate FS-2 was found to be optimal at this concentration of DMSO.12

Memapsin 25,12 enzyme stock (typically 6 µM, 50 µl aliquot) is added, followedby substrate (20 µl of 300 µM stock FS-2 in DMSO; see Reference 12) to initiatethe reaction (2 ml total volume). Fluorescence intensity over a 5-min period wasmonitored with excitation at 328 nm and emission at 393 nm, using a detector voltageof 700 V on an Aminco Bowman luminescence spectrometer. Fit of the linear portionof the signal to a linear model produces a typical signal of 10-3 fluorescence unitsper second (FU/sec). A typical time trace for hydrolysis of FS-2 by memapsin 2 isshown in Figure 4.1.

The determined rate of reaction is proportional to the rate of cleavage of molaramounts of substrate per unit volume, but requires conversion of the observed initial

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velocity (in FU/sec) using the response factor of known concentrations of fluoro-phore and the ratio of free flourophore to that of the cleavage product, due to theinner filter effect of intermolecular quenching.15 However, this correction is notnecessary in the determination of the inhibition constant Kiapp (see below) becauserelative initial velocities are determined at a fixed substrate concentration.

4.3 SYNTHESIS OF MEMAPSIN 2 INHIBITORS BASED ON APP SEQUENCE

Transition state theory indicates that an enzyme will bind most tightly to its substratewhen the substrate adopts a conformation approximating the transition state in theprogression toward product formation. Thus the enzyme induces or stabilizes thesubstrate in that conformation, lowering the activation energy to permit more fre-quent progression to product. Inhibition of this process is therefore best accom-plished with compounds that mimic the transition state of the scissile bond and arethus potent inhibitors of β-secretase.16

Peptide-based inhibitors of β-secretase have been described by our labora-tory.8,9,17 The synthesis of OM99-2 and OM00-3 consists of two major steps. Thefirst is the synthesis of a Leu*Ala dipeptide transition state isostere (that will bereferred to as the di-isostere; the asterisk represents the hydroxyethylene moiety)with appropriate protective groups. The second step is to use solid-phase peptidesynthesis to incorporate the di-isostere into the inhibitor.

The synthesis of the Leu*Ala di-isostere using commercial BOC-leucine as astarting material is outlined in Figure 4.2. The Fmoc-protected di-isostere (compound10 in Figure 4.2) is inserted in a coupling step as for other amino acid residues inthe peptide synthesis. In this manner, it is possible to develop inhibitors to exploitthe substrate specificity of memapsin 29 by incorporating various standard or non-standard amino acids into the peptide. Thus incorporation of the di-isostere in solid-phase peptide synthesis creates a molecule endowed with the potential to inhibitmemapsin 2 activity. Detailed steps for the synthesis of the Leu*Ala di-isostere arediscussed in the following sections. The compound numbers appearing in parenthe-ses in the headings below correspond to Figure 4.2.

4.3.1 N-(TERT-BUTOXYCARBONYL)-L-LEUCINE-N′′′′-METHOXY-N′′′′-METHYLAMIDE (3)

To a stirred solution of N,O-dimethylhydroxyamine hydrochloride (5.52 g, 56.6 mmol)in dry dichloromethane (25 mL) under N2 atmosphere at 0˚C, 1-methylpiperidine(6.9 mL, 56.6 mmol) is added dropwise. The resulting mixture is stirred at 0˚C for30 min. In a separate flask, N-(tert-butyloxycarbonyl)-L-leucine (BOC-leucine, 2)(11.9 g, 51.4 mmol) is dissolved in a mixture of THF (tetrahydrofuran, 45 mL) anddichloromethane (180 mL) under N2 atmosphere. The resulting solution is cooledto –20˚C. To this solution is added 1-methylpiperidine (6.9 mL, 56.6 mmol) followedby isobutyl chloroformate (7.3 mL, 56.6 mmol).

The resulting mixture is stirred for 5 min at –20˚C and the above solution ofN,O-dimethylhydroxyamine is added to it. The reaction mixture is kept at –20oC for

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30 min and then warmed to 23˚C. The reaction is quenched with water and the layersare separated. The aqueous layer is extracted with dichloromethane (3 × 100 mL).The combined organic layers are washed with 10% citric acid, saturated sodiumbicarbonate, and brine. The organic layer is dried over anhydrous Na2SO4 andconcentrated under the reduced pressure. The residue is purified by flash silica gelchromatography (25% ethyl acetate–hexane) to yield the title compound 3 as a paleyellow oil.

FIGURE 4.2 Scheme for synthesis of Leu*Ala di-isostere. Letters accompanying arrows referto reagents and conditions: (a) LiAlH4, Et2O, –40oC, 30 min (86%); (b) LDA, HC≡C-CO2Et,THF, –78oC, 30 min, then 4, –78oC, 1 hr (42%); (c) H2, Pd-BaSO4, EtOAc; (d) AcOH, PhMe,reflux, 6hr (74%); (e) LiHMDS, MeI, THF, –78oC, 20 min (76%); (f) aqueous LiOH, THF-H2O, 23oC, 10 hr; (g) TBDMSCl, imidazole, DMF, 24 hr (90%); (h) CF3CO2H, CH2Cl2, 0oC,1.5 hr; (i) Fmoc-OSu, aqueous NaHCO3, dioxane, 23oC, 8 hr (61%).

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4.3.2 N-(TERT-BUTOXYCARBONYL)-L-LEUCINAL (4)

To a stirred suspension of lithium aluminum hydride (770 mg, 20.3 mmol) in drydiethyl ether (60 mL) at –40oC under N2 atmosphere is added N-tert-butyloxycar-bonyl-L-leucine-N′-methoxy-N′-methylamide (5.05 g, 18.4 mmol) in diethyl ether(20 mL). The resulting reaction mixture is stirred for 30 min, after which the reactionis quenched with 10% NaHSO4 solution (30 mL). The resulting reaction mixture isthen warmed to 23˚C and stirred at that temperature for 30 min. The resulting solutionis filtered and the filter cake is washed with two portions of diethyl ether. Thecombined organic layers are washed with saturated sodium bicarbonate and brineand dried over anhydrous MgSO4. Evaporation of the solvent under reduced pressureyields the title aldehyde 4 (3.41 g) as a pale yellow oil. The resulting aldehyde isused immediately without further purification.

4.3.3 ETHYL (4S,5S)- AND (4R,5S)-5-[(TERT-BUTOXYCARBONYL)AMINO]- 4-HYDROXY-7-METHYLOCT-2-YNOATE (5)

To a stirred solution of diisopropylamine (1.1 mL, 7.9 mmol) in dry THF (60 mL)at 0oC under N2 atmosphere is added n-BuLi (1.6 M in hexane, 4.95 mL, 7.9 mmol)dropwise. The resulting solution is stirred at 0oC for 5 min and then warmed to 23˚Cand stirred for 15 min. The mixture is cooled to –78oC and ethyl propiolate (801 µL)in THF (2 mL) is added dropwise over a period of 5 min. The mixture is stirred for30 min, after which N-BOC-L-leucinal 4 (1.55 g, 7.2 mmol) in 8 mL of dry THFis added.

The resulting mixture is stirred at –78oC for 1 hr, after which the reaction isquenched with acetic acid (5 mL) in THF (20 mL). The reaction mixture is warmedto 23˚C and brine solution is added. The layers are separated and the organic layeris washed with saturated sodium bicarbonate and dried over Na2SO4. Evaporationof the solvent under reduced pressure provides a residue that is purified by flashsilica gel chromatography (15% ethyl acetate–hexane) to afford a 3:1 mixture ofacetylenic alcohols 5.

4.3.4 (5S,1′′′′S)-5-[1′′′′-[(TERT-BUTOXYCARBONYL)AMINO]-3′′′′-METHYLBUTYL]-DIHYDROFURAN-2(3H)-ONE (7)

To a stirred solution of the above mixture of acetylenic alcohols (1.73 g, 5.5 mmol)in ethyl acetate (20 mL) is added 5% Pd–BaSO4 (1 g). The resulting mixture ishydrogenated at 50 psi for 1.5 hr. After this period, the catalyst is filtered off througha plug of Celite and the filtrate concentrated under reduced pressure. The residue isdissolved in toluene (20 mL) and acetic acid (100 µL). The reaction mixture is refluxedfor 6 hr, after which the reaction is cooled to 23˚C and the solvent is evaporated toproduce a residue purified by flash silica gel chromatography (40% diethylether–hexane) to yield the (5S,1S′)-γ-lactone 7 (0.94 g, 62.8%) and the (5R,1S′)-γ-lactone 6 (0.16 g, 10.7%).

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4.3.5 (3R,5S,1′′′′S)-5-[1′′′′-[(TERT-BUTOXYCARBONYL)AMINO)]-3′′′′-METHYLBUTYL]-3-METHYL DIHYDROFURAN-2(3H)-ONE (8)

To a stirred solution of the lactone 7 (451.8 mg, 1.67 mmol) in dry THF (8 mL) at–78oC under N2 atmosphere is added lithium hexamethyldisilazane (3.67 mL, 1.0 Min THF) over a period of 3 min. The resulting mixture is stirred at –78oC for 30 minto generate lithium enolate. After this period, CH3I (228 µL) is added dropwise andthe resulting mixture stirred at –78˚C for 20 min. The reaction is quenched withsaturated aqueous NH4Cl solution and allowed to warm to 23˚C. The reaction mixtureis concentrated under reduced pressure and the residue extracted with ethyl acetate(3 × 100 mL). The combined organic layers are washed with brine and dried overanhydrous Na2SO4. Evaporation of the solvent affords a residue that is purified bysilica gel chromatography (15% ethyl acetate–hexane) to furnish the alkylated lac-tone 8 (0.36 g, 76%) as an amorphous solid.

4.3.6 (2R,4S,5S)-5-[(TERT-BUTOXYCARBONYL)AMINO]-4-[(TERT-BUTYLDIMETHYLSILYL)OXY ]-2,7-DIMETHYLOCTANOICACID (9)

To a stirred solution of lactone 8 (0.33 g, 1.17 mmol) in THF (2 mL) is added 1 Naqueous LiOH solution (5.8 mL). The resulting mixture is stirred at 23˚C for 10 hr,after which the reaction mixture is concentrated under reduced pressure and theremaining aqueous residue cooled to 0oC and acidified with 25% citric acid solutionto pH 4. The resulting acidic solution is extracted with ethyl acetate (3 × 50 mL).The combined organic layers are washed with brine, dried over Na2SO4 and con-centrated to yield the corresponding hydroxy acid (330 mg) as a white foam. Thishydroxy acid is used directly for the next reaction without further purification.

To the hydroxy acid (330 mg, 1.1 mmol) in anhydrous DMF is added imidazole(1.59 g, 23.34 mmol) and tert-butyldimethylchlorosilane (1.76 g, 11.67 mmol). Theresulting mixture is stirred at 23˚C for 24 hr. After this period, MeOH (4 mL) isadded and the mixture stirred for 1 hr. The mixture is then diluted with 25% citricacid (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined extractsare washed with water and brine and dried over anhydrous Na2SO4. Evaporation ofthe solvent produces a viscous oil that is purified by flash chromatography oversilica gel (35% ethyl acetate–hexane) to afford the silyl protected acid 9.

4.3.7 (2R,4S,5S)-5-[(FLUORENYLMETHYLOXYCARBONYL)AMINO]-4-[(TERT-BUTYLDIMETHYL SILYL)OXY]-2,7-DIMETHYLOCTANOIC ACID (10)

To a stirred solution of the acid 9 (0.17 g, 0.41 mmol) in dichloromethane (2 mL)at 0oC is added trifluoroacetic acid (500 µL). The resulting mixture is stirred at 0oCfor 1 hr and an additional 500 µL of trifluoroacetic acid is added to the reactionmixture. The mixture is stirred for an additional 30 min and the progress of thereaction monitored by thin layer chromatography (TLC). After this period, thesolvents are carefully removed under reduced pressure at a bath temperature not

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exceeding 5oC. The residue is dissolved in dioxane (3 mL) and NaHCO3 (300 mg) in5 mL of H2O. To this solution is added Fmoc-succinimide (166.5 mg, 0.49 mmol)in 5 mL of dioxane. The resulting mixture is stirred at 23˚C for 8 hr.

The mixure is then diluted with H2O (5 mL) and acidified with 25% aqueouscitric acid to pH 4. The acidic solution is extracted with ethyl acetate (3 × 50 mL).The combined extracts are washed with brine, dried over Na2SO4, and concentratedunder reduced pressure to yield a viscous oil residue. Purification of the residue byflash chromatography over silica gel affords the Fmoc-protected acid 10.

4.3.8 COUPLING OF DI-ISOSTERE IN SOLID-PHASE PEPTIDE SYNTHESIS

The solid-phase peptide synthesis protocol may be utilized for the synthesis ofinhibitors. The synthesis is initiated with Wang resin (0.3 mmol) precoupled with acarboxy terminal amino acid of choice and capped with a N-9-fluorenylmethyloxy-carbonyl(Fmoc) alpha amino-protecting group. The coupling of N-Fmoc-Ala, isostere10 is accomplished as follows. The removal of the N-Fmoc group from the peptidechain downstream of the di-isostere is carried out in 20% piperidine in dimethyl-foramide for 15 min. The peptide coupling reaction is accomplished with 2-(1H-benzotriazol-1-yl) 1,1,3,3-tetramethyluronium tetrafluoroborate, 1-hydroxybenzotri-azole and diisopropylethylamine (3.3 equivalents each) in N-methyl pyrolidine.

After the di-isostere coupling, other Fmoc-protected derivatives are coupled.After the last residue coupling step, the peptide is cleaved from the solid-state resinusing 95% trifluoroacetic acid, which also removes all the side chain-protectinggroups including the silyl-group of 10. The inhibitors are purified in reversed-phasehigh performance liquid chromatography (HPLC) using a C18 column equilibratedin 0.1% trifluoroacetic acid in H2O with a linear gradient of acetonitrile from 0 to25% over 25 min.

4.4 DETERMINATION OF INHIBITION CONSTANTS

Because the inhibitors of interest are very potent and have Ki values in the nM range,the inhibition constants cannot be determined accurately by conventional steady-state kinetics. The inhibition constant Kiapp is determined from nonlinear regressionof the model of Bieth.18 Proteolytic activity in the presence of inhibitor (Vi) and fullactivity free of inhibitor (V0) are measured with a constant concentration of theenzyme. The mixture of the enzyme and inhibitor is pre-equilibrated for 20 min andthe reaction is initiated by the addition of the substrate. The relative activity (a) isthe ratio of Vi/V0 and is determined at various concentrations of inhibitor. Theapparent inhibition constant, Kiapp, may be determined from a plot of relative activity(a) versus [I] (Figure 4.3) based on Equation 1:

a =− + + − + +1

0 0 0 0I E K I E K

iapp iapp(( ) −

2

0 0

0

4

2

I E

E

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Nonlinear regression of the data with the above equation to obtain Kiapp may beaccomplished using GraFit.19 Typically 5 to 10 determinations of relative activityover a range of [I] will produce an error of the fit to ±5 to 10%. Sensitivity of themodel to [E]0 requires that the concentration of the enzyme stock be determinedaccurately. Simple conversion of optical density measurements at 280 nm may notprovide accurate information. Rather it is preferred to determine the number of activesites by titration using a tight-binding inhibitor.20 The data from determination of[E]0 by this method may also be used to confirm the Kiapp value for the titratinginhibitor, using additional determinations at [I] beyond the range of the linearrelationship between a and [I]. The obtained Kiapp measurement may be dependentupon substrate concentration [12] and may be corrected by the relationship:

4.5 SUMMARY

Inhibitors of aspartic proteases may be developed from peptide substrate templates.In the case of memapsin 2, by employing the basic sequence of a good substratefrom the APP Swedish mutant and the principle of a transition-state mimic,21 potentinhibitors like OM99-2 can be developed. In this chapter we provide basic methodsfor such work. The synthesis of a transition-state isostere in the place of the scissilepeptide bond is essential for a tight-binding transition-state inhibitor. The syntheticprocedure of a blocked two-residue isostere has the advantage that it can be usedin standard solid-phase peptide synthesis of a long inhibitor. This approach is suitableto explore diverse amino acid sequences, including nonstandard amino acids.8,17

The second essential aspect in inhibitor development is the kinetic assay forinhibition potency. A fluorogenic substrate based on a slightly modified sequence ofthe APP Swedish mutant is described. This assay,12 based on the principle of fluores-cence resonance energy transfer, is very sensitive and has been reliably used to deter-mine competitive inhibition constants using a model for tight-binding inhibitors.8,17

FIGURE 4.3 Determination of Kiapp from a profile of relative activity vs. inhibitor concen-tration. Initial velocities (Vi) were determined at various concentrations of inhibitor andexpressed relative to the initial velocity of uninhibited control reaction (V0) (solid symbols).Nonlinear regression of the data (solid line) with Equation 1 determines Kiapp.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 200 400

[I], nM

Rel

ativ

e A

ctiv

ity

K K S Kiapp i m

= + ( )1

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REFERENCES

1. Vassar, R. et al. β-Secretase cleavage of Alzheimer’s amyloid precursor protein bythe transmembrane aspartic protease BACE. Science 286, 735–741, 1999.

2. Sinha, S. et al. Purification and cloning of amyloid precursor protein β-secretase fromhuman brain. Nature 402, 537–540, 1999.

3. Yan, R. et al. Membrane-anchored aspartyl protease with Alzheimer’s disease β-secre-tase activity. Nature 402, 533–537, 1999.

4. Hussain, I. et al. Identification of a novel aspartic protease (Asp 2) as β-secretase.Mol. Cell. Neurosci. 14, 419–427, 1999.

5. Lin, X. et al. Human aspartic protease memapsin 2 cleaves the β-secretase site ofβ-amyloid precursor protein. Proc. Natl. Acad. Sci. USA 97, 1456–1460, 2000.

6. Selkoe, D.J. Translating cell biology into therapeutic advances in Alzheimer’s disease.Nature 399A, 23–31, 1999.

7. Mullan, M. et al. A locus for familial early-onset Alzheimer’s disease on the longarm of chromosome 14, proximal to the alpha 1-antichymotrypsin gene. Nature Genet.2, 340–342, 1992.

8. Ghosh, A.K. et al. Design of potent inhibitors for human brain memapsin 2 (β-secre-tase). J. Am. Chem. Soc. 122, 3522–3523, 2000.

9. Turner, R.T., III et al. Subsite specificity of memapsin 2 (β-secretase): implicationsfor inhibitor design. Biochemistry 40, 10001–10006, 2001.

10. Hong, L. et al. Structure of the protease domain of memapsin 2 (β-secretase) com-plexed with inhibitor. Science 290, 150–153., 2000.

11. Hong, L. et al. Crystal structure of memapsin 2 (β-secretase) in complex with aninhibitor OM00-3. Biochemistry 41, 10963–10967, 2002.

12. Ermolieff, J. et al. Proteolytic activation of recombinant pro-memapsin 2 (pro-β-secretase) studied with new fluorogenic substrates. Biochemistry 39, 12450–12456,2000.

13. Ermolieff, J., Lin, X., and Tang, J. Kinetic properties of saquinavir-resistant mutantsof human immunodeficiency virus type 1 protease and their implications in drugresistance in vivo. Biochemistry 36, 12364–12370, 1997.

14. Co, E. et al. Proteolytic processing mechanisms of a miniprecursor of the asparticprotease of human immunodeficiency virus type I. Biochemistry 33, 1248–1254, 1994.

15. Liu, Y., Kati, W., Chen, C.-M., Tripathi, R., Molla, A., and Kohlbrenner, W. Use ofa fluorescence plate reader for measuring kinetic parameters with inner filter effectcorrection. Anal. Biochem. 267, 331–335, 1999.

16. Ghosh, A.K., Hong, L., and Tang, J. β-Secretase as a therapeutic target for inhibitordrugs. Curr. Med. Chem. 9, 1135–1144, 2002.

17. Ghosh, A.K. et al. Structure-based design: potent inhibitors of human brain memapsin2 (β-secretase). J. Med. Chem. 44, 2865–2868, 2001.

18. Bieth, J. Some kinetic consequences of the tight binding of protein-proteinase inhib-itors to proteolytic enzymes and their application to the determination of dissociationconstants, in Bayer Symposium V, Proteinase Inhibitors: Proceedings of the 2ndInternational Research Conference, Fritsch, H., Tschesche, H., and Greene, L.J., Eds.,Springer-Verlag, Berlin, 1974, p. 463.

19. Leatherbarrow, R.J. GraFit Version 3.0, Erithacus Software Ltd., Staines, U.K., 1990.20. Tomasselli, A.G. et al. Substrate analogue inhibition and active site titration of purified

recombinant HIV-1 protease. Biochemistry 29, 264–269, 1990.21. Marciniszyn, J., Jr., Hartsuck, J.A., and Tang, J. Mode of inhibition of acid proteases

by pepstatin. J. Biol. Chem. 251, 7088–7093, 1976.

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5
Assays for Amyloid Precursor Protein γ-Secretase Activity
William A. Campbell, Michael S. Wolfe, and Weiming Xia

CONTENTS

Abstract5.1 Introduction5.2 Main Scheme of Approaches5.3 Method

5.3.1 Assaying γ-Secretase Activity in Living Cells5.3.2 Subcellular Fractionation of Membrane Vesicles5.3.3 In Vitro γ-Secretase Activity Assay Using Endogenous Substrate5.3.4 Determining pH Dependence of γ-Secretase Activity Using

Vesicles5.3.5 Protease Inhibitor Profiling of γ-Secretase Activity Using

Fractions5.3.6 Cell Membrane Preparation for Exogenous Substrate Assay

5.3.6.1 Buffers5.3.7 M2 Flag Purification of E. coli-Generated γ-Secretase Substrates

5.3.7.1 Buffers5.3.8 In Vitro γ-Secretase Activity Assay Using Exogenous Substrate

5.4 DiscussionReferences

ABSTRACT

γ-Secretase cleavage, mediated by a complex of presenilin (PS), nicastrin, PEN-2,and APH-1, is the final proteolytic step in generating amyloid beta (Aβ) protein andthe Notch intracellular domain. Aβ and Notch are critical in the pathogenesis ofAlzheimer’s disease (AD) and in development, respectively. In addition to cleavingamyloid precursor protein (APP) and Notch, γ-secretase also cleaves over a dozenadditional type I transmembrane domain proteins. γ-Secretase activity can be mea-sured in vivo by collecting conditioned media from tissue cultured cells and in vitro

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press

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by incubating endogenous substrate with total microsomes or with Golgi/trans-Golginetwork (TGN)-enriched microsomal vesicles or incubating recombinant substratewith solubilized membranes. For APP, Western blotting or enzyme-linked immun-osorbent assay (ELISA) has been used to quantify the generation of Aβ40, Aβ42,and amyloid intracellular domain (AICD). These methods can be applied to studythe γ-secretase cleavage of any γ-secretase substrate in a variety of experimentalconditions.

5.1 INTRODUCTION

Genetic and neuropathological studies suggest that processing of amyloid precursorprotein (APP) to C99 and then to amyloid β protein (Aβ), the major component ofdestructive neuritic plaques found in brains of AD patients, plays an important rolein the neuronal loss that leads to AD.1 The final proteolytic event in generating Aβ,which is mainly a 40- or 42-residue peptide, is accomplished through presenilin(PS)-dependent γ-secretase cleavage of the C99 peptide.1 In addition to APP, γ-secre-tase also cleaves over a dozen type I transmembrane proteins such as the Notchproteins involved in cell fate determination, ErbB4 tyrosine receptor kinase andcadherins.2–5

The growing list of γ-secretase substrates includes APP, Notch,2 E-cadherin andN-cadherin,5,6 ErbB4 tyrosine receptor kinase,4 CD44,7,8 Nectin1α,9 Delta andJagged,10,11 LRP,12 DCC,13 APLP1 and APLP2,14–16 p75 neurotrophin receptor,17

Syndecan 3,18 glutamate receptor subunit 3,19 and colony stimulating factor 1.20 Thebiological significance of this cleavage is not clear in most cases, although onenormal function may be to release intracellular domains that regulate gene transcrip-tion in the nucleus, in at least some cases.

While absolute identification of the catalytic component of γ-secretase activityhas been elusive, mounting evidence points to PS1 and PS2. Numerous studies havedemonstrated that PS is necessary for γ-secretase cleavage and Aβ generation. Forexample, mutation of two critical aspartate residues in transmembrane (TM) domains6 and 7 of PS1 or PS2 abolishes Aβ generation in cultured cells21–23 and in transgenicmice.24 PS knockout neurons do not produce any Aβ.25,26 PS1 and PS2 bind to theimmediate substrates of γ-secretase, C99/C83, in the major sites of Aβ generation,i.e., Golgi/trans-Golgi network (TGN)-type vesicles.27

Finally, using aspartyl protease transition-state analogue γ-secretase inhibitorsto probe the active site of the enzyme revealed that these inhibitors bind directly toPS N- and C-terminal fragments (NTF and CTF).28,29 PS and PS homologues alsohave nonclassic protease motifs conserved from bacteria to humans,30,31 and thesequence motifs of PS are similar to a signal peptide peptidase.32 Signal peptidepeptidase forms a homodimer that is labeled by an active site-directed γ-secretaseinhibitor, indicating that the active sites of signal peptide peptidase and PS/γ-secre-tase are similar.33

PS1 and PS2 are homologous eight transmembrane domain-spanning proteinsthat undergo constitutive endoproteolysis by an unknown enzyme termed presenili-nase to generate functional stable heterodimers of NTF and CTF.34,35 Familial AD(FAD) mutations in PS1 or PS2 lead to increased production of the longer, more

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amyloidogenic version of Aβ, the 42-residue version, Aβ42.34,36–41 Knockout of PS1in mice is embryonic lethal.42 Conditional knockout of PS1 in the brain results indeficits of long-term potentiation and cognition43 while restricted expression of PS1in the brain in the background of PS1 knockout mice leads to skin tumorigenesis.44

Many studies have implicated the PS fragments, along with mature Nicastrin,APH-1 (anterior pharynx defective), and PEN-2 (presenilin enhancer), as the func-tional components of the γ-secretase complex. N-linked glycosylation of nicastrinin the Golgi apparatus is associated with its entry into the active γ-secretase complex,and this mature form interacts preferentially with the functional PS1 hetero-dimers.45–50 Down-regulation of APH-151,52 or PEN-251,53 by RNAi in cells is associ-ated with reduced levels of PS1 NTF and CTF heterodimers and deficient γ-secretasefunction. Overexpressing APH-1 stabilizes the full-length (FL) PS1, whereas reducingPEN-2 decreases endoproteolytic processing of PS1.54–56 Many reports have shownthat co-expression of PS1, Nicastrin, APH-1, and PEN-2 results in increased PS1endoproteolysis and γ-secretase activity, both in mammalian cells54–59 and in yeast.60

To measure γ-secretase activity, cell-based assays using the endogenous C99substrate were used initially. Living cells can be treated with γ-secretase inhibitorsand γ-secretase activity can be measured by release of soluble Aβ into the tissueculture medium. Using total membrane vesicles isolated from tissue culture cells,γ-secretase was found to be pH-dependent and showed maximal activity at pHbetween 6.3 and 6.4.61 After separation of intact, fully functional membrane vesiclesfrom cultured cells on discontinuous Iodixanol gradients, γ-secretase activity waspredominantly localized to Golgi/TGN-rich vesicles.62 PS bound to the immediatesubstrates of γ-secretase, the C-terminal fragments of APP, in these Golgi/TGN-richvesicles.27

Subsequently, γ-secretase activity was solubilized to partially characterize itsactivity using a recombinant substrate containing an initiating methionine, C99, anda Flag epitope (C100Flag). Anti-PS1 antibodies were found to immunoprecipitateγ-secretase activity from these solubilized membranes.63 Next, a Notch-based sub-strate, N100Flag, was created using an N-terminal methionine, 99 residues of theNotch1 sequence beginning from the ligand-dependent S2 cleavage site, and aC-terminal Flag sequence.64 The C100Flag and N100Flag substrates were then usedto further characterize the γ-secretase activity that cleaves APP and Notch.64,65 Withthe help of these substrates and the detergent-dependent assay, the presenilin–γ-secre-tase complex was isolated from solubilized membrane preparations in an activity-dependent manner using an immobilized active site-directed inhibitor, and thecomplex was found to contain Nicastrin and C83.64 A comparison of C100Flag andN100Flag proteolysis suggested that the responsible proteases are identical, witheach substrate preventing cleavage of the other, and both substrates being cleavedat two distinct regions in the transmembrane domain.65


Characterization of γ-secretase activity can be obtained by cellular assays of γ-secre-tase activity and by detergent solubilization of γ-secretase. The assays describedpresent reliable and reproducible methods to measure the cleavage of the C99

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fragment of APP to generate Aβ40, Aβ42, and AICD, and should be applicable tomeasure the γ-secretase cleavage of any γ-secretase substrate.

This protocol contains the procedures for analyzing γ-secretase activity in livingcells in vitro using endogenous substrate in total membrane microsomes and inGolgi/TGN-rich fractions and in vitro using solubilized membranes and E. coli-generated recombinant γ-secretase substrates. The first method describes measuringAβ secreted from living tissue culture cells. The second method describes thesubcellular fractionation of membrane vesicles on discontinuous Iodixanol gradientsto separate intact, functional ER-rich vesicles from Golgi/TGN-rich vesicles. In thethird method, γ-secretase activity is measured in the Golgi/TGN-rich fractions. Inthe fourth method, the optimal pH for γ-secretase activity is determined using totalmembrane vesicles. The next method describes protease inhibitor profiling to dis-cover the protease class of γ-secretase. The sixth method describes the preparationof solubilized cell membranes that retain functional γ-secretase enzymes. The sev-enth method describes the use of an M2 anti-Flag immunoaffinity isolation procedureto purify the γ-secretase substrates from E. coli. The last method details the γ-secretaseactivity assay using recombinant substrates. An outline of this scheme is presented inFigure 5.1. Subcellular fractionation of membrane organelles has been successfullyused to measure γ-secretase activity in Golgi/TGN-rich vesicles.27,57,62,66,67 Solubi-lized γ-secretase has been used successfully to measure the cleavage of recombinantsubstrates C100Flag and N100Flag.63–65,68

FIGURE 5.1 Methods discussed in this chapter.

Isolation of totalmicrosomal membranes

(Method 2)

Subcellular fractionation(Method 2) Cell membrane preparation

(Method 6)

γ−secretase activity inGolgi/TGN-rich fractions

(Method 3) Purification of substrates(Method 7)

γ−secretase activity insolubilized membranes

(Method 8)

Western blot and/or ELISA

Measuring secretedA β in living cells

(Method 1)

pH dependence(Method 4)

Protease inhibitor profiling(Method 5)

Endogenous substrate Exogenous substrate

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5.3 METHOD

5.3.1 ASSAYING γγγγ-SECRETASE ACTIVITY IN LIVING CELLS

The first method to measure γ-secretase activity presented here is accomplished bycollecting conditioned media from cultured cells treated either with a vehicle controlor a γ-secretase inhibitor. Treating living cells with a γ-secretase inhibitor causes anincrease in the immediate substrates of γ-secretase, the APP CTF’s C99 and C83, anddecreases the total amount of Aβ secreted into the tissue culture medium (Figure 5.2).

1. Culture cells to confluence.2. Make stock concentrations of a γ-secretase inhibitor in 100% dimethyl

sulfoxide (DMSO).3. Dilute the γ-secretase inhibitor or vehicle (DMSO) into the tissue culture

media to achieve the desired final concentration in 1% DMSO.4. After a 4-hr incubation, collect the conditioned medium and centrifuge it

at 10,000 × g for 5 min to pellet cells or cell debris.5. Remove the supernatant and store it at –80˚C until analysis by ELISA.6. The cells can be collected, lysed and subjected to immunoprecipitation

with APP polyclonal antibody C7 followed by Western blot analysis with

FIGURE 5.2 Treatment of living cells with a γ-secretase inhibitor increases APP CTFs(C-terminal fragments) and decreases the amount of Aβ secreted into the media. (a) Chinesehamster ovary cells overexpressing wild-type (wt) human APP were treated with increasingconcentrations (µM) of γ-secretase inhibitor compound 1,73,74 and cell lysates were immuno-precipitated with APP polyclonal antibody C7 followed by Western blotting with APP mon-oclonal antibody 13G8 to visualize the APP C terminal fragments (i.e., γ-secretase substrates).(b) Aβ levels in the conditioned media of cells treated with increasing doses of compound 1were determined by ELISA. Aβ levels in each experiment were normalized to mock-treated(1% DMSO) samples and averaged (n = 8). (Reprinted from Xia, W. et al. Neurobiol. Dis.2000, 7, 673–681. With permission.)

a.

0 10 20 30 40 50

Dose (uM)

14

b.

00.10.20.30.40.50.60.70.80.91.0

0

Dose (uM)

7.5 12.5 20 25 30 40 5010 15 17.5 22.5

Rel

ativ

e A

β To

tal

n = 8

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APP monoclonal antibody 13G8 to visualize the APP C-terminal frag-ments (i.e., γ-secretase substrates). Polyclonal antibody C769 and mono-clonal antibody 13G870 (gift of P. Seubert and D. Schenk) are directedagainst APP732–751 (APP751 numbering).

5.3.2 SUBCELLULAR FRACTIONATION OF MEMBRANE VESICLES

The fractionation employs discontinuous Iodixanol gradients that are used becausethey effectively separate ER- from Golgi/TGN-rich vesicles in a way that preservesvesicle structure and function, allowing detection of γ-secretase activity upon incu-bation of Golgi/TGN-rich vesicles at 37˚C.62,71 The viscosity of the gradient mediumis a major determinant of the sedimentation rate. The osmotic activity of the mediumis also important because subcellular organelles are osmotically sensitive. Thus,although sucrose, glycerol, and Ficoll are widely used for gradient fractionation ofcellular membranes, they are not ideal in osmolality and viscosity. The main advan-tage of using an Iodixanol gradient is that osmolality and viscosity remain relativelyconstant with changes in the density of the gradient. Under this mild iso-osmoticcondition, each organelle can be isolated functionally intact, without loss of water,as the density of the gradient increases.

1. Culture five 15-cm dishes until confluent (~1 × 108 cells).2. Detach the cells with 20 mM EDTA in PBS (8 mL/15-cm plate).3. Pellet the cells by spinning for 5 min at 4˚C, 1000 rpm. The cell pellet

can be frozen at –80˚C indefinitely.4. Resuspend the cell pellet in 3 mL of cold homogenization buffer (0.25 M

sucrose, 10 mM HEPES, 1 mM EDTA) with freshly added proteaseinhibitors.

5. Break open the cells with ten strokes of a Dounce homogenizer and passthe cells through a 27-gauge needle five times.

6. Pellet the nuclei and unbroken cells by centrifugation at 1500 × g for10 min at 4˚C. Save the postnuclear supernatant.

7. Extract the pellet again by resuspending in 4 mL of homogenization bufferand centrifuge at 1500 × g for 10 min at 4˚C. Save the postnuclearsupernatant.

8. Combine the two supernatants and centrifuge for 1 hr at 65,000 × g, 4˚C,to pellet total membrane vesicles.

9. For measuring γ-secretase activity in total vesicles, the vesicles are washedin 0.1 M sodium carbonate (pH 11.3) on ice to remove peripherallyassociated membrane proteins and centrifuged at 100,000 × g for 1 hr.The vesicle precipitate is then resuspended in incubation buffer (10 mMKOAc, 1.5 mM MgCl2). One portion is lysed with Laemmli sample buffer(10% SDS, 0.3 M Tris, 50% glycerol, 0.1% bromophenol blue, 10%β-mercaptoethanol) for Western blot or 2X guanidium HCl (1 M guanid-ium HCl, 2% NP-40, 2 mM EDTA) for ELISA for basal γ-secretaseactivity. The other portion is incubated at 37˚C for de novo γ-secretaseactivity (see below).

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10. For separation of ER- and Golgi/TGN-rich vesicles, the membrane vesiclesare used to prepare subcellular fractions by resuspending the microsomalpellet in 800 µL of homogenization buffer on ice. Different percentagesof Iodixanol, 5,59-[(2-hydroxy-1-3- propanediyl)-bis(acetylamino)]bis-[N,N-9-bis(2,3-dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide]are made by diluting with OptiPrep (Accurate Chemical, Westbury, NY,60% Iodixanol).

11. A gradient stock solution of 50% Iodixanol is prepared by diluting in0.25 M sucrose, 6 mM EDTA, 60 mM HEPES, pH 7.4, at a 5:1 ratio.Different densities of Iodixanol are established by diluting this stock with0.25 M sucrose homogenization buffer.

12. The Iodixanol gradient is poured in 13 mL Beckman SW41 centrifugetubes by careful underlayering, adding each layer under the first layerwith a long needle, as follows:

1 mL 2.5% Iodixanol2 mL 5% Iodixanol2 mL 7.5% Iodixanol2 mL 10% Iodixanol0.5 mL 12.5% Iodixanol2 mL 15% Iodixanol0.5 mL 17.5% Iodixanol0.5 mL 20% Iodixanol0.3 mL 30% Iodixanol

13. Load the resuspended vesicles on top of the gradient and centrifuge in anSW41 rotor at 200,000 × g for 2.5 hr at 4˚C. Decrease the accelerationand deceleration rates of the centrifuge so the gradient is not disturbed.

14. Collect 12 fractions ~1 mL at a time by puncturing the bottom of the tubewith a 22-gauge needle. Seal the top of the tube with Parafilm prior topuncturing to control the flow of the gradient from the tube. Store thefractions indefinitely at –80˚C.

5.3.3 IN VITRO γγγγ-SECRETASE ACTIVITY ASSAY USING ENDOGENOUS SUBSTRATE

The 12 fractions must be characterized by Western blotting with the ER-specificmarker calnexin and the Golgi/TGN-specific marker syntaxin 6 or by measuring theactivity of the Golgi-specific enzyme galactosyltransferase to determine which frac-tions are Golgi/TGN-enriched.62,66 Typically, the first three fractions contain calnexin-positive ER-rich vesicles, the fourth fraction contains a mixture of ER andGolgi/TGN vesicles, and fractions five through eight contain Golgi/TGN-rich vesi-cles that can be used for measuring γ-secretase activity.62 Instead of using eachindividual fraction, the first four fractions can be combined and considered ER-richvesicles; fractions five through eight can be combined and considered Golgi/TGN-rich vesicles (Figure 5.3a). These vesicles can now be incubated at 37˚C for mea-surement of de novo γ-secretase activity, for example, by Aβ-specific ELISA orWestern blotting for AICD after incubation.

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1. On ice, thaw the fractions found to be Golgi/TGN-rich. If adding γ-secre-tase inhibitors or a vehicle control, add them to each well or tube at roomtemperature before adding the fractions on ice. Typically these reactionsare run in a 96-well polymerase chain reaction (PCR) plate.

FIGURE 5.3 Generation of Aβx-40, Aβx-42, and AICD in Golgi/TGN-rich microsome fractions.(a) Microsomes isolated from Chinese hamster ovary cells stably expressing wt APP and wtPS1 (PS1wt-1) were fractionated on discontinuous Iodixanol gradients. The first four 1-mLfractions were combined, and an aliquot was lysed and run in lanes 1 and 2. The next four1-mL fractions were combined, and an aliquot was lysed and run in lanes 3 and 4. ER-enrichedmicrosomes and Golgi/TGN-rich microsomes were lysed and probed by Western blot for theER marker calnexin and the Golgi/TGN marker syntaxin 6. The first four fractions wereenriched in the ER marker calnexin, while the next four fractions were enriched in theGolgi/TGN marker syntaxin 6. (b) and (c) The Golgi/TGN-enriched vesicles were lysed attime zero or incubated at 37˚C for the indicated times and then lysed. The lysed vesicles werethen subjected to ELISA for measurement of de novo Aβx-40 and Aβx-42 generation. Aβ levelsin each experiment were normalized to basal levels (denoted by dotted line) obtained at time 0.Error bars represent the standard error of the mean in all figures. (d) Golgi/TGN-richmicrosomes were incubated at 37˚C up to 2 hr, lysed and probed by Western blot withpolyclonal antibody R57 that readily detects AICD. Duplicate samples incubated at 37˚C for120 min were presented. (Reprinted from Campbell, W.A. et al. J. Neurochem. 2003, 85,1563–1574. With permission.)

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2. Gently invert the Golgi/TGN-rich fractions and aliquot 50 µL into eachcold tube or well for 4˚C and 37˚C samples. To the control 4˚C aliquot,immediately add 25 µL of 3X Laemmli sample buffer for determinationof basal AICD levels, for example. If the goal is to measure Aβ by ELISA,then add 50 µL of 2X guanidinium HCl.

3. If adding γ-secretase inhibitors or DMSO, make sure that they are insolution and/or mixed well by heating the samples at 37˚C for 2 min andvortexing. This aliquot is incubated at 37˚C for 2 hr for determination ofde novo Aβ or AICD generation. The reaction is stopped by adding 25 µLof 3X Laemmli sample buffer or 50 µL of 2X guanidinium HCl followedby vortexing.

4. AICD levels in both aliquots can be probed by Western blot. Densitometryusing the AlphaEase™ software package (Alpha Innotech, San Leandro,CA) is used to quantify protein levels from at least three independentblots, and the level of newly generated AICD is calculated by subtractingthe amount of AICD observed at 4˚C from that obtained at 37˚C.

5. For determination of de novo Aβ generation in vitro by ELISA, the newlygenerated Aβ levels are calculated by subtracting the Aβ level observedat 4˚C from that obtained at 37˚C.

6. For inhibition experiments with γ-secretase inhibitors, the mean levels ofnewly generated AICD or Aβ in the absence of inhibitor (H2O, DMSOor methanol) is used as the denominator (100%), and the relative percent-age is obtained by comparing AICD or Aβ levels in the presence ofinhibitor against this denominator in each experiment. Negative valuesreflect reduced levels after incubation.

Results using this method in a time course experiment are presented in Figure 5.3bthrough Figure 5.3d. Generation of endogenous Aβx-40 and Aβx-42 as measured byELISA and generation of endogenous AICD as measured by Western blot all beginapproximately 10 min after incubation of Golgi/TGN-enriched vesicles at 37˚C.

5.3.4 DETERMINING PH DEPENDENCE OF γγγγ-SECRETASE ACTIVITY USING VESICLES

This method has been used to show that the optimal pH for γ-secretase activity is6.3 to 6.4 (Figure 5.4).61 Sodium citrate is added to total membrane vesicles toachieve a final pH gradient of 5.3 to 7.6. After removal of all cytosol and extensivewashing of these microsomal vesicles, not much specific ionic exchange shouldoccur among these membranes. The pH in the suspension should closely reflect theactual pH inside the vesicles.

1. Isolate total microsomal vesicles as described in method 5.3.2.2. Resuspend microsomal vesicles in 5 mL incubation buffer (10 mM KOAc,

1.5 mM MgCl2) plus 50 mM sodium citrate, pH 5.6.3. Titrate the pH by adding a battery of stock solutions of 1M sodium citrate

with increasing pH (from 4.5 to 7.4) to isolated microsomes in incubation

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buffers. This results in a concentration of 50 mM sodium citrate with thefinal pH ranging from 5.3 to 7.6, as measured by a microprobe.

4. For in vitro Aβ generation, total isolated microsomes in incubation bufferare divided into two aliquots. One aliquot is used for determination ofbasal Aβ levels by adding an equal volume of stop solution (2X guani-dinium HCl) and freezing at –80˚C.

5. The other aliquot is incubated at 37˚C for 4 hr followed by addition ofan equal volume of stop solution.

6. Aβ levels are determined in both aliquots by ELISA, and the newlygenerated Aβ levels are calculated by subtracting the Aβ level generatedat –80˚C from that at 37˚C.

5.3.5 PROTEASE INHIBITOR PROFILING OF γγγγ-SECRETASE ACTIVITY USING FRACTIONS

This method can be used to determine whether any drug of choice or which classof protease inhibitor can inhibit γ-secretase activity. For pharmacological inhibition

FIGURE 5.4 pH dependence of Aβ generation in isolated microsomal vesicles. Stock solu-tions of sodium citrate with different pH levels were added to isolated microsomal vesiclesfrom PS1WT-1 (a) or 293695SW (b) cells. Aliquots were incubated at various pH levels, andde novo Aβ generation was measured by ELISA. Relative Aβ levels generated at each pHwere normalized with respect to the highest value (averages shown as x, with standard errorsindicated by bars; PS1WT-1, n = 5; 293695SW, n = 6). A third-order polynomial curve fittingrevealed a bell curve with the peak at pH 6.3 to 6.4. (Reprinted from Xia, W. et al. Neurobiol.Dis. 2000, 7, 673–681. With permission.)

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of γ-secretase, gradient fractions are incubated with each protease inhibitor orγ-secretase inhibitor at the desired concentration or with DMSO, methanol, or H2Ovehicle alone for 2 hr at 37˚C.

1. On ice, thaw the fractions found to be Golgi/TGN-rich. These reactionscan easily be run in a 96-well PCR plate testing three concentrations ofa particular inhibitor over three orders of magnitude. Add the proteaseinhibitor to a final concentration within the effective range of its activityto each well or tube at room temperature before adding the fractions on ice.

2. The four classes of protease inhibitors with the names of the inhibitorsand their reported effective concentrations in parentheses are as follows:aspartyl (pepstatin A, 1 µM), metallo (1,10-phenathroline, 1 to 10 mM;EDTA, 1 to 10 mM; phosphoramidon, 1 to 10 µM), serine (Pefabloc, 0.4to 4.0 mM; PMSF, 0.1 to 1.0 mM), and serine/cysteine (leupeptin, 10 to100 µM).72

3. Gently invert the Golgi/TGN-rich fractions and aliquot 50 µL into eachwell on ice for 4 and 37˚C samples. To the control 4˚C aliquot, immedi-ately add 25 µL of 3X Laemmli sample buffer for determination of basalAICD levels, for example. If the goal is to measure Aβ by ELISA thenadd 50 µL of 2X guanidinium HCl.

4. Ensure that the protease inhibitor is in solution by vortexing well, followedby heating the samples at 37˚C for 2 min, vortexing again, and incubatingat 37˚C for 2 hr.

5. Fractions are then lysed and subjected to Western analysis or ELISA.

5.3.6 CELL MEMBRANE PREPARATION FOR EXOGENOUS SUBSTRATE ASSAY

Total membranes are prepared from a large batch of cells (e.g., Jurkat or HeLa) formeasuring endogenous γ-secretase activity. It is important to remember that in orderto maintain functional γ-secretase activity the membranes must be solubilized in acorrect detergent that will not disrupt the γ-secretase complex, e.g., CHAPSO (SoltecVentures, Beverly, MA).

1. Collect and pellet cells. For cells grown in suspension (such as Jurkat orHeLa cells), collect 6.25 × 107 cells per microsome preparation. A volumeof 2.5 L of HeLa cells at 1 × 106 cells/mL will yield “40x” microsomes.For adherent cells, collect cells from five 15-cm dishes per microsomepreparation. Centrifuge at ~3000 × g for 10 min. Cell pellets can be storedat –80˚C until needed. This is defined as a 1x microsome preparation.

2. Add 30 mL of MES buffer (with 100x protease inhibitors added freshly)to 120x of microsomes and fully resuspend the cells.

