Role of the Slingshot-Cofilin and RanBP9 pathways in Alzheimer's Disease Pathogenesis

142 Pages·2016·3.18 MB·Korean

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USF Tampa Graduate Theses and Dissertations USF Graduate Theses and Dissertations
10-12-2015
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Jung A Woo
University of South Florida, [email protected]
Follow this and additional works at: https://digitalcommons.usf.edu/etd
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Woo, Jung A, "Role of the Slingshot-Cofilin and RanBP9 pathways in Alzheimer's Disease Pathogenesis"
(2015). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/6047
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Role of the Slingshot-Cofilin and RanBP9 Pathways

in Alzheimer’s Disease Pathogenesis

by

JungA Alexa Woo

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
with a concentration in Neuroscience
Department of Molecular Medicine
Morsani College of Medicine
University of South Florida

Co-Major Professor: David E. Kang, Ph.D.
Co-Major Professor: Dave Morgan, Ph.D.
Chad A. Dickey, Ph.D.
Yu Chen, Ph.D.
Kevin Nash, Ph.D.

Date of Approval:
Oct 6, 2015

Keywords: Amyloid β, Alzheimer’s Disease, Cofilin, Mitochondrial Dysfunction, Synaptic
dysfunction, Slingshot, RanBP9

Copyright © 2015, JungA Alexa Woo
DEDICATION

박사를 마치면서 석사 연구를 시작하기도 전에 썼던 연구 계획서를 읽어보았다. 그곳에는 포부와
열정에만 차있는 아직은 미성숙한 내가 있었다. 학부과정에 배운 지식이 전부이지만 모든 연구를 수행함이
있어서 가장 중요한것은 끈기와 겸손, 그리고 열정이라고. 그리고 궁극적으로는 사람을 살리는, 사람을
이롭게 하는 학문을 하고 싶다고 말했던 25살의 나. 앞으로도 더 나은 연구를 통해 사람을 살리는 학문을 할
수 있는 과학자가 될 수 있기를 소망해본다.
이 길을 지나오기까지 무조건적인 지지와 사랑을 보내준 우리 부모님, 사랑하는 가족들, 긴
석.박사기간동안 과학자로써의 역량을 키울 수 있게 도와주신 지도교수님, 마지막으로 지혜와 능력을
허락해주신 하나님께 이 박사학위를 바칩니다.

