Elucidating mechanisms of protein aggregation in Alzheimer's Disease using antibody-based

164 Pages·2017·2.59 MB·English

University of Missouri, St. Louis
IRL @ UMSL
Dissertations UMSL Graduate Works
7-25-2017
Elucidating mechanisms of protein aggregation in
Alzheimer’s Disease using antibody-based
strategies.
Benjamin A. Colvin
University of Missouri - St. Louis, [email protected]
Follow this and additional works at:https://irl.umsl.edu/dissertation
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Neuroscience Commons, and theOther Biochemistry, Biophysics, and Structural Biology
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Recommended Citation
Colvin, Benjamin A., "Elucidating mechanisms of protein aggregation in Alzheimer’s Disease using antibody-based strategies." (2017).
Dissertations. 671.
https://irl.umsl.edu/dissertation/671
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Elucidating mechanisms of protein aggregation in Alzheimer’s Disease
using antibody-based strategies.

A Dissertation
Submitted to the Faculty
of
University of Missouri – Saint Louis
by
Benjamin Alexander Colvin

In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
in
CHEMISTRY
with an emphasis in Biochemistry
May 2017
University of Missouri – Saint Louis
Advisory Committee
Michael R. Nichols, PhD
Wesley Harris, PhD
James Bashkin, PhD
Keith Stine, PhD
Dedicated to the millions
who live with
Alzheimer’s Disease.
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ACKNOWLEDGEMENTS

My parents, Mr. Grant Colvin and Dr. Juanita Polito-Colvin. They encouraged my
rampant inquisitiveness from an early age, and they were always there for me no matter
what sort of trouble I got myself into.
The faculty at The Saint Louis Priory High School, especially the first great
chemistry teacher I ever had, Mr. Gleich.
The QC group at GSK – Saint Louis, where I had my first real job as a scientist.
From my experiences in the QC lab I developed an interest in chemical analysis which
would ultimately lead to me returning to school for this degree.
Dr. Nichols has been a wonderful PI to study under. He has been a role model for
me, especially his work ethic, determination and attention to detail – areas I know I still
need to improve. My lab mates, current and former, especially Shana Terrill-Usery, for
all the hours she spent training me and putting up with all my questions, and Elizabeth
Hood, for many useful discussions. My committee, for their time and critical
considerations.
The Graduate School, for the Dissertation Fellowship award. It’s finally done!
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TABLE OF CONTENTS

Page
LIST OF TABLES………………………………………………………………….. vii
LIST OF FIGURES…………………………………………………………………viii
ABBREVIATIONS……………………………………………………………………x
ABSTRACT………………………………………………………………………… xii
PUBLICATIONS……………………………………………………………………xvi
CHAPTER 1. INTRODUCTION…………………………………………………….. 1
1.1 The cost of Alzheimer’s disease dementia…………………………………….1
1.2 The classic Alzheimer’s disease histopathology………………………………1
1.3 Amyloid aggregation…………………………………………………………..4
1.4 Genetic evidence for the involvement of Aβ in AD pathology………………..4
1.5 The Amyloid Cascade Hypothesis…….……………………………………….8
1.6 Modeling AD progression...………………………………………………….10
1.7 Non-Aβ amyloid diseases…………………………………………………… 12
1.8 Tau, a microtubule-associated protein………………………………………. 12
1.9 Tau phosphorylation………………………………………………………….16
CHAPTER 2. METHODS…….……………………………………………………..19
2.1 Preparation of tau oligomers………………………………………………… 19
Microplate BCA protein concentration assay……………………………….. 20
2.3 Protein determination by 280 nm absorbance…...……………………………21
2.4 Aβ preparation………………………….…………………………………….21
2.4.1 Processing and aliquoting………………………………………………21
2.4.2 Preparation of Aβ42/40 mixtures………………………………………22
2.4.3 Preparation of Aβ protofibrils…………………………………………. 23
2.4.4 Preparation of Aβ42 oligomers…………………………………………23
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2.4.5 Preparation of purified Aβ42 monomers……………………………….24
2.4.6 SEC-purified monomer Aβ42/40 aggregation reactions……………….24
2.5 Size-exclusion chromatography……...…………………………………….. 24
2.6 Multi-angle light scattering………….………………………………………25
2.7 Care and maintenance of MALS instrumentation…………………………..27
2.8 Dynamic light scattering…………………………………………………….28
2.9 Thioflavin T (ThT) fluorescence……………………………………………28
2.10 Electron microscopy………………………………………………………...28
2.11 Circular dichroism…………………………………………………………..29
2.12 Aβ C-terminal selective ELISA……………………………………………. 29
2.12.1 Ab5-HrP conjugation………………………………………………… 31
2.12.2 Ab5-HrP concentration determination……………………………….. 32
2.12.3 Assay antibody concentration selection and validation……………… 34
2.13 Aβ indirect ELISA…………………………………………………………. 37
2.14 Aβ dot blot…………………………………………………………………..37
CHAPTER 3. CHARACTERIZATION OF SEEDED TAU OLIGOMERS………..39
3.1 Introduction………………………………………………………………….. 39
3.1.1 The basics of tau protein aggregation…………………………………. 39
3.1.2 Tau pathology in AD…………………………………………………...40
3.1.3 Prion-like spreading of tau pathology...……………………………….. 41
3.1.4 Identified tau oligomers in vivo and in vitro………………………….. 46
3.1.5 Templated oligomerization……………………………………………. 47
3.1.6 Challenges inthe study of tau oligomer………………………………..50
3.3 Results……………………………………………………………………….. 52
3.3.1 Instrument and sample preparation validations………………………...52
3.3.2 2N4R tau seeded with cross-linked oligomer…………………………. 57
3.3.3 Reconstitution and seeding of a 2N3R Tau pellet……………………...58
3.3.4 SEC-MALS comparison of seeded and unseeded 2N4R tau………….. 62
3.3.5 Detection of 2N3R oligomers and intriguing elutions………………… 66
3.4 Results discussion……..…………………………………………………….. 75
3.5 Technical discussion…….……………………………………………………77
3.5.1 Protein concentration determination……………………………………77
3.5.2 Sample preparation……………………………………………………..80
3.5.3 Preformed aggregates…………………………………………………..81
3.5.4 Care and maintenance of MALS instrumentation……………………...82
CHAPTER 4. LARGE SOLUBLE Aβ OLIGOMERS………………………………83
4.1 Introduction………………………………………………………………….. 83
4.1.1 Aβ aggregation, and aggregate pathologies…………………………… 83
4.2 Results……………………………………………………………………….. 89
4.2.1 Influence of buffer on purified Aβ42 monomer aggregation.………….89
4.2.2 Influence of temperature in low ionic strength aggregations…………. 93
4.2.3 The Aβ42/Aβ40ratio influences rapid protofibril formation…………. 96
4.2.4 Aβ40 as an inhibitor of Aβ42 aggregation……………………………. 98
4.2.5 Subtle inhibitory effect of Aβ40 on conversion to protofibril…………103
4.2.6 Protofibrils from Aβ42/Aβ40 monomers areprimarily Aβ42…….…..106
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4.3.6 At fixed Aβ42 concentration,minimal Aβ40 concentration effect……108
4.3 Discussion…………………………………………………………………...110
CHAPTER 5. A NOVEL ANTIBODY FOR Aβ42PROTOFIBRIL………………113
5.1 Introduction………………………………………………………………….113
5.1.1 The mammalian immune system…………….………………………..113
5.1.2 Innate immunity……………………………………………………….114
5.1.3 Adaptive immunity…………………………………………………….114
5.1.4 Macrophages…………………………………………………………..115
5.1.5 The neuroinflammatory response………………………………………115
5.2 Results……………………………………………………………………….119
5.2.1 Titer determinations……………………………………………………119
5.2.2 Selective affinity of AbSL for Aβ42 protofibril conformation………..122
5.2.3 Selective affinityof AbSL for Aβ42over Aβ40 protofibril…………..125
5.2.4 Probing the AbSL epitope…………………………………………….127
5.3 Discussion…………………………………………………………………...133
REFERENCES………………………………………………………………………135
VITA…………………………………………………………………………………148

