Biophysical Insights into the Role of Amyloid-Beta Misfolding in Alzheimer's Disease Pathogenesis

155 Pages·2015·11.3 MB·English

Biophysical Insights into the Role of Amyloid-Beta Misfolding in Alzheimer’s
Disease Pathogenesis
by

Samuel A. Kotler

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Biophysics)
in the University of Michigan
2015

Doctoral Committee:

Professor Ayyalusamy Ramamoorthy, Chair
Professor Carol A. Fierke
Professor Nicholas Kotov
Professor Sarah L. Veatch
© Samuel A. Kotler
2015
Acknowledgements
First and foremost, I would like to thank my adviser, Professor Ayyalusamy
Ramamoorthy, for his dedication and support throughout my time at the University of Michigan.
I am incredibly lucky to have had the guidance and mentorship while working in his group, and
it is because of these experiences that I developed into the researcher I am today. I appreciate his
trust in me at a very early point as a PhD student, allowing me the freedom to pursue the work I
enjoyed the most.
I would also like to acknowledge my committee Professors Carol Fierke, Nicolas Kotov,
and Sarah Veatch. Carol was willing step in as a committee member following the untimely
move of another. Additionally, from our very first interaction she showed great interest in my
work and provided meaningful advice. Nick challenged me to ask the questions that brought my
work to a broader focus. Last, but not least, of my committee members, Sarah was one of the
first professors I interacted with in the Biophysics program, allowed me to rotate in her lab,
taught me in class, and was a mentor to me throughout my time as a PhD Student.
I would additionally like to thank past and present members of the Ramamoorthy group
that have provided support and plentiful insights throughout my time at UM. Special thanks to
the RAMyloid team: Dr. Patrick Walsh, Amit Pithadia, Kyle Korshavn, Dr. Jeffrey Brender, Dr.
Janarthanan Krishnamoorthy, Dr. Anirban Bhunia, and Jennifer Chen. Additional thanks to Dr.
Kamal Mroue, Dr. Kazutoshi, Dr. Dongkuk Lee, Dr. Anirban, and Dr. Manoj Pandey who have
in particular contributed to my success.
ii
Finally, I would like to thank my family and friends for their unconditional support and
inspiration. I feel incredibly grateful to my fellow Biophysics graduate students, particularly
Carter Swanson and David Rowland who have been there with me from the start. I also want to
particularly thank Dr. Ricardo Capone for his continued encouragement, mentorship, and
friendship without which I would not be where I am today. I cannot express enough gratitude
towards my parents, brother, and sister who will always be my number one fans. And of course, I
want to give a very special acknowledgement to the person who supports me on a daily basis, my
girlfriend, Anna. Thank you for everything.

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Table of Contents

Acknowledgements ......................................................................................................................... ii

List of Figures ................................................................................................................................. v

List of Appendices ......................................................................................................................... ix

Abstract...... ..................................................................................................................................... x

Chapter 1:
 Introduction ................................................................................................................... 1

Chapter 2:
 Elucidating the Role of Lipid Composition on Aβ Aggregation and Aβ-Induced
Membrane Disruption ................................................................................................ 34

Chapter 3:
 Characterizing Structural Features of Amyloid-β Oligomers Through Combined
Approaches in Solution- and Solid-State NMR ........................................................ 61

Chapter 4:
 Elucidating the Mechanism of Amyloid-β Aggregation Using Fast-Acquisition NMR
Methods ..................................................................................................................... 89

Chapter 5:
 Conclusions and Future Outlook .............................................................................. 110

iv
List of Figures
Figure 1.1 Aβ peptides derive from the proteolytic cleavage of the amyloid precursor protein
(APP). ..................................................................................................................................... 2

Figure 1.2 The amyloid hypothesis for Alzheimer’s disease. ......................................................... 3

Figure 1.3 Various structures of Aβ in solution and detergents characterized by NMR. ............... 5

Figure 1.4 Model of GM1 ganglioside-clusters leading to the formation of toxic Aβ species. ..... 8

Figure 1.5 A two-step mechanism of membrane disruption by Aβ40. ......................................... 12

Figure 1.6 Measuring the process of amyloid aggregation can be probed by many biophysical
methods. ................................................................................................................................ 19

Figure 1.7 Pulse sequences to record 1H-X correlation spectra. ................................................... 21

Figure 1.8 Magic angle spinning can create solution-like conditions on a solid sample. ............. 23

Figure 1.9 Pulse sequence schemes for 2D RFDR-based (top) and NOESY (bottom) 1H/1H
chemical shift correlation experiments. ................................................................................ 25

Figure 2.1 Membrane disruption induced by Aβ40, as measured by the 6-carboxyfluorescein dye
leakage assay. ........................................................................................................................ 37

Figure 2.2 Kinetics of Aβ40 amyloid formation measured by ThT fluorescent emission. .......... 38

Figure 2.3 Ca2+ influx into LUVs after the addition of freshly dissolved Aβ40. ........................ 40

Figure 2.4 Zn2+ inhibits the pore activity of freshly dissolved Aβ40. ......................................... 42

Figure 2.5 Zn2+ ions cannot block the fiber-dependent step of membrane disruption. ............... 43

Figure 2.6 Membrane fragmentation induced by prolonged incubation with Aβ40. ................... 44