This dissertation is dedicated to God for giving me strength and wisdom to continue on
my research journey. I also dedicated to this thesis to my precious and beloved parents, Un-ha
Woo and Hyang-yeon Jung for their endless love, support, and encouragement. Without your
support, love and prayer, I would not have come this far. I love you Mom & Dad!
ACKNOWLEDGMENTS
지금까지 건강과 지혜와 능력을 허락해주신 하나님께 감사드립니다.
박사학위를 마치기까지 감사해야할 사람들이 참 많이 있습니다. 지도교수님, Dr. David Kang
교수님이 없었다면 지금의 연구자로써의 저는 없을 것입니다. 처음 파이펫 한번 제대로 잡아보지 못했던
제가 5 년만에 제 연구에 자부심을 가지고 치열하게 연구하는 과학자로 성장하게 해 주신 것 감사드립니다.
처음에 교수님을 뵜을때 나는 언제쯤 내 스스로 실험가설을 세우고 연구를 진행하고 논문을 쓰고 할 수
있을까 까마득하게만 느껴졌는데 돌이켜보니 어느새 저도 한 사람의 과학자가 되어 있네요. 감사하다는
말로는 표현이 안될 것 같습니다. 앞으로도 배운 지식으로 더 나은 과학자가 되겠습니다. 제 공동 지도교수가
되주신Dr. Dave Morgan, 학위 심사 위원분들, Dr. Yu Chen, Dr. Chad Dickey, Dr. Kevin Nash, 그리고
마지막으로 Emory 에서 제 박사학위 심사를 위해 먼 걸음 해주신 Dr. Allan Levey 께 모두 감사의 말씀
드립니다.
우리 실험실 가족들, 실험실 동료라고 정의하기엔 너무나 과분한 사람들. 모두 감사합니다. 우리
실험실 가족들이 없었다면 지금의 저, 저의 연구또한 없을 겁니다. Courtney Uhlar, Taylor Boggess, Richard
Witas, Emilio De Narváez, Shazia Majid, Parth Patel, Anusha Bukhari, Kamal Makati, Catherine Fang, Tian Liu,
Xingyu Zhao, Patrick LePochat, Drew Maslar, Josh Vega, 그리고 Evy Panero -모두 제가 동료 이상의
사람들입니다. 다들 너무나 고맙습니다. Courtney Penn (Soon to be Trotter), Hirah Khan- 너희와 같이 연구할
수 있었던 지난 시간들 감사하고 행복한 시간들이었어. 내게 가족만큼 소중한 존재가 되어줘서 감사해.
앞으로 우리 앞에 펼쳐질 과학자로써의 미래가 나는 더욱 기대돼. Justin Trotter, 그리고 Amirthaa
Suntharalingam, Joe Grieco 여기와서 얻게된 소중한 친구들, 앞으로도 좋은 연구로 함께 세상을 더 이롭게
하는 긍정적인 역할을 함께 감당하자! 한국에서 항상 지지와 격려를 아끼지 않고 학위 심사 발표 응원하러
미국까지 날아와 준 내 친구 유리 고마워,
마지막으로 사랑하는 우리 엄마아빠, 엄마아빠의 기도와 지지가 없었다면 여기까지 오지
못했을꺼예요. 멀리 있느라 제대로 효도도 못하는 딸이지만 앞으로도 엄마아빠가 자랑스러워 할 수 있는
딸이 될게요. 엄마아빠가 기도하시는데로 늘 초심을 잃지 않고 인류에 도움이 되고, 세상을 이롭게 하는
과학자가 되겠습니다. 성교 & 지은이 사랑한다. 앞으로도 행복하게 인생의 새 시작을 잘 감당하길 바란다.
사랑하는 우리 외할머니, 건강히 오래오래 사세요! 외숙모 외삼촌, 끊임없는 기도와 격려에 감사하고
사랑합니다.
제가 사랑하는 모든 이들에게 이 논문을 바칩니다.

I have so many people to thank for supporting me during my Ph.D. years. Foremost, I
would like to show my sincere gratitude to my advisor Dr. David Kang who has taught me how
to be a great scientist. Without your support, patience and immense knowledge, there is
absolutely no way I would have been where I am now. Having you as my mentor was the best
luck I’ve ever had. You offered me continuous advice and encouragement throughout all these
years. It has been such a great journey working with you. I remember I just had some ambiguous
cynical ideas about biological research. I was totally unexperienced without knowing what I
want to do in my life. With your guidance and support, now I know what I want, where I want to
go. (I still remember the day when I got surface LRP western blot data and how much you were
excited about!) Based on knowledge that I was given by you, I will always try to be a better
scientist in my career path. Truth to be told, there is NO way I can thank you enough for
everything Dr. Kang.
I acknowledge my gratitude to my co-Professor and committee members Dr. Dave
Morgan, Dr. Yu Chen, Dr. Chad Dickey, and Dr. Kevin Nash for the several years of support. I
also would like to express my sincere gratitude to Dr. Allan Levey at Emory University for
serving as my Ph.D. thesis outside chair.
In my daily life, I have been amazingly blessed with the best group of people with whom
I’ve had to pleasure to work with in my lab. Without these people, this work simply would
NEVER have happened. Courtney Uhlar, Taylor Boggess, Richard Witas, Emilio De Narváez,
Shazia Majid, Parth Patel, Anusha Bukhari, Kamal Makati, Catherine Fang, Tian Liu, Xingyu
Zhao, Patrick LePochat, Drew Maslar, Josh Vega, and Evy Panero-You guys are not just my
students or lab folks, but my family and my best friends. It has been such an honor and pleasure
to have all of you in my life. I really appreciate all your hard work and I will never forget all
those days and nights that we spent together in the lab. Special thanks go to Hirah Khan and
Courtney Penn (Soon to be Courtney Trotter). Thank you for being part of my life. You guys
have been the best friends and colleagues anyone could ask for. I loved our times together in lab.
Many thanks and appreciation to my dear friends, Justin Trotter in Dr. Sudhof’s lab: Thank you
for being an amazing friend Justin, You inspire me and you give me motivation to become a
better scientist. I appreciate our friendship! I miss you, Amirthaa Suntharalingam in Dr. Dickey
Lab: You are like my big little sister. I love you, and Joe Grieco in Dr. Weeber Lab: Thank you
for being a great friend and colleague.
My best friend who was willing to come from 6823miles away (From Korea) just for my
defense, Yuri Seo, I love you! Thank you for putting up with me for past more than 10 years.
Finally, I thank my beloved family for supporting me throughout all of my studies. Mom
and Dad, you showed me unconditional love. You taught me to always be grateful for life. You
have always respected and supported my endeavors with an open mind - even when I wanted to
go to military school to become an army official in Korea. SeongKyo, my beloved brother, I love
you and always miss you. I pray for your next chapter of your life with Jieun Kwon (My lovely
future sister-in-law). I am also thankful to my best aunt (Soon Kang) and uncle (Byungdae Jung),
thank you for all your prayers and support. I love you. My beloved grandma, I always miss you.
To all of you, thanks for always being there for me. God bless you!
TABLE OF CONTENTS