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LIST OF TABLES

Page
CHAPTER 3.
3.1 Validation experiments molecular weight summary…………………………. 56
3.2 SEC-MALS comparison of seeded and unseeded 2N4R Tau…………………64
3.3 SEC-MALS comparison of seeded and unseeded 2N3R Tau…………………70
3.4 SEC-MALS comparison of seeded and unseeded 2N3R Tau…………………72
3.5 2N3R seed oligomerization reactions preparation protocol comparison…….. 74
CHAPTER 4.
4.1 Summary of molecular weight data from Figure 4.5…………………………105
4.2 ELISA-determined Aβ42/Aβ40 molar ratios……………..…………………. 107
CHAPTER 5.
5.1 Summary of antibodies used in Figure 5.6………………….………………..130

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LIST OF FIGURES

Page
CHAPTER 1.
1.1 The quintessential Alzheimer’s Disease histology…………………………… 3
1.2 Pathways of APP processing………………………………………………… 7
1.3 A modern Amyloid Cascade Hypothesis……………………………………. 9
1.4 Integrated model of AD progression…………………………………………11
1.5 The tau protein………………………………………………………………..15
CHAPTER 2.
2.1 Ab5-HrP conjugation…………………………………………………………33
2.2 C-terminal Aβ ELISA method development…………………………………36
CHAPTER 3.
3.1 Brakk staging of tauopathies………………………………………………….45
3.2 Generation of tau oligomer via seeding………………………………………49
3.3 Method validation with yADh………………………………………………..54
3.4 2N3R tau sample preparation with DTT………………………………………55
3.5 Purification of 2N4R seeded with 2N4R cross-linked seeds…………………58
3.6 Characterization of 2N3R Tau seeded with 2N4R oligomer…………….……61
3.7 SEC-MALS comparison of 2N4R seeded and unseeded preparations……….65
3.8 Characterization of 2N3R tau monomer seeded with 2N4R oligomer….……..71
3.9 Characterization of 2N3R tau monomer seeded with 2N4R oligomer…………73
CHAPTER 4.
4.1 Monomer aggregation reactions in varied buffer conditions………….……… 92
4.2 Temperature-dependent kinetics, but similar secondary structures……………95
4.3 ThT fluorescence of Aβ monomer is influenced by Aβ40 content……………101
4.4 Effect of Aβ42 concentration when Aβ40 is absent…………………………..102
4.5 Subtle Aβ40 effects on protofibril polydispersity and conformation…………104
4.6 Weak concentration dependence of Aβ40 effects at constant Aβ42………….109
CHAPTER 5.
5.1 Aβ and microglial pathology in AD…………………………………………..118
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5.2 Indirect ELISA to determine antisera titers against Protofibril Aβ42…………121
5.3 Indirect AbSL ELISA selects strongly for Aβ42 PF..…………………………123
5.4 AbSL is selective for protofibril in a dot blot format…………………………124
5.5 AbSL has less affinity for Aβ40 protofibrils than Aβ42 protofibrils…………126
5.6 ELISA-style probe of AbSL epitope sequence involvement...……………….129
5.7 AbSL vs. Ab9 cis-competition ELISA………………………………………..131
5.8 AbSL vs. Ab2.1.3 trans-competition capture ELISA…………………………132

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