3. Pass the cells once through a French pressure cell at greater than 1000 psi.4. Pellet the nuclei and unbroken cells by centrifuging the samples at 3000 ×

g for 10 min (4550 rpm in a Sorvall SA600 rotor). Save the postnuclearsupernatant.

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5. Pellet total membranes by centrifuging the supernatant at 100,000 × g for1 hr (30,000 rpm in SW55 rotor; 25,000 rpm in SW41 rotor). Discard thesupernatant.

6. Microsome pellets as 10x or 20x pellets can be stored at –80˚C untilneeded.

7. Resuspend each 10x microsome pellet in 0.5 mL of ice cold bicarbonatebuffer. Pipet up and down at least 30 times. Incubate 20 min at 4˚C.

8. Pellet the washed membranes by centrifuging at 100,000 × g for 1 hr at4˚C and discard the supernatant.

9. Resuspend the membranes by adding 0.5 mL of 1% CHAPSO lysis bufferto a bicarbonate-washed 10x microsome pellet. Pipet up and down at least30 times. Vortex briefly and incubate for 1 hr at 4˚C.

10. Pellet the insoluble material by centrifugation at 100,000 × g for 1 hr at4˚C. Discard the pellet. Dilute the supernatant 1:3 with HEPES buffer(final CHAPSO concentration is 0.25%). This diluted lysate is defined assolubilized γ-secretase. Measure the protein concentration with BCAreagent. The concentration should be ~0.10 to 0.25 mg/mL. Samples canbe aliquoted and stored at –80˚C.

5.3.6.1 Buffers

MES Buffer: 50 mM MES, pH 6.0, 150 mM NaCl, 5 mM MgCl2, 5 mMCaCl2, 100X complete protease inhibitor cocktail (Roche) added freshly

Sodium Bicarbonate buffer: 0.1 M NaHCO3, pH 11.3CHAPSO lysis buffer: 1% CHAPSO, HEPES bufferHEPES buffer: 50 mM HEPES, pH 7.0, 150 mM NaCl, 5 mM MgCl2, 5 mM

CaCl2

5.3.7 M2 FLAG PURIFICATION OF E. COLI-GENERATED γγγγ-SECRETASE SUBSTRATES

To examine γ-secretase activity in vitro without the complication of cellular material,a recombinant substrate such as C100Flag can be generated to measure activity. Thesubstrate is expressed in E. coli and purified using an M2 anti-Flag immunoaffinityresin.

1. Inoculate a 5-mL Luria Broth (LB) culture (with 100 µg/mL ampicillin)of BL21 (DE3)-transformed E. coli by scraping a small amount of glycerolculture, shaking at 37˚C throughout the day and storing at 4˚C overnight.

2. Inoculate the small culture into 250 mL LB medium and grow at 37˚Cuntil OD600 = 1.0.

3. Induce protein expression by adding 1 mM IPTG (59.5 mg/250 mL) and100 µg/mL ampicillin into the cultures and return to 37˚C for 2 hr.

4. Collect the cell pellet by centrifuging the cells at 3000 × g for 10 min.5. Lyse 125 mL of cell pellet in 10 mL of 1% Triton X-100 lysis buffer with

protease inhibitors and pass through the French press twice at pressureabove 1000 psi.

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6. Spin down the insoluble pellet by centrifugation at 3000 × g for 10 min.7. Bind to the M2 anti-Flag resin for 2 hr by adding 2 mL of M2 anti-Flag

resin (4 mL of slurry) and rock for 2 hr at room temperature.8. Pour into a 2-mL column and wash the column with lysis buffer three

times with two column volumes (12 mL total).9. Elute the substrate with acidic glycine, collecting five fractions of 2 mL

each. Re-equilibrate the column immediately with 10 mL PBS, thenexchange into storage buffer. Store the column at 4˚C until reuse. Thecolumn can be used up to three times. Analyze each fraction on a 4 to30% Tris-glycine gel and stain with Coomassie blue (Gelcode Blue).

5.3.7.1 Buffers

Lysis buffer: 10 mM Tris, pH 7.0, 150 mM NaCl, 1% Triton X-100, 1Xbacterial protease inhibitor (Sigma, St. Louis, MO)

Elution buffer: 1% NP-40, 100 mM glycine, pH 2.7Storage buffer: 50% glycerol, phosphate buffered saline, 0.02% Na azide

5.3.8 IN VITRO γγγγ-SECRETASE ACTIVITY ASSAY USING EXOGENOUS SUBSTRATE

To measure γ-secretase activity in the solubilized membrane preparation, it is rela-tively straightforward to combine the substrate and the membranes. Since theC100Flag protein tends to form high molecular weight aggregates, perhaps due tothe presence of the Aβ sequence, SDS must be added to the substrate to disaggregateit. The assay must be performed in a detergent such as CHAPSO that is compatiblewith γ-secretase activity. The addition of phosphatidylcholine (PC) and phosphati-dylethanolamine (PE) at certain concentrations can augment γ-secretase activity.65

After incubation of the membranes at 37˚C, the three predominant cleavages madeby γ-secretase can be detected by either Western blot or ELISA using the appropriateantibodies.

1. Thaw bicarbonate-washed, detergent-solubilized HeLa cell membranes(0.25% CHAPSO HEPES) and substrate (C100Flag) on ice.

2. Disaggregate the substrate by heating in SDS. Add SDS to 0.5% finalconcentration, then heat for 5 min at 65˚C. Centrifuge at 14,000 rpm for2 min to pellet the insoluble C100Flag aggregates.

3. Aliquot into a 96-well plate or microfuge tubes. For each assay, aliquot50 µL of solubilized HeLa cell membranes at 0.150 mg/mL and add2.5 µL of PE (5 mg/mL in 1% CHAPSO–HEPES) and 5 µL of PC(10 mg/mL in 1% CHAPSO–HEPES) to a final concentration of0.025% PE and 0.100% PC. Now add 1 µL of C100FLAG. For time 0,add 15 µL of 4X Laemmli sample buffer prior to adding the substrate. Ifthe sample will be analyzed by Aβ ELISA, stop with 10 µL of 2.5% SDS.

4. Mix briefly by vortexing, cover with a 96-well plate sealer to preventevaporation and incubate for 4 hr at 37˚C.

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5. Stop the reaction by adding 15 µL of 4X Laemmli sample buffer or 10 µLof 2.5% SDS solution and mix.

6. Analyze by M2 anti-Flag western blot or Aβ ELISA. Load 20 µL ofsample onto an Invitrogen 4 to 20% Tris-glycine 10- or 12-well gel.Transfer and western blot with M2 anti-Flag antibody (Sigma) usingstandard protocols. Alternatively, analyze the C100Flag reaction for Aβx-40

and/or Aβx-42 proteolytic products by ELISA.7. Important points to consider about the enzyme: It is stable at 4˚C for at

least 1 week and survives three freeze–thaws, but detergents such asNP-40, Triton X-100 and SDS disrupt activity. Optimal pH is slightlyacidic to neutral (6.5 to 7.0) and CHAPSO, CHAPS, and Big CHAPdetergents are critical for retaining activity, preferably below the criticalmicelle concentration (0.25% for CHAPSO).

5.4 DISCUSSION

This chapter has described assays for measuring γ-secretase activity in whole cells,total membranes, Golgi/TGN-enriched membrane vesicles from cultured cells, andin a solubilized membrane preparation using recombinant substrates. We also dis-cussed methods for determining the optimal pH of γ-secretase activity and usingprotease inhibitor profiling for γ-secretase activity. While we have described theseassays to examine the γ-secretase-mediated cleavage of APP, certainly these proto-cols can be adapted to examine the cleavage of any γ-secretase substrate. Thesemethods have already been applied to Notch cleavage, for example.64,65 In any case,it is always necessary to include controls. For the Golgi/TGN-enriched vesicles, thecollected fractions must be characterized to confirm that they are Golgi/TGN-enriched and a γ-secretase inhibitor should be included during incubation as acontrol. For the substrate assays, it is also important to disaggregate the substrateand include a γ-secretase inhibitor as a control. The amounts of PE and PC addedto the assay may need to be optimized for each substrate.

In summary, the approaches described in this chapter provide methods to analyzeγ-secretase activity in whole cells, in vesicles isolated from cells, or in solubilizedmembranes. In this way, the γ-secretase cleavage of each substrate as well as theinherent characteristics of the enzyme itself can be analyzed.

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54. Hu, Y. & Fortini, M. Different cofactor activities in γ-secretase assembly: evidencefor a nicastrin-Aph-1 subcomplex. J. Cell Biol. 161, 685, 2003.

55. Luo, W.J. et al. PEN-2 and APH-1 coordinately regulate proteolytic processing ofpresenilin 1. J. Biol. Chem. 278, 7850, 2003.

56. Takasugi, N. et al. The role of presenilin cofactors in the γ-secretase complex. Nature422, 438, 2003.

57. Baulac, S. et al. Functional γ-secretase complex assembly in Golgi/trans-Golgi net-work: interactions among presenilin, nicastrin, Aph1, Pen-2 and γ-secretase sub-strates. Neurobiol. Dis. 14, 194, 2003.

58. De Strooper, B. Aph-1, Pen-2, and nicastrin with presenilin generate an active γ-secre-tase complex. Neuron 38, 9, 2003.

59. Kimberly, W. et al. γ-Secretase is a membrane protein complex comprised of prese-nilin, nicastrin, Aph-1, and Pen-2. Proc. Natl. Acad. Sci. USA 100, 6382, 2003.

60. Edbauer, D. et al. Reconstitution of γ-secretase activity. Nat. Cell Biol. 7, 7, 2003.61. Xia, W. et al. FAD mutations in presenilin-1 or amyloid precursor protein decrease

the efficacy of a γ-secretase inhibitor: a direct involvement of PS1 in the γ-secretasecleavage complex. Neurobiol. Dis. 7, 673, 2000.

62. Xia, W. et al. Presenilin 1 regulates the processing APP C-terminal fragments andthe generation of amyloid β-protein in ER and Golgi. Biochemistry 37, 16465, 1998.

63. Li, Y.M. et al. Presenilin 1 is linked with γ-secretase activity in the detergent solubi-lized state. Proc. Natl. Acad. Sci. USA 97, 6138, 2000.

64. Esler, W.P. et al. Activity dependent isolation of the presenilin-γ-secretase complexreveals nicastrin and a γ substrate. Proc. Natl. Acad. Sci. USA 99, 2720, 2002.

65. Kimberly, W.T. et al. Notch and the amyloid precursor protein are cleaved by similarγ-secretase(s). Biochemistry 42, 137, 2003.

66. Campbell, W. et al. Endoproteolysis of presenilin in vitro: inhibition by γ-secretaseinhibitors. Biochemistry 41, 3372, 2002.

67. Campbell, W.A. et al. Presenilin endoproteolysis mediated by an aspartyl proteaseactivity pharmacologically distinct from γ-secretase. J. Neurochem. 85, 1563, 2003.

68. Xu, M. et al. γ-Secretase: characterization and implication for Alzheimer diseasetherapy. Neurobiol. Aging 23, 1023, 2002.

69. Podlisny, M.B., Tolan, D., and Selkoe, D.J. Homology of the amyloid β-proteinprecursor in monkey and human supports a primate model for β-amyloidosis inAlzheimer’s disease. Am. J. Pathol. 138, 1423, 1991.

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70. Oltersdorf, T. et al. The Alzheimer amyloid precursor protein: identification of a stableintermediate in the biosynthetic/degradative pathway. J. Biol. Chem. 265, 4492, 1990.

71. Zhang, J. et al. Subcellular distribution and turnover of presenilins in transfectedcells. J. Biol. Chem. 273, 12436, 1998.

72. Beynon, R. and Salvesen, G., Proteolytic Enzymes: A Practical Approach. Beynon,R. and Bond, J., Eds., Oxford University Press, New York, 2001, p. 317.

73. Wolfe, M.S. et al. A substrate-based difluoro ketone selectively inhibits Alzheimer’sγ-secretase activity. J. Med. Chem. 41, 6, 1998.

74. Wolfe, M.S. et al. Peptidomimetic probes and molecular modeling suggest Alzhei-mer’s γ-secretase is an intramembrane-cleaving aspartyl protease. Biochem. 38, 4720,1999.

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6
Cell-Free Reconstitution of β-Amyloid Production and TraffickingDongming Cai, William J. Netzer, Feng Li, and Huaxi Xu
CONTENTS

Abstract6.1 Introduction6.2 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 System6.2.4 Preparation of Energy-Regenerating System for Cell-Free

Reconstitution System6.2.5 Aβ Generation in Cell-Free System Utilizing ERS in the Absenc

of Cytosol6.2.6 Detection of Aβ Production from Cell-Free System6.2.7 Formation of Nascent Secretory Vesicles in Cell-Free System

Supplemented with ERS and Cytosol6.2.8 Detection of βAPP in Different Fractions through

Immunoprecipitation6.3 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

AcknowledgmentsReferences

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press

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ABSTRACT

The production of β-amyloid is one aspect of the metabolism of βAPP. To achievean effective understanding of this process, its must be considered in terms of boththe enzymatic activities required for the proteolytic processing of βAPP and thetrafficking of βAPP and its metabolites within the secretory pathway. Whole cellsare often insufficient to elucidate the details of this process. We have thereforedeveloped a cell-free reconstitution system for β-amyloid and βAPP production andtrafficking. This experimental system has allowed us to study the effects of addingor removing salient cellular factors and to focus on βAPP processing as it occursin various subcellular compartments.

6.1 INTRODUCTION

Alzheimer’s disease (AD) is characterized by the excessive generation and accumu-lation of β-amyloid (Aβ) peptides. The amyloidogenic Aβ peptide is proteolyticallyderived from the β-amyloid precursor protein (βAPP) within the secretory pathwayby distinct enzymatic activities known as β- and γ-secretase.1,2 Full-length βAPP issynthesized in the endoplasmic reticulum (ER) and transported through the Golgiapparatus. The major population of secreted Aβ peptides is generated within thetrans-Golgi-network (TGN),3–5 also the major site of βAPP residence in neurons atsteady state. βAPP can be transported in TGN-derived secretory vesicles to the cellsurface if not first proteolyzed to Aβ or an intermediate metabolite.

At the plasma membrane βAPP is either cleaved by the α-secretase activity toproduce a soluble molecule, sβAPP,6 or alternatively, reinternalized within clathrin-coated vesicles to an endosomal/lysosomal degradation pathway.7,8 The endocyticcompartments have also been shown to contribute to Aβ generation.9,10 Thus, thedistribution of βAPP between the TGN and cell surface has a direct influence uponthe relative generation of sβAPP versus Aβ. This phenomenon makes delineation ofthe mechanisms responsible for regulating βAPP trafficking from the TGN/ERrelevant to understanding the pathogenesis of AD.

It has been suggested that AD pathology rests on the cellular processes thatregulate βAPP metabolism and its localization within the secretory compartment.Specifically, it was demonstrated that Aβ 1-40 and various N terminal truncated Aβvariants (x-40) are produced largely in the TGN and packaged into post-TGNsecretory vesicles. It was also demonstrated that Aβ1-42 (one of the most amy-loidogenic forms) and Aβx-42 are produced in the TGN and are also packaged intopost-TGN secretory vesicles.5 In addition, a fraction of the N-terminally truncatedAβ42, Aβx-42, can be generated and remain insoluble in the ER.5,11

These studies suggest that the accumulation of intracellular Aβ may be importantin AD pathology and that stable intracellular pools of insoluble Aβ may exist. Inaddition, studies by our group suggest that the α-secretase activity is up-regulatedby protein kinase C (PKC).12 This phenomenon was investigated by utilizing a cell-free system consisting of isolated TGN from PC12 cells, which allowed us to control

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the exposure of viable secretory membranes to cytosolic factors that could affectβAPP metabolism and vesicle trafficking.28 It was also demonstrated, using a similarexperimental approach, that activation of PKA increases trafficking of βAPP fromthe TGN, but through a mechanism that is distinct from PKC.13 Recently, we reportedthat 17β-estradiol (17β-E2), a gonadal steroid hormone known to reduce Aβ forma-tion both in cells and in AD transgenic mice,14,15 stimulates formation of vesiclescontaining βAPP from the TGN. Accelerated βAPP trafficking precludes maximalAβ generation within the TGN.16

The cell-free system described herein has been used successfully to investigatethe formation of nascent secretory vesicles containing various proteins such asprohormones, VSV G protein and βAPP, as well as proteolytic cleavage of βAPPin a variety of cells.5,17,18 This cell-fee reconstitution system has been well charac-terized, e.g., the integrity of Golgi stacks and nascent vesicles has been demonstratedby electron microscopy, and the intactness of the nascent vesicles has been shownby the resistance of luminal cargo proteins to proteinase K digestion.19

It is well known that trafficking of proteins to the cell surface from TGN/ERdepends on the recruitment of cytosolic trafficking factors and the precise regulationof phospholipid composition.20,21 In addition, vesicle biogenesis is also dependenton the correct conformation or folding of the cargo molecule or the proper interac-tions between each component of a complex cargo. One of the best examples is thatthe transport of VSV G protein from the ER requires oligomerization of its mono-mers, a process of protein–protein interaction strictly dependent upon adenosinetriphosphate (ATP) and temperature. Failure of proper oligomerization, by ATPdepletion or incubation at nonpermissive temperatures severely impairs the exit ofVSV G from the ER.22,23 Interestingly, our results also demonstrated an ATP require-ment for Aβ formation at the γ-secretase cleavage step.3,24 Therefore, supplementa-tion of cytosol and/or energy in the cell-free reconstitution system has been crucialfor analyzing the metabolism and trafficking of βAPP through the secretory pathway.

In addition, it has been well established that incubation of cells at 15 or 20oCleads to an accumulation of membrane and secretory proteins in the ER and TGN,respectively.17,25 These phenomena were first taken advantage of in cell-free systemsthat reconstituted prohormone cleavage and maturation which allow accumulationof prohormones in the respective compartments without being proteolyzed at thelow temperatures.17 In general, small peptide hormones are synthesized as longerpolyproteins that must be cleaved endoproteolytically at specific sites to yield themature peptide hormones. Using a cell-free system along with temperature blocksallowed both the cellular sites of prohormone maturation and necessary cellularfactors to be elucidated. Similar methods have been co-opted successfully for thestudy of βAPP processing.

The objective of this chapter is to provide a thorough outline of experimentalprocedures for preparation of the cell-free reconstitution system and the analysis ofAβ production as well as intracellular trafficking of βAPP from the TGN and theER in this system.

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6.2 METHODS

6.2.1 PULSE-LABELING OF N2A CELLS AND TEMPERATURE BLOCKING OF NASCENT PROTEIN TRANSPORT

1. Mouse neuroblastoma N2a cells overexpressing human βAPP variants aregrown in culture media up to 80% confluent (one 100-mm plate is suffi-cient to prepare two cell-free reactions).

2. Wash cells on plate in 2 ml Dulbecco’s Modified Eagle Medium (DMEM)media without methionine. Then starve cells in 2 ml DMEM withoutmethionine for 30 min at 37oC.

3. Cells are then labeled with [35S]methionine (500 µCi/ml) for 10 to 15 minat 37oC.

4. Wash cells with PBS (prewarmed or equilibrated to 20oC) three times.5. Add 2 or 3 ml complete media equilibrated to 20oC and incubate the cells

at 20˚C for 2 hr by floating plates in an undisturbed water bath. At thistemperature, the transport of proteins, including βAPP, from the ER tothe TGN is unimpaired. However, under these conditions, the egress ofsecretory vesicles from the TGN is blocked, thus allowing labeled full-length APP (and other newly synthesized, labeled membrane proteins) toaccumulate in the TGN.3,5,16 This experimental design is used when eitherTGN-specific vesicle biogenesis or βAPP metabolism is assayed.

6. Alternatively, to assay βAPP trafficking from the ER or APP metabolismin the ER, cells are labeled with [35S]-methionine for 4 hr at 15˚C toaccumulate βAPP within the ER and block its transport to the TGN.

6.2.2 PREPARATION OF PERMEABILIZED N2A CELLS THROUGH OSMOTIC SHOCK AND MECHANICAL SHEAR

For both TGN and ER temperature block experiments, cells are permeabilized afterthe 2- or 4-hr chase as follows.

1. Transfer plates to 4oC immediately.2. Cells are incubated at 4oC in 3 ml “swelling buffer” (10 mM KCl, 10 mM

HEPES, pH 7.2) for 10min.3. The buffer is discarded and replaced with 1 ml of “breaking buffer”

(90 mM KCl, 10 mM HEPES, pH7.2), after which the cells are brokenby scraping vigorously with a rubber policeman.

4. The permeabilized cells are centrifuged at 800 × g for 5 min to removecytosol, and washed in 5 ml of breaking buffer. The permeabilized cellsmust be washed and resuspended at least three times in order to removecytosol and resuspended in five pellet volumes of breaking buffer. Thisshould result in >95% cell breakage as evaluated by trypan blue staining.

5. Broken cells (cell-free system) are resuspended and incubated in a finalvolume of 300 µl containing 2.5 mM MgCl2, 0.5 µM CaCl2, and 110 mMKCl, an energy-regenerating system (ERS), with or without cytosol (15 µgproteins) prepared from N2a or other cells as needed.16,19 A protease

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inhibitor cocktail is added for vesicle budding experiments but not whenAβ production is assayed.

6.2.3 PREPARATION OF CYTOSOL FOR CELL-FREE RECONSTITUTION SYSTEM

1. Ten plates of N2a (or other desired) cells are cultured and collected intheir media when grown up to 90% confluent.

2. Pellet cells down by centrifugation at 800 × g for 5 min.3. Wash cell pellet with TEA buffer (10 mM triethanolamine, 140 mM

KOAc, pH 7.2) followed by a wash in homogenization buffer (25 mMHEPES–KOH, pH 7.2, 125 mM KOAc, and protease inhibitor cocktail).

4. Resuspend cells in 1 volume of pellet/5 volumes of homogenization buffer.5. Cells are then broken in a stainless ball-bearing homogenizer (clearance

18 µm) by passing the cell suspension through the homogenizer four orfive times.

6. A postnuclear supernatant is created by centrifuging at 800 × g for 5 minat 4˚C.

7. This postnuclear supernatant is further centrifuged at 100,000 × g for 1 hrat 4˚C.

8. The supernatant is collected and passed through a Sephadex G-25 columnpre-equilibrated in homogenization buffer to remove small molecules suchas nucleotides.

9. Aliquots of 50 µl are frozen in liquid nitrogen after protein concentrationdetermined by the Bradford assay and cytosol (~2 mg protein/ml) is storedat –80˚C for up to 6 months.

6.2.4 PREPARATION OF ENERGY-REGENERATING SYSTEM FOR CELL-FREE RECONSTITUTION SYSTEM

An energy-regenerating system (ERS) consists of 1 mM ATP, 0.02 mM GTP, 12 mMcreatine phosphate (CP), and 80 µg/ml creatine phosphokinase (CPK).

1. Aliquots of 100 mM ATP, 2 mM GTP, 600 mM CP, and 8 mg/ml CPKare prepared from powder and stored at –20˚C in water for later use.

2. To make 20X ERS, first mix ATP, GTP, and CP concentrated aliquots ata volume ratio of 1:1:2.

3. Titrate the pH of this mixture to 7 by adding a few µl of 1 N KOH andcheck the pH with pH paper.

4. Add one volume of CPK into the ERS mixture.5. Add 15 µl of 20X ERS to a 300-µl cell-free reaction system.

6.2.5 Aββββ GENERATION IN CELL-FREE SYSTEM UTILIZING ERS IN THE ABSENCE OF CYTOSOL

1. Permeabilized cells (cell-free system) are suspended in breaking bufferwith a final volume of 300 µl containing 25 mM HEPES, 2.5 mM MgCl2,0.5 µM CaCl2, and 110 mM KCl.

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2. Additionally, 15 µl of 20X ERS is added. The final concentration of ERSin the cell-free system will be 1 mM ATP, 0.02 mM GTP, 12 mM creatinephosphate, and 80 µg/ml creatine phosphokinase.

3. Vortex the reactions briefly to mix cells and reagents.4. Incubate the cell-free reactions at 37˚C in a water bath for 90 min.5. Gently vortex the reaction tubes every 15 min during incubation.

6.2.6 DETECTION OF Aββββ PRODUCTION FROM CELL-FREE SYSTEM

1. For Aβ detection in a cell-free system, spin reaction samples at 12,500 rpmfor 30 sec after incubation period.

2. Transfer supernatant to new Eppendorf tubes and add 100 µl of 3% SDS(with 8 µl β-mercaptoethanol/ml SDS solution) to each pellet. For super-natant, add 15 µl of 10% SDS to make a final of 0.5% SDS.

3. Vortex at full speed at 95oC for 5 min in an Eppendorf Thermomixer.4. Let samples cool down at room temperature.5. Sonicate pellet samples to shear DNA or facilitate sample solubilization

when necessary.6. Add 1 ml immunoprecipitation (IP) buffer (50 mM Tris HCl, pH 8.8,

150 mM NaCl, 6 mM EDTA, 2.5% Triton X-100, 5 mM methionine andcysteine, and 1 mg/ml bovine serum albumin) into all fractions and vortex.(Alternatively, an IP buffer consisting of 20 mM Tris HCl, pH 7.4, 300 mMNaCl, 5 mM EDTA, and 1% Triton X-100 is equally effective.)

7. Spin at 12,000 × g or at full speed for 10 min at 4oC in a Brinkmancentrifuge and collect supernatants. This step is absolutely necessary toeliminate an otherwise strong and ubiquitous background.

8. Immunoprecipitate Aβ using antibody 4G8 (Signet Laboratories, Inc.,Dedham, MA).

9. Immunoprecipitated Aβ can be resolved by 10 to 20% tricine gels and thentransferred to a 0.2 µm PVDF membrane and analyzed by autoradiography.

6.2.7 FORMATION OF NASCENT SECRETORY VESICLES IN CELL-FREE SYSTEM SUPPLEMENTED WITH ERS AND CYTOSOL

1. Permeabilized cells (cell-free system) will be incubated in a final volumeof 300 µl containing 2.5 mM MgCl2, 0.5 µM CaCl2, and 110 mM KCl,an energy-regenerating system consisting of 1 mM ATP, 0.02 mM GTP,10 mM creatine phosphate, 80 µg/ml creatine phosphokinase, and a pro-tease inhibitor mixture.

2. 15 µl cytosol prepared from N2a cells16,19 at a concentration of 1 to 2 µg/µl(15 to 30 µg proteins) is included in the cell-free system to initiate nascentvesicle budding from the TGN membrane or ER membrane.

3. Incubations are carried out at 37oC to initiate nascent vesicle release.4. Cell-free systems are incubated for various periods (15 to 120 min) to

observe the kinetics of protein trafficking and the production of Aβ fromboth membrane and vesicle fractions.

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5. Following incubation of cell-free systems, vesicle and membrane fractionscan be separated by centrifugation at 11,000 × g for 30 sec at 4oC in aBrinkman centrifuge.

6. The integrity of TGN stacks, ER membrane and derived vesicles can bedemonstrated by electron microscopy (Figure 6.1).

6.2.8 DETECTION OF ββββAPP IN DIFFERENT FRACTIONS THROUGH IMMUNOPRECIPITATION

1. Resuspend vesicle (supernatant) and membrane (pellet) fractions with1 ml IP buffer (50 mM Tris HCl, pH 8.8, 150 mM NaCl, 6 mM EDTA,2.5% Triton X-100, 5 mM methionine and cysteine, and 1 mg/ml bovineserum albumin).

2. Centrifuge resuspended fractions at 14,000 rpm for 10 min at 4oC in aBrinkman centrifuge.

FIGURE 6.1 Cell-free reconstitution system. Cells are labeled with [35S]methionine for10 min and then incubated at 20˚C, a temperature at which transport of proteins, includingβAPP, from the ER to the TGN is unimpaired. However, under these conditions, the egressof secretory vesicles from the TGN is blocked, thus allowing labeled βAPP (and othermembrane proteins) to accumulate in the TGN.3,5,16 This experimental design assures thatTGN-specific vesicle biogenesis is measured after reconstitution of the cell-free system.Alternatively, cells are labeled with [35S]-methionine at 15˚C to accumulate βAPP within theER. Cells are then permeabilized, followed by incubation at 37˚C to initiate vesicle release.Following incubation, vesicle and membrane fractions are separated by centrifugation at11,000 rpm for 30 seconds at 4oC in a Brinkman centrifuge. Vesicle (supernatant) andmembrane (pellet) fractions are diluted with IP buffer, and immunoprecipitated using antibodyagainst βAPP or Aβ and analyzed by SDS-PAGE. ER and TGN membranes can be separatedby sucrose gradient fractionation and verified by specific markers.

NN

N

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Sucrose GradientsFractionation

ER markers(Calnexin, BiP)

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β PP or A β detection(IP, SDS-PAGE, Mass-Spec.)

10 min labeling + 2 h incubating at 20°Or 4h labeling at 15°

HypotonicTreatment

Scraping

Cytosol,Energy,Ions, etc.

Incubationat 37° or 20°

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3. Collect supernatants and immunoprecipitate full-length βAPP and C-ter-minal fragments (CTFs) using antibody 3697,15 or antibody 4G8.

4. Immunoprecipitated βAPP can be resolved in 4 to 12% Tris–glycine gelsand analyzed by autoradiography. CTFs can be resolved in 10 to 20%Tricine gels and analyzed by autoradiography.

6.3 DISCUSSION

In summary, the procedures described herein provide detailed information about thepreparation of a cell-free reconstitution system for studying intracellular Aβ pro-duction as well as trafficking of βAPP through secretory compartments. Figure 6.1shows the outline of the experimental procedures. The discussion that followsfocuses on specific areas in detail.

6.3.1 ATP-DEPENDENT Aββββ GENERATION IN CELL-FREE RECONSTITUTION SYSTEM

Because our cell-free system allows us to reconstitute Aβ generation and producevarious C-terminal fragments of APP, we studied both β-secretase and γ-secretaseactivities independently and dissected the requirements and optimal conditions forboth. We showed that ATP is required for γ-secretase activity to yield Aβ (Figure 6.2).If the ATP-degrading enzyme apyrase was included in the cell-free system at 37oC,much less Aβ was generated compared to that in which an ATP regenerating systemwas present. In addition, we demonstrated that while ATP is required for γ-secretaseactivity, it appears less essential for β-secretase activity.

Therefore, supplementation of an energy-regenerating system in the cell-freereconstitution assay is crucial to study γ-secretase-dependent Aβ generation.

FIGURE 6.2 Gamma-secretase cleavage of βAPP in the cell-free reconstitution systemrequires ATP. (a) Aβ generation is reconstituted in the TGN which is incubated at 37oC inthe presence of an energy regenerating system (+ATP) or without an ATP regenerating systemin the presence of the ATP degrading enzyme apyrase (+Apyrase). (b) Quantitative analysisof (a).

20°C

βCTF(C99)

+Apyrase

+ATP

Aβ

a. b.

14.3

6.5

3.4

β−CTF

Aβ

19° APY ATP

1.0

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6.3.2 CYTOSOL-DEPENDENT ββββAPP TRAFFICKING FROM TGN/ER IN THE CELL-FREE RECONSTITUTION SYSTEM

The importance of cytosolic trafficking proteins in the genesis and budding of TGNvesicles is well established.26 It was not known, however, whether these traffickingfactors are regulated by estrogen. Utilizing the cell-free trafficking assay, we dem-onstrated the effects of estrogen on cytosolic regulation of TGN vesicle biogenesis.16

Cytosol prepared from N2a cells stimulated TGN vesicle budding between 1 ngof protein/ml and 1 mg of protein/ml with maximal stimulation reached at 100 ngof protein/ml (Figure 6.3a). Cytosol prepared from cells treated with estrogen (estro-gen-primed cytosol) stimulated budding nearly two times when compared with reac-tions using cytosol prepared from control cells (estrogen-naive cytosol) (Figure 6.3b,lane 5 vs. lane 7) and nearly two and a half times when compared to identicalreactions without cytosol (Figure 6.3b, lane 3 vs. lane 7). These results suggest thatsupplementing cytosol in the cell-free reconstitution system is necessary to initiatevesicle release from the secretory compartments and that estrogen treatment stimu-lates protein transport from the TGN through alteration of cytosol/TGN membranecomposition.

6.3.3 UTILIZING THE CELL-FREE RECONSTITUTION SYSTEM TO DEMONSTRATE PS1-REGULATED INTRACELLULAR TRAFFICKING AND SURFACE DELIVERY OF ββββAPP

The formation of βAPP-containing vesicles from the TGN and from the ER in PS1-/-

fibroblasts was assessed, by comparison to wild-type (wt) cells, using the cell-freereconstitution system. As shown in Figure 6.4, budding of βAPP-containing vesiclesfrom the TGN or from the ER was greatly increased at all time points examined inpreparations that lacked PS1 when compared to preparations from cells that expresswt PS1 (Figure 6.4a and Figure 6.4b).

Moreover, live staining of loss-of-function PS1 cells (∆M1,2)29 using mono-clonal antibody 6E10 revealed an obvious increase in the amount of surface-boundβAPP compared to PS1 wt cells (Figure 6.4c). The amounts of total surface glyco-proteins are identical in the two types of cells as judged by staining for Vicia villosaagglutinin (VVA). The amount of newly synthesized βAPP delivered to the cellsurface was further measured quantitatively in these cells by pulse-chase labelingin combination with cell surface biotinylation27 in intact cells. As shown in Figure6.4d, up to 14.3% of nascent βAPP was transported to the plasma membrane after120 min of chase, a value that is 48.9% greater than that in PS1 wt cells.

6.3.4 CHARACTERIZATION OF VESICLE AND MEMBRANE FRACTIONS BY SUCROSE GRADIENT AND ELECTRON MICROSCOPY

Following permeabilization and incubation, the cell-free reconstitution system canbe separated into vesicle and TGN/ER membrane fractions. The nascent vesicles aswell as the intact TGN stacks and ER membranes can be determined by electron

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microscopy (shown in Figure 6.1). Furthermore, ER and TGN membranes can beseparated by sucrose gradients fractionation and verified by organelle-specific markers.

In summary, the methods described in this chapter allow quantitative study ofthe kinetics of TGN- and ER-specific βAPP vesicle budding. By taking advantageof the fact that secretory pathway transit can be restricted at specific points bytemperature blocks and then reinitiated by removing those blocks, we successfullydemonstrated that budding of vesicles carrying βAPP as cargo diminishes the substratepool from which Aβ peptides can be derived.16 Utilizing the cell-free reconstitution

FIGURE 6.3 Cytosolic factors are up-regulated in response to 17β-E2. (a) Cell-free βAPPbudding assays in the presence or absence of cytosol prepared from cells incubated with17β-E2. Titration of cytosol demonstrates an increase in βAPP budding at all concentrationswhen using estrogen-primed cytosol versus estrogen-naive cytosol, with a maximum effectat 100 ng of protein/ml. (b) βAPP budding assays using no cytosol (–Cyt., lanes 3 and 4),estrogen-naive cytosol (lanes 5 and 6), and estrogen-primed cytosol (lanes 7 and 8) inuntreated (odd lanes) or estrogen-treated (even lanes) cells. (c) Cytosol derived from primaryhuman (lanes 1 and 2), rat (lanes 3 and 4), and mouse (lanes 5 and 6) neurons after incubationin either the absence (odd lanes) or presence (even lanes) of estrogen was used to stimulateβAPP budding in N2a cell-free assays. (d) Cell-free assays were performed in rat (left panel)and mouse (right panel) primary neurons. In each type, cells were incubated in either theabsence or presence of 200 nM 17β-E2 for 1 wk before cell-free assays were performed. Thebudding of βAPP was assayed using 369 as described in methods. Experiments performed at20˚C provided negative control.

a.

d.

RatCortical Neurons

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system, the study of intracellular Aβ generation and regulation of βAPP traffickingthrough secretory compartments becomes feasible, as does analysis of other Alzhe-imer’s-associated secretory molecules.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes of Health (F32AG23431 to DM and NS046673 to HX) and the Alzheimer’s Association (to HX).

FIGURE 6.4 (See color insert following page 114.) PS1 deficiency or loss of functionaccelerates βAPP trafficking from the TGN/ER and increases cell surface delivery. (a) and(b) Cell-free βAPP budding assays were prepared from PS1–/– fibroblasts expressing humanβAPPswe alone (PS1–/–) or coexpressing βAPPswe and wild-type human PS1 cells. Cellswere first labeled for 15 min with [35S]methionine at 37˚C and chased for 2 hr at 20˚C toaccumulate labeled βAPP in the TGN. Alternatively, cells were labeled for 4 hr at 15˚C toaccumulate labeled βAPP within the ER. Permeabilized cells were prepared and incubatedat 37˚C for various periods to allow the formation of post-TGN or post-ER vesicles. (c) LiveN2a cells were incubated with primary antibody 6E10 (1:100) at 4˚C for 1 hr to label cellsurface βAPP (shown in red in color insert), and FITC-conjugated VVA to stain all surfaceglycoproteins (shown in green in color insert). Cells were then fixed and visualized by confocalmicroscopy. (d) Cells were labeled with [35S]methionine at 37oC for 10 min and chased at20˚C for 2 hr to accumulate labeled βAPP in the TGN (total cell βAPP). Cells were thenincubated at 37oC for various periods to allow transport of βAPP to the plasma membrane.Cell surface proteins were then biotinylated at 4˚C for 15 min. Biotinylated and nonbiotiny-lated proteins were separated into two fractions by binding to streptavidin beads. βAPP wasimmunoprecipitated from each fraction and analyzed.

TGN Membrane TGN Vesicles

PS1WT

0’ 15’ 30’ 60’ 90’ 0’ 15’ 30’ 60’ 90’

a.

PS1-/-

b.ER Membrane ER Vesicles

PS1WT

PS1-/-

c.

WT

6E10 FITC-VVA

∆Μ1,2

d.

Chase Time (min)

Newly Synthesized βAPP on Cell Surface

WT

∆Μ1,2

TotalCell βAPP

0’ 15’ 30’ 60’ 90’ 0’ 15’ 30’ 60’ 90’

0’ 10’ 20’ 30’ 40’ 50’ 60’ 120’

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REFERENCES

1. Selkoe, D.J. The cell biology of beta-amyloid precursor protein and presenilin inAlzheimer’s disease. Trends Cell Biol. 8, 447–453, 1998.

2. Greenfield, J.P. et al. Cellular and molecular basis of beta-amyloid precursor proteinmetabolism. Front. Biosci. 5, D72–D83, 2000.

3. Xu, H. et al. Generation of Alzheimer beta-amyloid protein in the trans-Golgi networkin the apparent absence of vesicle formation. Proc. Natl. Acad. Sci. USA 94,3748–3752, 1997.

4. Hartmann, T. et al. Distinct sites of intracellular production for Alzheimer’s diseaseA beta40/42 amyloid peptides. Nat. Med. 3, 1016–1020, 1997.

5. Greenfield, J.P. et al. Endoplasmic reticulum and trans-Golgi network generate dis-tinct populations of Alzheimer beta-amyloid peptides. Proc. Natl. Acad. Sci. USA 96,742–747, 1999.

6. Sisodia, S.S. Beta-amyloid precursor protein cleavage by a membrane-bound pro-tease. Proc. Natl. Acad. Sci. USA 89, 6075–6079, 1992.

7. Caporaso, G.L. et al. Morphologic and biochemical analysis of the intracellulartrafficking of the Alzheimer beta/A4 amyloid precursor protein. J. Neurosci. 14,3122–3138, 1994.

8. Nordstedt, C. et al. Identification of the Alzheimer beta/A4 amyloid precursor proteinin clathrin-coated vesicles purified from PC12 cells. J. Biol. Chem. 268, 608–612,1993.

9. Soriano, S. et al. Expression of beta-amyloid precursor protein-CD3gamma chimerasto demonstrate the selective generation of amyloid beta(1-40) and amyloid beta(1-42)peptides within secretory and endocytic compartments. J. Biol. Chem. 274,32295–32300, 1999.

10. Perez, R.G. et al. Mutagenesis identifies new signals for beta-amyloid precursorprotein endocytosis, turnover, and the generation of secreted fragments, including Abeta42. J. Biol. Chem. 274, 18851–18856, 1999.

11. Cook, D.G. et al. Alzheimer’s A beta(1-42) is generated in the endoplasmic reticu-lum/intermediate compartment of NT2N cells. Nat. Med. 3, 1021–1023, 1997.

12. Buxbaum, J.D. et al. Processing of Alzheimer beta/A4 amyloid precursor protein:modulation by agents that regulate protein phosphorylation. Proc. Natl. Acad. Sci.USA 87, 6003–6006, 1990.

13. Xu, H. et al. Metabolism of Alzheimer beta-amyloid precursor protein: regulation byprotein kinase A in intact cells and in a cell-free system. Proc. Natl. Acad. Sci. USA93, 4081–4084, 1996.

14. Zheng, H. et al. Modulation of A (beta) peptides by estrogen in mouse models.J. Neurochem. 80, 191–196, 2002.

15. Xu, H. et al. Estrogen reduces neuronal generation of Alzheimer beta-amyloid pep-tides. Nat. Med. 4, 447–451, 1998.

16. Greenfield, J.P. et al. Estrogen lowers Alzheimer beta-amyloid generation by stimu-lating trans-Golgi network vesicle biogenesis. J. Biol. Chem. 277, 12128–12136,2002.

17. Xu, H. and Shields, D. Prohormone processing in the trans-Golgi network: endopro-teolytic cleavage of prosomatostatin and formation of nascent secretory vesicles inpermeabilized cells. J. Cell Biol. 122, 1169–1184, 1993.

18. Ling, W.L., Siddhanta, A., and Shields, D. The use of permeabilized cells to inves-tigate secretory granule biogenesis. Methods 16, 141–149, 1998.

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19. Musch, A. et al. Transport of vesicular stomatitis virus G protein to the cell surfaceis signal mediated in polarized and nonpolarized cells. J. Cell. Biol. 133, 543–558,1996.

20. Rothman, J.E. Mechanisms of intracellular protein transport. Nature 372, 55–63,1994.

21. Sweeney, D.A., Siddhanta, A., and Shields, D. Fragmentation and re-assembly of theGolgi apparatus in vitro. A requirement for phosphatidic acid and phosphatidylinositol4,5-bisphosphate synthesis. J. Biol. Chem. 277, 3030–3039, 2002.

22. de Silva, A.M., Balch, W.E., and Helenius, A. Quality control in the endoplasmicreticulum: folding and misfolding of vesicular stomatitis virus G protein in cells andin vitro. J. Cell Biol. 111, 857–866, 1990.

23. Doms, R.W. et al. Role for adenosine triphosphate in regulating the assembly andtransport of vesicular stomatitis virus G protein trimers. J. Cell. Biol. 105, 1957–1969,1987.

24. Netzer, W.J. et al. Gleevec inhibits beta-amyloid production but not Notch cleavage.Proc. Natl. Acad. Sci. USA 100, 12444–12449, 2003.

25. Beckers, C.J. and Balch, W.E. Calcium and GTP: essential components in vesiculartrafficking between the endoplasmic reticulum and Golgi apparatus. J. Cell Biol. 108,1245–1256, 1989.

26. Sollner, T.H. and Rothman, J.E. Molecular machinery mediating vesicle budding,docking and fusion. Experientia 52, 1021–1025, 1996.

27. Yan, R. et al. The transmembrane domain of the Alzheimer’s beta-secretase (BACE1)determines its late Golgi localization and access to beta-amyloid precursor protein(APP) substrate. J. Biol. Chem. 276, 36788–36796, 2001.

28. Xu, H., Greengard, P., and Gandy, S. Regulated formation of Golgi secretory vesiclescontaining Alzheimer beta-amyloid precursor protein. J. Biol. Chem. 270,23243–23245, 1995.

29. Leem, J.Y. et al. A role for presenilin 1 in regulating the delivery of amyloid precursorprotein to the cell surface. Neurobiol. Dis. 11, 64–82, 2002.

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7
Studying Amyloid β-Protein Assembly
Erica A. Fradinger, Samir Kumar Maji, Noel D. Lazo, and David B. Teplow

CONTENTS

7.1 Introduction7.2 Background7.3 Peptide Production and Preparation

7.3.1 Production of Aβ Peptides7.3.2 Preparation of Starting Peptide Stocks

7.3.2.1 HFIP Pretreatment7.3.2.2 NaOH Pretreatment7.3.2.3 Sample Clarification and Fibril Isolation7.3.2.4 Preparation of LMW Aβ by Size Exclusion

Chromatography7.3.2.5 Preparation of LMW Aβ by Filtration7.3.2.6 Preparation of Aggregate-Free Aβ by

Ultracentrifugation7.3.2.7 Preparation of Oligomeric Aβ42

7.3.2.8 Preparation of Aβ Protofibrils7.3.2.9 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 SEC7.4.1.3 Determination of Particle Diffusion Coefficients Using

QLS7.4.2 Fibril Formation

7.4.2.1 Thioflavin T (ThT) Binding7.4.2.2 Congo Red Binding7.4.2.3 Turbidity

7.4.3 Secondary Structure Determination7.4.3.1 CD Spectroscopy7.4.3.2 Fourier Transform Infrared Spectroscopy (FTIR)

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press

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7.4.4 Topographical Analysis7.4.4.1 8-Anilino-1-Naphthalenesulfonic Acid (ANS) Binding7.4.4.2 Intrinsic Fluorescence7.4.4.3 Electron Paramagnetic Resonance (EPR)7.4.4.4 Hydrogen–Deuterium Exchange

7.4.5 NMR Spectroscopy7.4.6 Morphological Analysis

7.4.6.1 Electron Microscopy7.4.6.2 Atomic Force Microscopy


7.1 INTRODUCTION

The amyloid β-protein precursor (AβPP), also referred to as the amyloid precursorprotein (APP), is a type I transmembrane glycoprotein.1 Through the actions ofspecific endoproteases, the ~110 to 135 kDa AβPP is post-translationally processed,producing a 40- to 42-amino acid amphipathic peptide, the amyloid β-protein (Aβ).2

Aβ is expressed ubiquitously in the human body as a normal part of cellular andorganismal physiology.3,4 Pioneering work by George Glenner two decades ago5,6

revealed that the protein deposits that are pathognomonic for Alzheimer’s disease(AD) are composed primarily of Aβ. Since then, tremendous advances have beenmade in our understanding of both Aβ and AβPP.

Genetic evidence revealed a causal link between AβPP and AD.1 In all kindredsthus far examined, mutations in genes encoding AβPP or in genes encoding proteinsinvolved in AβPP metabolism either cause increased production of the longer, moreamyloidogenic, 42-residue form of Aβ (Aβ42) or production of Aβ peptides containingamino acid substitutions affecting Aβ self-assembly.7 Aβ assembly thus is inextri-cably linked with AD. For this reason, therapeutic strategies for AD have focusedon controlling Aβ metabolism, Aβ-mediated neurotoxicity, or Aβ self-assembly.8

The centrality of Aβ in the neuropathogenesis of AD has made biochemical andbiophysical studies of Aβ aggregation exceedingly important. How is this done? Thegoal of this chapter is to provide readers the answer to this question, specificallywith respect to studies of Aβ folding and self-assembly. Moreover, Aβ assembly isan archetype for amyloid assembly. Therefore, the methods discussed here arerelevant and applicable to the study of many other amyloidogenic proteins andpeptides, of which ~25 have been defined clinically.