Figure 2.7 31P solid-state NMR of LUVs incubated with Aβ40. .................................................. 45

v
Figure 2.8 Absence of membrane defects after fiber formation as detected by paramagnetic
relaxation enhancement. ....................................................................................................... 47

Figure 3.1 Biophysical characterization of Aβ40 disordered oligomers. ..................................... 65

Figure 3.2 RFDR-based 2D 1H/1H chemical shift correlation spectra on solution samples of
freshly dissolved (red) and aggregated (blue) forms of Aβ40. ............................................. 67

Figure 3.3 MAS spectra of the filtered disordered Aβ40 oligomer. ............................................. 69

Figure 3.4 The disordered Aβ40 oligomer grows in size while maintaining its morphology. ..... 72

Figure 3.5 Comparison of 1H/15N HSQC spectra of the freshly dissolved (red) and the spin-X-
isolated disordered oligomer (blue) of Aβ40 (after 4 days) recorded from a 900 MHz
spectrometer. ......................................................................................................................... 74

Figure 3.6 Simultaneously occurring aggregation pathways of Aβ40. ......................................... 75

Figure 4.1 Aggregation of Aβ40 as monitored by real-time 1H-15N SOFAST HMQC. ............... 92

Figure 4.2 Real-time 2D NMR demonstrates residue-specificity of seeded Aβ40 aggregation. .. 93

Figure 4.3 Cartoon model showing possible modes of interaction of soluble Aβ40with Aβ40

fibers and the effect of the interaction on the NMR spectra. ................................................ 96

Figure 4.4 New peaks arising in HSQC spectra suggest slow exchange process in seeded Aβ40
aggregation. ........................................................................................................................... 97

Figure 4.5 Variation in longitudinal relaxation rates in the presence and absence of amyloid
fibers. .................................................................................................................................. 101

Figure 4.6 Residual dipolar couplings induced by the addition of amyloid fibers, which
spontaneously align parallel to the magnetic field. ............................................................. 103

Figure A.1 Timescales of dye leakage and fiber formation. ....................................................... 122

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Figure A.2 Membrane disruption induced by seeded fiber growth of Aβ40 measured by the 6-
carboxyfluorescein dye leakage assay. ............................................................................... 123

Figure A.3 Kinetics of calcium influx into LUVs after the addition of freshly dissolved Aβ40.
............................................................................................................................................. 123

2+ 2+
Figure A.4 Fura 2 is sensitive to membrane disruption by both Zn and Ca . ..................... 124

Figure B.1 Characterizing the aggregated Aβ sample. .......................................................... 125

1-40
Figure B.2 DLS studies on the spin-X-isolated oligomer and freshly dissolved samples of Aβ .
1-40
............................................................................................................................................. 126

Figure B.3 The two-dimensional RFDR-based (top) and NOESY (bottom) 1H/1H chemical shift
correlation experiments used in this study. ......................................................................... 127

Figure B.4 The aggregated Aβ sample shows strong up-field shifts. .................................... 127

1-40
Figure B.5 1D 1H MAS spectra acquired under increasing MAS rates on the filtered disordered
Aβ oligomer. ................................................................................................................... 128

1-40
Figure B.6 An overlay of the aliphatic and alpha proton regions of RFDR-based 2D 1H/1H
spectra acquired using mixing times of 20 ms (red) and 50 ms (black) on the aggregated
Aβ sample. ...................................................................................................................... 129

1-40
Figure B.7 Time-course 1D 1H MAS NMR spectra of the aggregated Aβ sample, containing
1-40
the disordered oligomer. ..................................................................................................... 130

Figure B.8 Two-dimensional 1H/1H NOESY experiments under 2.7 kHz MAS on the mixed fiber
and oligomer sample of Aβ . ........................................................................................... 131

1-40
Figure C.1 Chemical shift perturbations of Aβ over time following titration of Aβ fibers.
1-40 1-40
............................................................................................................................................. 135

Figure C.2 Time-course intensity changes of HSQC peaks of a seeded Ab40 sample at 10 ºC. 136

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Figure C.3 Time-course intensity changes of HSQC peaks of a seeded Aβ sample at 25 ºC. 137

1-40
Figure C.4 One-dimensional 1H slices taken from the glycine region of the real-time HSQC
measurements. ..................................................................................................................... 138

Figure C.5 Real-time HSQC experiments over an extended time period show that the most of the
Aβ monomer is pushed towards a fibril-bound state. .................................................... 139

1-40
Figure C.6 Assigned 1H-15N HSQC spectrum of the seeded Aβ sample at a late stage of the
1-40
aggregation process. ............................................................................................................ 140

Figure C.7 Time-course Diffusion Order SpectroscopY (DOSY) experiments performed on Aβ
1-
in the presence of a 10% fiber seed. ................................................................................ 141

40
Figure C.8 Sonication accelerates the formation of new peaks arising from the slow exchange
between free and fiber-bound forms of monomeric Aβ . ................................................ 142

1-40
Figure C.9 Time-course 1D 1H spectra of seeded Aβ aggregation. ....................................... 143

1-40

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List of Appendices
Appendix A.
  Supporting Information for Chapter 2 ............................................................ 120

Appendix B.
  Supporting Information for Chapter 3 ............................................................ 125

Appendix C.
  Supporting Information for Chapter 4 ............................................................ 132

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