Biophysical Understanding of Novel Synthetic AMYLOID-β (Aβ)

118 Pages·2016·2.71 MB·English

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Dissertations
Summer 8-2013
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Amit Kumar
University of Southern Mississippi
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Kumar, Amit, "Biophysical Understanding of Novel Synthetic AMYLOID-β (Aβ) Prions in Alzheimer's
Disease" (2013). Dissertations. 186.
https://aquila.usm.edu/dissertations/186
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The University of Southern Mississippi

BIOPHYSICAL UNDERSTANDING OF NOVEL SYNTHETIC

AMYLOID-β (Aβ) PRIONS IN ALZHEIMER`S DISEASE.

by

Amit Kumar

Abstract of a Dissertation
Submitted to the Graduate School
of The University of Southern Mississippi
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

August 2013
ABSTRACT
BIOPHYSICAL UNDERSTANDING OF NOVEL SYNTHETIC
AMYLOID-β (Aβ) PRIONS IN ALZHEIMER`S DISEASE.
by Amit Kumar
August 2013
Oligomers of amyloid-β (Aβ) peptide are the primary toxic agents that play a
pivotal role in the pathogenesis of Alzheimer’s disease (AD). Oligomers are the
intermediates formed during the Aβ aggregation process leading up to insoluble fibrils. It
is important to know that oligomers can also be formed via pathways that do not lead to
fibril formation. Such ‘off-pathway’ oligomers would have significantly longer half-lives
than the ‘on-pathway’ ones, which may result in prolonged toxicity to neuronal cells.
Furthermore, neither the mechanism of neurotoxicity nor the potential mechanisms of
propagation and proliferation to neighboring cells are well understood. Moreover, recent
in vivo studies on transgenic animal models have implicated a prion-like mechanism
involved in the propagation of toxic oligomeric seeds. Interfaces generated by lipids,
fatty acids and other surfactants are well known to affect A aggregation, especially in
inducing alternate pathways. In this study, the effect of saturated non-esterified fatty
acids (NEFAs) on the rate of A aggregation was studied. We have observed that
NEFAs were able to induce an alternate pathway of aggregation, which was depended on
NEFA concentrations. More importantly, in a narrow concentration range, NEFAs
induced the formation of 12-18mers (Large Fatty Acid-derived Oligomers; LFAOs),
which were isolable by size exclusion chromatography (SEC). We discovered that
LFAOs can behave like prions, undergoing self-propagation, by quantitatively converting
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monomeric Aβ into toxic LFAO assemblies in a template-assisted manner. We further
analyzed the prion-like behavior of LFAOs by the ‘protein misfolding via cyclic
amplification’ (PMCA) assay, as was done for prions. Together, our findings indicate
that LFAOs are unique Aβ prions and support the developing hypothesis that a common,
prion-type mechanism of infectivity could be an underlying conserved mechanism among
many neurodegenerative diseases.

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COPYRIGHT BY
AMIT KUMAR
2013
The University of Southern Mississippi

BIOPHYSICAL UNDERSTANDING OF NOVEL SYNTHETIC

AMYLOID-β (Aβ) PRIONS IN ALZHEIMER`S DISEASE.

by

Amit Kumar

A Dissertation
Submitted to the Graduate School
of The University of Southern Mississippi
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

Approved:

Vijay Rangachari
____________________________________
Director

Gordon Cannon
____________________________________

Douglas Masterson
____________________________________

Robert Bateman
____________________________________

Sandra Leal
____________________________________

Susan A. Siltanen
____________________________________
Dean of the Graduate School

August 2013
DEDICATION
I would like to dedicate this dissertation to my father Shri. Ram Narain Singh, my
mother Shrimati. Rajkumari, my brothers Anand and Rakesh, my sisters in law Maumita
and Sunita, my sister Premlata (Didiya), and my brother in law Akhileshwar Singh for
their endless love, blessing and support throughout my career, and especially to my
nephew Ashwin and nieces Akash, Rani and Gudiya for making me smile all the time.

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ACKNOWLEDGMENTS
Firstly, I would like to thank my graduate mentor, Dr. Vijay Rangachari, for
teaching me the fundamentals of research, guiding and motivating me all the time to give
my level best and, most importantly, being patient with me.
I would like to thank my committee members, Drs. Gordon Cannon, Douglas
Masterson, Robert Bateman and Sandra Leal, for their support, advice, and guidance
throughout my PhD career. I would also like to thank my past and current lab members,
especially Gaurav, Rebekah and Matthew for their friendship, support and happy times,
and also for making me do all their work.
I would also like to thank some of my very dear friends especially, Saurabh,
Deepak, Mayank (monu), Sarthak, Dr. Ankur Jain, Rahul (Spity), Manoj, Mridu,
Anirban, Souvik, Dhritiman, Adhiraj, Tejal, Kalyani, Dr. Srinivas Konda, Dr. Sunil
Pusarla, Dr. Balaraj Menon, Dr. Avijit Biswas, Min Bahadur, Sadhna, Divya, Kavita,
Shekhar, Rajni, Nabanita, Sudeshna, Natasha (Nattu), Sonal and Chandrani for their
unconditional friendship, beautiful memories and support.
I would also like to thank our collaborators Dr. Sarah Morgan, Dr. John Correia,
Dr. Ewa Bienkiewicz, Dr. Peter Fajer, Dr. Likai Song, and their lab members, especially
Lea Pasley and Daniel Lyon.
I would also like to thank Dr. Heinhorst, Ms. Sharon King, Ms. Tina Masterson
and Martha for their help. I would also like to thank the American Heart Association,
USM Department of Chemistry and Biochemistry and USM Graduate School for the
funding and support.

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TABLE OF CONTENTS
ABSTRACT .................................................................................................................... ii
DEDICATION ............................................................................................................... iv
ACKNOWLEDGMENTS ............................................................................................... v
LIST OF ILLUSTRATIONS ....................................................................................... viii
LIST OF ABBREVIATIONS .......................................................................................... x
CHAPTER
I. INTRODUCTION & LITERATURE REVIEW ........................................ 1 .
Aβ Aggregation
Oligomers in Aβ Aggregation
Amyloid Polymorphism and Structural Classification of Oligomers
Interfacial Aggregation and ‘Off-pathway’ Oligomers
Prion-type Propagation Mechanism – Conserved Among
Neurodegenerative Diseases?

II. MATERIALS & METHODS .................................................................. 16
Preparation of Aβ42 Monomers
Aβ Aggregation Reactions
Dynamic Light Scattering (DLS)
Polyacrylamide Gel Electrophoreses (PAGE) & Immunoblotting
Atomic Force Microscopy (AFM)
Analytical Ultracentrifugation (AUC)
Circular Dichroism (CD)
III. HYPOTHESIS ........................................................................................ 28
IV. RESULTS & DISCUSSION .................................................................... 30
The Effect of NEFAs on Aβ Aggregation
Physiochemical Properties and Self-propagation of LFAOs
Evaluation of LFAOs Prion-type Behavior

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V. CONCLUSIONS AND FUTURE WORK ................................................ 83
Conclusions
Future Work

REFERENCES .............................................................................................................. 87

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