7.2 BACKGROUND

The assembly of Aβ from a nascent monomer into the structure known as amyloid 9–11

is an exceedingly complex process.12–14 In addition to the seminal event of Aβ self-association, other proteins, glycosaminoglycans, lipids, and reactive oxygen speciesalso may affect amyloidogenesis. Ideally, one would like to construct an assemblyscheme incorporating all elements that contribute significantly to the process. To do

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so, reductionist approaches seek to understand each element with great mechanisticdetail. Logically, the most important element is Aβ itself. Determining the tertiarystructures of Aβ in its monomeric and assembled states, the quaternary structuresresulting from monomer self-association and the dynamics of the association pro-cesses are important goals. These data must be correlated with the biological behaviorsof specific assemblies in order to determine meritorious therapeutic targets.

A prerequisite for any study of Aβ assembly is pre facto consideration of whichassembly is to be studied. Aβ forms a variety of structures, including multiplemonomeric conformers,15 different types of oligomers,16–18 Aβ-derived diffusibleligands (ADDLs),19,20 annuli,21 micelles,22,23 protofibrils,24,25 fibrils,12 and sphe-roids.26,27 The structural relationships among these assemblies and the differencesin the assembly processes of wild type Aβ40, Aβ42, and mutant Aβ peptides are notentirely understood and are areas of active investigation.

Studies of Aβ comprise three different classes — static, dynamic, and biological.Static studies reveal features of the assembly process observable at defined times,e.g., the morphology of end-stage structures such as fibrils. Electron microscopy(EM), x-ray diffraction, solid state nuclear magnetic resonance spectroscopy (NMR),and some types of atomic force microscopy (AFM) are examples of useful techniquesfor studying static features of Aβ assembly. Dynamic studies reveal time-dependentchanges in peptide conformation and aggregation state. Dynamics studies can beaccomplished through continuous monitoring or iterative use of static techniques.Circular dichroism (CD) spectroscopy, Congo Red and thioflavin T dye binding,turbidity, quasielastic light scattering spectroscopy (QLS), hydrogen–deuteriumexchange, electron paramagnetic resonance spectroscopy (EPR), NMR, size exclu-sion chromatography (SEC), intrinsic fluorescence, Fourier transform infrared spec-troscopy (FTIR), and analytical ultracentrifugation are examples of techniques forstudying assembly dynamics. Biological studies elucidate assembly-specific effectson cellular function and typically involve vital assays (cell death) and metabolicassays (redox activity, transcriptional dynamics, signaling events, and post-transla-tional modifications).

The two most important questions common to all of these approaches are (1) howdoes one initially prepare the peptide for study and (2) how does one perform eachassay? The importance of question 1 with respect to the proper interpretation of dataproduced subsequently cannot be overemphasized. It also should be noted thatimplicit in question 2 is the issue of how structure–activity relationships can bedefined in studies of heterogeneous structures existing in rapid equilibria. In thesections that follow, we discuss issues associated with preparation of peptide stocksand provide selected examples of static and dynamic approaches for monitoringearly, mid-, and late-stage events in Aβ assembly.

7.3 PEPTIDE PRODUCTION AND PREPARATION

7.3.1 PRODUCTION OF Aββββ PEPTIDES

Aβ is produced either by solid-phase peptide synthesis (SPPS) or through recombi-nant DNA techniques, providing high purity peptide suitable for in vitro and in vivo

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experimentation. Unfortunately, substantial compositional variation has beenreported among Aβ preparations, resulting in experimental irreproducibility.28–31 Itis important that the experimentalist verifies that the peptide itself is chemically pureand that nonpeptide components are either absent from peptide stocks or inert. Mostpeptide lyophilizates are not 100% peptide by weight, but contain salts and othercomponents. For all the protocols that follow, when calculating initial peptide con-centration, peptide weight (and not total lyophilizate weight) must be used.

Intrinsic chemical purity is the state in which the primary structure (proteinsequence) of Aβ is correct. SPPS cannot produce an Aβ product that is 100% pure.Failure sequences, peptides missing one or more amino acids, are unavoidable,although with proper synthesis chemistry their relative amounts can be minimized.Oxidation of Met35 to its corresponding sulfoxide, Met(O)35, is a common reactionthat can occur during peptide workup and purification, especially in the presence offormic acid or oxidants. Synthesis-related amino acid racemization, and side-reac-tions during peptide cleavage and deprotection, may also be observed, but thesegenerally occur infrequently. Most peptide suppliers perform quantitative amino acidanalysis and mass spectrometry to characterize their products. However, becauseamino acid and simple mass analyses cannot determine primary structure, Edmanor mass spectrometric sequence analysis can be used to prove formally that thepeptide structure is correct. With respect to nonpeptide components of peptidepreparations, fluoren-9-ylmethoxycarbonyl (FMOC)-mediated SPPS and high per-formance liquid chromatography (HPLC) purification produce trifluoroacetic acid(TFA) salts of the resulting peptides. These salts along with chemical scavengersare often present in lyophilized peptide preparations and can complicate the initialsolvation and preparation of peptide stock solutions.

For recombinantly derived Aβ, primary structure changes are rare because ofthe high fidelity of the protein expression systems and the physiologic conditionsunder which these systems operate. In these systems, Aβ often is produced as afusion protein requiring post-translational processing with highly specific endopro-teases. It is important to ensure that the Aβ component of the fusion protein is notcontaminated by uncleaved fusion protein, the enzyme itself or by peptide fragmentsproduced through adventitious proteolysis. Because fusion protein cleavage is per-formed with biological buffers, buffer exchange or removal may be necessary priorto peptide use.

7.3.2 PREPARATION OF STARTING PEPTIDE STOCKS

One of the most common problems encountered in work with Aβ is irreproduciblebehavior of the peptide. Primary causes of irreproducibility are the initial structureand aggregation state of the peptide, both in the solid state31 and immediately aftersolvation. Several solvation methods have been developed in an effort to eliminatepreexisting aggregates and create conformationally uniform, monomeric, Aβ stocksolutions. A “magic formula” for achieving this goal has not been found. Dimethylsulfoxide (DMSO),32–34 TFA,35 trifluoroethanol (TFE),36 hexafluoroisopropanol(HFIP),33,37 and NaOH31 all have been used to solubilize Aβ. HFIP and TFE disrupthydrophobic interactions in aggregated amyloid preparations and stabilize α-helical

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structure,36,38,39 leading to disruption of preexistent β-sheet structure. HFIP pretreat-ment (Section 7.3.2.1) of Aβ has been shown to yield peptide solutions of uniformglobular morphology with predominantly α-helical and random coil secondary struc-tures and less than 1% β-sheet.33

Aβ prepared by SPPS can contain residual TFA. Dissolution of these peptidelyophilizates in water produces a strongly acidic solution (pH 3 to 4). Addition ofbiological buffers of neutral pH causes the solution pH to pass through the isoelectricpoint of Aβ (~5.5) — a point at which the solubility of Aβ is minimal and itspropensity to aggregate is maximal.38 The result is rapid aggregation that producesan inhomogeneous starting peptide solution. One strategy that addresses this problemis to predissolve Aβ under strongly alkaline conditions and then to relyophilize thesolutions prior to solvation in biological buffers. NaOH pretreatment (Section7.3.2.2) has been shown to improve significantly the yield of unaggregated Aβ.31

A number of postsolvation approaches have been developed to remove pre-existing aggregates. Filtration methods have been used to remove large and smallaggregates from starting solutions of Aβ.31 Filtration of Aβ through a 0.2-µm nylonMicro-Spin Whatman filter at 5000 × g for 10 min will remove fibrils, fibril aggre-gates, and other structures larger than 200 nm. However, for most experimentalneeds, this filtration is insufficient because assemblies of 200-nm size are quite largeand generally are already fibrillar. Filtration through filters with 10-kDa exclusionlimits (see Section 7.3.2.5) is a superior method that initially yields monomers anddimers. It is important to note that, in peptide regimes of µM concentration and higher,nascent Aβ monomers immediately establish a rapid equilibrium with higher orderoligomers.17,40

Most aggregate-free Aβ solutions therefore comprise an oligodisperse populationof assemblies. Nevertheless, because this population can be produced routinely andis not polydisperse, reproducible peptide assembly behavior can be observed. Anotherhighly effective procedure for preparing aggregate-free Aβ is SEC (Section 7.3.2.4).SEC allows the almost simultaneous collection of larger oligomeric populations,including protofibrils (Section 7.3.2.8).24 Although more laborious, ultracentrifugation(Section 7.3.2.6) also has been used to prepare aggregate-free, nominally monomeric,peptide stock solutions.41

In the sections that follow, we present protocols for solvating Aβ lyophilizates,clarifying turbid solutions (Section 7.3.2.3), producing aggregate-free Aβ solutions,isolating oligomeric assemblies (Section 7.3.2.7) and protofibrils, and preparingfibrils (Section 7.3.2.9). It should be noted that some controversy surrounds theoligomerization state of aggregate-free starting preparations of Aβ. Here we referto these preparations, which typically exist at concentrations of 10 to 50 µM, as lowmolecular weight (LMW) Aβ.42 We do so because techniques including QLS,24

chemical cross-linking,17,40,43 fluorescence resonance energy transfer,44 andultracentrifugation45,46 have shown that a monodisperse monomer population doesnot exist in these solutions. Readers should note that for many experimental needsthis issue may be academic because the primary concern is the ability to preparepeptide stock solutions identical in their distribution of peptide assembly states,whatever that distribution may be.

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7.3.2.1 HFIP Pretreatment

1. Dissolve Aβ peptide in 100% HFIP to a final concentration of 0.5 mM.2. Distribute 100 µl aliquots into 1.5 ml polypropylene microcentrifuge tubes.3. Remove HFIP by evaporation under a gentle stream of nitrogen or argon.4. Remove residual HFIP in vacuo, using a lyophilizer or a centrifugal

concentrator (e.g., Savant SpeedVac concentrator, Savant Instruments,Holbrook, NY).

5. The final product will be a peptide film within the microcentrifuge tube.If properly desiccated, the tubes can be stored for extended periods(months to years) at –20 or –80ºC.

Note: All work with HFIP should be conducted in a chemical fume hood withadequate protection. The peptide film produced by this procedure may not easilydissolve in water or aqueous buffers. Initial solvation in DMSO can facilitate com-plete resuspension.

7.3.2.2 NaOH Pretreatment

1. Suspend lyophilized Aβ in 2 mM NaOH at a concentration of 1 mg/ml.It is important to ensure that the pH of the resulting solution reaches atleast 10.5.31

2. Sonicate the suspension in a bath-type sonicator for 1 min (#1200-R,Branson Ultrasonics, Danbury, CT).

3. Lyophilize the resulting NaOH-treated peptide.4. Store the lyophilizate at –20°C until needed.

7.3.2.3 Sample Clarification and Fibril Isolation

For some experiments, the investigator may want to fractionate or remove largeaggregates from more buoyant structures, such as protofibrils, oligomers and LMWAβ. A simple method is low speed centrifugation. Elimination of large, experimen-tally irrelevant or undesired aggregates produces a “clarified” supernatant fluidcontaining protofibrils and smaller assemblies. The pellet contains aggregates andfibrils that may be used for morphological studies as well as seeding experiments.

1. Dissolve Aβ at a concentration of 2 mg/ml in Milli-Q water and then addan equal volume of double-strength (2X) buffer.

2. Sonicate the sample for 1 min in an ultrasonic water bath (#1200-R,Branson).

3. Centrifuge the sample for 10 min at 16,000 × g using a bench top micro-centrifuge.

7.3.2.4 Preparation of LMW Aββββ by Size Exclusion Chromatography

1. Prepare 10 mM sodium phosphate, pH 7.4, and then filter the solutionthrough a 0.22-µm polyethersulfone (or equivalent) membrane to removebacteria and any other large particulates.

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2. Using the buffer prepared in step 1, wash and equilibrate a 10/30 Superdex75 HR (Amersham Biosciences, Piscataway, NJ) column at a flow rate of0.5 ml/min until a flat ultraviolet (UV) trace is observed. The chromato-graphic pumping system per se is not relevant to the procedure. It needonly provide appropriate flow rates and include a detector capable ofdetermining absorbance in the UV range (200 to 300 nm).

3. Dissolve 350 to 400 µg of lyophilized Aβ in DMSO at a concentrationof 2 mg/ml.

4. Sonicate the dissolved peptide for 1 min in a bath sonicator (#1200-R,Branson).

5. Centrifuge the peptide solution at 16,000 × g for 10 min to remove anylarge aggregates.

6. Inject 160 to 180 µl of the supernate onto the equilibrated column.7. UV monitoring is used to detect peaks eluting from the column. We

routinely monitor at 254 nm, but wavelengths of 215 nm (peptide bondabsorbance) or 280 nm (tyrosine absorbance) are also satisfactory. A voidvolume peak elutes from the column first. A large peak composed ofLMW Aβ elutes late in the run with a retention time consistent withglobular standards of molecular mass 5 to 15 kDa. Column calibrationboth with globular and polymeric standards will provide the most accurateindications of apparent molecular weight.

8. Generally, only the apex (middle third, based on collection time) of theLMW peak is collected and used.

Note: LMW Aβ comprises an equilibrium mixture of low-order oligomers. It iscritical that this material be used immediately after its isolation if structure–activitycorrelations are required. Time delays and sample manipulation allow assembly oflarger structures, which can complicate interpretation of the experimental data.

7.3.2.5 Preparation of LMW Aββββ by Filtration

An alternative to SEC for preparation of LMW Aβ is filtration. This method issimpler than SEC and requires less time and fewer instrumental resources. The LMWAβ40 produced is qualitatively similar to that produced by SEC. However, theoligomerization states of Aβ42 differ within LMW fractions prepared by the twomethods.17 SEC-isolated LMW Aβ42 produces higher order oligomers, in additionto the relatively narrow (predominantly monomer through heptamer) distribution ofoligomers observed with filtered preparations.17

1. Wash Centricon YM-10 filters with 200 µl of 10 mM phosphate buffer,pH 7.4, by adding the solution to the filter and centrifuging at 16,000 ×g for 20 min.

2. Discard the filtrate and repeat step 1.3. Dissolve lyophilized NaOH-treated (see section 7.3.2.2) Aβ in Milli-Q

water at a concentration of 4 mg/ml.4. Add an equal volume of 20 mM phosphate buffer, pH 7.4.

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5. Sonicate for 1 min (#1200-R, Branson).6. Place the washed filter unit in a clean microcentrifuge tube, transfer the

peptide solution to the unit, and then centrifuge at 16,000 × g for 30 min.7. Collect the LMW Aβ filtrate.

Note: The use of filtered deionized, 18.2-MΩ water is suggested. A MilliporeMilli-Q Synthesis system is an excellent source for water. If the Aβ peptide availablehas not been NaOH-pretreated, pretreatment can be done prior to step 4 by adding1 M NaOH to the peptide solution to produce a final NaOH concentration of 6 mM.For experiments involving extended incubation times, 0.01% (w/v) sodium azide isadded to the buffers to prevent microbial growth.

7.3.2.6 Preparation of Aggregate-Free Aββββ by Ultracentrifugation

Döbeli et al.41 developed a method for the production of “aggregation competentmonomeric Aβ peptides.” This method uses fusion proteins produced in Escherichiacoli and processed through chemical cleavage of the Aβ component, reduction ofoxidized Met35, and multiple chromatographic steps. The final step, ultracentrifuga-tion, is done immediately prior to use of the peptide to maximize the content ofpeptide monomer. This centrifugation produces soluble Aβ,46 equivalent in principleto LMW Aβ.42 Full details of the preparative and analytical ultracentrifugationprocedures have been published.41,46 A brief protocol follows.

1. The starting peptide stock contains Aβ at a concentration of 50 to 100 µMin 12 mM Tris HCl, pH 8. The procedure is performed with Aβ42, but isapplicable to any Aβ peptide. The buffer composition is not critical, aslong as it does not accelerate Aβ self-association. Low ionic strengthbuffers without added salt (NaCl) are preferred.

2. The sample is introduced into the ultracentrifuge and spun at 320,000 ×g for 12 hr at 20°C.

3. The supernate contains soluble Aβ estimated to contain >80% monomers.The remainder of the peptide mass comprises dimers and higher orderoligomers.

7.3.2.7 Preparation of Oligomeric Aββββ42

Aβ42 has been found to oligomerize in a distinctly different manner from Aβ40.17 Inparticular, Aβ42 has a propensity to form metastable globular structures approxi-mately 5 to 6 nm in diameter. These have been termed paranuclei17 or ADDLs (Aβ-derived diffusible ligands).20 Aβ42 oligomers form immediately upon solubilizationof synthetic Aβ42 preparations, but because these oligomers exist in a rapid equilib-rium with monomers and larger structures, preparing well-defined oligomer popu-lations is problematic. The following method details how to prepare ADDLs32 andis essentially identical to that used by Stein et al.33 As with all methods for preparingoligomeric or polymeric assemblies, the actual morphologies of the peptides shouldbe confirmed by EM or AFM.

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1. Pretreat Aβ peptide with HFIP (Section 7.3.2.1).2. Resuspend peptide film in DMSO to a final concentration of 5 mM.3. Mix well by pipetting, then bath sonicate for 10 min (#1200-R, Branson).4. Add ice-cold phenol red-free F12 medium to the solution so that the final

peptide concentration is 100 µM.5. Mix well by vortexing for 30 sec.6. Incubate at 4°C for 24 hr.7. Centrifuge at 14,000 × g for 10 min.8. The supernate contains ADDLs.

7.3.2.8 Preparation of Aββββ Protofibrils

1. Dissolve Aβ in Milli-Q water at a concentration of 4 mg/ml.2. Add an equal volume of 0.2 M Tris-HCl, pH 7.4.3. Incubate at room temperature (22°C) for 48 hr.4. Centrifuge the solution at 16,000 × g for 5 min.5. Remove the supernate and chromatograph on a Superdex 75 column, as

described in Section 7.3.2.4.6. The peak eluting immediately after the void volume of the column will

contain protofibrils.

Note: The incubation time can be adjusted to provide the greatest yield ofprotofibrils. A 48-hr period generally produces equivalent amounts of protofibrilsand LMW Aβ40, but times vary for Aβ42 and peptides containing amino acid substi-tutions associated with familial forms of AD. The method described here is theclassic method for protofibril preparation. Solvent additives,47 alterations in buffercomposition,48 and changes in the primary structure of Aβ49,50 also have been usedto increase protofibril yield or stabilize the assembly.

7.3.2.9 Preparation of Aββββ Fibrils

Fibril preparation is the easiest task to accomplish in studies of Aβ because fibrilformation is the default pathway for Aβ assembly. Simple incubation of solubilizedAβ peptide at sufficient concentration in physiologic buffers will produce fibrilsunless extraordinary precautions are taken to eliminate preexisting nidi for fibrilnucleation. It should be noted, however, that many different polymeric structureshave been observed, including short, stubby fibers, single filaments, twisted assem-blies containing two or more subfilaments, ribbons, and sheets.51,52 Alterations intemperature, ionic strength, pH, solvent composition, and agitation can producedramatic differences in the morphology of the end-stage assembly. Therefore, thefinal product should be examined by EM or AFM to determine its morphology. Theprotocol that follows is one means for obtaining fibrils.

1. Dissolve lyophilized Aβ in sterile Milli-Q water at a concentration of 4mg/ml.

2. Add sufficient 1 M NaOH to produce a final concentration of 6 mM.

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3. Add an equal volume of 2× buffer. The buffer should be chosen based onthe goal of the specific experiment conducted.

4. Sonicate the peptide sample for 10 min at room temperature (#1200-R,Branson).

5. Pellet any large aggregates at 16,000 × g for 10 min.6. Transfer the supernate to a 1.5-ml microcentrifuge tube.7. Incubate at 37°C. Agitation will accelerate the fibril formation process

significantly.

7.4 MONITORING Aββββ ASSEMBLY

7.4.1 OLIGOMERIZATION

Data obtained from in vitro and in vivo studies correlating Aβ aggregation state andbiological activity support the hypothesis that small, oligomeric Aβ assemblies maybe key pathogenetic effectors of AD16,53–56 Therefore, understanding early Aβ assem-bly events is vital if rational approaches for AD therapy are to be developed.Achieving this understanding is complicated by the fact that assembly intermediatesare metastable. Techniques such as EM and AFM are not ideal for elucidatingassembly dynamics. The polydispersity of Aβ populations and the presence of largeaggregates make QLS analyses difficult. SDS gel electrophoresis destroys noncova-lent complexes, making this approach unsuitable.

To allow study of assembly intermediates, methods have been sought for theirstabilization. Recently, we introduced a method designated photo-induced cross-linking of unmodified proteins (PICUP) to the study of Aβ oligomerization.40,57

PICUP produces covalent bonds between unmodified peptides or proteins. To do so,a tris(bipyridyl)ruthenium(II) complex [Ru(Bpy)] is photo-oxidized in the presenceof an electron acceptor. This produces Ru(III), a powerful one-electron oxidizer thatabstracts electrons from reactive amino acids and results in free radical formation.The free radicals react quickly with neighboring groups, yielding covalently cross-linked peptides or proteins.

PICUP has several attractive characteristics. It requires very short reaction times(1 sec) and no pre facto peptide modifications. It can be performed at neutral pHand under isotonic conditions. It is highly efficient (80%), and is initiated by visible(not UV) light, thereby preventing UV-induced peptide damage. We discuss herethe use of PICUP to study peptide and protein oligomerization.

7.4.1.1 Determination of Oligomer Size Distributions Using PICUP

1. Prepare a 1 mM solution of tris(2,2′-bipyridyl)dichlororuthenium(II)[Ru(bpy)] in 10 mM sodium phosphate, pH 7.4. Protect this solution fromambient light.

2. Prepare 20 mM ammonium persulfate (APS) in 10 mM sodium phosphate,pH 7.4.

3. Prepare LMW Aβ. This can be done using SEC (Section 7.3.2.4), filtration(Section 7.3.2.5), or ultracentrifugation (Section 7.3.2.6).

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4. Add 18 µl of LMW Aβ into a thin-walled 0.2-ml polymerase chainreaction (PCR) tube.

5. Add 1 µl of Ru(bpy) and 1 µl of APS and mix well.6. Illuminate for 1 sec.7. Quench immediately by adding 1 µl of 1 M dithiothreitol in water.

Note: We find that a convenient and inexpensive illumination system is a 35-mmSLR camera body to which a small bellows is attached. A 200-W incandescent lampis an appropriate light source. This arrangement allows reproducible and preciseillumination times. The sample is placed in the bellows, the bellows is capped, andthe light source illuminates the shutter through the open film compartment (i.e., fromthe rear of the camera). Actuation of the shutter then allows the light to enter thebellows. Note also that instead of quenching with dithiothreitol, 5% (v/v) β-mer-captoethanol in SDS-PAGE sample buffer may be used.

7.4.1.2 Determination of Oligomer Size Using SEC

SEC or gel permeation chromatography is a technique that fractionates solutes onthe basis of their Stokes radii.58,59 SEC can be performed either in the presence orabsence of denaturants. The absence of denaturants is advantageous relative to SDSgel electrophoresis because many oligomeric assemblies are SDS-labile. SEC is anexcellent technique both for preparative work and for the analysis of particle size.

The dynamic range of SEC is large, ranging from molecular weights below athousand up to millions. However, the resolution is low, thus baseline separation ofsimilarly sized proteins generally requires at least a 2× molecular weight difference.Nevertheless, SEC provides the means to isolate homogeneous fractions of particularAβ assemblies and to monitor early Aβ oligomerization events.

The basic instrumental arrangement and sample manipulations involved in SECstudies of Aβ have been described in Section 7.3.2.4. Iterative chromatographicanalyses of Aβ assembly reactions can provide useful information on both the typesof oligomers formed and the kinetics of oligomer formation and maturation intolarge polymers. An extensive literature exists in these areas, to which readers arereferred to learn more about applications of SEC of special relevance to theirexperimental needs.17,24,27,40,50,60–62

7.4.1.3 Determination of Particle Diffusion Coefficients Using QLS

QLS, also known as dynamic light scattering or photon correlation spectroscopy, isan optical method for the determination of diffusion coefficients of particles under-going Brownian motion in solution.63,64 Diffusion coefficients are determined byparticle size, shape, and flexibility as well as by interparticle interactions. Theseparameters provide important information about the kinetics and structural transi-tions in a system and can be studied by QLS. Using the Stokes-Einstein equation,65

D = kBT/6π ηRH, diffusion coefficients (D) can be converted into hydrodynamic radii(RH) — conceptually similar to Stokes radii.

Thus QLS, like SEC, allows monitoring of protein assembly reactions. However,QLS provides a number of advantages. It is noninvasive; therefore reactions can be

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monitored in situ without the need for sample manipulation or consumption. QLSis rapid, requiring only minutes to acquire data, and is quantitative. QLS thus is anexcellent tool for studying protein aggregation and has been used by a number oflaboratories to examine Aβ assembly.17,22,61,66–69 In our laboratory, QLS studies haveprovided insight into nucleation and elongation rates of Aβ fibril formation,22,70 theexistence and structural organization of micelle-like Aβ intermediates,22,71 activationenergies for Aβ monomer addition to fibril ends,72 and sizes of the earliest Aβ42

oligomers (paranuclei).17 Although simple in principle, QLS requires substantialexpertise in optical and polymer physics. A complete discussion of the subject isbeyond the scope of this chapter and therefore interested readers are referred to arecent review on the theory and practice of QLS for studying protein aggregation.73

7.4.2 FIBRIL FORMATION

7.4.2.1 Thioflavin T (ThT) Binding

ThT is a fluorescent dye that binds to β-sheet-rich assemblies.74,75 Excitation of thebound dye at 450 nm produces strong fluorescence at 482 nm, providing a meansof monitoring formation of the extended sheets that comprise amyloid fibrils.76,77

1. Prepare a 100 µM stock solution of ThT in 10 mM phosphate buffer, pH 7.5.2. Mix 5 µl of the ThT stock with 100 µl of an Aβ sample.3. Record the fluorescence intensity four times, at 10-sec intervals after

90 sec of incubation. Use an excitation wavelength of 450 nm (slit width =5 nm) and an emission wavelength of 482 nm (slit width = 10 nm). Averagethe four readings to obtain a value for the ThT fluorescence.

4. Determine the “blank-corrected” fluorescence by measuring the fluores-cence of a peptide-free sample and subtracting this intensity from thoseof the peptide samples.

Note: Iterative measurement of ThT fluorescence during assembly should reveala time-dependent increase in intensity that peaks near the time of maximal fibrilformation and then declines. This decline, which is commonly observed, resultsfrom precipitation of fibrils and sequestration of ThT-reactive sites within supramo-lecular fibril aggregates.

7.4.2.2 Congo Red Binding

Congo red, like ThT, is a dye routinely used to detect β-sheet-rich assemblies.78,79

Upon binding, the absorption maximum of Congo red undergoes a “red shift,”changing from 490 to 540 nm. Because these wavelengths are in the visible range,amyloid binding can be monitored using simple spectrophotometric equipment (seeSection 7.4.2.2.1) and optical microscopes (see Section 7.4.2.2.2). In addition, Congored is birefringent. If macroscopic amyloid assemblies are viewed using polarizedlight, a yellow–green color is visible in the polarization plane. Cross-polarized light,obtained using two plane polarizers mounted at a 90° angle relative to each otherwill produce a classic Maltese cross in the presence of amyloid.80

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7.4.2.2.1 Spectrophotometric Monitoring of Congo Red Absorbance

1. Prepare a 20 µM stock solution of Congo red in 20 mM phosphate buffer,pH 7.4, containing 150 µM sodium chloride.

2. Mix 225 µl of the Congo red solution with 25 µl of Aβ. Controls shouldinclude Congo red alone and peptide alone.

3. Mix and incubate for 30 min at 22°C.4. Time-dependent changes in the absorption spectra of samples can be

determined by wavelength scans between 400 and 700 nm. Monitoringthe red shift of the Congo red intensity maximum is the most reliableindicator of the binding of the dye to amyloid.

5. Quantification of amyloid binding levels can be achieved by measurementof the absorbance of the Congo red:Aβ complex at 541 and 403 nm. Theamount of bound Congo red is then calculated according to the followingformula, where C is the concentration (µM) of the Congo red:Aβ complexand λA is the total absorbance of the sample at wavelength λ:

Note: Amyloid assemblies are quite large and therefore scatter appreciablequantities of light. This scattering reduces the measured light intensity in a wave-length-dependent manner and, depending on the formulae used for quantification,produces artificially high or low estimations of the amount of Congo red bound. Atlow Aβ concentrations (<5 µg/ml), the error is relatively small. However, at higherconcentrations, scattering must be taken into account. Klunk et al. studied theseissues extensively and developed protocols for determining absolute amounts offibrillar Aβ (for a recent review, see Klunk et al.79). These protocols use the absor-bance values for the Congo red (ACR) and peptide (AAβ) controls (step 2 above) toadjust for scattering. The adjustment produces the following formula, useful for Aβsamples with and without significant scattering:

where Aβfib is the concentration of fibrillar Aβ in µg/ml, 541A and 403A are the totalabsorbance values of the sample, 403ACR is the absorbance of the Congo red control,and r = 541AAβ /403AAβ is the ratio of scattering of the peptide control at 541 and 403 nm.

7.4.2.2.2 Microscopic Monitoring of Congo Red Binding

1. To stain macromolecular aggregates, samples are pelleted by centrifuga-tion at 16,000 × g for 30 min.

2. The resulting pellets then are stained by addition of 100 µl of 20 µMCongo red in phosphate buffer, gentle agitation, and incubation for 5 minat 22°C.

3. Excess Congo red is removed from the labeled aggregates by centrifuga-tion at 16,000 × g for 2 min, removal of the supernate, suspension of the

C = A 47,800 – A 38,100 .541 403( ) ( )

A = A 4780 – A 4780 r + A r 478fib

541 403 403CR

β ( ) ( ) 00 – 1 3810( ) ( ) ( )

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pellet in 100 µl of 50% (v/v) ethanol in water, recentrifugation, andrepetition of the wash procedure once.

4. The washed pellet is suspended in 50 µl of phosphate buffer and spreadevenly on a glass microscope slide.

5. Examination of the sample in a light microscope fitted with crossedpolarizers will reveal Maltese cross-like structures if amyloid fibrils arepresent.

Note: It should be noted that fibrillar preparations of Aβ are birefringent regard-less of whether they are treated with dyes. For example, at concentrations of 5 to10 mg/ml in the absence of dyes, striking birefringence can be observed in fibrillarAβ samples viewed using cross-polarized light.81 However, dye binding provides amore sensitive indicator of this optical property.

7.4.2.3 Turbidity

Turbidity, the scattering of light by large particles (scatterers) in solution,82 providesa simple, rapid method to examine Aβ fibril formation.38,49,83,84 Initially, when thepeptide exists in a monomeric or oligomeric form, the intensity of scattered light islow and the solution appears clear to the eye. As fibril assembly proceeds, scatterersize increases, producing significant apparent light absorption when samples aremonitored spectrophotometrically, and eventually producing a turbid solution witha typically milky appearance. A plot of absorbance versus time will reveal a qua-sisigmoidal curve. The initial, relatively flat portion of the curve, often called thelag phase, occurs during the period of initial peptide assembly. Scattering intensityvaries as the square of molecular weight; therefore, while the peptide aggregates arerelatively small, they scatter insufficient light to be detected. As the aggregates growlarger, the intensity of light scattered from them increases to the point that changesin absorbance are observable.

At this point the lag phase ends and a rapid increase in absorbance is observed,consistent with the aggregate growth. Eventually the average particle size reaches amaximum, at which point the turbidity remains constant. In samples in whichinterparticle interactions occur, large fibril masses may precipitate, causing a latedecrease in turbidity as these scatterers leave the illuminated area of the samplecuvette.

[Readers are cautioned not to equate the term “lag phase,” as associated withturbidity, Congo red, ThT, or similar measures of amyloid fibril formation, with thelag phase associated with nucleation-dependent polymerization reactions. The latteris a measure of the time required for the self-association of monomers to producefibril nuclei. The lifetimes of nuclei are exceedingly short because they are immedi-ately elongated through monomer addition. Nucleation is not measured directly inthe assays mentioned above.]

The following protocol is illustrative of the basic strategy for performing tur-bidity experiments. The precise sample preparation method can be varied to suit theparticular experimental needs of the investigator.

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1. Prepare the Aβ sample for study. If gross questions about fibril formationare being studied, one might, for example, prepare lyophilized Aβ peptidein 0.06 M NaOH at a concentration of 4 mg/ml.

2. Add an equal volume of 2× phosphate-buffered saline, pH 7.4, to producea final peptide concentration of 2 mg/ml.

3. Sonicate the peptide solution for 10 min in a bath sonicator (#1200-R,Branson).

4. Clarify the peptide solution by centrifugation at 16,000 × g for 10 min.5. Carefully transfer the supernate to a cuvette and place the cuvette in a

spectrophotometer.6. Monitor absorbance at 400 nm.

Note: Rates of fibril assembly can vary dramatically, depending on the peptidestudied and the conditions of the assembly reaction. Aggregate-free preparations cantake weeks or longer [one Aβ solution has remained monomeric for years (J. Lee,personal communication)] to assemble. Agitation, a pH near the pI of Aβ (5.5),increased salt concentration, increased temperature, and other manipulations canaccelerate the assembly kinetics.

7.4.3 SECONDARY STRUCTURE DETERMINATION

Amyloid formation involves reorganization of secondary structure elements withinthe amyloid protein monomer. This is a prerequisite for assembly of the classicextended sheet structures comprising amyloid fibrils.10,11 The reorganization processoccurs in two ways, via organization of initially unstructured peptide (e.g., α-synucleinor synthetic Aβ) or destabilization of initially structured protein (e.g., transthyretinor prion). During Aβ fibril formation, the unstructured monomer forms a partially-structured intermediate containing a significant amount of helix.18

This intermediate then undergoes a conformational transition to the β-sheet-richform present in fibrils. Monitoring peptide secondary structures provides the meansto study basic features of amyloid assembly and how changes in primary structure,solvent conditions and the presence of other proteins or chemicals affect amyloido-genesis. CD spectroscopy (Section 7.4.3.1) and Fourier transform infrared spectros-copy (FTIR; Section 7.4.3.2) are two common methods for monitoring proteinsecondary structure.

7.4.3.1 CD Spectroscopy

Circular dichroism (CD) is the difference in the absorption of left and right circularlypolarized light. Optically active samples exhibit CD, and thus the acquisition andanalysis of CD data allow inferences to be made about the structures within samples.In proteins, secondary structure elements, including α-helices, β-strands, β-turns,and disordered regions display characteristic wavelength-dependent CD. CD thus isa useful tool for determining protein secondary structure content.

CD also is used frequently to determine the stability of the folded states ofproteins by monitoring the levels of α-helix or β-sheet during variations in temperature

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or in the concentrations of chemical denaturants (usually urea or guanidinium hydro-chloride (Gu·HCl)). The simplest experiment monitors the two-state conformationalfolded↔unfolded transition. For example, temperature and Gu ·HCl denatur-ation–renaturation experiments have been used to study the effects of TFE on Aβ40

and Aβ42 helix stability and fibrillogenesis kinetics.85

1. Prepare LMW Aβ at a concentration of ~30 µM in 10 mM sodiumphosphate, pH 7.4.

2. Incubate the sample under the desired experimental conditions (see notebelow).

3. Transfer the sample into a quartz CD cuvette. A 0.1-cm path-length quartzcell (Hellma, Forest Hills, NY) is a useful compromise between thenecessities (due to generally low peptide concentrations) of having thelongest path length possible and the lowest solvent absorption possible.If the peptide sample is not maintained within the CD cuvette during theexperiment, transfers to and from the cuvette should be made as gentlyas possible (especially under nonagitated incubation conditions).

4. CD measurements can be performed on any standard spectropolarimeter,according to the manufacturer’s instructions. Jasco, Inc. (Easton, MD)and Aviv Associates (Lakewood, NJ) both supply suitable instruments.Measurements should be made at the same temperature used to incubatethe sample. Greater accuracy in spectral interpretation is obtained if alower wavelength limit of 185 to 190 nm can be achieved, although formany applications this is not essential, and for some, not obtainable.Multiple spectra should be acquired in order to increase the signal-to-noise ratio and obtain smooth spectra.

5. A CD spectrum from buffer alone should always be acquired and sub-tracted from the sample spectra.

6. Raw spectra typically are acquired as tables of absolute ellipticity vs.wavelength. To normalize for peptide concentration, it is preferable todetermine the mean residue ellipticity, [Θ]λ (deg cm2 dmol–1), accordingto the formula [Θ]λ = M Θλ /10dcn, where M is peptide molecular weight(g/mol), Θλ is ellipticity (degrees), d is path length (cm), c is peptideconcentration (g/ml), and n is the number of amino acids in the peptide.The resulting spectra then can be interpreted both qualitatively and quan-titatively through comparison with spectra from standard proteins or pep-tides. A variety of spectral deconvolution packages are available (e.g.,CDANAL86 and CDPro87) that will produce quantitative estimates of thelevels of specific secondary structure elements, including random coil,α-helix, β-sheet, and β-turn. Readers are cautioned, however, that noneof these methods is entirely accurate and therefore the numbers theyproduce must be considered approximations.

Note: Sample preparation method, buffer composition, incubation conditions,and CD acquisition parameters all may be varied to suit experimental needs. How-ever, a number of factors must be considered. Increased sensitivity is obtained byusing more concentrated peptide solutions. Buffers should be UV-transparent so that

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a wavelength range of sufficient magnitude can be scanned. Temperature must becontrolled carefully so that variations do not occur during sample manipulationoutside of and inside the CD instrument.

7.4.3.2 Fourier Transform Infrared Spectroscopy (FTIR)

Infrared spectroscopy is a well-established optical method for determining proteinsecondary structures.88–91 FTIR provides the means to determine protein conforma-tion within samples in multiple milieus, including in solution, in thin films, and assolids. The technique is based on the fact that absorption spectra of peptides andproteins acquired in the infrared wavenumber range (1600 to 1700 cm-1) producepeaks characteristic of specific secondary structure elements.89–95 These elementsinclude α-helix, 310-helix, extended chain/β-sheet, loop, β-turn, aggregated strands,antiparallel β-sheet, and disordered (random coil). Characteristic differences havebeen reported between β-sheet structures in monomeric and aggregated species. TheFTIR method has been used quite successfully for secondary structure determina-tions of Aβ samples.31.89,96

7.4.4 TOPOGRAPHICAL ANALYSIS

Protein folding in general, and amyloid protein assembly in particular involve theformation of tertiary and quaternary structures in which amino acids can be consid-ered exposed or buried with respect to solvent water. The packing of buried residuesis involved in the nucleation of intramolecular protein folding and the organizationand stabilization of folded structures both in protein monomers and large assemblies.Monitoring the topography of an amyloid protein provides important insights intothe mechanisms of its folding and assembly.

7.4.4.1 8-Anilino-1-Naphthalenesulfonic Acid (ANS) Binding

ANS, a fluorescent dye, binds to solvent-exposed hydrophobic surfaces, such asthose found in partially folded intermediate (molten globule) states.97 When bound,an 8- to 10-fold increase in ANS fluorescence intensity occurs. This large increase,along with the rather weak affinity ANS has for native or completely unfolded states,makes it a useful probe of protein folding. This is true for amyloid assembly as well,which involves the formation of partially folded (e.g., α-synuclein and Aβ) andpartially unfolded (e.g., transthyretin and prion) intermediates.

1. Prepare stock 100 µM ANS in 10 mM phosphate buffer, pH 7.5.2. Mix 50 µl of an Aβ sample with 50 µl of the ANS stock solution.3. Measure the fluorescence intensity immediately after mixing. ANS should

be excited at a wavelength of 370 nm. Its emission can be measured withinthe wavelength range of 400 to 500 nm. Fluorometers are available com-mercially from many sources. We use an F4500 spectrofluorometer (Hita-chi Instruments, Rye, NH).

4. Binding of ANS to hydrophobic patches causes a blue wavelength shift(toward lower wavelengths) for the fluorescence intensity maximum alongwith an absolute increase in the fluorescence intensity.

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Note: The Aβ sample can be studied in phosphate buffer or alternate bufferscan be used. In the latter case, the investigator should ensure that the fluorescenceproperties of the dye are unaffected.

7.4.4.2 Intrinsic Fluorescence

The fluorescence characteristics of the aromatic amino acids tryptophan (Trp),tyrosine (Tyr), and phenylalanine (Phe) provide the means to monitor the confor-mational dynamics of protein folding and assembly.98 Trp is a particularly attractiveamino acid for use in intrinsic fluorescence experiments because it fluorescesstrongly and the emission spectrum of the Trp indole ring is highly sensitive tosolvent polarity and to the presence of fluorescence quenchers. Changes in theimmediate vicinity of Trp that occur during protein folding and assembly thus areindicated clearly by alterations in Trp fluorescence. Tyr can also be used to probelocal environmental changes, although it fluoresces less strongly than Trp. Tyrfluorescence (λmax), in contrast to that of Trp, is almost insensitive to solvent polarity.However, Tyr fluorescence can be quenched by exposure of the phenol ring tohydrated peptide carbonyl groups. Quenching also may be produced through hydro-gen bond formation with peptide carbonyls or with carboxylate side chains ofaspartic or glutamic acid. Intrinsic tyrosine fluorescence thus reveals features of thelocal environment of the phenol group. Aβ40 and Aβ42 contain a single Tyr at position10; thus intrinsic fluorescence experiments can be done on wild-type peptides with-out modification to their primary structure.

1. Transfer 100 µl of Aβ solution into a rectangular 10-mm quartz microcu-vette. The fluorescence intensity of Tyr10 in 50 to 100 µM wild-type Aβis substantial (10,000 arbitrary units), thus Aβ assembly reactions carriedout in the micromolar concentration regime are amenable to study by thismethod. We routinely use 50 µM sodium phosphate, pH 7.5, containing0.01% (w/v) sodium azide as the buffer in this method.

2. Place the cuvette into a fluorometer and measure the fluorescence usingan excitation wavelength of 280 nm. Spectra can be acquired by scanningfrom 290 to 500 nm. For assay purposes, fluorescence at a wavelength of303 nm can be monitored. Slit widths used for excitation and emissionare 5 and 10 nm, respectively, with a scan rate of 240 nm/min.

3. The fluorescence emission spectrum of phosphate buffer (backgroundintensity) is subtracted from the emission spectrum of the Aβ samples.

7.4.4.3 Electron Paramagnetic Resonance (EPR)

EPR is a technique for monitoring changes in the spin state of electrons. It can beapplied to free radicals and other molecular species that possess unpaired electrons.Alterations in spin state reveal molecular features of the chemical environment,structure, and motional dynamics of the electrons under study. Interactions betweentwo spin systems can also provide distance information.

EPR is a powerful approach for probing the dynamics of protein folding andassembly.99–105 Typically, a spin label (a stable free radical such as a nitroxide) is

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covalently attached to a specific site in a protein. Cysteine residues, either presentnatively in the protein or placed at precise locations through genetic engineering, areconvenient sites for attachment. Analysis of EPR spectra during the assembly processallows correlation of local environmental features around the spin label with theglobal structural organization of the protein assembly. Factors controlling the EPRspectrum include the solvent-accessibility of the probe and the motional freedom ofthe spin-labeled side chain. It has been shown that backbone secondary structure andaspects of the tertiary structure can be determined from periodic variations in theaccessibility of nitroxide side chains in a sequential set of spin-labeled proteins.100 Italso has been established that the motional dynamics of the side chain reflected inthe EPR spectral line shape correlates with general features of the protein fold.100

An excellent example of the use of EPR for understanding the structural orga-nization of Aβ in amyloid fibrils comes from the work of Langen et al. who usedsite-directed spin labeling (SDSL).106 Nineteen different spin-labeled Aβ peptideswere synthesized and each was allowed to form fibrils. Multiple techniques wereused to demonstrate that the spin label did not alter the ability of each peptide tofold and assemble into fibrils. Information from spin–spin interactions betweenequivalent residues in different peptides revealed that the fibrils formed by both Aβ40

and Aβ42 have arrangements of their peptide backbones that are parallel and in-register. Motional analysis showed that the central region of Aβ, residues 14-38, isrelatively stable, whereas significant mobility exists at both the N and C termini.Interestingly, in contrast to some amyloids, Aβ40 and Aβ42 formed mixed fibrils uponcoincubation. The SDSL EPR technique is a general method. For example, it hasbeen used to study transthyretin amyloid formation.107

7.4.4.4 Hydrogen–Deuterium Exchange

Hydrogen exchange is a phenomenon in which covalently bound, but labile, hydro-gen atoms of proteins exchange (switch places) at a finite rate with solvent hydrogenatoms.108 Hydrogen exchange is a powerful probe of protein structure and dynamics.The technique typically involves exchange of labile protein hydrogens with deuteronsfrom solvent 2H2O (D2O) and thus is called hydrogen–deuterium exchange. Labilehydrogens in proteins are those bonded to nitrogen, sulfur or oxygen. The exchangeof backbone amide hydrogens is studied frequently because their exchange rates areamenable to monitoring and they play key roles in hydrogen bonding interactionsin many structural elements of proteins (e.g., α-helices and β-strands).

For a backbone amide hydrogen to exchange with a solvent deuteron, the amidehydrogen must be solvent-exposed and not involved in hydrogen bonding. Theserequirements make hydrogen exchange sensitive to structural rearrangements thatoccur during protein aggregation. As oligomeric and higher-order assemblies form,the amide hydrogens buried in the core of the assembly or hydrogen bonded inα-helices and β-sheets do not exchange readily with solvent deuterons. This phe-nomenon is known as protection. Kheterpal and coworkers have shown that 40% ofthe backbone amide hydrogens of protofibrils formed by Aβ40 are protected fromexchange.60 In contrast, 60% of the backbone amide hydrogens are protected inmature amyloid fibrils.109

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A typical hydrogen exchange experiment involves three main steps: (1) forma-tion and isolation of the assembly of interest; (2) exposure of the assembly to anexcess of D2O; and (3) detection of exchange. Detection techniques include NMRand mass spectrometry (MS). NMR can pinpoint at the residue level the site wherethe exchange took place. However, it is limited by the need for high, nonphysiolog-ical protein concentrations and by a molecular mass limit of ~35 kDa. MS canmeasure the increase in mass that results from the exchange of hydrogens(1 amu/hydrogen exchanged). Advantages of MS include lower (by orders of mag-nitude) required concentrations of protein and applicability to very high mass proteinassemblies and complexes. Furthermore, following endoproteolysis with the acidprotease pepsin, MS experiments can be used to identify peptides in which hydrogenexchange occurred and then to systematically fragment them to determine preciselywhich amides underwent exchange. In-depth discussions of theoretical and practicalaspects of the use of NMR and MS to monitor hydrogen exchange may be foundin recent reviews by Engen and Smith110 and Dempsey.111

7.4.5 NMR SPECTROSCOPY

Solution-state NMR is an extremely powerful technique for determining high-reso-lution structures of stable protein conformers and monitoring protein dynamics.NMR generally is performed at relatively high protein concentrations (1 mM) andrequires that the protein of interest be soluble and unaggregated. Because of thepropensity of Aβ to aggregate, these requirements have complicated NMR studiesof this peptide under quasiphysiologic conditions. NMR studies of full-length Aβhave been conducted in the presence of high concentrations of organic cosolvents,including HFIP112 and TFE,113 or detergents (e.g., sodium dodecyl sulfate).114 Underthese conditions, aggregation is blocked but formation of α-helices is facilitatedsignificantly.

Studies done using these solvent conditions thus have yielded insights intoα-helix formation and stabilization. An alternate approach to controlling Aβ self-association has been to conduct NMR experiments at low temperature (e.g., 5°C)or to use Aβ in which Met35 has been oxidized to its sulfoxide form.115 Thismodification has been shown to delay the oligomerization of Aβ.116,117 Results fromthese studies have revealed a predominantly random, extended-chain conformationfor Aβ40, Aβ42, Met35(O)Aβ40 and Met35(O)Aβ42, and turn- or bend-like structureslocated at Asp7–Glu11 and Asp23–Ser26.117

One of the most exciting advances in NMR-based studies of Aβ assembly hascome from studies of Aβ40 fibrils in the solid state.118 These studies were madepossible by improved technology (e.g., probes that allow rotation at speeds up to~30 kHz) and experiments that permit accurate measurements of intramolecular andintermolecular distances, backbone torsion angles, and chemical shifts that correlatewith secondary structures.119 A structural model of Aβ40 protofilaments, derived byenergy minimization with constraints based on solid-state NMR data, has been shownto be consistent with the overall dimensions and linear density derived from EMdata.118

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7.4.6 MORPHOLOGICAL ANALYSIS

In some ways, the fine-structure analyses discussed to this point may be likened tothe proverbial blind men describing an elephant.120 Each technique provides impor-tant information about a particular peptide subregion or assembly process, but doesnot provide a global view of the peptide assembly itself. In the following twosections, we discuss in general terms the use of electron microscopy (EM) andatomic force microscopy (AFM) for determining the morphologies of Aβ assemblies.

7.4.6.1 Electron Microscopy

Electron microscopy has been used extensively to examine the morphology of Aβassemblies. In one approach, the protein sample is spotted onto a small support(grid), allowed to adhere and then washed briefly with water. The assemblies thusimmobilized are chemically cross-linked with glutaraldehyde to stabilize them andthen stained with an electron-dense dye such as uranyl acetate or phosphotungsticacid. The dye provides contrast so that the assemblies can be visualized afterplacement inside the electron microscope. The images obtained show dark structuresagainst a white background; thus this technique is referred to as negative staining.In addition to direct examination of negatively stained preparations, metal casts ofthe assemblies can be constructed using a technique called rotary shadowing.121 Thisproduces very high contrast images.

Care must be exercised in the conduct and interpretation of EM studies becauseartifactual results are easy to produce. These artifacts arise for numerous reasons.Particulates in the EM stains can be interpreted as proteinaceous. Damage to thegrid surface may be interpreted as protein sheets. Specific subsets of assembliespresent in a protein sample may adhere to the grid, whereas others may not. Thisleads to erroneous conclusions about the distributions of morphologies present. Evenif all assemblies adhere, because only a small area of a grid is illuminated at anyone time, it is critical to sample a sufficient number of locations to ensure that arepresentative sampling of the bound assemblies is obtained. A substantial literatureexists concerning EM studies of Aβ. The assemblies examined include LMW Aβ,17,116

small oligomers,122–124 paranuclei,17,116 protofibrils,42,60,61,66,122,124 spheroids,26,27,125 andfibrils.24,38,66,121,125,126

7.4.6.2 Atomic Force Microscopy

AFM is a method for morphological analysis that complements EM. AFM providesthree-dimensional images of assemblies adherent to an atomically flat support,typically mica. AFM is a physical method in which a small probe is rastered acrossa mica surface. The probe either remains in continuous contact with the surface ortaps the surface at high frequency. As the probe encounters discontinuities in thesurface due to the presence of adherent material, its vertical position changes andthese changes are recorded by the instrument. Thus, after complete scanning of theAFM support, a relief map can be constructed in which extremely fine (nanometer)detail can be revealed.

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The resolution of this method is limited by the size and shape of the probe tip.Therefore, although the heights of objects can be measured accurately, widths canbe overestimated.25 A powerful feature of AFM that cannot be achieved by EM isthe continuous monitoring of protein assembly in the “solution phase.” Experimentscan be done in which fibril growth occurs on an AFM support immersed in solvent.127

This allows real-time monitoring of assembly growth and kinetics. Although obvious,it is important to appreciate that any AFM method can only reveal features of materialthat will adsorb to the mica surface. Thus, as with EM, conclusions about populationmorphologies must be made cautiously. AFM has been used extensively to studythe morphology of Aβ oligomers,20,33,128 protofibrils,12,128 and fibrils.128–131

7.5 DISCUSSION

Since the time of Virchow, when amyloid was thought to be a complex carbohydrate(and thus the misnomer “amyloid” was introduced), the study of amyloid proteinstructure, assembly and biological activity has been problematic. To a large degree,this is because of the strong propensity of amyloid proteins to self-associate, whichmakes the use of classical methods in structural biology either difficult or impossible.Aβ, in particular, has been a vexing subject of study because the native monomericstate of this peptide remains largely undefined and the peptide exists in equilibriumwith dimers and larger oligomers. Therefore, unlike proteins with well-defined nativefolds, Aβ folding and assembly proceeds from a poorly defined starting conformerto an incompletely defined amyloid fibril. Nevertheless, significant progress has beenmade in elucidating pathways of Aβ assembly. In this chapter, we have sought toprovide both the theoretical and practical tools necessary to study this enigmaticpeptide. We also have pointed out pitfalls that must be fully understood and consid-ered in experimental planning if valid and useful conclusions are to be derived fromstudies of Aβ. In this regard, the following maxim of Sir Isaac Newton (1642–1727)may have special applicability for studying amyloid protein assemblies:

To explain all nature is too difficult a task for any one man or even for any one age.’Tis much better to do a little with certainty, and leave the rest for others that comeafter you, than to explain all things.

ACKNOWLEDGMENTS

We thank Dr. Gal Bitan and Ms. Sabrina Vollers for critical comments. We gratefullyacknowledge the support of the National Institutes of Health (NS38328, AG18921,and NS44147) and the Foundation for Neurologic Diseases.

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8
Intracellular Accumulation of Amyloid β and Mitochondrial Dysfunction in Down’s Syndrome
Jorge Busciglio, Alejandra Pelsman, Pablo Helguera, and Atul Deshpande

CONTENTS

Abstract8.1 Introduction8.2 Experimental Procedures

8.2.1 Cell Culture8.2.2 Antibodies8.2.3 Immunocytochemical Procedures

8.2.3.1 Extraction8.2.3.2 Fixation8.2.3.3 Second Labeling Protocol

8.2.4 Western Blot.8.2.5 Inhibition of Energy Metabolism8.2.6 Assessment of Mitochondrial Function

8.2.6.1 MTS Assay8.2.6.2 JC1 Protocol

8.2.7 Cell Viability Assays8.2.7.1 Trypan Blue8.2.7.2 Propidium Iodide

8.2.8 Neuroprotection Assays8.3 Results

8.3.1 Detection of Intracellular Aβ in DS Astrocytes8.3.2 DS-Like Alterations in APP Processing Induced in Normal

Astrocytes by Energy Depletion8.3.3 Mitochondrial Dysfunction in DS Astrocytes8.3.4 APPs Rescues DS Cortical Neurons from Apoptosis


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ABSTRACT

Cortical astrocyte and neuronal cultures derived from fetal Down’s syndrome (DS)brain were utilized to demonstrate intracellular accumulation of insoluble Aβ42 andinhibition of mitochondrial energy metabolism consistent with impaired mitochon-drial function in DS. Similarly, energy depletion in normal astrocytes resulted inintracellular Aβ accumulation, suggesting an important role for mitochondrial dys-function in the generation and accumulation of intracellular Aβ. Energy deficits inDS cells also alter amyloid precursor protein (APP) processing, resulting indecreased APP secretion in vitro and in vivo. The survival of DS cortical neuronswas markedly increased by addition of recombinant APP or conditioned medium ofnormal astrocyte cultures, but not by conditioned medium depleted of secreted APP(APPs), suggesting that APPs may be a survival factor for human neurons. Theseresults indicate that mitochondrial dysfunction in DS brain cells leads to intracellulardeposition of Aβ42, reduced levels of APPs, and a chronic state of increased neuronalvulnerability.

8.1 INTRODUCTION

Down’s syndrome (DS) or trisomy 21 is the most common genetic cause of mentalretardation. The neuropathology of DS is complex and includes decreased brainweight and neuronal number, abnormal neuronal differentiation, and structuralchanges in dendritic spines.1 A distinct feature of DS is the onset of Alzheimer’sdisease (AD) by middle age.1,2 The development of AD in DS may be related tooverexpression of the amyloid precursor protein (APP) due to increased gene dosage,leading to increased Aβ generation.3,4

Deficits in mitochondrial function cause selective neuronal degeneration andmay be involved in a number of neurodegenerative disorders.5,6 DS cortical neuronsin culture exhibit intracellular accumulation of reactive oxygen species (ROS) andincreased lipid peroxidation leading to neuronal apoptosis.7 Interestingly, perturba-tions of mitochondrial homeostasis constitute an early and critical feature of apop-totic processes that precede free radical formation and neuronal death.8 Energydepletion and oxidative stress can also induce amyloidogenic changes in APP pro-cessing,9 suggesting a potential link between mitochondrial dysfunction, oxidativestress, and Aβ production. In this regard, dysfunction of mitochondrial electrontransport proteins and a close relationship between mitochondrial abnormalities andoxidative damage have been described in AD brains.10–12

To characterize the molecular events involved in DS neuropathology and thedevelopment of AD in DS, we analyzed Aβ generation and mitochondrial activityin normal and DS cortical astrocytes in culture and in the DS brain. The resultssuggest that mitochondrial dysfunction may play a significant role in the develop-ment of AD neuropathology in DS patients by promoting aberrant APP processingand intracellular accumulation of Aβ. Below is a detailed description of the tech-niques utilized for immunodetection of intracelllular Aβ and for the assessment ofmitochondrial function and cell viability in normal and DS cortical astrocyte andneuronal cultures.



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8.2 EXPERIMENTAL PROCEDURES

8.2.1 CELL CULTURE

Primary human cortical cultures were established from the cerebral cortices ofnormal and DS-aborted fetuses from embryonic weeks 17 through 21. Twelve DSbrain specimens and 12 age-matched controls were used to generate neuronal andastrocyte cultures. The protocol for tissue procurement complied with federal andinstitutional guidelines for fetal research. Cortical astrocyte cultures were preparedas previously described.13 Cells were plated on culture dishes or glass coverslips ata density of 100,000 cells/cm2 and maintained in 10% iron-supplemented calf serum(HyClone, Logan, UT) and D-MEM (Life Technologies, Invitrogen, Carlsbad, CA).For all experiments, astrocyte cultures were fixed or harvested between 30 and 35days in vitro. More than 95% of the cells present in the cultures stained positive forglial fibrillary acidic protein (GFAP). Normal and DS cortical neuronal cultures wereprepared as previously described.7,14

8.2.2 ANTIBODIES

C8 is a polyclonal antibody directed against residues 676–695 of APP which rec-ognizes holo-APP and C-terminal fragments. Alz90 (Roche, Basel, Switzerland) and8E15 (supplied by Dr. Peter Seubert, Elan Pharmaceuticals, Dublin, Ireland) aremonoclonal antibodies that recognize residues 511–608 and 444–592, respectively,and recognize APPs. Monoclonal antibodies α-Aβ42 and α-Aβ40 specifically recog-nize the free C terminus of Aβx-42 and Aβx-40, respectively.16,17 Other antibodies usedincluded anti-syntaxin 6, anti-early endosomal antigen 1 (Signal Transduction Lab-oratories, BD Biosciences, Canada), anti-LAMP1 (Signal Transduction Laborato-ries), mouse anti-α-tubulin and anti-α-tubulin isotype III (Sigma Chemical, St.Louis, MO, USA), and mouse anti-Cu/Zn superoxide dismutase (Sigma).

8.2.3 IMMUNOCYTOCHEMICAL PROCEDURES

Double immunofluorescence was performed on fixed cultures using two consecutiveprotocols (Figure 8.1). In the first step, intracellular Aβ in cultured astrocytes wasimmunolabeled using a tyramide signal amplification kit (TSA, NEN Life ScienceProducts, now PerkinElmer, Boston, MA). This technique is designed to enhancesignal labeling approximately 1000-fold over regular protocols using biotinylatedsecondary antibodies. The TSA indirect assay uses the capability of horseradishperoxidase (HPR) to generate reactive tyramide radicals with very short half-lives,resulting in biotinyl–tyramide depositions very close to the sites of enzymatic activities.

Biotin is detected by streptavidine–fluorophore conjugates, in this case strepta-vidine–Oregon green. In the second step, organelle-specific markers were immuno-labelled by standard procedures. For some experiments, the cultures were extractedprior to fixation.7 The purpose of this step is to extract soluble proteins whilepreserving fibrillar and/or insoluble cellular elements in the preparation. The proce-dure is as follows.



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8.2.3.1 Extraction

1. Wash cultures with prewarmed stabilizing buffer (0.13 M HEPES, pH6.9, 2 mM MgCl2, and 10 mM EGTA).

2. Extract cultures in the same buffer plus 0.2% Triton X-100 for 5 min at37˚C.

3. The cultures are fixed and immunocytochemistry is performed on theTX-100-resistant material remaining on the coverslip.

8.2.3.2 Fixation

1. Fix cultured cells in 4% paraformaldehyde and 0.12 M sucrose in PBSfor 20 min at 37˚C.

2. Wash in PBS 3X 5 min each.3. If a permeabilization step has not been performed prior to fixation, per-

meabilize with 0.2% Triton X-100 in PBS for 5 min at room temperature.3. The TSA procedure is performed according to manufacturer’s instructions

(refer to NEN Life Sciences TSA-Indirect technical brochure).

FIGURE 8.1 Flowchart of general procedure utilized for double immunofluorescence ofintracellular Aβ and organelle markers in cultured astrocytes.



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8.2.3.3 Second Labeling Protocol

1. Block for 1 hr in 5% BSA/PBS.2. Incubate overnight at 4˚C with corresponding primary antibody.3. Wash with PBS 0.1% Triton X-100 3X 5 min each.4. Incubate in Cy3-conjugated mouse or rabbit secondary antibody in 1%

BSA/PBS for 30 min at room temperature.5. Wash with PBS 0.1% Triton X-100 3X 5 min each.6. Mount coverslips with antifade mounting medium.

Fluorescence was visualized with an Olympus IX-70 inverted microscope or aZeiss LSM 410 confocal-scanning microscope. Specificity was confirmed by pre-absorption of the primary antibody with synthetic Aβ1-42 peptide which abolishedimmunoreactivity.

8.2.4 WESTERN BLOT

Cultures were washed with PBS and harvested in RIPA buffer at 4oC. The lysateswere centrifuged at 100,000× g for 60 min. Supernatants were mixed 1:1 with SDS-reducing sample buffer and boiled for 5 min, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrotrans-ferred to polyvinylidene difluoride membranes, blocked, incubated overnight at 4oCwith primary antibody and developed by enhanced chemiluminescence. To controlfor protein loading, tubulin levels were analyzed in all samples with anti-α-tubulin.Quantitative analysis was performed using a FastScan densitometer (MolecularDynamics, Sunnyvale, CA) as described.18 Briefly, a standard curve of pixel valueswas constructed by immunoblotting a serial dilution of purified tubulin. Volumeanalysis on the appropriate bands was performed using NIH Image software. Alldensitometric values used for analysis were within the linear range of pixel values.

8.2.5 INHIBITION OF ENERGY METABOLISM

To inhibit mitochondrial energy metabolism, cultures were incubated with carbonylcyanide phenylhydrazone (CCCP). CCCP is one of the most potent uncouplers ofboth oxidative phosphorylation and photophosphorylation. It produces strong blockingeffects on adenosine triphosphate (ATP) generation in mitochondria and chloro-plasts.19 Normal astrocytes were incubated with 80 µM of CCCP for 4 hr beforeharvesting for biochemical analysis. For immunofluorescence microscopy of intra-cellular Aβ, astrocytes were incubated with 20 µM CCCP for 3 days and with 80 µMCCCP for 4 hr before fixation. This longer incubation protocol does not reduceastrocyte survival as determined by propidium iodide exclusion assay. The longerincubation increases intracellular Aβ levels, enhancing detection by immuno-cytochemistry or Western blot.

8.2.6 ASSESSMENT OF MITOCHONDRIAL FUNCTION

Mitochondrial redox activity was analyzed in normal and DS astrocyte cultures using3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-


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tetrazole (MTS) salt and MTT reduction assays following the vendor’s protocol(Promega, Madison, WI). Both are colorimetric assays that measure the conversionof a tetrazolium component (MTS or MTT) into a colored formazan product. Thesetechniques are widely used to determine cell viability. MTS is cleaved in activemitochondria to a soluble formazan by mitochondrial succinate dehydrogenase whichis located in complex II (succinate–ubiquinone oxidoreductase complex). The sol-uble formazan product is released to the culture medium; consequently, cultures canbe assayed multiple times.

8.2.6.1 MTS Assay

1. Replace culture medium with phenol-free D-MEM. Let equilibrate for30 min.

2. Add 20 µl of a solution of MTS and PMS (phenazine methosulfate), anelectron coupler, as provided by the manufacturer, per 100 µl medium.

3. Return the culture to the incubator for 1 hr.4. Record absorbance at 490 nm using an enzyne-linked immunosorbent

assay (ELISA) plate reader.

In the MTT assay, the formazan product is insoluble. For that reason, cell lysisand formazan solubilization are performed in an extra step. Maximum absorbanceis read at 570 nm.

Mitochondrial transmembrane potential was analyzed using JC1 (MolecularProbes, Eugene, OR).20,21 JC1 is a positively charged carbocyanine dye taken intothe negatively charged inner mitochondrial membrane where it stays as a monomerat depolarized potential, producing a green fluorescence emission at 527 nm. In activemitochondria with higher membrane potentials, JC1 forms multimers called J aggre-gates that produce red fluorescence emissions at 590 nm. Hence, reduced mitochon-drial activity results in reduced JC1 aggregation and decreased red fluorescence.Normal and DS astrocyte cultures were incubated with JC1 and analyzed by fluo-rescence microscopy.

Cultures were treated with 0.25% trypsin for 5 min, dissociated and replated ata lower density (50,000 cells/cm2). After 24 hr, a dose–response curve was con-structed to determine a nonsaturating concentration of JC1 in normal astrocytecultures. This is important because at nonsaturating concentrations JC1 aggregationcorrelates with mitochondrial membrane potential, but at concentrations near orabove saturation, the levels of J aggregate fluorescence rise linearly, independent ofthe mitochondrial membrane potential. Under our experimental conditions, a 30-minincubation with 0.5 µM JC1 resulted in approximately 70% of normal astrocytesexhibiting red fluorescent J aggregates.

8.2.6.2 JC1 Protocol

1. Incubate normal and DS astrocytes with 0.5 µM JC1 for 30 min.2. Wash in warm PBS.3. Quantify the number of individual astrocytes exhibiting J aggregates

(emission at 590 nm) and the total number of astrocytes (emission at527 nm) by image analysis of 10 microscopic fields per culture.


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Equivalent cellular loading of the dye in normal and DS cultures was confirmedby quantification of JC1 emission intensity at 527 nm. Equivalent astrocyte cellnumbers in normal and DS cultures were further confirmed by scoring fluorescentnuclei after staining with Hoechst 33258. All experiments were performed blindedand in quadruplicate cultures. Approximately 450 cells were analyzed per experi-mental condition.

8.2.7 CELL VIABILITY ASSAYS

Cell viability assays can be used during the setup of new cultures and to evaluatepharmacological treatments (neuroprotective or neurotoxic assays). Cell viability incultures used for MTT, MTS, and JC1 assays was assessed using propidium iodideor trypan blue exclusion.7 These assays can be performed easily by microscopicanalysis.

8.2.7.1 Trypan Blue

Trypan blue is an acidic, water-soluble dye that cannot penetrate the intact plasmamembranes of viable cells. If loss of membrane integrity occurs, trypan blue canenter cells and stain intracellular elements. Under bright field microscopic observa-tion, live cells appear colorless and dead cells appear dark blue.

Trypan blue is added to cells at 0.2% final concentration in D-MEM or serum-free media for 2 to 3 min. The culture is washed 3X with PBS, and the cells areobserved under a bright field microscope at a final magnification of 100×. Thenumber of stained cells is recorded in five to six fields per well in triplicate orquadruplicate wells.

8.2.7.2 Propidium Iodide

Propidium iodide (PI) is a fluorescent dye that binds to double-stranded nucleicacids by intercalating between purines and pyrimidines, enhancing its fluorescentproperties by 20- to 30-fold. It does not bind to single-stranded nucleic acids. Theexcitation and emission wavelengths of PI are ~493 and ~632 nm, respectively. Bothexcitation and emission are shifted ~35 and ~15 nm, respectively, after binding tonucleic acids. Similar to trypan blue, PI is cell-impermeable and can only penetrateinside a cell when the plasma membrane is damaged as a consequence of apoptoticor necrotic processes.

After PI incubation, the culture is observed under fluorescent light using a630-nm filter. The nuclei of nonviable cells appear red due to PI staining. Hoechst33258, another dye that interacts with nucleic acids, can be used along with PI asa counterstain to assess the total number of cells in the culture. Hoechst has anexcitation in the range of 351 to 363 nm and emission between 390 and 480 nm.PI is a known mutagen and care should be taken to use and dispose of it properly.

Incubate cells with PI (10 µg/ml) in serum-free medium for 10 min. Wash cellsgently with PBS. Fix the cells with 4% paraformaldehyde at 37oC for 20 min.Incubate fixed cells with Hoechst 33258 (10 µg/ml in PBS) for 5 min. Wash cellswith PBS 3X. Mount the coverslip with antifade mounting media. Observe under



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fluorescence microscope with appropriate red filter for PI and UV filter for Hoechst33258.

8.2.8 NEUROPROTECTION ASSAYS

Treatment of DS neuronal cortical cultures growing in 24-well plates was initiatedat day 7, before the onset of neuronal degeneration.7 A recombinant form of APPcomprising amino acids 20 through 590 of APP695 and 17-mer reverse sequencepeptides corresponding to a neurotrophic–neuroprotective domain of APP, kindlysupplied by Dr. J.M. Roch22,23 (University of California, San Diego), were dilutedin PBS and immediately added to the culture medium at the indicated concentrations.NGF, BDNF, and NT3 (supplied by Dr. M. Greenberg, Division of Neuroscience,Children’s Hospital, Boston, MA) were added to the culture medium at a finalconcentration of 40 ng/ml. At days 9 and 11, a 30% partial medium change wasperformed and the treatment was repeated.

The cultures were fixed at day 14, immunostained with anti-α-tubulin class III,and the number of viable neurons was scored in quadruplicate cultures as previouslydescribed.7 More than 400 neurons were scored per experimental condition. Forsome experiments, DS neurons were incubated with conditioned medium of DSastrocyte cultures, normal astrocyte cultures or normal astrocyte-conditionedmedium previously depleted of secreted APP by immunoprecipitation with a mixtureof antibodies Alz-90 and 8E5.

8.3 RESULTS

8.3.1 DETECTION OF INTRACELLULAR Aββββ IN DS ASTROCYTES

To establish the presence and intracellular localization of Aβ, immunofluorescencemicroscopy was performed in DS astrocytes using monoclonal antibodies generatedagainst the C termini of Aβ42 and Aβ40.17 These antibodies specifically recognizedAβ42 and Aβ40 respectively, but not full-length APP.16 Antibody specificity was alsoconfirmed by immunocytochemical staining of synthetic Aβ1-42 and Aβ1-40 fibrils(data not shown).

Intracellular Aβ42-specific staining was successfully achieved using a tyramidesignal amplification system. Minimal background staining was detected in the cyto-plasms of normal astrocytes (Figure 8.2A). In contrast, DS astrocytes had significantAβ42-positive labeling in a vesicular distribution and often forming clusters in dif-ferent regions of the cytoplasm (Figure 8.2B and Figure 8.2C, arrows). Aβ42 immu-nofluorescence was completely abolished by preincubation of the primary antibodywith synthetic Aβ1-42 peptide (data not shown). Aβ42 was also detected in DS astro-cytes extracted with Triton X-100 prior to fixation (Figure 8.2C), strongly suggestingthe presence of insoluble, detergent-resistant intracellular Aβ42 aggregates. Aβ40

immunoreactivity was not detected in DS astrocytes. An electrophoretic gel systemthat separates Aβ40 from Aβ42

24 confirmed the presence of intracellular Aβ42 in DScortical cultures (data not shown).



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Western blotting and immunocytochemical analysis of Cu–Zn superoxide dis-mutase, another protein overexpressed in DS, and tubulin, an abundant cytoplasmicprotein, showed no significant differences in protein levels and intracellular local-ization in DS cultures (data not shown), suggesting a specific mechanism in the

FIGURE 8.2 (See color insert following page 114.) Intracellular accumulation of aggregatedAβ42 in DS astrocytes. Immunofluorescence of normal (NL) and DS astrocyte cultures usingα-Aβ42. Note the appearance of Aβ42 labeling in a vesicular-like distribution in DS (arrows;B and C) but not in NL astrocytes (A). Detergent-insoluble aggregates of Aβ42 appear in DSastrocytes extracted with Triton X-100 before fixation (C). Scale bar: 20 mm.



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accumulation and aggregation of intracellular Aβ (Aβi). Sequential double immun-ofluorescence showed that Aβi partially colocalized with APP, which is associatedwith subcellular compartments of the secretory pathway (Figure 8.3A). Aβi stainingwas particularly intense in perinuclear regions where it partially colocalized withthe Golgi-resident protein syntaxin 6 (Syn), the early endosome antigen 1 protein(EEA1) and the lysosomal marker LAMP1 (Figure 8.3B through Figure 8.3D). Thus,intracellular Aβ42 accumulates in subcellular compartments associated with APPprocessing.

8.3.2 DS-LIKE ALTERATIONS IN APP PROCESSING INDUCED IN NORMAL ASTROCYTES BY ENERGY DEPLETION

Alterations in APP metabolism observed in DS astrocytes16 are very similar tochanges in APP processing induced by energy depletion.9 To further characterizethe potential role of energy deficits in APP metabolism in the central nervous system(CNS), APP processing was analyzed in normal human astrocytes treated with themitochondrial electron chain uncoupler CCCP.9,25

Energy depletion in normal astrocytes significantly increased the amount ofcellular APP by 20.2 ± 4.3% (p < 0.002) and C99 by 50.6 ± 8.7% (p < 0.001) andreduced the levels of C83 and secreted APP (APPs) by 46.7 ± 7.7% (p < 0.001) and49.6 ± 2% (p < 0.0001), respectively (Figure 8.4A and Figure 8.4B). Secreted Aβlevels were also reduced by CCCP treatment (data not shown). These changes arereminiscent of the alterations in APP processing in DS astrocytes.

Immunocytochemical analysis of normal astrocytes after exposure to CCCPshowed the presence of Aβ42 aggregates throughout the cytoplasm from perinuclearregions to the cell membrane (arrows, Figure 8.4C through Figure 8.4F). Aβ42

aggregates were resistant to solubilization with Triton X-100 (Figure 8.4F) and,similar to Aβ42 aggregates in DS astrocytes, partially co-localized with ER, Golgiand endosomal markers (data not shown). Thus, DS and energy-depleted normalastrocytes exhibit strikingly similar profiles of APP processing, including increasedlevels of cellular APP and C99, decreased levels of APPs and C83, and intracellularaccumulation of detergent-resistant aggregates of Aβ42.

8.3.3 MITOCHONDRIAL DYSFUNCTION IN DS ASTROCYTES

The similarity in the alterations in APP processing observed in DS and CCCP-treatednormal astrocytes raised the possibility that mitochondrial energy deficits could leadto altered APP processing in DS astrocytes. We investigated mitochondrial functionby assessing mitochondrial transmembrane potential (∆Ψm) in viable DS and normalastrocytes using the fluorescent probe JC1.20,21 Normal and DS astrocyte cultureswere incubated with JC1 and analyzed by fluorescence microscopy. Under theseconditions, 72.9 ± 7.3% of normal astrocytes exhibited JC1 red fluorescence, indic-ative of active mitochondria (Figure 8.5A through Figure 8.5D).

The number of DS astrocytes exhibiting J aggregates was significantly reducedto 42 ± 5.6% (Figure 8.5C and Figure 8.5D) despite similar cellular loading of JC1as measured by JC1 emission intensity at 427 nm. Normal astrocytes treated with


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CCCP showed a dramatic decrease in the number exhibiting J aggregates (16.7 ±4.9%, Figure 8.5D). Thus, DS astrocytes exhibit significantly reduced mitochondrialtransmembrane potential.

To further examine mitochondrial function in DS, we assessed reduction of MTSreagent as an indicator of mitochondrial redox activity.26 MTS-reducing activity was

FIGURE 8.3 (See color insert following page 114.) Double labeling of Aβ42 and differentsubcellular markers. Double labeling shows partial colocalization of Aβ42 (Aβ) with APP(APP) in a DS astrocyte. APP immunostaining was performed with antibody Alz-90 (A).Double labeling shows partial colocalization of Aβ42 with the early endosomal antigen 1(EEA1) in endosomal vesicles (B), with syntaxin 6 in the Golgi compartment (C), and withLamp1 in lysosomal vesicles (D). Scale bar: 20 mm. (Panels A, B, and C reprinted fromBusciglio, J. et al., 2002. With permission. Panel D prepared by the authors.)



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diminished by approximately 30% in DS cultures (p < 0.01, Figure 8.5E). A similarresult was obtained using another mitochondrial redox assay utilizing the insolubletetrazolium compound MTT (data not shown). Trypan blue and propidium iodideexclusion assays showed no significant differences in the number of viable cells innormal and DS cultures (Figure 8.5F), indicating that the decrease in mitochondrialredox activity in DS cultures was not due to decreased cell viability. Furthermore,analysis of cytochrome C levels by Western blotting did not show a significantdifference between normal and DS astrocytes, suggesting that mitochondrial proteincontent was not different in DS cultures (data not shown). Taken together, theseresults suggest that mitochondrial energy metabolism is impaired in DS astrocytes.

8.3.4 APPS RESCUES DS CORTICAL NEURONS FROM APOPTOSIS

Previous studies suggest that APPs may generate both neurotrophic and neuropro-tective activities.27 After the first week in culture,7 DS cortical neurons exhibitedincreased degeneration, accumulation of intracellular Aβ (Figure 8.2 and Figure 8.3)and decreased APP secretion.16 To determine whether APPs increase DS neuronalsurvival, cortical neurons were incubated with recombinant APP spanning residues20 to 590 of APP69523 or a 17-mer peptide comprising the putative neurotrophicdomain of APPs corresponding to amino acids 310 through 335.22,28 Incubation ofDS cultures with either APP or the 17-mer peptide dramatically increased DS

FIGURE 8.4 (See color insert following page 114.) Mitochondrial dysfunction in normalastrocytes induces aberrant APP processing and intracellular Aβ accumulation. (A) Normalastrocytes treated with CCCP exhibit increased cellular APP (APP) and decreased levels ofsecreted APP (APPs). Samples were separated in a 4 to 20% gradient gel, electroblotted andincubated with antibodies C8 (cellular) and Alz-90 (secreted). (B) treatment with CCCPreduced C83 and increased C99 levels in normal astrocytes. Shown are Western blots withantibody C8 which recognizes APP C terminal fragments and 6E10 which recognizes C99but not C83. (C through F) Accumulation of intracellular Aβ42 in CCCP-treated astrocytes.Immunofluorescence was performed on CCCP-treated and control astrocytes with antibodyα-Aβ42. Nuclei were counterstained with propidium iodide. Intensely labeled aggregates ofAβ42 were localized throughout the cytoplasm after treatment with CCCP (arrows; D and F)and were detergent-insoluble (F). Scale bar: 20 mm. (Reprinted from Busciglio, J. et al., 2002.With permission.)



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neuronal survival (Figure 8.6A). In contrast, no significant effect was observed witha reverse sequence control peptide or with neurotrophic factors NGF, BDNF, andNT3 (Figure 8.6A). Thus, APPs (but not other neurotrophic factors) prevents thedegeneration of DS neurons. APP did not significantly affect neuronal survival innormal cortical cultures (Figure 8.6A).

To confirm a role for diminished APP secretion in DS neuronal degeneration,DS cortical neurons were incubated in culture with conditioned medium of normalor DS astrocytes from days 7 through 14. Normal astrocyte-conditioned mediumdramatically increased DS neuronal survival, whereas DS astrocyte-conditionedmedium did not increase survival (Figure 8.6B). DS cultures incubated with condi-tioned medium of normal astrocytes depleted of APP by immunoprecipitation didnot exhibit increased neuronal survival (Figure 8.6B). Taken together, these resultsindicate that APPs acts as neuroprotectives agent for DS cortical neurons and thatreduced levels of APPs may compromise neuronal survival in DS and AD.

8.4 DISCUSSION

These experiments demonstrate intracellular Aβ accumulation in DS astrocytes thatcan be replicated in normal astrocytes by inhibition of mitochondrial energy metab-olism. Moreover, mitochondrial function is impaired in DS astrocytes, as indicated

FIGURE 8.4 (continued)



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FIGURE 8.5 (See color insert following page 114.) Impaired mitochondrial function in DS astro-cytes. (A) Normal and DS astrocytes were labeled with the mitochondrial transmembrane potential-sensitive dye JC1 that fluoresces green (527 nm) or red (590 nm), depending on whether mitochondriaare relatively less or more activated. A normal astrocyte (NL) labeled with JC1 shows inactive andactive mitochondria labeled green and red, respectively, in the color insert. (B and C) DS astrocyteslabeled with JC1 and visualized with the red channel show reduced numbers of cells with activemitochondria (arrows) and more cells with inactive mitochondria (arrowheads) relative to normalastrocytes. Astrocyte nuclei are labeled with Hoechst 33258 (blue channel in color insert). Scale bars:20 mm. (D) Quantitative analysis shows a significant decrease in the number of DS astrocytesexhibiting red JC1 labeling (42 ± 5.6%) compared to normal astrocytes (72.9 ± 7.3%). CCCP-treatmentof normal astrocytes markedly reduces the number of cells exhibiting red JC1 labeling (16.7 ± 4.9%).Values represent mean ± SEM; n = 4 independent experiments. *p < 0.01 relative to control byStudent’s t test. (E) Reduced mitochondrial redox activity in DS astrocytes. MTS-reducing activity inDS cultures was significantly reduced relative to normal cultures. Values represent mean ± SEM; n =4 independent experiments. *p < 0.01 relative to control by Student’s t test. (F) Trypan blue exclusionassay shows no significant differences in cell viability between normal and DS cultures. Valuesrepresent mean ± SEM; n = 4 independent experiments. (Panels B and C reprinted from Busciglio,J. et al., 2002. With permission. Panels A, D, E, and F prepared by the authors.)



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by reduced mitochondrial redox activity and membrane potential. We suggest thatimpaired energy metabolism in DS cells gives rise to increased β-secretase cleavageof APP 16 and altered trafficking of Aβ, resulting in intracellular accumulation ofaggregated Aβ42. Thus, impaired mitochondrial energy metabolism may contributeto AD pathogenesis in DS. We have also observed a similar impairment in mito-chondrial function in primary cultures of DS fibroblasts (unpublished results), sug-gesting that mitochondrial dysfunction may be widespread in DS.

We found intracellular Aβ in a detergent-resistant form in DS astrocytes and innormal astrocytes treated with the mitochondrial uncoupler CCCP. Intracellular Aβ42

partially colocalizes with APP and appears predominantly in the Golgi complex as

FIGURE 8.6 (A) Secreted APP prevents the degeneration of DS cortical neurons. DS corticalneuronal cultures were incubated in culture with recombinant APP (200 nM), an APP-derivedtrophic peptide (17-mer, 500 nm), the reverse sequence peptide (17-mer-rev, 500 nm) orneurotrophic factors NGF (40 ng/ml), BDNF (40 ng/ml) or NT3 (40 ng/ml) from days 7 to14. Neuronal viability at day 14 is expressed as percent of the neuronal number at day 7(100%). Neuronal viability in untreated DS cultures was reduced to 45 ± 4.8%. RecombinantAPP and the 17-mer peptide prevented DS neuronal degeneration, whereas the 17-mer reversepeptide, NGF, BDNF, and NT3 did not affect DS neuronal survival. Values represent mean± SEM; n = 4 independent experiments. *p < 0.01 by Student’s t test. (B) A significantincrease in neuronal survival is observed in DS cultures incubated with conditioned mediumof normal astrocyte cultures (c.m./NL APP(+)) but not with conditioned medium of DSastrocyte cultures (c.m./DS) or with conditioned medium of normal astrocytes depleted ofAPP by immunoprecipitation (c.m./NL APP(-)). Neuronal viability determined at day 14 inculture is expressed as percent of the neuronal number at day 7 (100%). Values representmean ± SEM; n = 3 independent experiments. *p < 0.01 by Student’s t test.



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well as in endosomal and lysosomal vesicles. It has previously been shown thatintracellular Aβ42, APP and C-terminal fragments accumulate in a detergent-insol-uble form in APP-overexpressing cells incubated with 20 µM synthetic Aβ1-42.29,30

Our results suggest that a similar phenomenon may occur under physiologicalconditions in DS astrocytes and in normal astrocytes under conditions of impairedenergy metabolism. We have also detected detergent-insoluble APP that cosedimentswith intracellular Aβ in detergent-resistant pellets of DS cell lysates and brainhomogenates (unpublished results). These experiments provide evidence for analternative processing pathway in DS cells in which Aβ42, APP, and potentiallyamyloidogenic fragments accumulate and form detergent-resistant aggregates.16

A neuroprotective function of the secreted ectodomain of APP has been sug-gested by several studies.27,28,31 Our results show that APPs is significantly reducedin DS astrocytes in culture and in the DS brain.16

DS cortical neuronal cultures that exhibit reduced levels of APPs show a dramaticrecovery in neuronal survival after incubation with recombinant APP or the 17-merpeptide corresponding to the neurotrophic domain of APP. DS neuronal survival wasalso significantly increased by incubation with conditioned medium of normal astro-cytes, but not by the same medium depleted of APPs by immunoprecipitation. Incontrast, NGF, BDNF, and NT3 did not improve DS neuronal survival. Takentogether, these results suggest that APPs may be a survival factor for human neuronsand underscore the potential pathological relevance of reduced APPs levels in DSand AD.

In summary, we suggest that chronic APP overexpression may impair mitochon-drial function, which in turn increases intracellular accumulation of Aβ and reducessecretion of neuroprotective APPs. Impaired mitochondrial function may result froma direct toxic effect of Aβ,32 APP accumulation in mitochondria,33 or from themetabolic cost of clearing aggregated proteins through chaperones and the degra-dative apparatus — processes that are highly energy-dependent. Future studiesdirected to analyze in detail the relationship between mitochondrial metabolism andneurodegeneration in DS are warranted.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes of Health (HD38466)and the Alzheimer’s Association to J.B. This chapter was reprinted partially fromJ. Busciglio et al., Altered metabolism of the amyloid beta precursor protein isassociated with mitochondrial dysfunction in Down’s syndrome, Neuron, 33, 677,2002. With permission from Elsevier.

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13. Busciglio, J. et al. Generation of beta-amyloid in the secretory pathway in neuronaland nonneuronal cells, Proc. Natl. Acad. Sci. USA, 90, 2092, 1993.

14. Busciglio, J., Yeh, J., and Yankner, B.A. β-Amyloid neurotoxicity in human corticalculture is not mediated by excitotoxins, J. Neurochem., 61, 1565, 1993.

15. McConlogue, L. et al. Differential effects of a Rab6 mutant on secretory versusamyloidogenic processing of Alzheimer’s beta-amyloid precursor protein, J. Biol.Chem., 271, 1343, 1996.

16. Busciglio, J. et al. Altered metabolism of the amyloid beta precursor protein isassociated with mitochondrial dysfunction in Down’s syndrome, Neuron, 33, 677,2002.

17. Lippa, C.F. et al. Deposition of beta-amyloid subtypes 40 and 42 differentiatesdementia with Lewy bodies from Alzheimer disease, Arch. Neurol., 56, 1111, 1999.

18. Pigino, G. et al. Presenilin-1 mutations reduce cytoskeletal association, deregulateneurite growth, and potentiate neuronal dystrophy and tau phosphorylation, J. Neu-rosci., 21, 834, 2001.

19. Heytler, P.G. Uncoupling of oxidative phosphorylation by carbonyl cyanide phenyl-hydrazones. I. Some characteristics of m-Cl-CCP action on mitochondria and chloro-plasts, Biochemistry, 2, 357, 1963.

20. Reers, M. et al. Mitochondrial membrane potential monitored by JC-1 dye, MethodsEnzymol., 260, 406, 1995.

21. Reers, M., Smith, T.W., and Chen, L.B. J-aggregate formation of a carbocyanine as aquantitative fluorescent indicator of membrane potential, Biochemistry, 30, 4480, 1991.

22. Roch, J.M. et al. Increase of synaptic density and memory retention by a peptiderepresenting the trophic domain of the amyloid beta/A4 protein precursor, Proc. Natl.Acad. Sci. USA, 91, 7450, 1994.

23. Roch, J.M. et al. Bacterial expression, purification, and functional mapping of theamyloid beta/A4 protein precursor, J. Biol. Chem., 267, 2214, 1992.



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24. Wiltfang, J. et al. Improved electrophoretic separation and immunoblotting of beta-amyloid (A beta) peptides 1-40, 1-42, and 1-43, Electrophoresis, 18, 527, 1997.

25. Fries, E. and Rothman, J.E. Transport of vesicular stomatitis virus glycoprotein in acell-free extract, Proc. Natl. Acad. Sci. USA, 77, 3870, 1980.

26. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: applicationto proliferation and cytotoxicity assays, J. Immunol. Methods, 65, 55, 1983.

27. Mattson, M.P. Cellular actions of beta-amyloid precursor protein and its soluble andfibrillogenic derivatives, Physiol. Rev., 77, 1081, 1997.

28. Bowes, M.P. et al. Reduction of neurological damage by a peptide segment of theamyloid beta/A4 protein precursor in a rabbit spinal cord ischemia model, Exp.Neurol., 129, 112, 1994.

29. Yang, A.J. et al. Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Aβ1-42,J. Biol. Chem., 274, 20650, 1999.

30. Yang, A.J. et al. Intracellular A beta 1-42 aggregates stimulate the accumulation ofstable, insoluble amyloidogenic fragments of the amyloid precursor protein in trans-fected cells, J. Biol. Chem., 270, 14786, 1995.

31. Mattson, M.P. et al. Evidence for excitoprotective and intraneuronal calcium-regu-lating roles for secreted forms of the beta-amyloid precursor protein, Neuron, 10,243, 1993.

32. Casley, C.S. et al. Beta-amyloid inhibits integrated mitochondrial respiration and keyenzyme activities, J. Neurochem., 80, 91, 2002.

33. Anandatheerthavarada, H.K. et al. Mitochondrial targeting and a novel transmem-brane arrest of Alzheimer’s amyloid precursor protein impairs mitochondrial functionin neuronal cells, J. Cell Biol., 161, 41, 2003.


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9
Linking Alzheimer’s Disease, β-Amyloid, and Lipids: A Technical Approach
Marcus O.W. Grimm, Andreas J. Paetzold, Heike S. Grimm, Eva G. Zinser, Thomas Ruppert, and Tobias Hartmann

CONTENTS

9.1 Introduction9.2 Cholesterol and Aβ .

9.2.1 Cell Culture and Cholesterol Depletion9.2.2 Protein Analysis9.2.3 Results

9.3 Sphingolipids and AD9.3.1 Cell Culture9.3.2 Enzymatic Assay9.3.3 Lipid Extraction9.3.4 Phosphorus Determination9.3.5 Scintillation Count

9.4 Analysis of Lipids by Mass Spectrometry9.4.1 Theoretical Background9.4.2 Sample Preparation9.4.3 Sample Application9.4.4 Measurement

9.4.4.1 Instrumentation9.4.4.2 TOF pos Measurement .9.4.4.3 Precursor Ion Scans on m/z = 184.1, 188.1, and 264.3

9.4.5 Data Interpretation9.4.6 Key Steps and Modifications

9.4.6.1 Sample Preparation9.4.6.2 Sample Application

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press



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9.4.6.3 TOF pos Measurement9.4.6.4 Precursor Ion Scan

9.4.7 ResultsAcknowledgmentReferences

9.1 INTRODUCTION

Until recently, four genes harboring point mutations that significantly affect Alzhe-imer’s disease (AD) pathogenesis have been identified.1 All these mutations resultin increased β-Amyloid 42 (Aβ42) levels.2 Three of these genes, the amyloid pre-cursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2), are involved in themolecular pathway of AD. The fourth gene, apolipoprotein E (ApoE), suggests apossible link to lipid pathways. ApoE encodes a lipid-binding protein that transportslipids between cells and is therefore an important factor in lipid homeostasis.3 HumanApoE exists in three major alleles. In epidemiological studies, the ε4 allele increasesthe risk for hypercholesterolemia and decreases the age of onset of AD.4 Moreover,ApoE transgenic and knockout mice show altered Aβ deposition indicating thatApoE and lipids may play a significant role in Aβ pathology.5

Alterations in lipid homeostasis have long been recognized to severely affectneuronal function and cause neurodegenerative diseases.6 Table 9.1 presents a sum-mary of the most common lipid-related diseases.

Enzymes of the sphingolipid and ganglioside pathways seem to be involved inneurodegenerative disorders. e.g., in Niemann-Pick disease, the sphingomyelin levelis drastically increased by a lack of the sphingomyelin-degrading enzyme, the acidsphingomyelinase (Figure 9.1). However, it should be noted that these diseases areoften fatal during childhood or early adulthood and therefore little is known abouttheir relevance to AD.

APP and all APP secretases are transmembrane proteins. Therefore it is con-ceivable that lipids may also play a fundamental role in Alzheimer’s disease. It hasrecently been shown that intramembrane proteolysis of APP is influenced by thephysical characteristics of the membrane. For example the γ-cleavage seems to takeplace in the middle of the transmembrane domain of APP.7 Aβ was still generatedwhen residues within the transmembrane domain were mutated,8 whereas the cleav-age site shifted by an altered length of the transmembrane domain, which led to achanged ratio of Aβ42 to Aβ40. C terminal insertion of two residues and N-terminaldeletion of two residues strongly altered the ratio.

As the length of the transmembrane domain corresponds to the membranediameter, it is obvious that a changed membrane thickness may produce drasticconsequences for the Aβ42/Aβ40 ratio.8 Accordingly, a membrane that has a smallerdiameter should shift toward Aβ42 production, which is in fact the case for compar-isons of plasma and endoplasmic reticulum (ER) membranes. In summary, this modelsuggests that the ratio of pathogenic Aβ42 and nonpathogenic Aβ40 is modulated bymembrane composition.



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9.2 CHOLESTEROL AND Aββββ

Animals fed a cholesterol-enriched diet showed increased accumulation of Aβ intheir brains.9 Similarly, reduced cellular cholesterol levels resulted in decreased Aβproduction.10 Primary neurons treated with cholesterol lowering drugs, e.g., statins,produced significantly reduced Aβ levels.11 In order to evaluate whether unspecificside effects of the statins or indeed cholesterol depletion caused reduced Aβ production,cholesterol plasma membrane levels were decreased with methyl-β-cyclodextrin(CDX).12 While statins reduced cholesterol levels by inhibition of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, CDX physically extracted cho-lesterol from the plasma membranes.

These mechanistically unrelated approaches led to similarly decreased Aβ levels,indicating that cholesterol levels and not a side effect of the statins are responsible foraltered Aβ production.13 Treatment of guinea pigs with a high dosage of Simvastatininduced a reversible decrease in cerebrospinal fluid and brain tissue Aβ levels.14

Similar results were found using the cholesterol synthesis inhibitor BM15.766 whichinhibits ∆7-reductase, a later step in cholesterol synthesis15,16 (Figure 9.2).

TABLE 9.1Lipid-Related Neuronal Disorders and Affected Lipids and Enzymes

Disease Affected Lipids Enzymatic Defects

Faber disease Ceramide Acid ceramidase Niemann-Pick disease Sphingomyelin Sphingomyelinase Krabbe disease Galactosylceramide,

Galactosylsphingosine Galactosylceramidase

Gaucher disease Glucosylceramide, Glucosylsphingosine

Glucosylceramidase

Fabry disease Digalactosylceramide α-Galactosidase A Tay-Sachs disease GM-ganglioside β-Hexosaminidase A Sandhoff disease GM-ganglioside β-Hexosaminidases A and B Metachoromatic leukodystrophy Sulfatide Arylsulfatase A (sulfatidase) Multiple sulfatase deficiency Sulfatide Arylsulfatases A, B, and C Sulfatidase activator deficiency (sap-B deficiency)

Sulfatide, globotriaosylceramide, digalactosylceramide, GM3-ganglioside

Sulfatidase activator (SAP-1, sap-B)

SAP-2 deficiency Glucosylceramide SAP-2 (sap-C) SAP precursor deficiency All glycolipids with short

sugarchains, e.g., Cer, GlcCer, LacCer, GalCer, DigalCer, Sulfatide

SAP precursor, sap-A, B, C, and D

GM1-gangliosidosis GM1-ganglioside GM1-ganglioside, β-Galactosidase

GM2-gangliosidosis (B1 variant) GM2-ganglioside β-Hexosaminidase A GM2-gangliosidosis (AB variant) GM2-ganglioside β-Hexosaminidase A


FIGURE 9.1

compounds appear in large type; enzyme names appear in smallertype.

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Biochemical pathway of sphingomyelin synthesis. The names of the

Group.

FIG

pear in bold type; enzyme names appear in normal type.Inh

s one of the final steps of the pathway.

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URE 9.2 Biochemical pathway of cholesterol synthesis. The names of the compounds apibitors are noted at places of action. BM15.766 is blocking the ∆7-reductase that catalyze

r & Francis Group.

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The following sections will describe the experimental approaches to loweringcholesterol levels in cell cultures.

9.2.1 CELL CULTURE AND CHOLESTEROL DEPLETION

As mentioned above, cholesterol level can be decreased by inhibition of HMG-CoAreductase as a committed step enzyme in cholesterol biosynthesis or by physicaldepletion of plasma membrane cholesterol. To maximize the effect on Aβ production,these approaches can be used in combination.

Hippocampal neurons were prepared from 17-day-old fetal rats.17 The neuronswere cultivated for 5 days at 90% humidity and 5% CO2 atmosphere at 37˚C inN2MEM.

Subsequently 4 µM lovastatin (Calbiochem, La Jolla, CA) or simvastatin (Merck,Whitehouse Station, NJ) and 0.25 mM mevalonate (Sigma Chemical, St. Louis, MO)were added for 48 or 72 hr. By supplementing the medium with 5 mM mevalonate,biochemical pathways for nonsteroidal products were allowed to proceed. Withoutthis supplementation cellular toxicity increased.14,18

The neurons were infected with SFV-APP695 for 60 to 90 min, depending onthe virus titer. Exposition times longer than 90 min are not recommended becauseof increasing cytotoxicity. After washing the cells with N2MEM and incubation for2 hr, the cells were treated with 5 mM CDX (Sigma) for 10 min. With longer exposuretimes or higher concentrations of CDX, Aβ levels dropped below the detection limit.After 3.5 to 4.5 hr inside the nontoxic window of SFV infection, cells were harvestedand Aβ levels were analyzed. As a control, cells were treated in the same way inthe absence of CDX and statins. To analyze the effects of statins without cyclodex-trin, cyclodextrin treatment was omitted as a variation of the experiment. Moreover,to avoid analyzing effects limited to hippocampal neurons, mixed cortical neuronsfrom brain were used.

9.2.2 PROTEIN ANALYSIS

Cell culture media were collected and cell extracts were prepared in 2% (v/v) NonidetP-40, 0.2% (w/v) SDS, and 5 mM EDTA supplemented with complete proteaseinhibitor (Roche, Basel, Switzerland). In order to detect equal intensities of allanalyzed Aβ-forms, cell lysates were divided differently for Aβ42 (79%) and Aβ40

(19%). The remaining 2% was saved for direct loading of cell lysates for detectionof APP and total Aβ. The conditioned media were split 90% for Aβ42 and 10% forAβ40 production. Adjusting Aβ levels to equal ranges of band intensities for Aβ40

and Aβ42 allows a more precise determination of Aβ ratio because densiometricanalysis of the Western blot can be performed from individual blots and expositiontimes.

For immunoprecipitation, monoclonal antibodies G2-10 (2 µg/ml) for Aβ40 andG2-11 (4 µg/ml) for Aβ42 were used. The monoclonal W0-2 antibody directed againstamino acids 4 through 10 of human Aβ was used for total Aβ and APP detection.Quantitative immunoprecipitation was performed according to Schröder et al.19

Precipitates were analyzed on 10 to 20% Tris-Tricine polyacrylamide gels (Novex,


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San Diego, CA). Quantitative Western blotting was performed modified to Ida et al.29

For blocking 3% (w/v) milk powder instead of BSA was used. G2-10 and G2-11precipitates were detected with W0-2 antibody. Quantification was done afterenhanced chemoluminescence (ECL) development by densiometric scans.

9.2.3 RESULTS

De novo synthesis of cholesterol was blocked by both lovastatin and simvastatin.Additionally, plasma membrane cholesterol was extracted by CDX. In the condi-tioned media, cholesterol depletion by lovastin and CDX decreased Aβ40 levels to50% (±13%). Aβ42 levels were decreased to 29% (±10%) compared to untreatedcells. Similar results were obtained for intracellular Aβ levels. Aβ40 levels weredecreased to 42% (±18%) and intracellular Aβ42 levels dropped to 33% (±15%)compared to untreated cells. A more detailed description of the results can be foundin Fassbender et al.14

9.3 SPHINGOLIPIDS AND AD

As described above, sphingolipids and gangliosides play important roles in manyneurodegenerative disorders. Sphingolipids and cholesterol play crucial roles in lipidraft biology and modulate the physical states of membranes.20,21 Moreover, it hasbeen recently found that sphingolipid levels and composition are altered in ADbrains, indicating a role for this lipid class in AD pathology.22

The following section describes experiments analyzing the committed step reac-tion of the sphingolipid pathway catalyzed by serine–palmitoyl–transferase (SPT).This enzymatic reaction catalyzes the condensation of serine and palmitoyl-CoA, areaction that produces 3-ketodihydrosphingosine (KDS). SPT belongs to a familyof pyridoxal 5′-phosphate (PLP)-dependent α-oxoamine synthases (POAS). Mam-malian SPT is a heterodimer of 53-kDa LCB1 and 63-kDa LCB2 subunits localizedat the ER.

The enzymatic assay is based on radioactive incorporation of 14C-serine. While14C-serine is water soluble, the originated products of SPT and the enzymes of thesphingolipid pathway are liposoluble. As a control, the same approach is performedwith 1 mM myriocin. Myriocin is a selective inhibitor of the SPT. Under theseconditions no activity should occur.

9.3.1 CELL CULTURE

Cells were cultured in DMEM containing 5 to 10% fetal calf serum (FCS) at 37˚C,90% humidity, and 5% CO2 atmosphere. Cells were grown until a 90% confluentcell layer appeared.

9.3.2 ENZYMATIC ASSAY

Cells were washed three times with Buffer 1 containing 100 mM HEPES, pH 7.3.After harvesting, the cells were homogenized in 100 mM HEPES, pH 7.3, supple-mented with complete protease inhibitor (Roche). Homogenization and the following


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steps were performed at 4˚C in order to decrease protein degradation. The homoge-nates were adjusted to equal protein levels. For each assay, a protein concentrationof 5 to 7 mg/ml in a final volume of 3000 µl is recommended. Lower proteinconcentrations result in enzymatic activity that is too low for proper analysis. Thereaction is started by adding 300 µl of Buffer 2 containing 500 µM pyridoxalphos-phate (Sigma), 100 mM HEPES, pH 7.3, 3 mM palmitoyl-CoA, and 10 µCi/mlL-serine (Amersham Biosciences, Uppsala, Sweden) at 37˚C in glass tubes (Wheaton,Millville, NJ).

To stop the reaction 400 µl of the reaction mixture is transferred in glass tubescontaining 3.75 ml freshly prepared solvent CHCl3:MeOH:HCl (5:10:0.075). Thereaction is stopped after 0, 2, 4, 8, 16, 32, 64, and 96 min. It is very important touse freshly prepared solvent because the very low vapor pressure of chloroformleads to a high rate of evaporation of chloroform and changed ratios of the differentsolvents. Glass tubes instead of plastic ones must be used because lipids adhere toplastic surfaces.

9.3.3 LIPID EXTRACTION

The lipid extraction was based on the method of Bligh and Dyer23 with somemodifications that are briefly described. All steps were performed at room temper-ature. The samples were vortexed in the glass tubes for 60 min. After 1 ml CHCl3

was added, the samples were vortexed again for 10 min. Phase separation occurredby the addition of 1 ml distilled H2O. The resulting hydrophilic and lipophilic phaseswere thoroughly mixed by vortexing for 30 min, after which the phases wereseparated by centrifugation for 10 min at 3000 × g. The lower lipophilic phase wastransferred to a new glass tube. The transfer of the lower lipophilic phase must beperformed quantitatively without contaminating the organic phase with the inter-phase or hydrophilic phase. The interphase and the hydrophilic phase contain nucleicacids, carbohydrates, and proteins, whereas the lipophilic phase contains lipids,especially sphingolipids and phospholipids. The procedure is carried out two moretimes starting again with the addition of CHCl3:MeOH:HCl (5:10:0.075).

After the last extraction, the lipophilic phase containing the extracted lipids, isevaporated under continuous nitrogen flow. The samples were resolved again in200 µl CHCl3:MeOH:HCl (5:10:0.075) by vortexing for 60 min; 50 µl are neededfor phosphorus determination and the remaining 150 are applied for scintillationcount.

9.3.4 PHOSPHORUS DETERMINATION

It should be noted that phosphate-based detergents must not be used for glasswarewhen this method is employed. All glassware should be washed with distilled H2Oimmediately before use. As noted earlier, the lipophilic phase contains most of thelipids and especially phospholipids. The phospholipid concentrations of the differentsamples should not differ after extraction.


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Analyzing phosphorus concentration that directly corresponds to phospholipidconcentration reveals the quality of the extraction and points out errors due toextraction. In order to quantify the inorganic phosphorus concentration, the phospho-lipids were digested by refluxing in perchloric acid to release inorganic phosphate.The inorganic phosphate was then converted to phosphomolybdic acid which wasreduced to a blue compound for spectrometric determination.24

To prepare the chromogenic solution, 16 g ammonium molybdate is dissolvedin 120 ml water (Reagent A), after which 40 ml of hydrochloric acid and 10 mlmercury are added to 80 ml of Reagent A and thoroughly mixed. The supernatantis used as Reagent B; 40 ml of Reagent A is combined with 200 ml concentratedsulfuric acid and added to Reagent B to produce Reagent C. Subsequently, 25 mlof Reagent C is added to 45 ml methanol, 5 ml chloroform, and 20 ml water. Thischromogenic solution can be stored at 4˚C for several weeks.

The next step is adding 13 µl chromogenic solution to 50 µl sample and thesolution is heated at 100˚C for 75 sec. After the samples are thoroughly mixed andcooled for 5 min to room temperature, 500 ml nonane is added. The samples arebriefly mixed and incubated for 15 min. The tubes are centrifuged for 3 min at3000 × g before the absorbance of the supernatant at 730 nm is compared with theblank where 50 µl CHCl3:MeOH:HCl (5:10:0.075) is used instead of the sample.Very high amounts of cholesterol influence the phosphorus determination.

9.3.5 SCINTILLATION COUNT

The remaining 150 µl of the sample is used for scintillation counting. First, 2.5 mlof scintillation mix is added and thoroughly mixed for 1 hr, after which its radio-activity is determined in a scintillation counter (Beckmann LS 6000IC).

Figure 9.3 shows serine–palmitoyl–transferase (SPT) activity in murine fibro-blast cells.

FIGURE 9.3 Serine-palmitoyl-transferase (SPT) activity in murine fibroblast cells.


© 2005 by T

9.4 ANALYSIS OF LIPIDS BY MASS SPECTROMETRY

Alzheimer’s disease is linked to lipid homeostasis. Therefore this is a reliable methodto identify and quantify sphingomyelin and ceramide. In recent years, mass spec-trometry has become a well-established biochemical method, particularly after thegenomic research led to the use of mass spectrometry to identify different proteins.The subsequent sections will focus on the usage of mass spectrometry in lipid analysis.

9.4.1 THEORETICAL BACKGROUND

Electrospray ionization mass spectrometry (ESI-MS) is a very efficient tool for theanalysis of lipid extracts.25–28 Lipids dissolved in an appropriate solvent are introducedthrough a small-diameter needle to which a high voltage is applied. At the needletip, the surface of the solution becomes highly charged and droplets are formed toincrease the surface. The droplets shrink due to evaporation of solvent whichincreases the charge density at the surface. When the surface charge density exceedsthe Rayleigh stability limit, electrostatic repulsion greater than the surface tensionof the droplet results in an explosion of the droplet and expulsion of gas phase ions.These ions are positively or negatively charged, depending on the polarity of theapplied voltage.

In the following method, lipids are characterized by a quadrupole time-of-flightmass analyzer (Q-TOF) consisting of a quadrupole that serves as a mass filter, acollision cell for collision-induced fragmentation and a TOF analyzer. Mass deter-mination of positive charged lipid ions is performed using only the TOF analyzer(TOF pos). Ions are accelerated in an electric field and the time is determined untilthe ions reach the detector. From the time of flight, the mass-to-charge ratio (m/zvalue) of the ions is calculated.

Structural information of a particular lipid ion is obtained by selecting the ionsusing the first quadrupole. Only these ions enter the collision cell. After collisionwith nitrogen molecules, the generated fragment masses are determined by the TOFanalyzer (product ion scan). Using the first quadrupole in a scanning mode, ions ofa complex mixture are allowed to enter the collision cell sequentially, depending ontheir mass. After fragmentation, the TOF analyzer is used to detect the appearanceof a characteristic fragment and the masses of components are calculated from whichthis fragment is derived (precursor ion scan or PIS).

Fragmentation of sphingomyelins yields a fragment of m/z = 184.1 that ischaracteristic for the phosphorylcholine moiety (see Figure 9.4 and References 25and 26). This fragment is not characteristic for sphingomyelins because it also resultsfrom fragmentation of any representative of phosphatidylcholines (PCs). However,although monoprotonated sphingomyelins have odd nominal m/z values, phosphati-dylcholines have nominal m/z values because of a formal exchange of an amidegroup (15 Da) for an oxygen atom (16 Da). Furthermore, fragmentation of sphingo-myelin and of ceramides leads to a fragment of m/z = 264.3 (see Figure 9.4),representing the di-dehydro-sphingenine body. This is characteristic for ceramidesand is built up by the fusion of serine and palmitoyl-CoA in the sphingolipidbiosynthesis pathway (compare Figure 9.1). By comparison of a simultaneous PIS


orylcholine (sphingomyelin 16:0) in the mass rangeled ×10,0 above the spectrum. Each peak is labeledmentation of sphingomyelin 16:0.

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FIGURE 9.4 (A) TOF spectrum of a positive mode fragmentation of N-palmitoyl-D-erythro-sphingosyl-phosphfrom m/z = 70 to 720. The mass range which is extended 10 times (does not include the peak at 184.07) is labewith its centroid mass. (B) Compounds with corresponding masses including their paths of formation during frag

by Taylor & Francis Group.

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on m/z = 184.1 and 264.3 in the presence of an external standard, it is possible todetermine and quantify sphingomyelins and ceramides.

9.4.2 SAMPLE PREPARATION

The procedure of sample preparation is the same as described above. However, therecommendation is to use 25 to 50% of the cells as in the case of thin layerchromatography. For mass spectrometry, the solvent should be CHCl3:MeOH:HCl(5:10:0.075). If necessary evaporate a different solvent under nitrogen atmosphereand resolve the sample in CHCl3:MeOH:HCl (5:10:0.075).

The addition of approximately 1.8 pmol 1,2-dimyristoyl-sn-glycero-3-phospho-choline-1,1,2,2-D4 (Avanti Polar Lipids, Inc., Alabaster, AL) as an external standardwill produce a reference peak of m/z = 682.5 in TOF pos spectra.

9.4.3 SAMPLE APPLICATION

1. Rinse a shortened nano-ESI needle (Econo12, New Objective, Woburn,MA) with approx. 5 µl of sample.

2. Subsequently fill the needle with 5 to 10 µl of sample.3. Feed it into the holder of the ESI source.4. The needle tip should be placed about 1 mm from the MS orifice.

9.4.4 MEASUREMENT

9.4.4.1 Instrumentation

Spectrometric analysis was performed on a Q-TOF Pulsar mass spectrometer (PESIEX, Weiterstadt, Germany) equipped with a nano-ESI source (MDS Protana,Odense, Denmark).

9.4.4.2 TOF pos Measurement

1. Charge nano-ESI needle with a potential of 700 V.2. Set declustering potential to 40 eV.3. Set TOF pos range to m/z = 600 to 900.4. While monitoring the spectrum, note whether total ion count (TIC) cor-

relates with extracted ion count (XIC) of reference peak (m/z = 682.5)and at least one peak representing sphingomyelin, e.g., m/z = 703.3 or731.3.

5. Maintain a stable spectrum for at least 2 min.

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

1. Set TOF range to m/z = 170 to 270.2. Enable peak enhancement for m/z = 264.3.3. Set collision energy to 60 eV.4. Run PIS in ranges of m/z = 500 to 690 and 670 to 820, respectively.



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9.4.5 DATA INTERPRETATION

TOF pos spectra were calculated by Analyst QS software (Applied Biosystems,Foster City, CA) by averaging a 2-min period of the profile. Precursor ion scanswere calculated in the same way for at least 5 min. Further interpretations weremade after exporting Analyst QS data into MS Access. A database was programmedto pair same peaks from different spectra with a ∆m/z of 0.05 and normalizeintensities to the reference peak of the added external standard m/z = 682.5 or 188.1,respectively.

9.4.6 KEY STEPS AND MODIFICATIONS

9.4.6.1 Sample Preparation

The reduced number of cells is recommended to reduce the possibility of formationof precipitates on the tip of nano-ESI needle. Our dilution experiments indicate thatthese precipitates are not based on high concentrations of lipids or salts.

9.4.6.2 Sample Application

Rinsing of the needle is recommended to achieve a stable spray in a shorter time.

9.4.6.3 TOF pos Measurement

It is essential for quantification that the spray is stable, i.e., the spectrum does notchange in the course of testing. Additionally, the precursor ion scan needs a reason-able signal input.

9.4.6.4 Precursor Ion Scan

The characteristic fragment ions of phosphorylcholines, m/z = 184.1, and ceramide,m/z = 264.3, were selected for discrimination of phosphatidylcholines from ceram-ides and/or sphingomyelins.

9.4.7 RESULTS

Fragmentation of the external standard mentioned above leads to a peak at m/z =188.1 instead of m/z = 184.1. A precursor ion scan on m/z = 188.1 results in amaternal peak of m/z = 682.5 and its first and second isotope peaks (Figure 9.5).This clear-cut result shows the suitability of the external standard and the conditions.

ACKNOWLEDGMENTS

We would like to thank Inge Tomic for technical support and Marco Duering forhelpful advice.


oach to analyzing a lipid extract derived from a celling the stability of the spray. (B) PIS of the fragment 188.1) of standard (1,2-dimyristoyl-sn-glycero-3-

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FIGURE 9.5 Partial screen shot from Analyst QS software. It is an example of a precursor ion scan (PIS) apprhomogenate of a retransfected preseniline double knockout cell line. (A) Total ion counts per time (TIC) representm/z = 184.1 (phosphatidylcholine headgroup). (C) Result of the simultaneous PIS of the head group (m/z =phosphocholine-1,1,2,2-D4). (D) Peaks generated by PIS of di-dehydro-sphingenine (m/z = 264.3).

by Taylor & Francis Group.


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REFERENCES

1. De Strooper, B. and Annaert, W. Proteolytic processing and cell biological functionsof the amyloid precursor protein. J. Cell Sci. 113, 1857–1870, 2000.

2. Sinha, S. and Lieberburg, I. Cellular mechanisms of beta-amyloid production andsecretion. Proc. Natl. Acad. Sci. USA 96, 11049–11053, 1999.

3. Mahley, R.W. and Rall, S.C., Jr. Apolipoprotein E: far more than a lipid transportprotein. Annu. Rev. Genomics Hum. Genet. 1, 507–337, 2000.

4. Corder, E.H. et al. Gene dose of apolipoprotein E type 4 allele and the risk ofAlzheimer’s disease in late onset families. Science 261, 921–923, 1993.

5. Bales, K.R. et al. Lack of apolipoprotein E dramatically reduces amyloid beta-peptidedeposition. Nat. Genet. 17, 263–264, 1997.

6. Nathan, B.P. et al. Differential effects of apolipoproteins E3 and E4 on neuronalgrowth in vitro. Science 264, 850–852, 1994.

7. Grziwa, B. et al. The transmembrane domain of the amyloid precursor protein inmicrosomal membranes is on both sides shorter than predicted. J. Biol. Chem. 278,6803–6308, 2003.

8. Lichtenthaler, S.F. et al. The intramembrane cleavage site of the amyloid precursorprotein depends on the length of its transmembrane domain. Proc. Natl. Acad. Sci.USA 99, 1365–1370, 2002.

9. Sparks, D.L. et al. Induction of Alzheimer-like beta-amyloid immunoreactivity in thebrains of rabbits with dietary cholesterol. Exp. Neurol. 126, 88–94, 1994.

10. Simons, M. et al. Cholesterol depletion inhibits the generation of beta-amyloid inhippocampal neurons. Proc. Natl. Acad. Sci. USA 95, 6460–6464, 1998.

11. Cucchiara, B. and Kasner, S.E. Use of statins in CNS disorders. J. Neurol. Sci. 187,81–89, 2001.

12. Kilsdonk, E.P. et al. Cellular cholesterol efflux mediated by cyclodextrins. J. Biol.Chem. 270, 17250–17256, 1995.

13. Frears, E.R. et al. The role of cholesterol in the biosynthesis of beta-amyloid. Neuro-report 10, 1699–1705, 1999.

14. Fassbender, K. et al. Simvastatin strongly reduces levels of Alzheimer’s disease beta-amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proc. Natl. Acad. Sci.USA 98, 5856–5961, 2001.

15. Refolo, L.M. et al. Hypercholesterolemia accelerates the Alzheimer’s amyloid pathol-ogy in a transgenic mouse model. Neurobiol. Dis. 7, 321–3331, 2000.

16. Refolo, L.M. et al. A cholesterol-lowering drug reduces beta-amyloid pathology ina transgenic mouse model of Alzheimer’s disease. Neurobiol. Dis. 8, 890–899, 2001.

17. Tienari, P.J. et al. Intracellular and secreted Alzheimer beta-amyloid species aregenerated by distinct mechanisms in cultured hippocampal neurons. Proc. Natl. Acad.Sci. USA 94, 4125–4130, 1997.

18. Fassbender, K. et al. Effects of statins on human cerebral cholesterol metabolism andsecretion of Alzheimer amyloid peptide. Neurology 59, 1257–1258, 2002.

19. Schroder, J. et al. Cerebral changes and cerebrospinal fluid beta-amyloid in Alz-heimer’s disease: a study with quantitative magnetic resonance imaging. Mol. Psy-chiatr. 2, 505–507, 1997.

20. Roy, D. and Mukhopadhyay, C. GD1a in phospholipid bilayer: a molecular dynamicssimulation. J. Biomol. Struct. Dyn. 18, 639–646, 2001.

21. Kappel, T. et al. Gangliosides affect membrane-channel activities dependent on ambi-ent temperature. Cell. Mol. Neurobiol. 20, 579–590, 2000.


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22. Cutler, R.G. et al. Involvement of oxidative stress-induced abnormalities in ceramideand cholesterol metabolism in brain aging and Alzheimer’s disease. Proc. Natl. Acad.Sci. USA 101, 2070–2075, 2004.

23. Bligh, E.G and Dyer, W.J. A rapid method of total lipid extraction and purification.Can. J. Med. Sci. 37, 911–917, 1959.

24. Hundrieser, K.E., Clark, R.M., and Jensen, R.G. Total phospholipid analysis in humanmilk without acid digestion. Am. J. Clin. Nutr. 41, 988–993, 1985.

25. Brugger, B. et al. Quantitative analysis of biological membrane lipids at the lowpicomole level by nano-electrospray ionization tandem mass spectrometry. Proc. Natl.Acad. Sci. USA 94, 2339–2344, 1997.

26. Ekroos, K. et al. Charting molecular composition of phosphatidylcholines by fattyacid scanning and ion trap MS3 fragmentation. J. Lipid Res. 44, 2181–2192, 2003.

27. Han, X. and Gross, R.W. Electrospray ionization mass spectroscopic analysis ofhuman erythrocyte plasma membrane phospholipids. Proc. Natl. Acad. Sci. USA 91,10635–10639, 1994.

28. Kerwin, J.L., Tuininga, A.R., and Ericsson, L.H. Identification of molecular speciesof glycerophospholipids and sphingomyelin using electrospray mass spectrometry.J. Lipid Res. 35, 1102–1114, 1994.

29. Ida, N. et al. Analysis of heterogeneous A4 peptides in human cerebrospinal fluidand blood by a newly developed sensitive Western blot assay. J. Biol. Chem. 271,22908–22914, 1996.



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10
Regulation of Amyloid Precursor Protein Processing by Lithium
Xiaoyan Sun and Akihiko Takashima

CONTENTS

Abstract10.1 Introduction10.2 Experimental Procedures

10.2.1 Lithium Treatment in Transfected Cultured Cells10.2.2 Aβ ELISA10.2.3 Preparation of fibrillar Aβ (fAβ)10.2.4 Microinjection of fAβ into Tau Transgenic Mice10.2.5 Lithium Administration

10.3 Results10.3.1 Establishment of Selective Sandwich Aβ ELISA10.3.2 Effect of LiCl on Aβ Secretion in Association with GSK-3β

Activity from APP C100-Transfected COS7 Cells10.3.3 Effect of GSK-3β on fAβ-Induced Tau Pathology In Vivo

10.4 DiscussionReferences

ABSTRACT

Secreted Aβ is produced during normal cell metabolism and it is regulated by manyfactors. Quantification of Aβ generation is an important approach to studying APPprocessing. In this chapter, we demonstrate that lithium, a potent GSK-3 inhibitor,can regulate amyloid secretion in COS7 cells overexpressing APPC100 demonstratedby a sandwich enzyme-linked immunosorbent assay (ELISA). Finally, we show thatlithium can effectively inhibit tau pathology induced by Aβ injection into the hippo-campus of a transgenic mouse model.

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10.1 INTRODUCTION

Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β peptide (Aβ)and accumulation of hyperphosphorylated tau protein in patients’ brains. Accumulatingevidence indicates that several kinases play an important role in the development ofAD pathology. Among these kinases, GSK-3β is reported to affect amyloid precursorprotein (APP) processing and tau pathology. In 1988, GSK-3β was found to phospho-rylate PHF-like epitopes on tau.1 Later studies demonstrated that GSK-3β is a leadingcandidate kinase responsible for tau hyperphosphorylation in AD. In addition to itsroles in tau phosphorylation, GSK-3β is also reported to be involved in APP processing.

Aplin et al. showed that GSK-3β is capable of phosphorylating the cytoplasmicdomain of APP at Thr743 in vitro. Coexpression of GSK-3β and truncated APPinduces an increase in maturation of APP,2 indicating that GSK-3β may regulateAPP processing via a phosphorylation mechanism. The relationship between APPprocessing and GSK-3β is further explained by several recent studies. Morfini et al.reported that GSK-3β colocalizes with membrane-bound organelles and phospho-rylates kinesin light chain (KLC) C termini.3 These results indicate that GSK-3βmay be directly involved in APP function in neurons because APP is known to bindthe C termini of KLC and is subjected to fast anterograde axonal transport.4,5 In1998, our group first observed that presenilin 1 binds GSK-3β, indicating that GSK-3β may regulate amyloid generation of APP processing.6

To study the role of GSK-3β in amyloid generation, we used lithium, a potentGSK-3β inhibitor, to inhibit the activity of GSK-3β, and examined amyloid secretionfrom cells overexpressing APPC100. Furthermore, we injected fibrillar Aβ into thebrains of transgenic mice in the presence or absence of lithium in vivo. We observedthat lithium could reduce Aβ secretion in the cells and attenuate the degree of Aβ-induced tau pathology in mice.


10.2.1 LITHIUM TREATMENT IN TRANSFECTED CULTURED CELLS

A cDNA (APP C100) encoding 99 amino acids from the C-terminal end of humanAPP was cloned into the expression vector pCIneo with the cytomegalovirus pro-moter. For the transient expression of APP C100, COS7 cells were grown andmaintained in D-MEM with 10% fetal calf serum (FCS).

1. COS7 cells are seeded into 12 wells with 80 to 90% confluence 1 daybefore transfection. On the following day, Lipofectamine 2000 (Gibco,now Invitrogen, Carlsbad, CA) is used to perform transfection accordingto the manufacturer’s instructions.

2. Lithium chloride (LiCl) at a concentration of 1 M is freshly prepared usingserum-free medium each time. Final concentrations of 5, 10, and 20 mMLiCl are used in the experiment.

3. Twenty-four hours after transfection, cells are conditioned in serum-freemedia for 12 or 24 hr in the presence or absence of LiCl. The media arecollected for subsequent Aβ measurements.

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10.2.2 Aββββ ELISA

Sandwich Aβ ELISAs were developed to quantitate Aβx-40 and Aβx-42 as reportedpreviously.7 The end-specific monoclonal antibodies, anti-Aβx-40 (MBC40) andanti-Aβx-42 (MBC42), were used as the capture antibodies. Biotinylated monoclonalanti-N-terminal Aβ secondary antibodies 2H8 and 6E10 were used as the detectionantibodies. Streptavidin-conjugated alkaline phosphatase and AttoPhos (AmershamBiosciences, Uppsala, Sweden) were used as the reporter systems. AttoPhos fluo-rescence was obtained with 444-nm excitation (emission at 555 nm).

1. NUNC Maxisorb immunoassay plates (Nalge NUNC International, Den-mark) are coated with 0.3 µg/well (100 µl) capture antibodies in filteredPBS overnight at 4ºC and sealed with sealer.

2. On the next day, plates are blocked with 300 µl/well Block ACE (Dain-ippon Pharmaceutical, Osaka, Japan, #UK-B80, 1:4 dilution of originalsolution with deionized water) for 2 hr at room temperature. Wash theplates with PBS-T (PBS with 0.05% Tween-20) once. Load the samplesin the wells (100 µl/well).

3. Incubate samples with coated antibodies overnight at 4ºC. Wash with PBS-Ttwice.

4. The plates are then incubated in a solution of the detector antibody for2 hr at 4ºC.

5. Wash the plates with PBS-T twice followed by treating the plates withalkaline phosphatase for 1.5 hr (streptavidin-conjugated alkaline phos-phatase, Amersham, 1:5000) at 4oC.

6. Wash plates with TBS-T twice. The signal is amplified by adding 100 µlAttoPhos (freshly prepared) and measured with a Fluoroskan (Thermo Lab-systems, Vantaa, Finland). Several scans are needed to achieve the best signal.

7. To construct standard curves, Aβ1-40 and Aβ1-42 peptides (AnaSpec, SanJose, CA) are dissolved in dimethyl sulfoxide (DMSO, 1 mg/ml). Furtherserial dilutions are performed by using Block Ace (1:10 dilution of originalsolution with 0.05% Tween-20) or 3% BSA with 0.05% Tween-20.

10.2.3 PREPARATION OF FIBRILLAR Aββββ (FAββββ)

1. Aβ1-40 peptide (U.S. Peptide, Rancho Cucamonga, CA) is dissolved in 100%1,1,1,3,3,3-hexafluoro-2-propanol buffer to a final concentration of 1 mg/ml.

2. Just prior to performing the experiment, aliquots of Aβ are evaporated,redissolved in HEPES buffer (Sigma), and the resulting 5 µM Aβ-40solution is incubated for 6 hr at 37˚C.

3. Aβ aggregation and toxicity are assessed with Western blotting and ThT,MTT, and histochemical staining as described previously.8

10.2.4 MICROINJECTION OF FAββββ INTO TAU TRANSGENIC MICE

1. Three-month-old V337M and wild-type (WT) tau transgenic (Tg) mice andnon-Tg littermates are anesthetized with 25 mg/kg pentobarbital (AbbottLaboratories, Chicago, IL) and positioned in a stereotaxic apparatus.

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2. An injection needle of a Hamilton syringe is lowered into area CA1 ofboth hippocampi (coordinates: –1.9 mm from bregma, ±1.0 mm frommidline, –1.9 mm dorsoventral).

3. Next, 1 µl of fAβ, sAβ, or polyglycine is injected with a 10-µl Hamiltonsyringe driven by a mini-pump (Motorized Stereotaxic Injector, Stoelting,Wood Dale, IL) at a rate of 0.05 µl/min. The needle is kept at the injectionsite for 20 min and then slowly withdrawn.

4. Intrahippocampal circuitry involved in the fAβ injection and the retro-grade labeled cells is assessed by stereotaxic injection of 50 nl of fluoro-gold dextran, a neuronal tracer (Fluoro-Chrome, Englewood, CO) intoCA1 as previously described.9 All microinjection operations are carriedout on a clean, specialized bench; none of the mice in our tests developedinfections.

10.2.5 LITHIUM ADMINISTRATION

1, fAβ-injected V337M Tg mice receive daily intraperitoneal injections(400 µl) of 0.3 M LiCl for 14 or 28 days. Control fAβ-injected V337MTg mice receive a single 400-µl injection of 0.3 M NaCl.

2. LiCl injections begin 1 day before fAβ injection into area CA1 of thehippocampus, and continue until mice are euthanized.

3. On the final day of treatment, the animals are killed immediately after thebeginning of the 12-hr light cycle and analyses are carried out.

10.3 RESULTS

10.3.1 ESTABLISHMENT OF SELECTIVE SANDWICH Aββββ ELISA

A selective sandwich Aβ ELISA was developed to assay secreted Aβ from cells asdescribed above. To determine the sensitivity and specificity of the assay, the syn-thetic peptides were diluted with loading buffer in a range from 10 ng/ml to3.125 pg/ml and loaded into a 96-well plate. Figure 10.1a shows the standard curvesobtained from this experiment. Both Aβ-140 and Aβ-142 had a detection limit of3.125 pg/ml (0.078 fmol/well).

No cross-reactivity was observed between Aβ1-40 and Aβ1-42, even with higherconcentration loadings of the peptides (10 ng/ml). The specificity of MBC40 andMBC42 was further confirmed in Western blot analysis. Because APP is the precursorof Aβ, the cross-reactivity of these capture antibodies (MBC40 and MBC42) to thefull-length and APP C-terminal fragments (CTFs) was investigated.

We overexpressed human APP695 and APPC99 in COS7. After immunoprecip-itation with an anti-APPC antibody (anti-APPC), the immunoprecipitates of APP-expressing cells were immunoblotted by another anti-APPC antibody (61C), 6E10,MBC40, and MBC42. Both 61C and 6E10 labeled the full-length APP as expected(Figure 10.1b). MBC40 and MBC42 did not label full-length APP. Additionally, theimmunoprecipitates of the cells expressing APPC-terminal fragment 99 (APPC100)were immunoblotted with 61C, 6E10, MBC40, and MBC42. The results demonstrated

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that 61C detected CTFs of APP and 6E10 detected APPC100 as expected. Again,MBC40 and MBC42 failed to label APPCTFs (Figure 10.1c). The results demon-strated that our Aβ ELISA has a good sensitivity and specificity for selectivelydetecting Aβ40 and Aβ42.

10.3.2 EFFECT OF LICL ON Aββββ SECRETION IN ASSOCIATION WITH GSK-3ββββ ACTIVITY FROM APP C100-TRANSFECTED COS7 CELLS

As a first step for determining the regulation of secreted Aβ by GSK-3, APP C100was transiently expressed in COS7 cells. Western blot analysis of the detergentextract from the transfected cells shows a band with a molecular weight of approx-imately 14 kDa, corresponding to APP C100 (data not shown). Aβ1-40 and Aβ1-42were then measured using the selective Aβ sandwich ELISAs. In agreement withthe results from the APP C100 expression experiment, abundant secretion of Aβwas detected from the transfected cells. The concentrations of Aβx-40 and Aβx-42,respectively, were 296.39 ± 3.063 pg/ml and 9.071 ± 0.628 pg/ml (data not shown).The percentage of Aβx-42 compared to total Aβ was approximately 5%, demon-strating that most of the Aβ was in the Aβx-40 form.

FIGURE 10.1 Sensitivity and specificity of sandwich ELISAs for both Aβx-40 and Aβx-42.(a) Standard curves for Aβ ELISAs. Both anti-Aβ40 and anti-Aβ42 detect the correspondingsynthetic peptides at 3.125 pg/ml (0.078 fmol/well). No cross-reactivity was observed evenat peptide concentrations up to 10 ng/ml. Replications show duplicates of each peptide loadedin the experiments. (b) The upper panel of the Western blot shows that MBC40 and MBC42did not label the immunoprecipitated full-length APP from APP-overexpressed COS7 cells.The lower panel of the Western blot shows that MBC40 and MBC42 did not label the immu-noprecipitated APP C terminal fragments from APP C100-overexpressed COS7 cells. Lane 1,anti-APP antibody 61C; lane 2, anti-Aβ antibody 6E10; lane 3, MBC40; and lane 4, MBC42.

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Previous studies showed that LiCl inhibits the activity of GSK-3β.10 In order tostudy the role of GSK-3β in Aβ generation, various concentrations of LiCl wereadded to APP C100-transfected cells. To demonstrate that any decrease in Aβsecretion was not due to LiCl toxicity resulting in cell death, cell counts wereperformed at the end of the 24-hr treatment period. No clear cellular toxicity wasobserved at any LiCl concentration tested (data not shown).

LiCl applied at concentrations of 0, 5, 10, and 20 mM decreased both Aβ40 andAβ42 secretion in a dose-dependent manner (Figure 10.2a). The LiCl-associateddecrease in Aβ secretion was statistically significant. The effect of lithium on Aβsecretion was also confirmed in primary neuronal cultures (data not shown). Inter-estingly, examination of cell lysates revealed a slight, dose-dependent increase incellular APP C100 as a result of LiCl treatment (Figure 10.2b).

10.3.3 EFFECT OF GSK-3ββββ ON FAββββ-INDUCED TAU PATHOLOGY IN VIVO

Because we observed that inhibition of GSK-3β reduced Aβ secretion, we furtherexamined inhibition of GSK-3β by lithium on Aβ-induced tau pathology in vivo. InP301L mutant tau Tg mice, fibrillar Aβ42 injection into brains was reported toaccelerate the formation of neurofibrillary tangle (NFT)-like tau pathology.11 Weinjected Aβ into the brains of 3-month-old V337M mutant tau Tg mice (V337M Tg)in which no tau pathology was apparent.12 We found that the injection of fAβ intothe CA1 of V337M Tg robustly induced hyperphosphorylated tau in the hippocampiof the mice as demonstrated by biochemical and immunohistochemistry studies(Figure 10.3).

Since we previously showed that Aβ-induced tau hyperphosphorylation couldbe blocked by the inhibition of GSK-3β in hippocampal primary culture,13,14 westudied the effect of inhibition of GSK-3β in tau pathology of Aβ-injected V337M

FIGURE 10.2 Aβx-40 and Aβx-42 secretions in APP C100-transfected cells following LiCltreatment for 24 hr (a) Bar graphs showing dose-dependent reductions of Aβx-40 and Aβx-42.Aβ secretion was measured by sandwich Aβ ELISAs. *p < 0.05, **p < 0.01, ***p < 0.001.(b) Western blot showing slight enhancement of APP C100 expression with increasing LiClconcentration.

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Tg. Inhibition of GSK-3β by LiCl administration could attenuate the AK-3-inducedtau phosphorylation and aggregation (Figure 10.3). Furthermore, inhibition of GSK-3βin these mice can rescue memory loss (data not shown), suggesting that GSK-3β playsan essential role in Aβ-induced pathological and behavioral changes even in vivo.

10.4 DISCUSSION

AD patients with different genetic backgrounds exhibit the same neuropathology,namely amyloid senile plaques, NFTs formed by accumulations of paired helicalfilaments (PHFs), and loss of neurons. Converging evidence suggests that blockadesof both amyloid and tau pathologies are key steps in treating this disease. In thischapter, we have described assays for studying amyloid generation and tau phos-phorylation. The sandwich Aβ ELISA discussed here shows high sensitivity andspecificity. It can serve as an assay to screen the effects of different drugs on Aβgeneration and measure endogenous Aβ from human and animal samples.

To extend this assay, different combinations of capture antibodies and detectorantibodies can be developed to differentiate the roles of several species of Aβ40 andAβ42 on this disease. The method showing induction of hyperphosphorylated tauby microinjection of fAβ into transgenic mice builds the bridge between amyloidand tau pathology. It is a useful approach to explore the fAβ-induced downstreamevents in vivo.

FIGURE 10.3 (See color insert following page 114.) The effects of GSK-3β inhibition onthe Aβ-induced formation of NFT-like tau pathology. (a) Immunohistochemistry study show-ing that the Aβ-induced tau pathology was blocked in the hippocampus of M337V Tg micecoinjected with Aβ and lithium as compared to mice injected with Aβ alone. (b) Western blotanalysis showing that hyperphosphorylated tau was reduced in the hippocampal tissues ofmice coinjected with Aβ and lithium as compared to mice injected with Aβ alone.

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The mechanism underlying the development of both amyloid deposition and tauhyperphosphorylation in AD still is not clear, although we hypothesize that somefactors might facilitate both amyloid and tau pathologies. The precise identities ofthese factors are still unknown. One promising candidate is GSK-3, an important“housekeeping” kinase that plays multiple roles in the central nervous system.15

Particularly, GSK-3β is shown to phosphorylate tau protein in vitro and in vivo andinteract with presenilin 1.6,16 We propose then, that under certain pathological con-ditions, GSK-3β functions as a mediator underlying amyloid overproduction andsubsequent tau hyperphosphorylation.

For many years, GSK-3β has been well recognized to have the ability to phos-phorylate tau protein. The association of GSK-3β with amyloid has not beenreported. In 1996, Klein and Melton first reported that lithium potently inhibitsGSK-3β activity.10 Additional data from in vitro and intact cell systems show thatLiCl specifically inhibits GSK-3β. LiCl at concentrations of 10 to 20 mM reducestau phosphorylation in cells coexpressing tau and GSK-3β.17 To examine the role ofGSK-3β on amyloid secretion, we applied LiCl at concentrations of 5 to 20 mM toAPP C100-transfected cells. Decreased Aβ secretion was observed at concentrationsas low as 5 mM, a concentration within the optimal range for the inhibition ofGSK-3β activity.

The results support our hypothesis that GSK-3β is involved in amyloid secretion.Later studies confirmed our findings showing that inhibition of GSK-3β in vivocould eliminate plaque formation in APP Tg mice as well.18 The molecular mecha-nism underlying the regulation of Aβ secretion by GSK-3β is not clear. The obser-vation that LiCl causes a slight accumulation of APP C100 indicates that GSK-3βmight affect γ-secretase activity. This is consistent with the finding that GSK-3βspecifically binds presenilin 1 and phosphorylates presenilin.6,19 Alternatively, APPphosphorylated by GSK-3β might have a different accessing ability to the processingenzymes, resulting in altered amyloid generation.

In addition to assaying the role of GSK-3β in APP processing, we furtherextended our study to tau pathology in tau Tg mice. As reported previously in P301Lmutant tau Tg mice,11 we observed that Aβ injection accelerated hyperphosphory-lated tau and caused neurodegeneration in V337M Tg mice. We found in Aβ-injectedV337M Tg mice that activation of GSK-3β was paralleled to tau pathology andassociated with accelerated neurodegeneration. Administration of lithium in thesemice not only reduced Aβ-induced formation of NFTs, but also rescued synapticloss, neuronal loss, and subsequent memory impairment.

The findings indicate that inhibition of GSK-3β by lithium possibly eliminatesamyloid and tau pathologies in AD. In view of the results presented here, severalgroups report that AD neuropathology is not observed in individuals diagnosed withdemented schizophrenia, since lithium is commonly used in the treatment of variouspsychiatric disorders including schizophrenia.20,21 Chronic administration of lithiumwith other medications, therefore, may reduce overproduction of amyloid initiatedby various factors. Exploring the action of LiCl on GSK-3β should play a significantrole in developing clinical therapies for AD.

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REFERENCES

1. Ishiguro, K. et al. A novel tubulin-dependent protein kinase forming a paired helicalfilament epitope on tau. J. Biochem. (Tokyo) 104, 319–321, 1988.

2. Aplin, A.E. et al. Effect of increased glycogen synthase kinase-3 activity upon thematuration of the amyloid precursor protein in transfected cells. Neuroreport 8,639–643, 1997.

3. Morfini, G. et al. Glycogen synthase kinase 3 phosphorylates kinesin light chainsand negatively regulates kinesin-based motility. EMBO J. 21, 281–293, 2002.

4. Kamal, A. et al. Axonal transport of amyloid precursor protein is mediated by directbinding to the kinesin light chain subunit of kinesin-I. Neuron 28, 449–459, 2000.

5. Koo, E.H. et al. Precursor of amyloid protein in Alzheimer disease undergoes fastanterograde axonal transport. Proc. Natl. Acad. Sci. USA 87, 1561–1565, 1990.

6. Takashima, A. et al. Presenilin 1 associates with glycogen synthase kinase-3beta andits substrate tau. Proc. Natl. Acad. Sci. USA 95, 9637–9641, 1998.

7. Sun, X. et al. Intracellular Abeta is increased by okadaic acid exposure in transfectedneuronal and non-neuronal cell lines. Neurobiol. Aging 23, 195–203, 2002.

8. Yoshiike, Y. et al. New insights on how metals disrupt amyloid beta-aggregation andtheir effects on amyloid-beta cytotoxicity. J. Biol. Chem. 276, 32293–32299, 2001.

9. Zappone, C.A. and Sloviter, R.S. Commissurally projecting inhibitory interneuronsof the rat hippocampal dentate gyrus: a colocalization study of neuronal markers andthe retrograde tracer fluoro-gold. J. Comp. Neurol. 441, 324–344, 2001.

10. Klein, P.S. and Melton, D.A. A molecular mechanism for the effect of lithium ondevelopment. Proc. Natl. Acad. Sci. USA 93, 8455–8459, 1996.

11. Gotz, J. et al. Formation of neurofibrillary tangles in P301l tau transgenic miceinduced by Abeta 42 fibrils. Science 293, 1491–1495, 2001.

12. Tanemura, K. et al. Neurodegeneration with tau accumulation in a transgenic mouseexpressing V337M human tau. J. Neurosci. 22, 133–141, 2002.

13. Takashima, A. et al. Exposure of rat hippocampal neurons to amyloid beta peptide(25-35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation oftau protein kinase I/glycogen synthase kinase-3 beta. Neurosci. Lett. 203, 33–36, 1996.

14. Takashima, A. et al. Activation of tau protein kinase I/glycogen synthase kinase-3beta by amyloid beta peptide (25-35) enhances phosphorylation of tau in hippocampalneurons. Neurosci. Res. 31, 317–323, 1998.

15. Grimes, C.A. and Jope, R.S. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Progr. Neurobiol. 65, 391–426, 2001.

16. Spittaels, K. et al. Glycogen synthase kinase-3beta phosphorylates protein tau andrescues the axonopathy in the central nervous system of human four-repeat tautransgenic mice. J. Biol. Chem. 275, 41340–41349, 2000.

17. Hong, M. et al. Lithium reduces tau phosphorylation by inhibition of glycogensynthase kinase-3. J. Biol. Chem. 272, 25326–25332, 1997.

18. Phiel, C.J. et al. GSK-3 regulates production of Alzheimer’s disease amyloid-βpeptides. Nature 423, 435–439, 2003.

19. Kirschenbaum, F. et al. J.V. Glycogen synthase kinase-3 beta regulates presenilin 1C-terminal fragment levels. J. Biol. Chem. 276, 30701–30707, 2001.

20. Arnold, S.E., Franz, B.R., and Trojanowski, J.Q. Elderly patients with schizophreniaexhibit infrequent neurodegenerative lesions. Neurobiol. Aging 15, 299–303, 1994.

21. Arnold, S.E. et al. Prospective clinicopathologic studies of schizophrenia: accrualand assessment of patients. Am. J. Psychiatr. 152, 731–737, 1995.

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11
Immunocytochemical Analysis of Amyloid Precursor Protein and Its Derivatives
Gunnar Gouras and Reisuke H. Takahashi

CONTENTS

11.1 Introduction11.2 Experimental Approaches

11.2.1 Immunocytochemistry: Fixation11.2.2 Immunocytochemistry: Controls11.2.3 Immunocytochemistry for APP and Aβ11.2.4 Immuno-Electron Microscopy for APP and Aβ

11.3 Conclusion11.4 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 Microscopy11.4.4 Protocol IV: Immuno-Electron Microscopy with Gold


11.1 INTRODUCTION

Histochemical analysis of postmortem human brains from subjects who sufferedfrom dementia led to the discovery of Alzheimer’s disease a century ago. Thehistological lesions that characterized this common age-related dementia were theaccumulation of senile plaques (SPs) and neurofibrillary tangles (NFTs). Subse-quently, NFTs were found in a variety of degenerative diseases of the brain, whileplaques remained more unique to Alzheimer’s dementia. The biochemical analysisof plaques in the 1980s led first to the identification of the fundamental component

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of plaques, the β-amyloid peptide (Aβ),1 followed by the identification of its longerprecursor protein, the amyloid precursor protein (APP).2

Since APP was cloned and sequenced, molecular biology studies have providedmultiple insights into the biology of APP and Aβ. Histochemical studies have takena backseat in basic Alzheimer’s research, appearing often as simply descriptive. Fewbiological studies are wholly accepted before in vivo support is provided by datafrom the diseased human brain. The combination of antibodies with histology pro-vides a powerful tool to confirm changes in a given protein in a diseased brain.Indeed, immunohistochemistry (or immunocytochemistry, ICC) is similar to a West-ern blot in that the antibody must find its specific antigen. This is followed byvisualization of a linked secondary antibody against the primary antibody via achemical reaction (chromophore).

In contrast to Western blot, where the protein antigen is located on a chargedmembrane, ICC detects the protein in its native site within the brain. A difficulty inanalyzing ICC in neuroscience is that an understanding of the brain cytoarchitectureand anatomy is important. Although viewed by biochemists as less accurate, ICCprocedures are more direct in taking the primary antibodies to their respectiveantigens and avoid some of the intermediate steps required for biochemical proce-dures such as protein extraction and immunoprecipitation.

11.2 EXPERIMENTAL APPROACHES

11.2.1 IMMUNOCYTOCHEMISTRY: FIXATION

ICC generally requires fixation of tissue prior to incubation with the primary anti-body. Fixation allows for the preservation of the brain cytoarchitecture, although itcan also introduce problems. The fixation procedure can lead to chemical cross-linking which may disrupt the antigenic site of a protein. Trial and error allow forthe assessment of optimal ICC conditions for a given antigen. The most commonfixative is 4% paraformaldehyde. Weaker fixative procedures such as alcohol-baseddehydration with methanol can be employed for antigens that are more sensitive tostandard fixation. Stronger fixatives, used especially for electron microscopy (EM)to further preserve the ultrastructural morphology, often include glutaraldehyde withparaformaldehyde.

ICC generally requires treatment with a detergent to allow for sufficient pene-tration of antibodies into the tissue. Triton X-100 is the most common detergent forICC. No detergent or a lower concentration of Triton or saponin can be used ifdetergent needs to be minimized (1) because a more lipid-associated antigen wouldbe extracted and thereby lost or (2) in the case of immuno-EM, to avoid damage tothe cytoarchitecture that can be observed at an ultrastructural level after treatmentwith detergent. For ICC and immuno-EM, a balance must be found between theoptimal level of fixation and detergent used. As can occur with Western blot, ICCmay not adequately detect certain antigens.

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11.2.2 IMMUNOCYTOCHEMISTRY: CONTROLS

Potential problems with ICC, as with all antibody-based assays, include potentialfalse cross-reactivity of the antibody and detection of a reaction product not relatedto the antibody. The latter can be explored by adding excess antigen (such as Aβpeptide) prior to addition of the primary antibody — a technique known as peptidecompetition. Loss of staining after peptide competition indicates that the antibodysees its antigen, but does not provide information on whether cross-reactivity withan unwanted antigen is present because the antibody is sequestered from the potentialcross-reactivity by the excess antigen. The best control for antibody specificity isfailure to observe the immunolabeling with a given antibody on knockout mousetissue that lacks the corresponding antigen.

11.2.3 IMMUNOCYTOCHEMISTRY FOR APP AND Aββββ

With regard to immunostaining of Aβ peptides and APP (full-length and APPC-terminal fragments), an understanding of the epitopes of a given antibody and theprocessing of APP is important. See Protocols I and II for ICC for Aβ or APP onfixed and frozen and paraffin-embedded brain tissue, respectively. C-terminal-spe-cific APP antibodies can be used to study full-length APP and various C-terminalfragments of APP. N-terminal APP antibodies can be used to study full-length APPand various secreted forms of APP (sAPP). In addition, the significant sequencehomology of APP with other members of the APP–APLP (amyloid precursor-likeprotein) family of proteins (APLP1 and APLP2) may lead to cross-reactivity of someN-terminal APP antibodies with APLPs. The C terminus of APP is less homologousand therefore C-terminal antibodies tend to be specific for APP.

One also must be aware when using anti-Aβ antibodies that antibodies raisedagainst the Aβ domain of APP, which includes many of the widely used Aβ antibodiesfor ICC, also see full-length APP and Aβ-containing APP CTFs (such as βCTF).Moreover, an understanding of the complexity of APP processing allows appreciationof the fact that one may also be assaying various lesser known APP and Aβ fragments.For example, the β-secretase BACE was demonstrated to cleave both at AβAsp1and at AβGlu11, generating Aβ1-40/42 and Aβ11-40/42 peptides, respectively.3,4

Indeed, the first plaques observed with AD pathology are composed of N-truncatedAβ and these peptides aggregate more readily than full-length Aβ. Therefore whenusing an antibody against the N terminus of Aβ, one may be missing an importantpool of N-truncated Aβ.5

We should note that just because an antibody was raised against a certain Aβpeptide domain, the actual epitope is not necessarily the entire domain. For example,the widely used monoclonal antibody 6E10 (Signet Laboratories, Dedham, MA)against the N terminus of Aβ was raised against Aβ1-16, but has been epitope-mapped to Aβ5-10. Thus, 6E10 will not see N-truncated Aβ11-40/42 peptides.

A more recent area of research reports that Aβ antibodies recognize more thanplaques in AD brains or in transgenic mice that develop plaque pathologies. Specifically,

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intraneuronal Aβ accumulation by ICC has been observed with AD pathogenesis.6–8

A prerequisite for observing intraneuronal Aβ with concomitant plaque pathology(Figure 11.1) is allowing the immunoperoxidase reaction product enough time tovisualize more than the most abundant signal coming from plaques. This is analogousto allowing a Western blot to develop for a sufficient time so that both the full-lengthAPP band and the less abundant Aβ band can be seen when employing an antibodydirected against the Aβ domain of APP on cell lysate. Intraneuronal Aβ can beespecially appreciated prior to plaque development when the “internal” peptidecompetition from the high concentration of Aβ in plaques is less, such as in a Down’ssyndrome brain destined to develop plaque pathology.6,9,10

An additional complexity is that Aβ antibodies have different affinities, depend-ing on the aggregation state of Aβ. If formic acid is employed to expose even theβ-pleated Aβ within plaques, antibodies that prefer monomeric Aβ reveal remarkablymore plaque staining. Increasing evidence links Aβ oligomers with the pathogenesisof AD,11 and Aβ oligomer-specific antibodies are being developed to better studyAβ oligomers in brains.

11.2.4 IMMUNO-ELECTRON MICROSCOPY FOR APP AND Aββββ

Generally, milder fixation and detergent conditions are required for immuno-EMthan for ICC. After incubation with the primary antibody, staining methods for

FIGURE 11.1 Immunohistochemistry of human AD brain with an Aβ42-specific antibody.Note plaque and intraneuronal Aβ42 staining.

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immuno-peroxidase and immuno-gold are different. See Protocols III and IV fordetails on both methods.

With regard to fixation, glutaraldehyde preserves ultrastructural morphology, butat the same time it can prevent the primary antibodies from recognizing antigensbecause aldehyde residues may block antibody–antigen interactions. To avoid thesequalities of glutaraldehyde, some employ acrolein for fixation.12 Acrolein is a milderfixative reagent than glutaraldehyde, especially for neuronal tissues, and does notblock antibody–antigen binding as much as glutaraldehyde. Mice or rats are perfusedwith 3.75% acrolein and 2% paraformaldehyde to preserve the ultrastructure andantigenicity. For human brain, biopsy tissue is rapidly immersion-fixed with 1.875%acrolein and 2% paraformaldehyde. After perfusion, brain tissue is cut (40 µm thick)on a vibrating microtome and treated with sodium borohydride to remove extraaldehyde sites.13,14

A treatment with a detergent is generally required to allow antibodies to penetrateto the antigen in the tissue. Triton X-100 (~0.05%) is commonly used as a detergentfor immuno-EM. Freeze–thaw methods can also be used for EM instead of detergent.To allow for penetration of the antibody, tissue is physically destroyed by liquidfreon and liquid nitrogen. In some cases, both methods may be required to detectantigen. An optimal combination must be found for the given antibody and antigen.

We employed Aβ42 C-terminal-specific antibodies and APP C-terminal-specificantibodies to study localization in brains of Aβ42 and APP, respectively. Employingwell-characterized C-terminal Aβ42-specific antibodies MBC42 (kindly providedby Dr. H. Yamaguchi, Gunma University, Gunma, Japan) and AB5078P (Chemicon,Temecula, CA), specific localization of Aβ42 to multivesicular bodies and endosomalvesicles was observed (Figure 11.2).15 In contrast, antibodies to the C terminus ofAPP predominantly labeled the Golgi apparatus.15,16

FIGURE 11.2 Immuno-gold electron microscopy demonstrating Aβ42 localization espe-cially on the outer limiting membranes of multivesicular bodies in a neuron of a normal mouse.

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11.3 CONCLUSION

Immunohistochemical methods are powerful tools for investigating the anatomicaldistribution of a given antigen, such as APP and its derivatives in brain. Immuno-EMcan be especially useful in exploring the cell biology and subcellular neuropathologyof Alzheimer’s disease.


11.4.1 PROTOCOL I: IMMUNOSTAINING PROTOCOL FOR PARAFFIN-EMBEDDED SECTIONS*

Day 1

1. Deparaffinize in xylene I, 5 min.2. Deparaffinize in xylene II, 5 min.3. Deparaffinize in xylene III, 5 min.4. 100% ethyl alcohol, 3 min.5. 100% ethyl alcohol, 3 min.6. 95% ethyl alcohol, 3 min.7. 70% ethyl alcohol, 3 min.8. 50% ethyl alcohol, 3 min.9. 0.1 M PB, 3 min × 2.

10. 0.1 M Tris-buffered saline (TBS), 3 min × 2.11. 99% formic acid, 5 min (for optimal plaque detection with Aβ antibodies

only).12. Wash with 0.1 M PBS, 5 min × 3.13. 3% hydrogen peroxide in 0.1 M TBS, 30 min.14. Wash with 0.1 M TS, 2 min × 3.15. Block in 0.5% bovine albumin serum (BSA) in 0.1 M TBS for 30 min

(steps 15 through 17 usually performed with 0.01 to 0.03% Triton).16. Wash once with 0.1 M TS.17. Incubate with primary antibody in 0.1% BSA at 4°C overnight (in hydra-

tion chamber).

Day 2

1. Wash in 0.1 M TS, 10 min ×3.2. Incubate in secondary antibody (1:400) in 0.1% BSA for 60 min at room

temperature.3. Prepare ABC-HRP (avidin–biotin horseradish peroxidase complex) solution

(Vectastain Elite ABC kit): 1 drop of reagent A and 1 drop of reagent Bper 2.5 ml of 0.1 M TS; let it sit at least 30 min.

4. Wash in TBS, 10 min × 3.5. Incubate in ABC-HRP solution for 30 min.

* Utilizing Vectastain ABC kit, Vector Laboratories, Burlingame, CA.

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6. Wash in 0.1 M TBS, 10 min × 3.7. Add 5 µl 30% hydrogen peroxide per 50 ml diaminobenzidine (DAB,

Sigma-Aldrich, St. Louis, MO) solution (22mg/100ml).8. Develop in and time the DAB reaction; check for staining under a light

microscope (or use kit from Vector).9. Rinse 1 min with distilled water.

10. Wash once with 0.1 M TBS.11. Wash with 0.1 M PB.12. Place in dessicator, 30 min.13. Dehydrate in series of increasing alcohol concentrations (50, 75, and 95%)

for 3 min each; 100% for 5 min × 2, Xylene I, Xylene II, and Xylene IIIfor 10 min each.

14. Coverslip with DPX mounting medium and let dry overnight.

11.4.2 PROTOCOL II: IMMUNOPEROXIDASE STAINING PROTOCOL FOR FLOATING SECTIONS*

Day 1

1. Wash with 0.1 M PBS, 5 min × 3.2. 99% formic acid, 5 min (for optimal plaque detection with Aβ antibodies

only).3. Wash with 0.1 M PBS, 5 min × 34. 3% hydrogen peroxide in 0.1 M PBS, 30 min.5. Wash with 0.1 M PBS, 5 min × 3.6. Block in 0.5% BSA in 0.1 M PBS for 30 min (steps 6 through 8 usually

performed with 0.01 to 0.03% Triton).7. Wash once with 0.1 M TBS.8. Incubate with primary antibody in 0.1% BSA overnight at 4ºC.

Day 2

1. Wash with 0.1 M PBS, 5 min × 3.2. Incubate in secondary antibody (1:400) in 0.1% BSA for 60 min at room

temperature.3. Prepare ABC-HRP (horseradish peroxidase) solution (Vectastain Elite

ABC kit) (1 drop of reagent A and 1 drop of reagent B per 2.5 ml of0.1 M PBS); let sit at least 30 min.

4. Wash in 0.1 M PBS, 5 min × 3.5. Incubate in ABC-HRP solution for 30 min.6. Wash in 0.1 M PBS, 5 min × 3.7. DAB solution: 5 ml dH2O, 2 drops solution, 4 drops DAB solution, 2 drops

hydrogen peroxide mixed well (or make DAB solution as in Protocol I).8. Time DAB reaction and check for staining under light microscope.9. Wash once with 0.1 M PBS.

* Utilizing Vectastain ABC kit, Vector Laboratories, Burlingame, CA.

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10. Mount sections on coated glass slices; let sections dry.11. Dehydrate in series of increasing alcohol concentrations (50, 75, and 95%)

for 3 min each; 100% for 5 min × 2, Xylene I, Xylene II, and Xylene IIIfor 10 min each.

12. Coverslip with DPX.

11.4.3 PROTOCOL III: IMMUNOPEROXIDASE ELECTRON MICROSCOPY

Day 1

1. Perfusion fixation with 3.75% acrolein/2% paraformaldehyde in 0.1 MPB; for brain biopsy, fix tissue with 1.875% acrolein/2% paraformalde-hyde in 0.1 M PB.

2. Cut brain tissue with Vibratome (40 µm).3. Wash in 0.1 M PB.4. 1% sodium borohydride in 0.1 M PB for 30 min.5. Wash in 0.1 M PB until bubbling stops.6. Wash in 0.1 M TBS, 10 min × 2.7. Block in 0.5% BSA/0.1 M TS for 30 min.8. Wash in 0.1 M TBS, 1 min × 3.9. Incubate sections with primary antibody in 0.1% BSA/0.1 TS overnight

at 4ºC and for a second night at room temperature. Add Triton X-100(~0.05%) if needed.

Day 2

1. Wash in 0.1 M TBS, 10 min × 3.2. Incubate with secondary antibody (1:400) in 0.1% BSA/0.1 M TBS for

30 min.3. Prepare ABC-HRP (horseradish peroxidase) solution (Vectastain Elite

ABC kit); 2 drops of reagent A and 2 drops of reagent B per 10 ml of0.1 M TBS; shake immediately and let it sit at least 30 min.

4. Wash in 0.1 M TBS, 10 min × 3.5. Incubate in ABC solution for 30 min.6. Wash in 0.1 M TBS, 10 min × 3.7. During washes, prepare DAB solution and add 22 mg DAB to 100 ml

0.1 M TBS and stir. Just before using, add 10 µl of 30% hydrogen per-oxide.

8. Time DAB reaction and check for staining under light microscope.9. Wash in 0.1 M TBS, 2 min × 2.

10. Wash in 0.1 M PB, 2 min × 3.11. Carefully lay sections flat in shallow wells containing 0.1 M PB and

replace with 2% osmium tetroxide in 0.1 M PB. Incubate 1 hr.12. Wash in 0.1 M PB, 3 min × 3.13. 30% ETOH, 5 min.14. 50% ETOH, 5 min.

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15. 70% ETOH, 5 min.16. 95% ETOH, 5 min.17. 100% ETOH, 10 min × 2.18. Propylene oxide 10 min × 2.19. Propylene oxide:EPON (mixed 1:1) rotate overnight at room temperature.

Day 3

1. Fresh EPON; rotate 2 hr.2. Flat embedding at 60ºC for 18 to 24 hr. Embed sections between two

sheets of aclar fluorhalocarbon film. Transfer as little EPON as possibleto the sheet with the section, and vigorously squeeze the sections together,removing air bubbles and pushing EPON away.

11.4.4 PROTOCOL IV: IMMUNO-ELECTRON MICROSCOPY WITH GOLD

Day 1

1. Same as Protocol III.2. Concentration of primary antibody is generally three to four times higher

than for immunoperoxidase method.

Day 2

1. Wash once in 0.01 M PBS.2. Block in washing buffer (0.1% gelatin, 0.8% BSA in 0.01 M PBS, pH 7.4)

10 min.3. Incubate with gold-conjugated IgG in washing buffer (1:50) for 2 hr.4. Wash in washing buffer, 5 min.5. Wash in 0.01 M PBS 5 min × 3.6. Incubate in 2% glutaraldehyde in 0.01 M PBS for 10 min.7. Wash in PBS.8. Wash in 0.2 M citrate buffer (0.2 M citrate sodium, pH 7.4). During

washing, mix silver reagents A and B (Amersham Biosciences, Uppsala,Sweden) at a 1:1 ratio.

9. Silver intensification reaction; check for staining under a light microscope.10. Wash in citrate buffer to stop reaction.11. Carefully lay sections flat in shallow wells containing 0.1 M PB.12. Incubate with 2% osmium tetroxide in 0.1 M PB for 1 hr.13. Wash in 0.1 M PB, 3 min × 3.14. 30% ETOH, 5 min.15. 50% ETOH, 5 min.16. 70% ETOH, 5 min.17. 95% ETOH, 5 min.18. 100% ETOH, 10 min × 2.19. Propylene oxide, 10 min × 2.20. Propylene oxide:EPON (mixed 1:1); rotate overnight at room temperature.

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Day 3

1. Same as Protocol III.

ACKNOWLEDGMENTS

We thank Dr. Noel Calingasan, Department of Neurology and Neuroscience, WeillMedical College of Cornell University, for reviewing this chapter.

REFERENCES

1. Glenner, G.G. and Wong, C.W. Alzheimer’s disease and Down’s syndrome: sharingof a unique cerebrovascular amyloid fibril protein. Biochem. Biophys. Res. Commun.122, 1131, 1984.

2. Kang, J. et al. The precursor of Alzheimer’s disease amyloid A4 protein resemblesa cell-surface receptor. Nature 325, 733, 1987.

3. Vassar, R. et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein bythe transmembrane aspartic protease BACE. Science 286, 735, 1999.

4. Cai, H. et al. BACE1 is the major beta-secretase for generation of Abeta peptides byneurons. Nat. Neurosci. 4, 233, 2001.

5. Gouras, G.K. et al. Generation and regulation of beta-amyloid peptide variants byneurons. J. Neurochem. 71, 1920, 1998.

6. Gouras, G.K. et al. Intraneuronal Abeta42 accumulation in human brain. Am. J.Pathol. 156, 15, 2000.

7. D’Andrea, M.R. et al. Consistent immunohistochemical detection of intracellularbeta-amyloid 42 in pyramidal neurons of Alzheimer’s disease entorhinal cortex.Neurosci. Lett. 333, 163, 2002.

8. D’Andrea, M.R. et al. The use of formic acid to embellish amyloid plaque detectionin Alzheimer’s disease tissues misguides key observations. Neurosci. Lett. 342, 114,2003.

9. Gyure, K.A. et al. Intraneuronal abeta-amyloid precedes development of amyloidplaques in Down syndrome. Arch. Pathol. Lab. Med. 125, 489, 2001.

10. Busciglio, J. et al. Altered metabolism of the amyloid beta precursor protein isassociated with mitochondrial dysfunction in Down’s syndrome. Neuron 33, 677, 2002.

11. Selkoe, D.J. Nature, 426, 900, 2003.12. Leranth, C. and Pickel, V.M. Folding proteins in fatal ways, in Neuroanatomical Tract

Tracing Methods, Vol. 2, Plenum, New York, 1989, p. 120.13. Milner T.A. et al. Hippocampal 2a-adrenergic receptors are located predominantly

presynaptically but are also found postsynaptically and in selective astrocytes.J. Comp. Neurol. 395, 310, 1998.

14. Chan, J., Aoki, C., and Pickel, V.M. Optimization of differential immunogold–silverand peroxidase labeling with maintenance of ultrastructure in brain sections beforeplastic embedding. J. Neurosci. Methods 33, 113, 1990.

15. Takahashi, R.H. et al. Intraneuronal Alzheimer abeta42 accumulates in multivesicularbodies and is associated with synaptic pathology. Am. J. Pathol. 161, 1869, 2002.

16. Caporaso, G.L. et al. Morphologic and biochemical analysis of the intracellulartrafficking of the Alzheimer beta/A4 amyloid precursor protein. J. Neurosci. 14, 3122,1994.

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12
Pathological Detection of Aβ and APP in Brain
Chica Mori and Cynthia A. Lemere

CONTENTS

12.1 Introduction12.2 Methods

12.2.1 Tissue Processing Protocols12.2.1.1 Paraffin Tissue12.2.1.2 Frozen Tissue

12.2.2 Immunohistochemistry12.2.2.1 Paraffin Sections12.2.2.2 Frozen Sections

12.2.3 Congo Red12.2.4 Thioflavin S Staining Protocol12.2.5 Double Immunofluorescent Labeling12.2.6 Double Labeling with DAB and Other Colored Markers

12.3 Results and Discussion12.3.1 Aβ40 vs. Aβ42 in Down’s Syndrome12.3.2 Aβ40 vs. Aβ42 in Familial Alzheimer’s Disease (FAD)12.3.3 Intraneuronal Aβ in Down’s Syndrome12.3.4 Intraneuronal Aβ in APP Transgenic Mice12.3.5 APP Immunoreactivity12.3.6 Colocalization of Aβ with Glia

References.

12.1 INTRODUCTION

In 1906, a German physician named Alois Alzheimer described the two majorpathologic features, amyloid plaques and neurofibrillary tangles (NFTs), found inthe brain of an elderly woman with a dementia-causing neurodegenerative disorderthat later became known as Alzheimer’s disease (AD). Subsequently, various histo-logical dyes and stains including silver were used to identify these lesions in autop-sied brain tissue. In 1984, Glenner and Wong purified and chemically identifiedamyloid-β protein (Aβ), a 4-kD monomer, from meningeal blood vessels.1 Later,the sequence was extended from 24 to 40 amino acids2 and found to be identical tothe amino acid compositions of senile plaques purified from human AD brains.3,4

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In the 1990s, several mutations in the amyloid precursor protein (APP) genewere correlated with early onset AD5,6 or hereditary cerebral hemorrhage7,8 in a smallnumber of families, setting the stage for the prominent roles of APP and Aβ in ADpathogenesis. In addition, persons with Down syndrome (DS), trisomy 21, developAD-like pathology, including plaques and NFTs, due primarily to the gene dosageeffect of having three copies of the APP gene encoded on chromosome 21.9

Prior to the mid-1980s, plaque and vascular amyloid were identified in brainsections using silver stains or dyes that bound β-sheet amyloid fibrils, such as Congored and thioflavin S (Figure 12.1). Upon the identification of the Aβ amino acidsequence, Aβ synthetic peptides were manufactured and Aβ-specific antibodies pro-duced by injecting synthetic Aβ peptide into rabbits to generate polyclonal antibodies(Pab) or into mice to generate monoclonal antibodies (Mab). Consequently, Aβ-specific antibodies proved exceptionally sensitive for immunohistochemically detect-ing Aβ protein on brain tissue sections. As a result, the extent of Aβ deposition inaged humans and in particular, AD patients, turned out to be much greater than thatpreviously observed using histological methods. One reason for this unveiling of Aβdeposition was that the some Aβ antibodies were able to identify more than onlyfibrillar amyloid; they also recognized nonfibrillar (i.e., diffuse, prefibrillar) Aβ deposits.

The objective of this chapter is to provide extensive technical details for thepreparation of brain tissue sections and the identification of Aβ protein and itsprecursor, APP, by various histological and immunohistochemical (IHC) methods.These methods may be applied to autopsied human, mouse or monkey brainsalthough the cross-reactivities of certain antibodies should be checked for eachspecies. In addition to specific protocols for immunohistochemistry and histologicalstains, examples of how they were used in our lab are illustrated. Table 12.1 liststhe antibodies we used in immunohistochemistry studies.

FIGURE 12.1 (See color insert following page 114.) Detection of fibrillar amyloid by thiofla-vin S and Congo red staining. (a) Thioflavin S dye binds to β-pleated Aβ in plaques (arrow-heads) and blood vessels (arrows), as demonstrated in this formalin-fixed paraffin brain sectionof an 18-month-old APP transgenic mouse. Thioflavin S staining is visualized using a fluo-rescent microscope. Magnification ×10. (b) Congo red also binds fibrillar amyloid and pro-duces a birefringence that alternates between yellow and green under polarized light. Plaques(arrowheads) and vascular Aβ (arrows) are observed in a paraffin section of temporal cortexfrom a human AD brain. Magnification ×20.

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12.2 METHODS

12.2.1 TISSUE PROCESSING PROTOCOLS

Fresh tissue must be fixed, dehydrated, and infiltrated with paraffin prior to paraffinsectioning. Tissue for cryosectioning should be embedded immediately in ornithinecarbamyl-transferase (OCT, Sakura Finetek, Torrance, CA) or fixed and then embed-ded in OCT and frozen at –80ºC. This section provides information about tissueprocessing and slide preparation for immunohistochemistry.

12.2.1.1 Paraffin Tissue

12.2.1.1.1 HumansFresh postmortem human brain is cut into small blocks of tissue and then fixed in10% neutral buffered formalin (pH 7.0 to 7.6) for 1 hr or more at room temperature.Tissue specimens are thoroughly washed in Tris buffer solution (TBS; 50 mM Tris,150 mM NaCl) to remove excess formalin, placed in cassettes, and stored in TBSat 4ºC until processing.

TABLE 12.1Immunohistochemistry Antibodies Used in Authors’ Laboratory

Antibody Mab or Pab Source Dilution Pretreatment

R1282 (Aβ) Pab Selkoe (Boston, MA) 1:1000 FA6E10 (Aβ) Mab Signet (Dedham, MA) 1:1000 FA21F12 (Aβ42) Mab Elan Corp. (Dublin, Ireland) 1:1000 FAC42 (Aβ42) Pab T.C. Saido (Tokyo, Japan) 1:250 FABC42 (Aβ42) Pab H. Yamaguchi (Gunma, Japan) 1:500 FAMBC42 (Aβ42) Mab H. Yamaguchi 1:1000 FAQCB42 (Aβ42) Pab BioSource (Camarillo, CA) 1:100 FA2G3 (Aβ40) Mab Elan Corp. 1:1000 FABC40 (Aβ40) Pab H. Yamaguchi 1:500 FAMBC40 (Aβ40) Mab H. Yamaguchi 1:1000 FAQCB40 (Aβ40) Pab BioSource 1:100 FAN3pE (Aβ pyroglut 3) Pab T.C. Saido 1:200 FA, FA/MWN1D (Aβ1-5) Pab T.C. Saido 1:150 FA, FA/MW3D6B (Aβ1-5) Mab Elan Corp. 1:200–500 FA, FA/MW8E5 (APP 444-592) Mab Elan Corp. 1:1000 MWC8 (APP, C terminus) Pab Selkoe 1:1000 MW22C11 (APP, N terminus) Mab Chemicon (Temecula, CA) 1:200 MWAT8 (PHF tau) Mab Innogenetics (Ghent, Belgium) 1:50 MWGFAP (human astrocyte) Pab Dako (Carpenteria, CA) 1:1000 —GFAP (mouse astrocyte) Mab Sigma (St. Louis, MO) 1:500 —CD45 (microglia) Mab Serotec (Raleigh, NC) 1:5000 MWHLA-DR (microglia) Mab NeoMarkers (Fremont, CA) 1:100 MW

Mab = monoclonal antibody. Pab = polyclonal antibody. FA = formic acid. MW = microwave.

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The specimens are dehydrated through graded ethanol, cleared of lipids inHistosolve (Shandon, Pittsburgh, PA), and infiltrated with two changes of hot paraffin(~60ºC; temperature varies among brands). The paraffin-saturated specimens areplaced into paraffin-containing embedding cassettes, cooled, and stored at roomtemperature for sectioning.

Paraffinized specimens are sectioned 10 to 12 microns thick and placed on glassslides. Slides are dried overnight, baked at 60ºC for 1 hr, and stored at roomtemperature for immunohistochemistry studies.

12.2.1.1.2 MicePerfuse mice in 4% paraformaldehyde (PFA), remove brain, and place it in PFA.Follow step 2 and 3 in human tissue protocol.

12.2.1.2 Frozen Tissue

Fresh brain specimens (mouse and human) are embedded in OCT. Mouse tissuescan be fixed in sucrose or formaldehyde before OCT embedding. They are snap-frozen in liquid nitrogen, then stored at –80ºC for cryosectioning.

Specimens should be stored at –20ºC prior to sectioning. They should be sectioned10 microns thick at –20ºC and placed on glass slides. The slides should be stored at–20ºC for immunohistochemistry studies. Long-term storage should be at –80ºC.

12.2.2 IMMUNOHISTOCHEMISTRY

Immunohistochemistry (IHC) studies are used to visualize proteins. Generally, par-affin sections produce better protein morphology with immunohistochemistry thanthey do in frozen sections. However, formalin fixation prior to paraffin embeddingof tissues can mask the antigen and may require antigen retrieval via boiling or withenzyme or acid digestion. These processes may expose antigen, but they can alsomake the sections brittle. In general, pretreating paraffin sections with formic acidprior to staining (see below) enhances Aβ immunoreactivity.

Microwave pretreatment enhances tau, neurofilament and APP immunoreactiv-ity. Frozen sections do not require such a step and surface antigens are well preservedcompared to formalin fixed sections. Hence, cytokine staining works better in frozensections than in paraffin sections. The disadvantages of frozen sections are poorantigen morphology and the need for a freezer that can provide storage at –80ºC.This section provides general protocols for immunohistochemistry studies. Protocolsfor antigen-enhancing pretreatments are also provided.

12.2.2.1 Paraffin Sections

1. Deparaffinize first in two changes (3 min each) of Histoclear (NationalDiagnostics, Atlanta, GA) followed by 3 min in a 50:50 mixture ofHistoclear and 100% ethanol, and then rehydrate in a graded series ofethanol concentrations (95, 75, and 50% ethanol) ending in water.

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2. Quench endogenous peroxidase activity in 0.3% hydrogen peroxide inmethanol for 8 min (99 ml methanol and 1 ml 33% H2O2). Pour outmixture. Rinse twice in distilled water for 3 min each time.

3. Perform antigen-enhancing pretreatment if necessary.Microwave pretreatment — Dilute Antigen Retrieval Citra (Bio-Genex, San Ramon, CA) ×10 with super-Q water. Put slides into aglass dish filled with Citra. Place the dish in a plastic box filled with1 in water. Microwave and bring to a boil, then perform cyclic boilingfor an additional 5 min. Cool to room temperature.Formic acid pretreatment — Add 88% formic acid (Fisher, FairLawn, NJ) onto sections for 10 min. Wash twice in distilled water for3 min each time.

4. Block in 10% serum (from the animal species in which the secondaryantibody was generated) for 20 min at room temperature or 4°C overnight.Dilute serum in TBS.

5. Incubate sections in primary antibodies for 1 hr at room temperature or4ºC overnight.

6. Wash gently in TBS.7. Incubate sections in secondary antibodies (goat anti-rabbit for polyclonal

antibodies and horse anti-mouse for monoclonal antibodies; Vector Lab-oratories, Burlingame, CA) for 30 min at room temperature (1 ml serum[goat for polyclonal, horse for monoclonal], 9 ml TBS, and 45 µl second-ary antibody).

8. Gently wash in TBS.9. Incubate in horseradish peroxidase–avidin (Elite ABC kit, Vector Labo-

ratories) 30 min at room temperature (10 ml TBS, 90 µl solution A and90 µl solution B).

10. Gently wash in 50 mM Tris.11. Develop in diaminobenzidine (DAB; Sigma Immunochemicals, St. Louis,

MO). Dissolve 2 mg DAB into 100 ml of 50 mM Tris; add 33 µl of 33%H2O2 to activate.

12. Gently wash in distilled water. 13. Counterstain with hematoxylin; differentiate with acid alcohol (1 ml con-

centrated HCl per 100 ml 70% ethanol).14. Dehydrate in distilled water, 50% ethanol, 75% ethanol, 95% ethanol, two

changes of 100% ethanol, 50:50 mixture of 100% ethanol and Histoclear,and two changes of Histoclear.

15. Coverslip with Permount (Fisher Scientific, Pittsburgh, PA).

12.2.2.2 Frozen Sections

1. Air dry the slides for 15 min at room temperature.2. Add a drop of acetone (stored at –20ºC) or methanol to a section and let

it air dry for 15 min at room temperature.3. Place in TBS for 5 min.4. Follow paraffin sectioning protocol (12.2.2.1) and continue.

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12.2.3 CONGO RED

Congo red recognizes the beta sheet conformations of Aβ fibrils. Images of Congo-red stained sections are observed using polarized light microscopy.

1. Deparaffinize and rehydrate.2. Stain in Mayer’s hematoxylin for 10 min.3. Wash quickly in distilled water.4. Differentiate with acid alcohol.5. Wash in three changes of super-Q water.6. Wash in freshly alkalinized sodium chloride solution (100 ml saturated

NaCl in 80% ethanol and 1 ml 1% NaOH) for 20 min.7. Filter Congo red (100 ml saturated NaCl in 80% ethanol, 0.2 g Congo

red, and 1 ml aqueous NaOH) and use it to stain within 15 min.8. Place in three changes of 100% ethanol.9. Dehydrate slides in graded concentration of ethanol and Histoclear.

10. Coverslip with Permount.

12.2.4 THIOFLAVIN S STAINING PROTOCOL

Thioflavin S (Sigma, St. Louis, MO) is a dye that binds beta-pleated fibrils. It detectsfibrillar amyloids in plaques and blood vessels. Sections are observed under afluorescent microscope.

1. Deparaffinize tissue in Histoclear and rehydrate in graded ethanol to water.2. Rinse three times in distilled water.3. Incubate in filtered (filter fresh before each use) 1% aqueous thioflavin S

for 8 min at room temperature (2 g thioflavin S in 200 ml distilled water).4. Decant thioflavin S and wash for 3 min in 80% ethanol.5. Decant ethanol and wash again for 3 min in 80% ethanol.6. Decant ethanol and wash for 3 min in 95% ethanol.7. Wash with three changes of distilled water.8. Coverslip in aqueous mounting media (Hydromount, National Diagnos-

tics, Atlanta, GA).

12.2.5 DOUBLE IMMUNOFLUORESCENT LABELING

Double immunofluorescent labeling is used to visualize two different proteins in asingle section. Signals are detected using a fluorescent microscope. For example,one antigen may be detected as red and the other as green. When two antigenscolocalize, the signal appears yellow. This technique is helpful in determining thespatial relationship of two proteins. It also allows us to visualize cellular ingestionof proteins such as when microglia phagocytose Aβ.

Day 1

1. Deparaffinize and rehydrate.2. Apply any pretreatments.

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3. Immerse sections in 0.1 M Tris buffer (pH 7.6) for 5 min (20 ml 1 M Trisand 180 ml distilled water).

4. Block in 2% goat serum (serum from the animal species in which thesecondary antibody was generated) in 0.1 M Tris for 5 min (200 µl serumin 9.8 ml Tris buffer).

5. Prepare antibodies. For double labeling, combine the two primary anti-bodies 50:50 in volume. Dilute antibodies in 2% serum (same species asused in step 4) in 0.1 M Tris.

6. Flick off and wipe off excess fluid and apply antibodies.7. Incubate overnight (16 to 20 hr) at 4°C in humidified chamber.

Day 2

1. Warm slides to room temperature.2. Rinse sections with 0.1 M Tris.3. Immerse sections in 2% goat serum (serum from the animal species in

which the secondary antibody was generated) in 0.1 M Tris for 5 min.4. Dilute secondary antibodies (Molecular Probes, Laiden, OR). [Combina-

tion 1 = AlexaFluor 488 goat anti-rabbit (green) and rhodamine red goatanti-mouse (red). Combination 2 = AlexaFluor 488 goat anti-mouse(green) and rhodamine red goat anti-rabbit (red)]

5. Centrifuge the mixture of red and green secondary antibodies for 30 sec.Apply the supernatant (1.5 µl AlexaFluor 488, 1.5 µl rhodamine red in997 µl of 2% serum in 0.1 M Tris).

6. Incubate at room temperature for 2 hr.7. Rinse secondary antibodies from sections with 0.1 M Tris.8. Apply Sudan black B for 10 min at room temperature. Make fresh batch

if Sudan black is more than 2 months old. Add 0.3 g (0.3%) Sudan blackB to 100 ml 70% ethanol. Wrap foil around container, add liquid, powderand a stirring bar. Stir container wrapped in foil for 2 hr using automixer.Store at 4ºC.

9. Rinse sections three times with 0.1 M Tris.10. Wash in distilled water.11. Fix in 10% formalin for 1 hr at room temperature. Wrap foil around glass

container and its lid.13. Wash twice in distilled water, 10 min each time.14. Coverslip with Hydromount. Apply clear nail polish around the edge of

the coverslip to prevent sections from drying out. Put slides in closedfolder at 4°C.

12.2.6 DOUBLE LABELING WITH DAB AND OTHER COLORED MARKERS

The principle for DAB labeling is the the same as for immunofluorescent doublelabeling in that each antigen is detected as a different color and this allows assessmentof colocalization of two different antigens. Typically, one antigen is stained by DAB(brown) and the other with an alkaline phosphatase (AP) substrate kit (Vector). Thekit allows a choice of red, blue or black. The order of staining is irrelevant in many

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cases although one particular order of staining may be better than another for certainantigens. In general, DAB staining can be followed by AP staining and vice versa.There are four possible combinations:

1. DAB (antigen 1) followed by AP (antigen 2)2. DAB (antigen 2) followed by AP (antigen 1)3. AP (antigen 1) followed by DAB (antigen 2)4. AP (antigen 2) followed by DAB (antigen 1)

It is important to determine which procedure works best for the antigens ofinterest. In the procedure below, AP staining is followed by DAB staining.

Day 1

1. Deparaffinize and rehydrate.2. Quench endogenous peroxidase activity in 0.3% hydrogen peroxide in

methanol for 8 min. Pour out mixture. Rinse twice in distilled water, 3 mineach time.

3. Perform antigen-enhancing pretreatments.4. Block with 10% serum (from the animal species in which the secondary

antibody was generated) in TBS.5. Apply primary antibody and incubate 1 hr at room temperature.6. Gently wash in TBS.7. Apply 45 µl secondary antibody to 10 ml of 10% serum in TBS; incubate

30 min at room temperature.8. Gently wash in TBS.9. Prepare ABC for AP staining (45 µl solution A and 45 µl solution B) in

5 ml TBS. Allow to sit 20 min.10. Apply ABC onto sections and incubate 30 min at room temperature.11. Gently wash in Tris.12. Prepare AP substrate kit in your color of interest (red, blue, or black).

A. 5 ml of 100 mM Tris at pH 8.2. It is very important to adjust the pHbetween 8.2 and 8.5 to achieve development.

B. Add a drop of levamisole. Shake well.C. Add 90 µl of solution 1. Shake well.D. Add 90 µl of solution 2. Shake well.E. Add 90 µl of solution 3. Shake well.

13. Rinse under cold running water to stop the reaction from proceeding further.14. Perform antigen-enhancing pretreatments.15. Block with 10% serum in TBS.16. Apply the other primary antibody. Incubate overnight at 4°C. If you are

in a hurry, you can incubate 1 hr at room temperature and continue on.

Day 2

1. Warm slides to room temperature if they have been incubated overnightat 4°C.

2. Gently wash in TBS.

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3. Apply secondary antibody (45 µl secondary antibody in 10 ml of 10%serum in TBS); incubate 30 min at room temperature.

4. Prepare ABC for DAB development (45 µl solution A and 45 µl solutionB in 5 ml TBS). Allow to sit 20 min.

5. Apply ABC and incubate 30 min at room temperature.6. Gently wash in Tris.7. DAB development.8. Counterstain with hematoxylin and differentiate with acid alcohol.9. Dehydrate.

10. Coverslip with Permount.

12.3 RESULTS AND DISCUSSION

12.3.1 Aββββ40 VS. Aββββ42 IN DOWN’S SYNDROME

Down syndrome (DS), trisomy 21, results in an extra copy of the APP gene locatedon chromosome 21. Most DS patients develop full-blown AD pathologies by the ageof 40 or 50.10,11 Plaque deposition begins decades earlier than it does in nonDSindividuals. Thus, DS provides a model for studying the temporal progression of theneuropathogenesis of AD. The generation of Aβ C-terminal-specific antibodies (rec-ognizing Aβ42 and Aβ40, specifically) has allowed further insight into the sequenceand morphological characterization of the deposition of different species of Aβ.

Multiple reports have demonstrated that Aβ42 is deposited into plaques prior toAβ40. For example, extracellular accumulation of Aβ was reported in diffuse Aβ42immunoreactive plaques as soon as 12 years of age in DS brain; Aβ40 immunolabelingwas not observed (Figure 12.2a and Figure 12.2b).12,13 At older ages, DS brain showedincreased numbers of plaques (Figure 12.2c and Figure 12.2d), aggregation of Aβand compaction of plaques that contained Aβ40 in addition to Aβ42 (Figure 12.2e,Figure 12.2f, Figure 12.3a, and Figure 12.3b).12,14 Biochemical studies of vascularamyloid reported the Aβ deposits contained predominantly Aβ ending at valine 40.15

IHC studies showed that blood vessels in young DS patients had weak Aβ42 immuno-reactivity in smooth muscle cells but with increasing age, vascular Aβ deposits werecomposed mainly of aggregated Aβ40 with some colocalization of lesser amountsof Aβ42 (Figure 12.4).

12.3.2 Aββββ40 VS. Aββββ42 IN FAMILIAL ALZHEIMER’S DISEASE (FAD)

Although 80 to 90% of total Aβ consists of Aβ40 and only 10 to 20% is Aβ42,Aβ42 is selectively deposited first in senile plaques and is more prone to fibrillizationthan Aβ40. In addition, Aβ42 aggregates into insoluble amyloid fibrils much fasterthan Aβ40.16 A study of Colombian patients with early onset FAD caused by anE280A mutation in the presenilin 1 (PS1) gene showed that abundant Aβ42 depo-sition and gliosis occurred about 30 years earlier than in sporadic AD patients.17 Inthe PS1-FAD patients, robust increases of Aβ42 were observed in plaques and bloodvessels in cortex, hippocampus, and cerebellum, but Aβ40 was not increased relativeto that found in sporadic AD cases (Figure 12.5).

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Over 90 different PS-1 mutations have been cited in the literature. The recentlyidentified early-onset PS-1 mutations, Y256S and Q222H, also exhibited elevatedAβ1-42 levels in brain homogenates. In particular, the PS1 mutation Y256S, with

FIGURE 12.2 Aβ C-terminal immunostaining in young DS brain. Antibody BC42 that spe-cifically recognizes residue 42 at the C terminus of Aβ revealed many diffuse plaques(arrowheads) in brains of 12-year-old (a), 17-year-old (c), and 29-year-old (e) DS subjects.Plaques were not detected in adjacent sections with antibody BC40 that specifically recognizesresidue 40 at the Aβ C terminus at ages 12 (b) and 17 (d), whereas a small number of plaqueswere detected at age 29 (f). In addition to the many discrete plaques observed in the 12- and17-year-old brains, larger patches of Aβ deposition (arrowhead in e) were detected at age 29by BC42. BC40 darkly stained the cores of a small number of plaques (large arrowheads inf) in the 29-year-old brain. Weak BC40 reactivity (small arrowheads in f) was also detectedby BC40 in occasional plaques that stained intensely with BC42 (e). Bar = 200 µm. (Reprintedfrom Lemere C.A. et al., in Neurobiology of Disease, Vol 3, 1996, p. 19. With permissionfrom Elsevier.)

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an age of onset at 25 years, showed markedly high Aβ42 levels and some increasein Aβ40 levels.18 The other PS1 mutation, Q222H, with an average age of onset at35 years, showed an elevation in Aβ42 but not Aβ40. In numerous studies in bothhuman FAD and in APP and PS1/APP transgenic mice, the level of Aβ42 in thebrain seemed to correlate with severity of AD in neuropathology and age of onset.

12.3.3 INTRANEURONAL Aββββ IN DOWN’S SYNDROME

Although the origin of extracellular A in cerebral plaques still remains to be eluci-dated, increasing evidence suggests that intraneuronal A may be involved as an earlyevent of AD pathogenesis.13,19-23 Immunohistochemical studies have demonstratedAβ42 IR-positive neurons particularly in young DS cases. Gouras et al. observedcytoplasmic staining in neurons using antibody QCB42 in a 3-year-old DS case.23

FIGURE 12.3 Colocalization of BC42 and BC40 immunostaining within a plaque. Adjacent8-µm sections of temporal cortex from a 47-year-old DS patient were immunostained withAβ C-terminal antibodies BC42 (a) and BC40 (b). Bar = 25 µm. (Reprinted from Lemere,C.A. et al., in Neurobiology of Disease, Vol 3, 1996, p. 26. With permission from Elsevier.)

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FIGURE 12.4 BC42 vascular immunostaining in DS brain. (a) Smooth muscle cell cytoplas-mic BC42 immunoreactivity (arrowheads) in a leptomeningeal blood vessel in the temporalcortex of a 16-year-old DS patient. (b and c) Adjacent 8-µm sections of temporal cortex froma 73-year-old DS patient were immunostained with BC42 (b) and BC40 (c). Large arrowheadsshow colocalization of BC42 and BC40. Small arrowheads show lack of colocalization. Bars =100 µm. (Reprinted from Lemere C.A. et al., in Neurobiology of Disease, Vol 3, 1996, p. 26.With permission from Elsevier.)

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FIGURE 12.5 Aβ42 is detected in much greater quantity than Aβ40 in PS1-FAD brains.(a) Large numbers of Aβ42-IR plaques are present in frontal cortex, including compactedplaques in all layers (small and medium arrowheads) and a diffuse Aβ42 band in layer IV(large arrowhead). (b) A few compacted Aβ40-IR plaques (small arrowheads) and bloodvessels occur in an adjacent section to the one shown in a. Note that most of the Aβ40-positiveblood vessels are also Aβ42-IR (asterisk). (c) Many intensely Aβ42-IR plaques (arrowhead)are present just outside the dentate gyrus and in CA1 and subiculum in the hippocampus.(d) A subset of Aβ42-containing plaques are also Aβ40-IR (for example, arrowheads in d andc) in a section adjacent to that shown in c. (e) Numerous Aβ42-IR plaques occur in cerebellum,including diffuse plaques in the molecular layer (large arrowhead) and compacted plaques inthe molecular, Purkinje cell and granule cell layers (small arrowheads). Many leptomeningealblood vessels are also Aβ42-IR (asterisk). (f) A minority of compacted plaques in the Purkinjecell layer (left arrowhead) and molecular layer (right arrowhead) and leptomeningeal bloodvessels (for example, asterisk) are labeled by Aβ40 antibody in an adjacent section to thatshown in e. Sections a through d are from a 47-year-old patient with an E280A PS-1 mutations;sections e and f are from a 62-year-old patient with the same PS-1 mutation. Scale bars =500 µm. (Reprinted from Lemere C.A. et al., in Nature Medicine, Vol 2, 1996, p. 1147. Withpermission from Nature, http://www.nature.com/.)

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Another DS study reported TUNEL-positive neurons immunolabeled for Aβ42,indicating apoptosis in neurons containing abundant Aβ42.20

Our study also detected intraneuronal Aβ42 immunolabeling in very young(3 and 4 years old) DS cases (Figure 12.6a). However, Aβ42-immunoreactive neu-rons decreased to very few or none with aging and increasing plaque deposition(Figure 12.6b through Figure 12.6d).13 Intraneuronal Aβ was nearly absent in DSbrains displaying dystrophic neurites, cored plaques, and neurofibrillary tangles.

12.3.4 INTRANEURONAL Aββββ IN APP TRANSGENIC MICE

Intraneuronal Aβ seems to be an early step in AD pathogenesis as supported by DSand other nonDS studies. For example, using immuno-gold EM and monoclonalantibody (Mab) MBC42, Takahashi et al. reported Aβ42 localization to multivesic-ular bodies of neurons in Tg2576 mice with human APP Swedish 670/671 mutation.21

They speculate that Aβ42 accumulation in presynaptic and postsynaptic compart-ments causes synaptic dissolution. Using triple transgenic mice models with APP,PS1, and tau mutations, Oddo et al. reported impairment in synaptic plasticitycorrelating with intraneuronal Aβ accumulation.19 However, their study utilized Mabs4G8 and 6E10 to detect anti-Aβ; both Mabs are known to cross-react with APP.Thus, it is possible that intraneuronal staining observed using those antibodies was APP.

12.3.5 APP IMMUNOREACTIVITY

Amyloid precursor protein (APP) involves two series of endoproteolytic cleavagesby secretases to produce Aβ. The first cleavage by a recently identified novel mem-brane-bound aspartyl protease, β-secretase (BACE1), generates an APP C-terminalfragment known as C99.24 Next, a γ-secretase complex containing PS1 and nicastrincleaves the C99 fragment.25 Confocal microscopy demonstrated the presence ofBACE1 and APP in late-Golgi.26 In our IHC studies using APP-specific antibodies,we found a vesicular staining pattern of APP in subcellular compartments or some-times around the nuclei of neurons in human DS and AD brains and in miceoverexpressing mutant APP (Figure 12.7a through Figure 12.7c). APP antibodiesalso recognized neuritic processes in compacted plaques in human and transgenicmouse brains (Figure 12.7b and Figure 12.7c).

12.3.6 COLOCALIZATION OF Aββββ WITH GLIA

Pro-inflammatory responses occur in pathologically vulnerable areas of the ADbrain. Activated microglia and reactive astrocytes cluster within or in proximity toneuritic plaques. When activated, those inflammatory cells show ramified processesand interdigitate into the plaques. Microglia cells interacting with Aβ were observedby double immunofluorescent IHC using Pab R1282 (a general Aβ antibody) andMab CD45 (to detect activated microglia) in PSAPP transgenic mice regardless ofwhether the animals had been immunized against Aβ peptide (Figure 12.8).

Mounting evidence from animal studies suggests that fibrillar amyloid peptidetriggers the activation of microglia and astrocytes that clearly colocalize with Aβplaques.27,28 It seems as though inflammatory cells remove Aβ by phagocytosis, as

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demonstrated in vitro29 and in vivo.30 Moreover, microglia-mediated cytokines suchas TNF-α and IL-6 exhibit neurotoxic as well as neuroprotective effects. The para-doxical roles of inflammatory cells in the neurodegenerative AD brain need to beclarified further.

FIGURE 12.6 Aβ42 staining with PabC42 in DS subjects of increasing ages.(a) Intense cytoplasmic neuronal stain-ing (arrows) in the absence of extracel-lular Aβ IR was evident at 3 years. Scalebar = 10 µm. (b) Temporal cortices ofmany young DS patients such as the 17-year-old shown here contained both dif-fuse plaques (large arrowhead) and neu-ronal staining (small arrowhead). Scalebar = 50 µm. (c) Fully matured plaques(arrow) along with some diffuse plaques(arrowhead) were present as early as29 years of age. Neuronal staining wasvery infrequent in this subject and gen-erally seemed to decline with age asplaques matured (see Table 12.1). Scalebar = 20 µm. (d) At 62 years of age,mature cored plaques were present(arrow) and intraneuronal Aβ42 wasnearly absent. Scale bar = 10 µm.(Reprinted from Mori C. et al., in Amy-loid: J. Protein Folding Disorders, Vol 9,2002, p. 95. With permission from Par-thenon/CRC Press/Taylor & Francis,http://www.tandf.co.uk.)

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FIGURE 12.7 APP immunoreactivity. (a) APP Mab 8E5 detected neuronal cytoplasmicvesicles (arrows) in frontal cortex of a 52-year-old DS subject. Original magnification × 100.(b) Mab 8E5 detected cytoplasmic APP in neurons (arrows) and plaque-associated dystrophicneurites (arrowheads) in temporal cortex of a 75-year-old AD patient. Original magnification× 32. (c) Neuritic plaques (arrowheads) and intraneuronal human APP (arrows) were immu-nolabeled by Mab 8E5 in entorhinal cortex of an 18-month-old APP transgenic mouseoverexpressing a familial mutation in human APP. Original magnification × 32.

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FIGURE 12.8 (See color insert following page 114.) Double immunofluorescent labeling ofplaques (R1282) and microglia (CD45) showed similar patterns of colocalization in Aβimmunized (bottom) and untreated (top and middle) PSAPP mice after 8 weeks of Aβimmunization. A dramatic reduction in plaque burden was seen in the 13-week-old mice aftertreatment. CD45-immunoreactive microglia colocalized with compacted plaques in treatedand untreated mice. However, because the numbers of plaques were significantly reduced inthe Aβ immunized mice, the number of labeled microglia was also reduced. Images wereobtained using a Zeiss Axiovert 100 M laser-scanning confocal microscope (LSM510).(Reprinted from Lemere C.A. et al., Modulating amyloid-beta levels by immunotherapy: Apotential therapeutic strategy for the prevention and treatment of Alzheimer’s disease, in Saido,T.C., Ed., Amyloid-beta Metabolism and Alzheimer’s Disease, Landes Bioscience, George-town, TX, 2003, p. 155. With permission from Landes Bioscience, http://www.Eurekah.com.)

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REFERENCES

1. Glenner, G.G. and Wong, C.W. Alzheimer’s disease: initial report of the purificationand characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys.Res. Commun., 120, 885, 1984.

2. Hardy, J. Framing β-amyloid. Nature Genet., 1, 233, 1992.3. Joachim, C.L. et al. Protein chemical and immunocytochemical studies of menin-

govascular β-amyloid protein in Alzheimer’s disease and normal aging. Brain Res.,474, 100, 1988.

4. Levy, E. et al. Mutation of the Alzheimer’s disease amyloid gene in hereditary cerebralhemorrhage, Dutch-type. Science, 248, 1124, 1990.

5. Masters, C.L. et al. Amyloid plaque core protein in Alzheimer disease and Downsyndrome. Proc. Natl. Acad. Sci. USA, 82, 4245, 1985.

6. Mullan, M. et al. A pathogenic mutation for probable Alzheimer’s disease in the APPgene at the N-terminus of β-amyloid. Nature Genet., 1, 345, 1992.

7. Neve, R.L. et al. Expression of the Alzheimer amyloid precursor gene transcripts inthe human brain. Neuron, 1, 669, 1988.

8. Selkoe, D.J. et al. Isolation of low-molecular-weight proteins from amyloid plaquefibers in Alzheimer’s disease. J. Neurochem., 146, 1820, 1986.

9. van Broeckhoven, C. et al. Amyloid β-protein precursor gene and hereditary cerebralhemorrhage with amyloidosis (Dutch). Science, 248, 1120, 1990.

10. Wisniewski, K.E., Wisniewski, H.M., and Wen, G.Y. Occurrence of neuropathologicalchanges and dementia of Alzheimer’s disease in Down’s syndrome. Ann Neurol., 17,278, 1985.

11. Mann, D.M. et al. A morphological analysis of senile plaques in the brains of non-demented persons of different ages using silver, immunocytochemical and lectinhistochemical staining techniques. Neuropathol. Appl. Neurobiol., 16, 17, 1990.

12. Lemere, C.A. et al. Sequence of deposition of heterogeneous amyloid beta-peptidesand APOE in Down’s syndrome: implications for initial events in amyloid plaqueformation. Neurobiol. Dis., 3, 16, 1996.

13. Mori, C. et al. Intraneuronal Aβ42 accumulation in Down’s syndrome brain. Amyloid:J. Protein Folding Disord., 9, 88, 2002.

14. Iwatsubo, T. et al. Amyloid beta protein (Aβ) deposition: A beta 42(43) precedes Abeta 40 in Down’s syndrome. Ann. Neurol., 37, 294, 1995.

15. Joachim, C.L. et al. Protein chemical and immunocytochemical studies of menin-govascular β-amyloid protein in Alzheimer’s disease and normal aging. Brain Res.,474, 100, 1988.

16. Jarret, J.T., Berger, E.P., and Lansbury, P.T., Jr. The carboxy terminus of the betaamyloid protein is critical for the seeding of amyloid formation: implications for thepathogenesis of Alzheimer’s disease. Biochemistry, 32, 4693, 1993.

17. Lemere, C.A. et al. The E280A presenilin 1 Alzheimer mutation produces increasedAbeta 42 deposition and severe cerebellar pathology. Nat Med., 2, 1146, 1996.

18. Miklossy, J. et al. Two novel presenilin-1 mutations (Y256S and Q222H) are asso-ciated with early-onset Alzheimer’s disease. Neurobiol. Aging, 24, 655, 2003.

19. Oddo, S. et al. Triple-transgenic model of Alzheimer’s disease with plaques andtangles: intracellular Abeta and synaptic dysfunction. Neuron, 39, 409, 2003.

20. Busciglio, J et al. Altered metabolism of the amyloid β precursor protein is associatedwith mitochondrial dysfunction in Down’s syndrome. Neuron, 33, 677, 2002.

21. Takahashi, R.H. et al. Intraneuronal Alzheimer Aβ42 accumulates in multivesicularbodies and is associated with synaptic pathology. Am. J. Pathol., 161, 1869, 2002.

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22. Gyure, K.A. et al. Intraneuronal Aβ-amyloid precedes development of amyloidplaques in Down syndrome. Arch. Pathol. Lab. Med., 125, 489, 2001.

23. Gouras G.K. et al. Intraneuronal Aβ42 accumulation in human brain. Am. J. Pathol.,156, 15, 2000.

24. Vassar, R. et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein bythe transmembrane aspartic protease BACE. Science, 286, 735, 1999.

25. Yu, G. et al. Nicastrin modulates presenilin-mediated notch/glp-1 signal transductionand beta APP processing. Nature, 407, 48, 2000.

26. Yan, R. et al. The transmembrane domain of the Alzheimer’s beta-secretase (BACE1)determines its late Golgi localization and access to beta-amyloid precursor protein(APP) substrate. J. Biol. Chem., 276, 39, 36788, 2001.

27. Benzing, W.C. et al. Evidence for glial-mediated inflammation in aged APP(SW)transgenic mice. Neurobiol. Aging, 20, 581, 1999.

28. Schenk, D. et al. Immunization with amyloid-beta attenuates Alzheimer disease-likepathology in the PDAPP mouse. Nature, 400, 173, 1999.

29. Paresce, D.M., Ghosh, R.N., and Maxfield, F.R. Microglial cells internalize aggregatesof the Alzheimer’s disease amyloid beta-protein via a scavenger receptor. Neuron,17, 553, 1996.

30. Bard, F. et al. Peripherally administered antibodies against amyloid beta-peptide enterthe central nervous system and reduce pathology in a mouse model of Alzheimer’sdisease. Nat. Med., 6, 916, 2000.

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13
Creating APP Transgenic Lines in Mice
Stanley Jones Premkumar Iyadurai and Karen Hsiao Ashe

CONTENTS

13.1 Introduction13.1.1 Origins of Gene Manipulation and Gene Transfer into Mouse

Genome13.1.2 Basic Principles for Consideration in Generating Transgenic

Mice13.1.3 APP Gene Structure: Its Isoforms and Promoters Used in

Creating APP Transgenics13.2 Experimental Procedures

13.2.1 Steps in Generating APP Transgenic Mice by Microinjection13.2.1.1 Strain Selection13.2.1.2 Isolation of Target DNA from Bacterial Host13.2.1.3 Purification of DNA for Microinjection13.2.1.4 Preparation of DNA for Microinjection13.2.1.5 Microinjection with Admixed DNAs13.2.1.6 Indentification of Founders13.2.1.7 Maintenance and Analysis of Founders

13.3 Concluding RemarksReferences

13.1 INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that primarilyimpairs memory function.1 Pathologically, AD is characterized by amyloid-contain-ing neuritic plaques and intraneuronal fibrillary tangles.2 While the biological causesand exact biological mechanisms by which the progressive neurodegeneration isinitiated are not very clear, it has been shown that mutations in some specific genesmay be important in these events. For example, mutations in genes encoding theamyloid precursor protein (APP),3–9 presenilin-1 (PS1) and presenilin-2 (PS2)10–13

have been associated with cases of familial AD.

0-8493-2245-6/05/$0.00+$1.50© 2005 by CRC Press

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Further analyses have shown that mutations in all the aforesaid genes result inincreased amounts of a specific cleavage product of APP, namely, Aβ.14–17 Interest-ingly, Aβ has been identified as a component of neuritic plaques in AD brains18 andlends support to Aβ serving as a key modulator in the progression of AD. Anothergene encoding the apolipoprotein E (apo E) is also thought to be a modifier of theAlzheimer’s disease phenotype.19

Although AD was first described almost a century ago,20 the sequential eventsthat lead to AD and memory dysfunction are not clearly defined and remain hard toelucidate for several reasons. At present, AD is a clinical diagnosis; definitivediagnosis is available only upon autopsy. Because of the variations in presentationand overlapping disease symptoms with other neurological disorders, AD poses achallenge for clinical diagnosis. For moral and ethical reasons, antemortem biopsyof brain tissue for diagnosis is not a viable option and studying the pathogenesis inhumans has been very difficult. However, the identification of genes associated withAD and the advent of molecular biological tools for gene transfer have made itpossible for AD researchers to study the human AD-causing genes in vivo in basicmodel organisms such as mice, rats, flies, worms, and yeasts.

One approach is to study the homologous genes in a given model system, forexample, studying the mouse homologue of the human PS gene in mice. Anotherapproach is to transfer and express the human gene of interest in a model organism,for example, expressing the human APP gene and studying the consequences ofexpressing it in mice. The latter technique has been termed the transgenic mouseapproach for modeling human diseases.

13.1.1 ORIGINS OF GENE MANIPULATION AND GENE TRANSFER INTO MOUSE GENOME

The laboratory mouse remains the well-studied genetic model organism that isevolutionarily closest to the human. With the rediscovery of Mendel’s laws in the1900s and subsequent interest in experimental mouse developmental biology, thefield of mouse genetics was firmly established by the 1960s.21 The first report ofintroduction of foreign DNA into a transgenic mouse as a way of manipulating themouse genome emerged from Jaenisch and Mintz in 1974.22 These researchersshowed that purified SV40 DNA, when injected into the blastocoel cavities of mouseblastocysts, was capable of integrating into the genomes of the embryonic cells.

Following the discovery that microinjection of cloned herpes simplex virusthymidine kinase (tk) DNA into the nuclei of cultured fibroblasts led to stableexpression of the tk gene,23,24 Gordon et al.25 reported the first successful pronuclearinjection into a one-celled mouse embryo and demonstrated that the transgenicmouse showed expression in somatic tissues. Integration of such foreign DNA intosomatic and germline tissues was reported immediately afterward.26 Since then, ahuge number of genes, including human genes, have been introduced into the mousegenome.

The establishment of human disease conditions in mice revolutionized theresearch arena, especially in understanding complex diseases such as AD. While no

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genetic disease model in mice can supplant the need for studying the disease inhumans and human clinical trials, studies in model systems allow researchers togain some understanding of the pathology and coordinate efforts to approach thedisease in humans.

The ability to transfer human genes to mice raised the possibility of studyingmutant disease-causing human genes in the context of the mouse genome andestablishing model disease paradigms. Research has shown that mouse disease modelsystems, albeit partial model systems, mimic human disease processes and serve asvaluable tools for understanding pathogenesis, progression, prevention, and treatment.

Several human disease models exist in transgenic mice, including AD,27 Parkin-son’s disease,28 Huntington’s disease,29,30 amyotropic lateral sclerosis,31 and others.32,33

The key first step in developing a model system is to identify specific genes involvedin the disease condition. This step is usually carried out by following family pedi-grees manifesting a given disease and using molecular biological approaches. Oncea gene is identified and cloned, one can study the basic biologic functions of thegene in heterologous systems, such as mice, by expressing that human gene in mice.For example, in a familial form of early-onset AD observed in Sweden, the offendinggene mutation was identified in the amyloid protein precursor (APP) gene.3 ThecDNA expressing the “Swedish mutation” of the APP gene was later cloned andtransferred to mice.34 The principles and methodology of generating transgenic micewill be dealt in detail in the sections to follow.

In addition to the transgenic technology, two other methodologies have beenused to manipulate the mouse genome — knock-out and knock-in mice — to under-stand the pathogenetic mechanisms underlying human diseases. Both technologiesutilize the properties of homologous recombination and embryonic stem (ES) cells.ES cells are derived from cells of the inner cell masses in developing blastocystsearly in embryonic development.21 ES cells can be isolated and cultured35,36 andmanipulated to include foreign genes in culture.37 Such manipulated ES cells canbe reinjected into a developing mouse blastocyst and then implanted into a recipient.

If the manipulated ES cells go on to differentiate into the germline, among othertissues, a germline transgenic mouse is generated. The knock-out strategy involvesreplacement of the wild-type gene with a disrupted version of a given gene (usuallywith neoR) or with a truncated version of the gene, so that the resulting gene at itsnative location is nonfunctional or dysfunctional. The knock-in strategy involvesreplacement of the wild-type gene, with a specifically engineered mutation of thesame gene. The advantage of generating knock-in and knock-out models is that thegiven gene is expressed from its native location in its own transcriptional context,usually mimicking the native gene’s expression abundance levels. Patterns andabundance expression at endogenous levels have often been insufficient to producedesirable phenotypes. Moreover, since both knock-in and knock-out techniques relyon extensive homology of DNA sequences for homologous recombination, replacingnative mouse genes with mutant human genes is limited to genes with high degreesof DNA homology. The generation of transgenic animals by microinjection over-comes these limitations and thus remains a superior strategy compared to otherstrategies discussed.

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13.1.2 BASIC PRINCIPLES FOR CONSIDERATION IN GENERATING TRANSGENIC MICE

An ideal disease model in any model organism recreates the typical features of thehuman disease and its progression. To this end, effective design of the transgene,its expression profile and strength of expression are very important factors. Thereforespecial consideration should be given to the transgene construction — whether touse the native gene or the complete cDNA and the relevant choice of promoters forfavorable expression — with expected expression profiles in a spatial and a temporalfashion, and with expression strong enough to recreate the mutant phenotype.

In the case of cDNA-based transgenic mice, the choice of one isoform amongothers derived from the same gene is critical. While creating the transgenic mousewith the full native gene circumvents the problem of isoform selection, selectivecDNA expression may be advantageous when the given cDNA is more closely relatedto the development of the disease, for example, an isoform with neuron-specificexpression (as opposed to ubiquitous expression) to study a neurological disorder.Additionally, one can use specific promoters to drive expression in a certain subsetof cell types in mice. It should also be borne in mind that since transgenic mice aregenerated by insertion of the gene of interest at a random site in the mouse genome,the gene is influenced by “position effect” — the expression of the gene is influencedby the location where it is integrated.38 For the same reason, it should be expectedthat the expression levels in independent germline transgenic mice carrying the sametransgene will be different. Based on this consideration, it is essential to generatemultiple, independent transgenic lines to ensure that one of them will providesufficient expression to produce the mutant phenotype.

Traditionally, several types of promoters have been used in directing expressionof transgenes in transgenic mouse lines. The promoters derived from the so-called“housekeeping” genes have been known to direct ubiquitous expression. Thesepromoters include the β-actin promoter,39,40 the mouse metallothionein promoter,41,42

the HMG CoA reductase promoter,43 and the histone H4 promoter.44

It should be noted that although the promoters are “ubiquitous,” they may notdirect the same levels of expression in all tissues. Another class of promoters, theconditional promoters, has also been used successfully. In their case, the transgeneof interest is silent until it is activated by specific manipulation, such as heat shockor by an inducer. Specific examples of this class of promoters include metallothioneinpromoter (inducers: Zn and Cd), hsp68 promoter (inducer: heat shock), lac operator-promoter (inducer: IPTG), and tet operon promoter (inducer: tetracycline). Otherstrategies for conditional expression involving FLP sites and FLP recombinase45

loxP sites and P1 Cre recombinase46 are beyond the scope of this chapter.

13.1.3 APP GENE STRUCTURE: ITS ISOFORMS AND PROMOTERS USED IN CREATING APP TRANSGENICS

APP, whose function is not completely clear, is the precursor of Aβ which is a corecomponent of senile plaques in AD brains.47 The APP gene contains 19 exons andis known to undergo alternative splicing.48 APP is known to have at least five major

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isoforms of sizes 639,49 695, 714, 751, and 770 amino acids.47,50 The longest isoform,APP770, contains a 56-amino acid domain (encoded by exon 7), which sharessequence homology with and can function like Kunitz-type serine protease inhibitors(KPIs)51 as well as an adjacent 19-amino acid domain (encoded by exon 8) withhomology to the MRC OX-2 antigen1,52 found on the surfaces of neurons and certainimmune cells. 53

Brain tissue expresses little APP770. APP751 contains the KPI region but lacksthe MRC OX-2 domain. APP751 is expressed variably in the brain at low, interme-diate, or high levels, depending on the region involved. APP695 lacks both of theabove domains and is primarily produced in neurons that constitute the primarysources of APP in the central nervous system. APP714 contains amino acids ofAPP695 form plus an addition of 19 amino acids of unknown significance, and isnot present in the brain. APP639 also lacks exon 2 and is highly expressed in liverand least expressed in the brain.49

Several transgenic AD models have been generated using expression of modifiedAPP, PS1, and tau genes. Most groups have focused on generating transgenic mouselines carrying mutant APP or PS1 genes. In this section, the discussion will belimited to selected APP transgenics.

The first APP transgenic model of AD in mice was created by expressing humanwild-type APP751 driven by a neuron-specific enolase promoter in inbred JU micethat developed age-related impairments in memory tests.54,55 Although no amyloidplaques were reported, diffuse Aβ/APP deposits and abnormal tau immunoreactivitywere observed in aged animals.56

The second APP transgenic model of AD in mice was created by Games andcolleagues.57 These authors expressed a recombinant transgene derived from portionsof APP cDNA and genomic DNA fragments such that alternative splicing of exons7 and 8 could be functional. The recombinant transgene was driven by the platelet-derived growth factor β-chain promoter.57 In addition, the transgene containedV717F, the “Indiana” mutation. These mice exhibited amyloid deposition around6 to 9 months, but neurofibrillary tangles were absent.58,59 Tg2576 was generated byinjecting APP cDNA containing the “Swedish mutation” (K670N, M671L) underthe control of the hamster prion protein promoter.59 The prion protein promoterdrives expression pan-neuronally in the brain.60 Plaque deposition and cognitivedysfunction in an age-dependent fashion have been described.61

Another set of transgenic lines, TgAPP22 and TgAPP23, were generated usingAPP751 cDNA containing the Swedish mutation, under the neuron-specific Thy-1promoter.62 Thy-1 promoter also directs expression throughout the central nervoussystem. TgCRND8 mice were generated by expressing APP695 cDNA containingboth the Swedish and Indiana mutations under the control of the pan-neuronalhamster prion protein promoter.63 These mice developed memory deficits at the earlyage of 3 months. APP knock-in or yeast artificial chromosome-based mice have alsobeen generated and they tend to develop pathologies later in life.64

The utility of transgenic and knock-out mice in modeling neurological diseasesis outlined in Aguzzi et al.65 The knock-in models have more recent origins, relativelyspeaking. Most of the knock-in models of AD have been described in the presenilin

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genes. Mutations in PS1 and PS2 have been linked to familial AD. The first AD-modeling knock-in mice were reported by Guo and colleagues.99 These researchersexchanged the wild-type PS1 sequences for a mutant PS1 sequence to make amethionine-to-valine change at the 146th amino acid position.66 Although the micedid not exhibit any overt phenotype, they were hypersensitive to seizure-inducedsynaptic degeneration and necrotic neuronal death in the hippocampus. Another PS1knock-in (PS1P264L), using a similar strategy was reported by Siman et al.67

Apolipoproteins E4 and E3 are alleles of apo E that seem to modify AD pathol-ogy, although the precise function of apo E in the AD pathway is unknown. Tounderstand the roles of apo E4 and apo E3 mutations in cholestrol metabolism insynaptic plasma membranes, knock-in models of apo E4 and E3 were generated byreplacing the wild-type apo E by homologous recombination.68 These authors foundthat the apo E4 knock-in mice showed abnormal cholestrol distribution in thesynaptic plasma membrane, as compared to wild-type and Apo E3 knock-in miceand suggested that the pathogenic effects of apo E4 might be mediated by the alteredsynaptic plasma membrane.

To better understand the role of APP in development, native mouse APP wasdeleted from the mouse genome by homologous recombination.69 The resultant “APPnull” mice were viable and fertile, showing mild deficits in locomotor activity andreactive gliosis, indicating a role for APP in normal neuronal function.

Efforts have been successful in developing an AD model system containingmodified APP transgenes that can be induced to express at will in mice. We haveused the tetracycline activator–tetracycline-response element (TRE) system toexpress APP and tau transgenes in our laboratory. In brief, constructs containingmodified APP and tau transgenes were cloned behind the TRE. The constructs werethen microinjected into pronuclei of one-celled embryos and transgenic mice wereproduced. These mice were the transgenic responders.

The activator mice were created by Mayford et al.70 (a kind gift of Dr. EricKandel, Columbia University, New York). The mice expressed the tetracycline trans-activator (tTA) in the forebrain, as dictated by the calcium–calmodulin-dependentkinase II (CaMKII) promoter regulatory elements, behind which the tTA was cloned(CKII-tTA).70 The CKII-tTA fusion ensures that the tTA expression mimics the nativeCKII expression profile in a spatial- and temporal-specific fashion. Under normalcircumstances, the responder gene is silent. However, in mice that are doubly trans-genic for CKII-tTA and TRE-responder (APP or tau), the APP or tau expression, asappropriate, was observed.

As expected, the CKII-tTA/TRE responder-mediated induced gene expressionin the double transgenic mice could be eliminated by addition of tetracycline to thediet or water. Thus, in the CKII-tTA/TRE responder double transgenics, in theabsence of tetracycline, the responder gene was expressed; in the presence of tetra-cycline, the responder gene was repressed. Models such as these will be effectivein answering a variety of questions related to time-specific expression of modifiedAPP and tau transgenes and the consequences.

The quest for creating a “complete” model for AD is ongoing. Ideally, such amodel would develop senile plaques and neurofibrillary tangles in a time-dependentfashion and show time-dependent cognitive decline, similar to conditions observed

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in humans. Such a model does not exist. However, attempts have been made tocombine available transgenic lines containing mutations in APP, PS1 and tau genesby conventional breeding. Some of these lines are very promising.

In the course of breeding mice carrying the tauP301L mutation and Tg2576, wehave shown that mice doubly transgenic for tauP301L and APPK670N, M671L exhibitamyloid plaques and neurofibrillary tangles (data not shown). Bringing transgenestogether by conventional breeding is limited by lack of maintenable homozygosity(due to insertional mutations from the transgene), strain effects on the transgene,etc. An ingenious approach to creating a closer-to-real model was taken by Oddoet al.71 They first generated a PS1 knock-in mutant transgenic mouse (PS1M146V) andthen microinjected APPK670N, M671L and tauP301L transgenes into single-cell embryosderived from the homozygous PS1M146V mice. They were able to show that amyloiddeposition precedes tangle formation in this triple transgenic model.72 In the yet-to-be developed “ultimate model,” tau pathology should be generated from wild-typetau since mutations in tau are not associated with AD.73


13.2.1 STEPS IN GENERATING APP TRANSGENIC MICE BY MICROINJECTION

13.2.1.1 Strain Selection

It is conceivable that the expression of a transgene and its behavior in one strainbackground may be different from the expression in another strain because differenthost strains contain different modifier alleles that may interact with the transgeneor the gene at the point of insertion. This effect is true in the case of mice expressingAPP transgenes as well. Our laboratory generated Tg(HuAPP695.SWE)2576 miceby microinjecting C57B6j × SJL F2 eggs.60 The Tg2576 transgene array could notbe transferred onto the C57B6j inbred background because the proportion of micedying prematurely increased and the fraction of transgene-positive mice that wereweaned fell significantly below the expected 50% as the percentage of C57B6j-derived alleles increased. 74

Interestingly, when these mice were crossed with F1 hybrids of C57B6j x SJL,the fraction of mice living long term increased, indicating that the SJL-derived allelesprotected against the lethal effects of APP overexpression. Similarly, when APPtransgenes were expressed in FVB/N mice, premature death was usually precededby a variety of neurologic signs including neophobia and thigmotaxic behavior.34 Itwas later shown that the influence of FVB/N-derived alleles was enough to causebehavioral abnormalities when a transgene generated in the C57B6j x SJL mice wascrossed to FVB/N mice.60

Concentrations of APP that produce amyloid plaques in outbred transgenic lineswere lethal for inbred FVB/N or C57B6j mice. From our experience, we chose to createtransgenic mice that overexpress APP by microinjecting C57B6j x SJL F2 embryos. Itshould also be noted that because of the relatively poor reproductive performance ofinbred mice, the production of fertilized eggs and the generation of transgenic miceand their subsequent breeding are more efficient when F2 zygotes are used for micro-injection.75 However, we have successfully used FVB/N mice for generation of TRE

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responder transgenic mice. FVB/N mice offer the advantages of being an inbred strainwith relatively large sized pronuclei in fertilized eggs, being fairly resistant to lysisduring injection and exhibiting good reproductive characteristics.76

13.2.1.2 Isolation of Target DNA from Bacterial Host

Isolation of DNA for the purposes of microinjection is a critical step. Usually thesource of the DNA is plasmid DNA and occasionally, cosmid DNA. Since the dissolvedDNA is injected ultimately into a viable zygote, the DNA should not contain anychemical or compound that will be toxic to the early zygote and its development.First, it is essential to grow the bacteria harboring the plasmid or the cosmid in theright host. We found that the use of Escherichia coli host strains DH5α and XL-1Blue produce similar results.

However, it should be borne in mind that if special host factors are required formaintenance of the plasmid or cosmid, it may be appropriate to use a special, specifichost. We routinely harvest bacteria after 15 to 16 hours of growth at 37ºC. Storingbacterial pellets at –20ºC does not seem to affect the quality of the isolated DNA.

The purity of DNA and the solution in which the DNA is suspended are ofparamount importance. For this reason, traditional DNA purification methodologiesinvolving cesium chloride gradients or ethidium bromide are not usually performed.Subjecting the DNA to restriction enzyme digestion and DNA sequencing analysiscan readily determine purity of the DNA samples. Pure DNA that is free of proteinsand other contaminants will be readily cut with restriction enzymes and may bereadily sequenced with standard methods.

Our laboratory has successfully used commercially made kits (QIAGEN;http:www.qiagen.com) that employ modified alkaline lysis procedures to isolate pureDNA. In brief, bacterial cell lysis is achieved, followed by binding the DNA to acolumn and eluting the DNA. The quality of DNA can be cross-checked by effectiverestriction analysis and/or the amenability of the DNA to sequencing. In addition,in our laboratory, we have obtained better results with kits that yield endotoxin-freeDNA. In protocols for endotoxin-free DNA, the additional step of entoxin removalmust be performed before the DNA is bound to the column for elution.

What is the biggest construct size one can use to generate transgenic mice? Theanswer: as big as anyone can make it. Precedents in the literature document stableintegration of a 50-kb bacteriophage λ clone,77 a 60-kb cosmid insert,78 and a 70-kbfragment produced by in vitro ligation of two cosmid inserts.79 As in yeasts,coinjection of large sequences with overlapping homologous regions allows homol-ogous recombination to effectively reconstruct the linear sequence.80 In addition,yeast artificial chromosomes that contain 300 to 400 kb of DNA have also beensuccessfully injected to produce transgenic mice by pronuclear injection.81 We suc-cessfully generated several lines of transgenic mice by injecting cosmid DNAs upto 50 kb in size.

Whether introns are necessary for favorable expression of the desired geneproduct is not clear. It has been shown that the levels of gene expression from cDNA-based constructs are significantly lower than those obtained with genomic sequences,

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which includes introns and exons.82 It has been suggested that enhancers in theintronic sequences may be one reason for this observation. However, it has also beennoted that the addition of heterologous introns to cDNA-based constructs can yieldincreases in gene expression levels.44,83

13.2.1.3 Purification of DNA for Microinjection

For microinjection into fertilized oocytes, plasmid or cosmid DNAs are furtherprocessed to generate specific DNA fragments without any native bacterial DNAvector sequences. Although prokaryotic cloning vector sequences have no apparenteffects on the integration frequency of microinjected genes, it is important to notethat they can severely inhibit the expression of eukaryotic genes introduced into themouse germline.84–87

Brinster et al.75 showed that both linear and supercoiled DNAs are capable ofintegration into the mouse genome; however, linear DNA integrated at a greaterfrequency (25% vs. 5%). The specific processing goals include (1) using specificrestriction endonucleases that strictly flank the DNA of interest, (2) separating theDNA fragments on a gel, (3) eluting the DNA fragment from the gel, and(4) reconcentrating the DNA fragment if necessary. Care should be taken to makesure that the DNA fragment of interest does not contain any site for the specificrestriction endonuclease used to remove the bacterial vector sequences, even withinthe intronic regions. Restriction digests are performed per standard conditions;however, the conditions of electrophoresis are modified so that the DNA is notcontaminated with ethidium bromide used in standard gel separation methodologies.

In fact, several modifications are employed in the electrophoresis protocol. First,the appropriate percentage agarose gel is cast without included ethidium bromide.The gel contains a slab well and at least two normal wells. The bulk of the restrictionenzyme-cut DNA is loaded to the slab well and a small-volume sample is loadednext to a lane of standard molecular weight markers. After appropriate hours ofrunning the gel, the marker and the small-volume sample lanes are cut and separatelystained with ethidium bromide. Once the bands are visualized, they are aligned tothe original gel and the DNA fragment of interest is cut out.

The cut-out DNA fragment still embedded in the gel is placed into treateddialysis tubing containing 0.1X TAE. The DNA fragment is eluted from the agarosegel onto the 0.1X TAE by placing the tubing inside an electrical field, usually thegel electrophoresis chamber. For a starting amount of 20 µg DNA, the gel is run for2 hr. The eluted DNA remains in solution inside the dialysis bag and is thentransferred to a fresh Eppendorf tube. If the eluted volume is too high (greater than2 ml), pure butanol can be used to reduce the aqueous volume.

The eluted DNA in solution is then precipitated by standard techniques usingpotassium acetate and resuspended in 5 mM Tris, 0.1 mM EDTA, pH 7.4. Theconcentration of the eluted DNA can be quantitatively estimated by measuring theoptical density of the resultant solution at 260 nm and 280 nm. Alternatively,comparison of band intensities to similar-sized λ-Hind III fragments of knownquantity can be done to estimate the quantity of the eluted DNA fragment.

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13.2.1.4 Preparation of DNA for Microinjection

DNA samples for microinjection should be free of contaminants such as traces ofphenol, ethanol or other proteinaceous debris. When the concentration of the DNAsolution containing the fragment of interest is established, the DNA is diluted to aconcentration of 4 µg/ml in 5 mM Tris, 0.1 mM EDTA, pH 7.4. For smaller constructs(<10 kb), we use a concentration of 2 µg/ml. After dilution, we have found it usefulto centrifuge the solution in a table-top device at full speed (13,000 rpm) for 30 min.

This approach helps prevent clogged needles during injection. If necessary, theDNA sample may also be run through a 0.2-µm filter. We have also found it usefulto ensure that the DNA to be microinjected is fresh — injected on the day ofreconstitution. Such DNA can be injected without problems for at least a week inthe microinjection facility as long as it is stored at 4ºC. In general, concentrationsabove 1 µg/ml75 or 1 to 3 µg/ml of linear DNA yield a DNA integration efficiencyof 20 to 40%. We have obtained an integration efficiency of about 20%.

13.2.1.5 Microinjection with Admixed DNAs

It is known that DNA injected into the nucleus is incorporated into the native mousegenome. Therefore, in theory, two distinct DNA fragments, if injected, should alsobe incorporated into the mouse genome, presumably independent of each other. Itwas reported earlier that two genes mixed and coinjected into mouse eggs generatedtransgenic mice carrying both transgenes.88 It was also noted that both transgenescointegrated at the same site in the genome in a head-to-tail fashion.

We successfully used this principle to generate a mouse containing both APP(Swedish and London [V717I] mutations) and tauP301L mutations. In brief, DNAfragments representing modified APP and modified tau transgenes were coinjectedinto fertilized mouse oocytes. Few of the transgenic mice generated from thesecoinjection studies contained both transgenes. Some had one or the other transgene,as confirmed by Southern blot hybridization (data not shown). By careful breedingand analysis, we have been able to separate these sublines and maintain them.

We were also able to show that in mice containing both transgenes, both trans-genes were expressed, as observed by Western blot analysis using antibodies againstboth APP and tau (data not shown). Successful recovery of multiple transgene miceusing this technique raises the possibility that it may be a faster method for generatingmultiple transgene mice instead of the conventional method of developing individuallines of mice and breeding them. However, the integration sites of the individualtransgenes may be different and the expression levels from these constructs may bedifferent. Given this caveat, gene dosage studies using double transgenics developedin this way may be of lesser value than those developed the conventional way.

Another clever methodology involving knock-in and coinjection strategies toproduce triple transgenic mice containing altered APP, PS1, and tau genes wasdiscussed earlier.71,72

13.2.1.6 Indentification of Founders

Following microinjection, fertilized embryos are implanted into surrogate dams andallowed to deliver. It should be noted that the integration of injected foreign DNA

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into mouse nuclear DNA is a chance event and usually occurs about 10 to 20% ofthe time. Therefore, it is essential to identify the mice that are products of transgenicembryos. Polymerase chain reaction (PCR) with specific primer sets for the transgeneis a sensitive enough test to screen for founders in the progeny.

We routinely use PCR as the first step screen using specific primers that yieldamplification products ranging between 200 and 1000 base pairs in size. To controlfor genomic DNA quality and amplification conditions, we also amplify a region ofthe mouse prion protein gene using specific primers in the same PCR reactionmixture. PCR-positive founders are subjected to maintenance breeding and analysisas warranted.

PCR is a very sensitive test and utmost care should be taken while preparingthe DNA and reagents and setting up the reactions. Standard precautions to avoidcross-contamination should be used. It is useful to include a reaction without DNAas a negative control every time a PCR screening is performed. PCR-positivefounders are further subjected to slot blot hybridization (to rule out false positivesfrom the PCR screen) and Southern blot hybridization (to determine the integrity ofthe inserted transgene).

We routinely perform copy number estimation by slot blot hybridization on DNAderived from progeny of founders. This step is necessary since it is possible that thefounder may have more than one insertion point, and might carry more than onearray of transgene in those locations. If indeed this is the case, the progeny willshow bands of varied intensities on a slot blot or bands of differing sizes on aSouthern blot, implying segregation of variable copy number transgenes in multiplelocations. We occasionally encountered situations where the transgene inserted inup to three locations and serial breeding to isolate stable sublines was necessary.

13.2.1.7 Maintenance and Analysis of Founders

After a founder is established, it is mated with mice of the opposite sex (of thedesired strain and background) for maintenance breeding. We usually combine twoor three females with one male for mating. The mice are allowed for mate for 1 or2 weeks. The pregnant females are separately housed and allowed to give birth. Thelitter is weaned at about 3 weeks of age, separated according to sex and housedappropriately. At weaning, the distal portions of their tails are snipped and collectedto provide material for DNA extraction for analysis. DNA collected from all theprogeny is subjected to PCR screening with specific primers to identify transgenicand the nontransgenic mice.

DNA from mice to be used for specific experiments including behavioral, RNA,and protein analysis is further subjected to slot blot hybridization to ensure thepresence or absence of the transgene. This step is necessary because PCR is sosensitive that false positives must be ruled out. Specific sublines identified andisolated from multiple-insert founders are typically followed by Southern blothybridization with appropriate probes after appropriate restriction enzyme digestionand electrophoresis.

Specific consideration should be given to experiments involving behavioralanalysis where large balanced groups of transgenic and nontransgenic animals are

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required. Specific matings should ensure that approximately equal numbers of sex-and age-matched transgenic and nontransgenic animals are born. While planning forbehavioral experiments and calculating numbers of animals required, it is necessaryto provide concessions for unexpected smaller litter size, lethality, and infertility.

In the case of mice that carry regulatable transgenes, the activator line (CK-tTAmice) and the responder line (various transgenes downstream of TRE) are maintainedseparately and the transgene status is followed by performing PCR on DNA samplesfrom these mice. When necessary, these lines are bred to each other to generate“double transgenic” mice. Ultimately, the transgene status of each mouse involvedin the study is confirmed by slot blot hybridization.

13.3 CONCLUDING REMARKS

A concerted, multidisciplinary, multifaceted approach involving genetics, molecularpathology, cell biology, biochemistry, neurophysiology, and pharmacology is nec-essary to understand complex diseases such as AD. The ability to create transgenicmouse models of AD has become a powerful tool for researchers aiming to under-stand the pathogenesis, development, prevention and treatment of AD. Several trans-genic AD models with specific characteristics exist and several newer models arebeing generated via novel methodologies.

The ability to breed transgenic AD mice that develop amyloid plaques, neu-rofibrillary tangles and age-dependent cognitive decline will undoubtedly allowresearchers to test strategies to prevent amyloid plaque and neurofibrillary tangleformation and ultimately identify agents that prevent, delay or treat cognitive decline.Transgenic AD mice can also be useful as traditional genetic screens to identifygenes that may be involved in modulating the manifestations of AD. Transgenicmice expressing APP and other genes related to AD will enable scientists to posequestions regarding the pathogenesis of AD and test relevant hypotheses relating tothe memory dysfunction associated with AD. Exciting years lie ahead, when thesweet fruits of the hard labor involved in generating these transgenic AD mice willbe realized.

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27. Janus, C., Chishti, M.A., and Westaway, D. Transgenic mouse models of Alzheimer’sdisease, Biochim. Biophys. Acta, 1502, 63, 2000.

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28. Maguire-Zeiss, K.A. and Federoff, H.J. Convergent pathobiologic model of Parkin-son’s disease, Ann. NY Acad. Sci., 991, 152, 2003.

29. Sathasivam, K. et al. Transgenic models of Huntington’s disease, Philos. Trans. R.Soc. Lond. B Biol. Sci., 354, 963, 1999.

30. Bates, G. Huntington aggregation and toxicity in Huntington’s disease, Lancet, 361,1642, 2003.

31. Shibata, N. Transgenic mouse model for familial amyotrophic lateral sclerosis withsuperoxide dismutase-1 mutation, Neuropathology, 21, 82, 2001.

32. Jankowsky, J.L. et al. Transgenic mouse models of neurodegenerative disease: oppor-tunities for therapeutic development, Curr. Neurol. Neurosci. Rep., 2, 457, 2002.

33. Wong, P.C. et al. Genetically engineered mouse models of neurodegenerative diseases,Nat. Neurosci., 5, 633, 2002.

34. Hsiao, K.K. et al. Age-related CNS disorder and early death in transgenic FVB/Nmice overexpressing Alzheimer amyloid precursor proteins, Neuron, 15, 1203, 1995.

35. Evans, M.J. and Kaufman, M.H. Establishment in culture of pluripotential cells frommouse embryos, Nature, 292, 154, 1981.

36. Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos culturedin medium conditioned by teratocarcinoma stem cells, Proc. Natl. Acad. Sci. USA,78, 7634, 1981.

37. Robertson, E. et al. Germ-line transmission of genes introduced into cultured pluri-potential cells by retroviral vector, Nature, 323, 445, 1986.

38. Isola, L.M. and Gordon, J.W. Transgenic animals: a new era in developmental biologyand medicine, Biotechnology, 16, 3, 1991.

39. Balling, R. et al. Craniofacial abnormalities induced by ectopic expression of thehomeobox gene Hox-1.1 in transgenic mice, Cell, 58, 337, 1989.

40. Beddington, R.S. et al. An in situ transgenic enzyme marker for the midgestationmouse embryo and the visualization of inner cell mass clones during early organo-genesis, Development, 106, 37, 1989.

41. Palmiter, R.D. et al. Metallothionein–human GH fusion genes stimulate growth ofmice, Science, 222, 809, 1983.

42. Iwamoto, T. et al. Aberrant melanogenesis and melanocytic tumour development intransgenic mice that carry a metallothionein/ret fusion gene, EMBO J., 10, 3167, 1991.

43. Mehtali, M., LeMeur, M., and Lathe, R. The methylation-free status of a housekeepingtransgene is lost at high copy number, Gene, 91, 179, 1990.

44. Choi, T. et al. A generic intron increases gene expression in transgenic mice, Mol.Cell Biol., 11, 3070, 1991.

45. O’Gorman, S., Fox, D.T., and Wahl, G.M. Recombinase-mediated gene activationand site-specific integration in mammalian cells, Science, 251, 1351, 1991.

46. Lasko, P.F. Molecular movements in oocyte patterning and pole cell differentiation,Bioessays, 14, 507, 1992.

47. Kang, J. et al. The precursor of Alzheimer’s disease amyloid A4 protein resemblesa cell-surface receptor, Nature, 325, 733, 1987.

48. De Strooper, B. and Annaert, W. Proteolytic processing and cell biological functionsof the amyloid precursor protein, J. Cell Sci., 113 (Pt 11), 1857, 2000.

49. Tang, K. et al. Identification of a novel alternative splicing isoform of human amyloidprecursor protein gene, APP639, Eur. J. Neurosci., 18, 102, 2003.

50. Golde, T.E. et al. Expression of beta amyloid protein precursor mRNAs: recognitionof a novel alternatively spliced form and quantitation in Alzheimer’s disease usingPCR, Neuron, 4, 253, 1990.

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51. Sandbrink, R., Masters, C.L., and Beyreuther, K. Beta A4-amyloid protein precursormRNA isoforms without exon 15 are ubiquitously expressed in rat tissues includingbrain, but not in neurons, J. Biol. Chem., 269, 1510, 1994.

52. Clark, M.J. et al. MRC OX-2 antigen: a lymphoid/neuronal membrane glycoproteinwith a structure like a single immunoglobulin light chain, EMBO J., 4, 113, 1985.

53. Rockenstein, E. et al. The neuroprotective effects of Cerebrolysin™ in a transgenicmodel of Alzheimer’s disease are associated with improved behavioral performance,J. Neural Trans., 110, 1313, 2003.

54. Quon, D. et al. Formation of beta-amyloid protein deposits in brains of transgenicmice, Nature, 352, 239, 1991.

55. Moran, P.M. et al. Age-related learning deficits in transgenic mice expressing the751-amino acid isoform of human beta-amyloid precursor protein, Proc. Natl. Acad.Sci. USA, 92, 5341, 1995.

56. Higgins, L.S. et al. Early Alzheimer disease-like histopathology increases in fre-quency with age in mice transgenic for beta-APP751, Proc. Natl. Acad. Sci. USA,92, 4402, 1995.

57. Games, D. et al. Alzheimer-type neuropathology in transgenic mice overexpressingV717F beta-amyloid precursor protein, Nature, 373, 523, 1995.

58. Irizarry, M.C. et al. Abeta deposition is associated with neuropil changes, but notwith overt neuronal loss in the human amyloid precursor protein V717F (PDAPP)transgenic mouse, J. Neurosci., 17, 7053, 1997.

59. Irizarry, M.C. et al. APPSw transgenic mice develop age-related A beta deposits andneuropil abnormalities, but no neuronal loss in CA1, J. Neuropathol. Exp. Neurol.,56, 965, 1997.

60. Hsiao, K. et al. Correlative memory deficits, Abeta elevation, and amyloid plaquesin transgenic mice, Science, 274, 99, 1996.

61. Westerman, M.A. et al. The relationship between Abeta and memory in the Tg2576mouse model of Alzheimer’s disease, J. Neurosci., 22, 1858, 2002.

62. Andra, K. et al. Expression of APP in transgenic mice: a comparison of neuron-specific promoters, Neurobiol. Aging, 17, 183, 1996.

63. Chishti, M.A. et al. Early-onset amyloid deposition and cognitive deficits in transgenicmice expressing a double mutant form of amyloid precursor protein 695, J. Biol.Chem., 276, 21562, 2001.

64. Kulnane, L.S. and Lamb, B.T. Neuropathological characterization of mutant amyloidprecursor protein yeast artificial chromosome transgenic mice, Neurobiol. Dis., 8,982, 2001.

65. Aguzzi, A. et al. Transgenic and knock-out mice: models of neurological disease,Brain Pathol., 4, 3, 1994.

66. Guo, Q. et al. Increased vulnerability of hippocampal neurons to excitotoxic necrosisin presenilin-1 mutant knock-in mice, Nat. Med., 5, 101, 1999.

67. Siman, R. et al. Presenilin-1 P264L knock-in mutation: differential effects on abetaproduction, amyloid deposition, and neuronal vulnerability, J. Neurosci., 20, 8717,2000.

68. Hayashi, H. et al. Cholesterol is increased in the exofacial leaflet of synaptic plasmamembranes of human apolipoprotein E4 knock-in mice, Neuroreport, 13, 383, 2002.

69. Zheng, H. et al. Beta-amyloid precursor protein-deficient mice show reactive gliosisand decreased locomotor activity, Cell, 81, 525, 1995.

70. Mayford, M. et al. Control of memory formation through regulated expression of aCaMKII transgene, Science, 274, 1678, 1996.

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71. Oddo, S. et al. Triple-transgenic model of Alzheimer’s disease with plaques andtangles: intracellular Abeta and synaptic dysfunction, Neuron, 39, 409, 2003.

72. Oddo, S. et al. Amyloid deposition precedes tangle formation in a triple transgenicmodel of Alzheimer’s disease, Neurobiol. Aging, 24, 1063, 2003.

73. Andorfer, C. et al. Hyperphosphorylation and aggregation of tau in mice expressingnormal human tau isoforms, J. Neurochem., 86, 582, 2003.

74. Carlson, G.A. et al. Genetic modification of the phenotypes produced by amyloidprecursor protein overexpression in transgenic mice, Hum. Mol. Genet., 6, 1951, 1997.

75. Brinster, R.L. et al. Factors affecting the efficiency of introducing foreign DNA intomice by microinjecting eggs, Proc. Natl. Acad. Sci. USA, 82, 4438, 1985.

76. Taketo, M. et al. FVB/N: an inbred mouse strain preferable for transgenic analyses,Proc. Natl. Acad. Sci. USA, 88, 2065, 1991.

77. Costantini, F. and Lacy, E. Introduction of a rabbit beta-globin gene into the mousegerm line, Nature, 294, 92, 1981.

78. Taylor, L.D. et al. A transgenic mouse that expresses a diversity of human sequenceheavy and light chain immunoglobulins, Nucleic Acids Res., 20, 6287, 1992.

79. Strouboulis, J., Dillon, N., and Grosveld, F. Developmental regulation of a complete70-kb human beta-globin locus in transgenic mice, Genes Dev., 6, 1857, 1992.

80. Pieper, F.R. et al. Efficient generation of functional transgenes by homologous recom-bination in murine zygotes, Nucleic Acids Res., 20, 1259, 1992.

81. Choi, T.K. et al. Transgenic mice containing a human heavy chain immunoglobulingene fragment cloned in a yeast artificial chromosome, Nat. Genet., 4, 117, 1993.

82. Brinster, R.L. et al. Introns increase transcriptional efficiency in transgenic mice,Proc. Natl. Acad. Sci. USA, 85, 836, 1988.

83. Palmiter, R.D. et al. Heterologous introns can enhance expression of transgenes inmice, Proc. Natl. Acad. Sci. USA, 88, 478, 1991.

84. Chada, K. et al. Specific expression of a foreign beta-globin gene in erythroid cellsof transgenic mice, Nature, 314, 377, 1985.

85. Krumlauf, R., Hammer, R.E., Tilghman, S.M., and Brinster, R.L. Developmentalregulation of alpha-fetoprotein genes in transgenic mice, Mol. Cell Biol., 5, 1639,1985.

86. Townes, T.M. et al. Erythroid-specific expression of human beta-globin genes intransgenic mice, EMBO J., 4, 1715, 1985.

87. Hammer, R.E. et al. Diversity of alpha-fetoprotein gene expression in mice is gen-erated by a combination of separate enhancer elements, Science, 235, 53, 1987.

88. Behringer, R.R. et al. Synthesis of functional human hemoglobin in transgenic mice,Science, 245, 971, 1989.

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14
Generation of Amyloid Precursor Protein Knockout Mice
Hui Zheng

CONTENTS

14.1 Introduction14.2 Experimental Procedures

14.2.1 Construction of APP Gene Targeting Vector14.2.2 Embryonic Stem Cells: Culturing and Maintenance

14.2.2.1 Growing SNL Cells and Preparation of Feeders14.2.2.2 Culturing and Electroporating Embryonic Stem Cells

14.2.3 Selection of Homologous Recombinant Clones14.2.3.1 Selection of Recombinant Colonies14.2.3.2 Expansion and Duplication of Recombinant Clones14.2.3.3 Freezing ES Cells in 96-Well Plates14.2.3.4 Mini-Southern Analysis of ES Clones to Identify Gene

Targeting Events14.2.3.5 Preparation of Gene-Targeted Clones for Blastocyst

Injection14.2.4 Generation of Gene (APP) Knockout Mice

14.2.4.1 Microinjection, Assessment of Chimerism, and Test forGermline Transmission

14.2.4.2 Breeding and Generation of Homozygous APP Knockout Mice


14.1 INTRODUCTION

Alzheimer’s disease (AD) is the most common cause of dementia in the agedpopulation. It is characterized pathologically by the deposition of β-amyloid plaques,the accumulation of neurofibrillary tangles and the losses of neurons and synapsesin selected areas of the brain. Senile plaques are extracellular deposits of heteroge-neous substances of which the major components are the 40 to 42 amino acid

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peptides referred to as β-amyloid peptides (Aβ) derived by proteolytic cleavage ofthe amyloid precursor protein (APP). Approximately 10% of AD cases are familialand cosegregate with autosomal dominant inheritance of mutations in genes includ-ing APP,1 presenilin 1 (PS1),2 and the PS1 homolog, presenilin 2 (PS2).3

APP is an integral membrane glycoprotein consisting of an extracellular domain,a single transmembrane domain and a short cytoplasmic tail. About two-thirds ofthe Aβ peptide is extracellular and the remaining sequence is embedded in themembrane. The APP gene located on the long arm of chromosome 21 spans approx-imately 400 kb and contains at least 18 exons.4,5 Alternative splicing generates APPmRNAs encoding several isoforms that range from 365 to 770 amino acid residues.The major Aβ peptide-encoding proteins are 695, 751, and 770 amino acids (referredto as APP695, APP751, and APP770). APP751 and APP770 contain domains homol-ogous to the Kunitz-type serine protease inhibitors (KPIs) and are expressed in mosttissues examined. The APP695 isoform lacks the KPI domain and is predominantlyexpressed in neurons.

APP is processed by at least three proteinases termed α-, β-, and γ-secretases.α-Secretase cleavage occurs inside the Aβ domain, thus precluding intact Aβ peptideformation. The identity of the α-secretase has not been unambiguously establishedbut the ADAM family of metalloproteases known as ADAM 10 and 17 (the latteris also known as TACE) has been reported to exhibit the activities.6,7 β-Secretase(BACE1) cleaves APP at the amino terminus of Aβ. The enzyme has been clonedand its characteristics extensively studied.8

These processing events generate large soluble APP derivatives (called APPsαand APPsβ, respectively). Following the extracellular cleavages, γ-secretase pro-cesses APP at the carboxyl-terminus of Aβ to produce either a 3-kDa product (p3in combination with the α-secretase) or Aβ (in concert with BACE1 cleavage),respectively. The γ-secretase activity seems to be executed by a high molecularweight complex of which presenilin (PS1 or PS2) is an essential component.9 In additionto the γ-cleavage that yields Aβ40 and Aβ42, presenilin-dependent proteolysis alsooccurs at the Leu 49–Val 50 position (counted from Aβ) downstream of the γ-siteand near the membrane intracellular boundary (termed ε-site). This ε-cleavagereleases an APP intracellular domain (AICD).9,10

Because of the central role of APP in AD pathogenesis, the expression patterns,biochemical properties, and potential functions of APP have been the subjects ofintensive studies in vitro. Within the nervous system, APP is present on cell surfacesand in axons, dendrites, and vesicles.11 It undergoes rapid anterograde transport12–14

and is targeted to the synaptic sites of both central and peripheral nervous sys-tems.15–17 The major activities reported include vesicular transport, transcriptionalregulation, synaptogenesis, trophic activity, and cell motility.9

The extracellular domain of APP has been proposed to exhibit neurotrophic,synaptogenic, and growth-promoting properties.18–21 Two distinct activities of theAPP intracellular domain (AICD) have been reported. First, it binds to kinesinmolecular motors and has been shown to regulate axonal transport of prepackagedvesicles.22–24 Second, the APP IC interacts with various adaptor proteins includingFe65, X11, and mDAB1.9 Following presenilin-dependent intramembrane proteol-ysis, AICD can be released, and the AICD/Fe65 complex can translocate to the

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nucleus25,26 where, in concert with the histone acetyltransferase Tip60, it functionsas a potent transcriptional regulator.27 This processing and signaling mechanism isanalogous to the Notch signaling pathway28 and several potential downstream targetshave been proposed.29–31

Several disease-causing missense mutations in the human APP gene have beenidentified.32 Among these, the KM-to-NL double (Swedish or APPsw) mutation ispresent immediately upstream of the BACE cleavage site and seems to enhance theBACE1 cleavage to increase both Aβ40 and Aβ42 levels. The Val mutation down-stream of the γ-secretase cleavage site results in a specific increase of the Aβ42peptide.33,34

These mutations apparently affect the γ-secretase activity that absolutely requiresPS1 and PS2.35–37 Consistent with this activity, mutations in presenilins lead to aspecific increase in Aβ42 production,38 although the molecular mechanism of thisapparent “gain-of-misfunction” effect is not well understood. Interestingly, thePS1L166P familial AD mutation results in an increase in Aβ42 (γ-cleavage) and adecrease in AICD (ε-cleavage).10 Likewise, the same mutation leads to impairedNotch intracellular domain (NICD) production.10 The effect of the presenilin ADmutation may be partial loss of function with respect to the signaling pathways ofNotch and, potentially, APP.

To understand the in vivo function of APP and its processing products, wegenerated an APP null mutation in mice using gene targeting in embryonic stem(ES) cells.39 This technology was developed based on (1) the successful isolation ofpermanent in vitro ES cell lines from preimplantation blastocyst stage embryos40;(2) documentation indicating that these cell lines, after sustained in vitro manipula-tion and growth, were capable of recolonizing embryos and contributing to thegermlines41; and (3) a demonstration that many mammalian cells42 including ES43

have a remarkable ability that allows transfected foreign DNA to locate and recom-bine with its homologous chromosomal counterpart.42,43

This process, referred to as homologous recombination or gene targeting, allowsvirtually any genetic modification of endogenous genes such as inactivation of agene of interest (gene knock-out), subtle and precise genetic modification (geneknock-in) and large chromosomal deletions (chromosome engineering).44 This chap-ter discusses only procedures and considerations related to the generation of theAPP knockout mice. Readers interested in general aspects of gene targeting tech-nology are referred to reviews in References 45 through 48.


The generation of APP knockout mice requires the following steps:

1. Construct an APP gene targeting vector that, upon homologous recombi-nation into the endogenous APP locus, can completely inactivate the gene.

2. Introduce the vector into ES cells and identify ES clones with the disruptedAPP gene.

3. Microinject the targeted APP clones into mouse blastocyst stage embryos,transplant the injected embryos into a foster mother, and bring them to

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term to generate chimeric mice with contributions from both donor EScells and host embryos.

4. Identify and breed germline chimeras capable of transmitting the APP-targeted allele to their offspring. The offspring will contain one copy ofthe wild-type allele and one copy of the APP-targeted allele and are calledAPP heterozygous (APP+/–) mice.

5. Interbreed the APP heterozygous mice to create animals that are eitherwild-type (APP+/+), APP+/–, or homozygous for the APP mutant allele(APP–/–).

These procedures are illustrated in Figure 14.1 and described below.

14.2.1 CONSTRUCTION OF APP GENE TARGETING VECTOR

A typical gene targeting vector of replacement type consists of two segments ofsequences homologous to the endogenous gene. The genomic sequences betweenthe two segments that encode essential functional units of the gene are deleted andreplaced by a selectable marker. The most common marker is the neomycin resis-tance gene (neo). Selection of transfected cells with geneticin antibiotic (G418)allows identification of total integration events, including both homologous recom-bination and random integration. In most circumstances, random integration is thepredominant event.

Many factors can influence the ratio of homologous recombination to randomintegration, for example, target locus, length of homology, and degree of polymor-phism between the vector sequence and the endogenous chromosomal DNA. As ageneral rule, given a target locus, the longer the homologous sequence, the higherthe targeting frequency.

To minimize polymorphic variations and optimize homologous recombination,the DNA in the targeting vector should be isolated from the same strain as that ofthe ES cells. In addition to positive selection using the neomycin resistance gene, anegative selectable gene, HSV-tk, can be added at the end of the linearized targetingvector. Incorporation of HSV-tk by random integration, not homologous recombi-nation (lost due to homologous crossover, Figure 14.2a), renders the cells sensitiveto antiviral agents 1-(2′-deoxy, 2′-fluoro-β-δ-arabinofuranosyl)-5-iodouracil (FIAU)and gancyclovir and, as a result, enriches gene-targeting events.49

Inactivation of the 400-kb APP gene which undergoes alternative splicingrequires a careful design of the targeting vector to ensure that an APP null allele iscreated. We chose to delete the APP promoter and first exon on the assumption thatthe mutation would prevent APP gene transcription. If an alternative promoter andATG initiation codon were used, transport of the protein to the membrane shouldhave been impaired because the signal peptide would have been deleted.

Using this rationale and the general gene-targeting principle described above,we used a 1.0-kb HindIII–PvuII fragment of the mouse APP promoter50 as a probe, andscreened a genomic-phage library made from the 129Sv strain (the strain AB2.1 EScells are derived from) of DNA. We isolated the APP promoter and surrounding regions

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and constructed an APP targeting vector by ligating a 1.4-kb BglII–XhoI fragmentfrom the 5′ portion of the APP promoter and a 7.1-kb XhoI–BglII fragment fromthe 3′ portion of exon 1. A 1.5-kb XhoI–SalI PGK–neo cassette was inserted betweenthe segments, deleting a 3.8-kb XhoI–XhoI fragment encoding the APP promoter andthe first exon. The PGK–neo cassette was inserted in an opposite orientation to avoidpotential transcriptional read-through to the APP sequence by the PGK promoter.

FIGURE 14.1 Gene targeting outline. (1) A gene-targeting construct (double helix) con-taining a genetic modification and a positive selectable marker (asterisk) is introduced intomouse embryonic stem (ES) cells by electroporation. (2) Cells that have integrated the gene-targeting construct are resistant to drug selection due to the presence of the positive selectablemarker in the genome. These cells form colonies (circles). Clones that result from accuraterecombination at the target locus are recognized by Southern hybridization or PCR.(3) Targeted clones are injected into developing embryos at the blastocyst stage and embryosare implanted into the uteri of surrogate mothers. Mice that have contributions from thegenetically modified ES cells are recognized by their chimeric coat colors because theblastocysts are derived from black mice (light circles), whereas the ES cells used for targetingare derived from a pigmented agouti mouse (dark circles). (4) Mouse strains are establishedby mating chimeras to black C57BL/6 mice. Germline transmission results in offspring thatare agouti color consisting of one half wild-type mice and the other half targeted, heterozygousmice. (5) Heterozygous APP+/– mice are interbred to produce mice that are wild-type (APP+/+),heterozygous (APP+/–), or homozygous (APP–/–) for the targeted APP allele. (Reprinted fromMills, A.A. and Bradley, A. Trends Genet., 17, 331, 2001. With permission from Elsevier.)

APP+/-X APP+/-

APP+/+APP+/-APP/-

1)

2)

3)

4)

5)

Electroporation

Selection

Blastocyst injection

Chimera

Targeted progeny Wild-type progeny

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The final vector was made by ligating a 2-kb XhoI fragment of pMC–TK to theend of the 7.1-kb homologous sequence (Figure 14.2a). The targeting vector waslinearized by digestion with NotI prior to electroporating into ES cells.

In addition to the DNA sequences used for vector construction, at least one andpreferably two DNA fragments of 300 to 1000 bp outside the vector should beisolated and tested against a genomic blot to make sure that they are free of repetitivesequences. They will be used as probes to screen for gene-targeting events fromtotal recombinant clones by Southern blot analysis. Because the probe does nothybridize to the vector DNA, only the endogenous allele, either wild-type or dis-rupted by the vector, will be recognized on a Southern blot.

To properly identify the gene-targeted clones, a restriction digestion schemeshould be in place to distinguish the wild-type and targeted alleles upon hybridizationwith the probe. For targeting the APP locus, we used EcoRI digestion becauseintroduction of a new EcoRI site by PGK–neo insertion alters the restriction patternsof the targeted allele (Figure 14.2).

FIGURE 14.2 Targeted disruption of the APP gene in ES cells. (a) The targeting vectorcontains the following, from left to right: a 1.4-kb segment preceding the APP promoter(hatched 5′ rectangle); a PGK–neo expression cassette inserted in an orientation opposite thatof the APP gene; a fragment of 7.1 kb homologous to the first intron of the APP gene (hatched3′ rectangle); and a HSV-tk gene (closed box) for negative selection with FIAU. Probes used(black boxes below physical map) for detecting targeted events are 1.0-kb XbaI-BglII (5′)and 0.8-kb BglII-NcoI (3′) probes. Digestion with EcoRI is used to separate the wild-typeand targeted APP alleles. R = EcoRI. X = XbaI. B = BglII. N = NcoI. Pr = mouse APPpromoter. E1 = exon 1 of the mouse APP gene. (b) Southern blot analysis of representativetargeted clones (76, 123, 174, 196) using the 5′-probe. AB2.1 wild-type ES cells.

PGKneo

R

HSV-tk

RR R

Homologous Recombination

PGKneo

R

B NX B

R RB NX B

ATG

9.0 kb 9.5 kb

9.0 kb6.5 kb

Targeting Vector

Wild-type APP Locus

Targeted APP Locus

a.

b.kbAB2.1

76 123174

196

Pr E1

9.0

6.5

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Hybridization with the 5′-probe creates a 9.0-kb wild-type band and a 6.5-kbtargeted band that can be separated easily and distinguished by Southern blotting.The homologous recombination event can be further confirmed using the 3′-probethat yields 9.5- and 9.0-kb fragments by the wild-type and targeted alleles, respec-tively (not shown).

14.2.2 EMBRYONIC STEM CELLS: CULTURING AND MAINTENANCE

The key requirement to successful generation of gene knockout mice is that boththe parental and targeted ES clones retain their totipotencies to contribute to thesomatic lineage and germline following microinjection into host embryos. Progres-sive culturing and cloning of ES cells — necessary procedures for gene targetingexperiments — may produce ES cells that have lost their totipotency but are mor-phologically indistinguishable from normal ES cells. These cells, however, can onlyproduce low-percentage chimeras that are not capable of transmitting through thegermline. Therefore, maintaining ES cells under optimal conditions is extremelyimportant.

The culture of ES cells requires a complex mixture of growth factors providedby fetal bovine serum and mitotically inactivated fibroblast feeder cells. The rightcombination of ES and feeder pair is essential to ensure germline transmission. TheAB1 and AB2.1 ES cells growing on clonal SNL76/7 feeder cell layers representan excellent system (reviewed in Reference 51). Both ES lines are derived from the129Sv strain of blastocysts and the feeder cells are generated by transfecting theSTO fibroblasts with a neomycin marker and leukemia inhibitory factor (LIF, anessential component for keeping ES cells in an undifferentiated state), hence thename SNL. The detailed culture procedure is described below.

14.2.2.1 Growing SNL Cells and Preparation of Feeders

All tissue culture reagents can be purchased from Gibco/Invitrogen (Carlsbad, CA)unless otherwise noted. Disposable plastic ware should be used for culturing andprocessing the cells. Although products from various suppliers can be used, we useCostar plates (Corning, Inc., NY).

SNL medium — 91% dimethyl sulfoxide (DMSO, high glucose, no pyruvate),7% fetal bovine serum (FBS), 1% L-glutamine, 1% antibiotics (penicillin and strep-tomycin mix).

SNL cells — These are easy to culture and fast growing. They can be thawedand cultured under routine tissue culture conditions using the above SNL medium.Split the cells every 3 days at a ratio of 1:8.

Feeder preparation — Add 260 µl of 0.5 mg/ml mitomycin C solution* (Sigma,St. Louis, MO) to a 100-mm dish containing confluent SNL cells in 12 ml medium.Incubate the cells at 37ºC for 3 hr. Coat appropriate dishes where the feeder cellsare to be plated with a 1% gelatin solution. Remove gelatin before plating the feederlayer. Wash the mitomycin C-treated cells twice with PBS. Trypsinize the cells using2.5% trypsin/EDTA at 37oC for 5 min. Spin down and resuspend the cells in SNL

* Add 4 ml PBS to a 2-mg bottle. Block the solution from light. Use within 2 weeks.

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medium. Count the cells and dilute to 3.5 × 105/ml. Plate 12 ml on a 100-mm gelatin-treated dish, 4 ml/60-mm dish, 2 ml/6-well dish, and 0.5 ml/24-well dish. For 96-wellplates, dilute the cells to 1.15 × 105/ml and plate 150 µl/well using a multichannelpipettor.

Feeder Maintenance — The feeder cells can be maintained in a 37ºC incubatorfor at least 5 weeks. Feed the cells with fresh SNL medium once every 2 weeksafter plating. In general, older feeder plates are better than new ones and 3- to4-week-old plates are best. For one gene targeting experiment, 5 × 100 mm and10 × 96-well plates are needed and should be prepared prior to electroporation.Feeder plates of other sizes are used for expanding ES stocks and selected clonesand should be prepared as planned.

14.2.2.2 Culturing and Electroporating Embryonic Stem Cells

ES cell medium — 83% DMSO (high glucose, no pyruvate), 15% FBS, 1%L-glutamine, 1% antibiotics (penicillin and streptomycin mix), 1% β-mercaptoethanol.

Serum testing — Use of high-quality FBS is crucial for optimal culturing ofES cells. The serum should be tested and compared against a control with a suc-cessful track record. Sera from several companies and lots should be tested. We havegood success with HyClone products (Logan, UT) in general.

For serum testing, plate ES cells onto 60-mm feeder plates at 2000 cells/plate.Five plates should be used for each batch of serum: four with 15% serum and onewith 30%. Incubate plates at 37ºC for 10 to 14 days. Use one 15% plate each fordaily examination of colony size and morphology. At the end of incubation, theremaining plates should be washed twice with PBS and stained with methylene bluesolution (2% w/v in 70% EtOH) for 5 min. The remaining plates will be washedonce with 70% EtOH and allowed to dry.

The number of colonies on each plate will be counted. Average the three 15%serum counts for each batch. Compare the 30% and 10% values of each serum lotwith those of the control serum. Select the batch that yields a plating efficiencyequal to or greater than the appropriate control sample and shows no toxicity at30%. A large stock should be purchased to avoid frequent testing. Alternatively, ES-qualified sera are available from several companies. The ones specifically tested forthe AB1/AB2.1-SNL system can be used to culture the ES cells.

ES cell culturing — The derivation, general morphology and growth propertiesof ES cells are illustrated in Chapter 4 of Robertson’s book.52 The doubling time ofES cells is in the range of 18 to 24 hr. The following rules should be followed whenculturing AB1 or AB2.1 ES cells:

1. The cells should grow on a feeder layer with serum-tested ES medium atrelatively high frequency (30 to 80% confluent). They should be splitevery 3 to 5 days. Always feed the cells with fresh medium 2 to 3 hr priorto passaging.

2. The cells have a high tendency to secrete acidic material and color themedium yellow. Extremely acidic pH is harmful to the cells and thereforethe medium should be replaced every 2 days under normal growth con-ditions or whenever the color becomes yellowish. All medium used should

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be orange (pH 7.4). Old, pinkish medium has a pH of 7.6 or higher andshould be avoided.

3. The ES cells grow in patches. To maintain the cells in an undifferentiatedstate, it is extremely important to adequately break them with trypsin. Use2.5% trypsin–EDTA, incubate the cells at 37ºC for 15 min, and pipettethe cells vigorously five to eight times after neutralizing the trypsin withan equal volume of ES medium.

4. Long-term passaging of ES cells will reduce their germline transmissionpotential.

A large stock of ES cells at relatively early passages (below 20) should be frozenin liquid nitrogen. A freshly thawed vial should be used for each gene targetingexperiment.

Freezing and thawing ES cells — The general rule of slow freezing and quickthawing applies. ES cells to be frozen should be grown in log phase and fed 2 to 3hr before trypsinization. Wash the plates twice with PBS. Add 2.5% trypsin–EDTA(1 ml/60-mm dish). Incubate at 37ºC for 15 min. Add an equal volume of ES medium.Pipette cells vigorously five to eight times with a Pasteur pipette. Transfer thesolution to a 15-ml conical tube and spin the cells down using a low speed centrifugeat 1000 rpm for 7 min. Resuspend cells in ES medium at 1 to 2 × 107/ml. Adddropwise an equal volume of 2X freezing medium (50% DMEM, 30% FBS, 20%DMSO) while mixing. Aliquot into 1-ml cryotubes. Place the tubes in a polystyrenebox and cool in a –80ºC freezer overnight. Transfer the vials to liquid nitrogen thenext day.

Thawing — Retrieve a frozen vial from storage and place directly in a 37ºCwater bath until the solution is completely thawed. Sterilize the vial with 70% EtOHand transfer the cells to a 15-ml conical tube containing prewarmed ES medium.Spin cells down; resuspend in 4 ml ES medium and plate onto a 60-mm feeder dish.

Electroporation — The targeting vector (25 µg) should be linearized at oneend by restriction enzyme digestion and resuspended in Tris-EDTA, pH 7.5, at1 µg/µl. Low-passage (<20) ES cells should be grown in log phase and fed 2 to 3 hrprior to electroporation. The cells are trypsinized and resuspended in PBS at 1.2 ×107/ml using the protocol described above. Add DNA to 800 µl of cells and mix byinverting. Transfer the mixture to a Gene Pulser cuvette with a 4-mm electrode gap(Bio-Rad Laboratories, Hercules, CA) and electroporate at 230 V, 500 µF using aBio-Rad Gene Pulser apparatus with a capacitance extender. The pulse time underthis condition is 0.6 to 0.8 msec. Transfer the treated cells to a bottle containing 60ml ES medium. Evenly distribute onto 5 × 100 mm feeder plates at 12 ml/plate.

14.2.3 SELECTION OF HOMOLOGOUS RECOMBINANT CLONES

14.2.3.1 Selection of Recombinant Colonies

Twenty-four hours after electroporation, G418 (200 µg/ml active ingredient, from1000X stock) is added to the plates. If HSV-tk is present in the vector, FIAU isadded at a final concentration of 0.4 µM. Change media every other day for 8 to

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10 days. Nontransfected cells start to die at 4 to 5 days with the appearance of G418-or G418/FIAU-resistant colonies soon afterward.

Under the electroporation conditions described above, we expect the transfectionefficiency to be in the range of 1 × 10–4 to 5 × 10–3. Therefore, with 1 × 107 cellselectroporated, approximately 1000 to 5000 total G418-resistant colonies should bepresent. With FIAU selection, we usually achieve a two- to fivefold enrichment andthus the number of G418/FIAU double-resistant clones is expected to be reducedby two- to fivefold. Most of the colonies should have round shapes and be denselypacked with darker central cores. They should be large enough to be picked 8 to10 days after G418 selection. Prolonged growth of ES colonies leads to differenti-ation and should be avoided. Differentiated cells can be distinguished from normalES cells since they are usually loosely packed and exhibit different morphologies.

14.2.3.2 Expansion and Duplication of Recombinant Clones

The G418- or G418/FIAU-resistant colonies are picked for further expansion andmini-Southern analysis.53 Place 25 µl of 0.25% trypsin–EDTA into each well of around-bottom 96-well plate using a multichannel pipette. Wash plates with coloniestwice with PBS. Leave 1 to 2 ml in plates the second time. Using a 200-µl pipetteset at 20 µl, pick colonies from the washed plates and transfer to each trypsin-containing well. Carefully mark the wells to make sure each well contains only onecolony. Our picking speed is ~100 to 150 colonies/hr. Therefore, when a 96-well iscompleted, enough time has elapsed to allow complete trypsinization and no furtherincubation is necessary.

Multichannel pipettes will be used for most of the procedures from this pointon. One row at a time from earlier-picked colonies, add 25 µl of ES medium perwell and pipette up and down a dozen times to disaggregate the cells. Transfer theentire solution to a 96-well feeder plate. Repeat with another dish. We normally pick400 colonies per experiment to ensure positive identification of a minimum of twoto four targeted clones (assuming a targeting frequency of 1/100). Change to freshES media containing G418 the next day and every other day afterward and allowcolonies to grow (~4 to 6 days). ES cells should be visible under a light microscopeon the third day. The average rate of colony recovery (percentage of wells withmedium color change) is around 70 to 80%.

The ES clones will then be duplicated: one set to gelatin plates for DNA isolationand mini-Southern analysis (see below) and the other set to feeder plates for freezingand storage. Feed the wells 2 to 3 hours before treatment. Wash cells twice withPBS. Add 50 µl 0.25% trypsin–EDTA per well and incubate at 37ºC for 10 min.Add 100 µl ES medium to each well and mix by pipetting up and down about10 times. Evenly transfer the solution to one gelatin-coated plate and one feederplate and fill each well with 150 µl additional ES medium.

14.2.3.3 Freezing ES Cells in 96-Well Plates

The feeder plate will be frozen as the master. Trypsinize the plate as above. Neutralizeand disaggregate the cells with 50 µl ES medium. Add dropwise 100 µl 2X freezing

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medium (50% DMEM, 30% FBS, 20% DMSO) to each well, followed by 50 µlsterilized (0.2 µm filtered) light paraffin oil to prevent degassing and evaporation.Tape the lid to the bottom of the plate and place in a polystyrene box. Cover withlid, freeze, and store at –80ºC.

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

Allow cells on gelatin plates continue to grow until most wells are near confluency.Wash twice with PBS. Add 50 µl of lysis buffer per well (10 mM Tris, pH 7.5,10 mM EDTA, 10 mM NaCl, 0.5% sarcosyl, and 1 mg/ml proteinase K added fresh).Incubate overnight at 60ºC in a humid atmosphere (e.g., a sealed container with apresoaked sponge). The next day, prepare a mix of NaCl and EtOH (1.5 µl of 5 MNaCl to 100 µl of cold absolute ethanol; salt will precipitate, so mix well). Add100 µl per well. Let the plate stand at room temperature 30 min. Gently invert theplate to discard the solution. Wash twice with 70% ethanol.

After the final washing, invert the plate and let it semi-dry for 15 min. Whilethe plate dries, prepare a restriction digest cocktail containing 1X restriction buffer,1 mM spermidine, 100 µg/ml bovine serum albumin and 10 to 15 units of enzymeper well). Add 30 µl of restriction digest cocktail to each well and incubate at 37ºCovernight in a humid atmosphere.

Add 4 to 5 µl of loading dye per well the next day and load onto a large 0.7 to1.0% agarose gel containing three rows (33 wells per row). Each gel can hold 96samples (one 96-well) plus one molecular weight marker per row. Run gel at 80 Vfor 3 to 5 hr, depending on the predicted size differences. Proceed with standardSouthern blot analysis.

14.2.3.5 Preparation of Gene-Targeted Clones for Blastocyst Injection

The targeted clones identified by above mini-Southern analysis will be expandedfor blastocyst injection. Feeder plates of 6 and 24 wells should be prepared. Toretrieve cells from frozen 96-well plates, place plates directly from –80ºC freezerinto 37ºC incubator. It takes 10 to 15 min for the wells in the center of the plate tothaw. After all the wells have thawed, carefully identify and mark the wells withtargeted clones and remove the entire solution to a 24-well feeder plate pre-equili-brated with 2 ml of ES medium. It is important for maximum recovery to vigorouslypipette the thawed cells and rinse once to dislodge them from the bottom of theplate where they settle during the freezing process. Change with fresh medium thesecond day.

Split the cells after 4 or 5 days to 6-well feeder plates. If the cell density is low,split to two 24-wells. It is not advisable to grow ES cells too long without passaging.Cells grown on 6-well plates can be divided for several purposes: further splittingand expansion, freezing to make a permanent stock, and isolating DNA to double-check the genotype. Clones with good growth characteristics and morphology will

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be chosen for microinjection. The viability of cells after trypsinization determinesthe quality of chimeras. Badly maintained cultures make poor chimeras.

The cloning efficiency at the time of microinjection is much more critical thanfor routine maintenance of the culture. The cells should be in log phase growth toensure maximum viability post-trypsinization. This is best achieved by plating thecells at a high density 24 to 32 hr prior to injection. Cells from a 24-well plate aremore than sufficient for a day of injection. Feed the cells 2 to 3 hr before splitting.After trypsinization for 15 min, add an equal volume of ES medium with 20 mMHepes (pH 7.4). Add an equal volume of FBS and pipette vigorously five to eighttimes with a Pasteur pipette. Transfer to 1-ml screw cap tube and place on ice.

14.2.4 GENERATION OF GENE (APP) KNOCKOUT MICE

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

Microinjection of targeted ES cells to create chimeric mice is a complicated processand requires sophisticated instruments. It is usually done by a core facility and isnot recommended for inexperienced researchers. This chapter will not discuss theprocedure. Interested individuals are referred to Bradley’s article about productionand analysis of chimeric mice (Chapter 5 in Robertson’s book).52

Pigmentation markers are commonly used to identify chimeras and to quantifythe extent of the chimerism. A common strain of host embryo is C57BL/6 (black,aa). The ES cells are 129Sv (agouti coat color, AA). Chimerisms are seen as areasof brown hairs on black backgrounds in mice 10 days and older. The percentage ofchimerism is a good prediction for germline transmission. A chimera with goodchance of germline transmission should have a chimerism of 70% or higher. How-ever, while a poor chimera most likely will not transmit through the germline, ahigh percentage chimera does not guarantee germline transmission.

Usually high percentage male chimeras are used to test for germline transmis-sion. There are two reasons:

1. Because the ES cells are derived from XY males, if the host embryo is afemale, it is possible for the ES cells to convert the host from female tomale. These sex-converted males are definitive germline transmitters ifthey are fertile.

2. In contrast to females that need to produce offspring one litter at a time,a male can simultaneously breed with multiple partners and a large numberof offspring can be generated in a short time. This is particularly importantif the chimera is a low germline transmitter, i.e., only a small percentageof pups are ES-derived.

Coat color is again used as a convenient and convincing test for germlinetransmission. When the 129Sv (AA) and C57BL/6 (aa) mosaic chimeras are bredwith C57BL/6 (aa) counterparts, the offspring can be either Aa (if 129Sv allele

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contributes to germline) or aa (if germ cells carry two C57BL/6 alleles) with regardto the pigmentation locus. Since agouti (A) is dominant over black (a), an Aacombination will confer agouti coat color and, therefore, germline transmission canbe determined by the presence of agouti pups in the offspring. The resulting miceare on a mixed C57BL/6 and 129Sv background. Due to the convenience and fastbreeding scheme, this is the most commonly used genetic background for initialphenotypic characterization of the mutant mice.

14.2.4.2 Breeding and Generation of Homozygous APP Knockout Mice

Because only one ES allele is targeted, 50% of the agouti offspring are expected tocarry the targeted allele and the other 50% wild-type. They can be distinguished,upon tail biopsy at weaning age, by Southern blot analysis described above (Figure14.3a). However, it is advisable to develop a much faster and easier PCR method.This can facilitate the genotyping efficiency tremendously. In the case of APP, wedesigned the following trimer mix (Figure 14.3b):

FIGURE 14.3 Southern blot (a) and PCR (b) analysis of representative offspring from het-erozygous mating of wild type (+/+), heterozygous (+/–), and homozygous (–/–) APP knock-out mice.

+/+ +/+ +/- +/- -/- -/-

+/+ +/- +/--/- -/-

kb

bp

470

250

9.0

6.5

a.

b.

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Primer 1 — APP wild-type sense oligo (2.2 kb into intron 1, deleted intargeted allele): 5′-CTG CTG CAG GTG GCT CTG CA-3′

Primer 2 — APP wild-type/targeted antisense oligo (2.45kb into intron 1,present in both alleles): 5′-CAG CTC TAT ACA AGC AAA CAA G-3′

Primer 3 — Neo antisense oligo (in proximal neo promoter 2540–2522,specific to targeted allele): 5′-CCA TTG CTC AGC GGT GCT GTC CAT-3′

Expected band pattern — APP wild-type allele (primers 1 and 2): 250 bp;APP targeted allele (primers 2 and 3): 470 bp; heterozygous: 250 bp + 470 bp

Heterozygous mice identified can be crossbred to yield mice that are wild-type(APP+/+), heterozygous (APP+/–), or homozygous (APP–/–) for the targeted APPallele (Figure 14.3). If the homozygous mice are viable as in the case of APP, theexpected ratios are 25, 50, and 25%, respectively. If embryonic or early postnatallethality arises as is the case for PS1 knockout,54 homozygous mice may not berecovered at weaning age. Genotyping and characterization at an earlier age or duringembryonic development are required.

After the homozygous knockout mice are identified, it is important to performexpression analysis to determine whether a null allele has been created. This can bedone by various methods — the most convincing are Northern and Western blotting(Figure 14.4). By deleting the promoter and the first exon of APP, we were able toeliminate the APP transcript (Figure 14.4a) and protein (Figure 14.4b) completely.

The homozygous APP-null mice and their littermate controls are subjected tovarious biochemical, immunohistochemical, and electrophysiological studies.39,55–57

Although the APP-null mice are fertile and the mutant mice can be obtained bybreeding the homozygous null mice, it is advisable to perform heterozygous inter-crosses to obtain littermates as controls. Because genetic backgrounds can influencethe phenotype under study, to minimize the variability caused by the genetic back-ground, it is recommended to back-cross the mutant mice onto the C57BL/6 strainfor at least 5 and preferably 10 generations. In fact, the APP-null mice availablefrom Jackson Laboratories (Bar Harbor, ME) have been back-crossed for more than10 generations and are considered congenic. It is particularly important to back-cross the animals if a series of behavioral evaluations are planned.

ACKNOWLEDGMENTS

The author is indebted to Allan Bradley, who fostered an ideal training and researchenvironment. I also wish to thank many of my colleagues at Merck ResearchLaboratories who contributed to the generation of APP knockout mice.

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FIGURE 14.4 APP expression analysis. (a) Top: Northern blotting of total brain RNA fromwild-type (+/+), heterozygous (+/–), and homozygous (–/–) APP mice using full-lengthAPP695 cDNA as a probe. Bottom: Mouse β-actin cDNA hybridization for loading control.(b) Top: Western blot analysis of brain APP protein using an APP C-terminal antibody. Bottom:Anti-β-actin antibody staining for loading control.

APP

β-actin

APP

β-actin

+/+ +/+ -/- -/-

+/+ +/+ -/- -/-a.

b.

116

97.4

285

185

+/- +/-

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COLOR FIGURE 6.4 PS1 deficiency or loss of function accelerates βAPP trafficking fromthe TGN/ER and increases cell surface delivery.

COLOR FIGURE 8.2 Intracellular accu-mulation of aggregated Aβ42 in DS astrocytes.

COLOR FIGURE 8.3 Double labeling ofAβ42 and different subcellular markers.

TGN Membrane TGN Vesicles

PS1WT

0’ 15’ 30’ 60’ 90 ’ 0’ 15’ 30’ 60’ 90 ’

a

PS1 -/-

bER Membrane ER Vesicles

0’ 15’ 30’ 60’ 90 ’

PS1WT

PS1 -/-

0’ 15’ 30’ 60’ 90 ’

c

WT

6E10 FITC -VVA

∆Μ1,2

d

Chase Time (min) 0’ 10’ 20’ 30’ 45’ 60’ 120 ’

Newly Synthesized βAPP on Cell Surface

WT

TotalCell βAPP

∆Μ1,2

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COLOR FIGURE 8.4Mitochondrial dysfunc-tion in normal astrocytesinduces aberrant APPprocessing and intracel-lular Aβ accumulation.

COLOR FIGURE 8.5Impaired mitochondrialfunction in DS astrocytes.

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COLOR FIGURE 10.3 The effects of GSK-3β inhibition on the Aβ-induced formation ofNFT-like tau pathology.

a.

Aß injection

AT100

Aß injection+LiCl

AT100

b.Aß Aß+LiClVehicle

AT180

Vehicle Aß Aß+LiCl

Tau-C

SDS-insoluble tau

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COLOR FIGURE 12.1 Detection of fibrillar amyloid by thioflavin S and Congo red staining.

COLOR FIGURE 12.8 Double immunofluorescent labeling of plaques (R1282) and micro-glia (CD45) showed similar patterns of colocalization in Aβ immunized (bottom) anduntreated (top and middle) PSAPP mice after 8 weeks of Aβ immunization.

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