List of Tables ............................................................................................................................................... iv

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

Abstract ....................................................................................................................................................... vii

Chapter 1 Introduction .................................................................................................................................. 1
Alzheimer’s disease (AD) overview ................................................................................................ 1
Amyloid plaques and neurofibrillary tangles ................................................................................... 2
Genetic basis of Alzheimer’s disease (AD) ..................................................................................... 3
Early Onset AD ................................................................................................................... 3
Late Onset AD .................................................................................................................... 4
Molecular basis of Alzheimer’s disease (AD) ................................................................................. 5
Soluble Aβ oligomer hypothesis and hyper phosphorylation of tau ................................... 5
Amyloid β and its receptors ................................................................................................ 7
Neuroinflammation in AD ................................................................................................ 10
Mitochondrial dysfunction in AD ..................................................................................... 10
Cytoskeletal aberrations in AD ......................................................................................... 14
Role of Cofilin in F-Actin dynamics and mitochondrial dysfunction ............................... 15
RanBP9 in APP processing, mitochondria-mediated neurotoxicity, and integrin
signaling ...................................................................................................................... 17

Chapter 2 Slingshot-Cofilin activation medicates Mitochondrial and synaptic dysfunction via
Amyloid β ligation to β1 integrin conformers ...................................................................................... 26
Background .................................................................................................................................... 26
Materials and Methods ................................................................................................................... 28
Cells, cDNA constructs, siRNA sequences, and transfection ........................................... 28
Antibodies and reagents .................................................................................................... 29
Cell/tissue lysis and Immunoblotting ................................................................................ 30
Cell surface biotinylation assay ........................................................................................ 30
Annexin V/PI apoptosis assay .......................................................................................... 31
FITC-Aβ oligomer binding assay ..................................................................................... 31
Purification of β1-integrin-Fc and binding to Aβ oligomers ............................................ 31
Mice and human samples .................................................................................................. 32
Immunofluorescence ......................................................................................................... 33
JC-1, Mitosox-Red, and H2DCF imaging ........................................................................ 34
Stereotaxic surgery ........................................................................................................... 34
Electrophysiology in mouse brain slices ........................................................................... 35
Fear conditioning, open field, and rotarod behavior ......................................................... 36
Statistical analysis & graph ............................................................................................... 37
Results .......................................................................................................................................... 37
i
Aβ oligomers induces loss of cell surface β1-integrin but not TfR or PrPc and reduces
β1-integrin activation ................................................................................................. 37
β1-integrin allosteric modulation or activation reduces Aβ oligomers binding to
neurons, HT22 cells, and Purified β1-integrin integrin .............................................. 38
Essential role of β1-integrin in Aβ oligomer-induced cofilin activation and reciprocal
requirement of cofilin in Aβ oligomers induced depletion of surface β1-integrin
and FCs ....................................................................................................................... 39
Essential roles of cofilin and β1-integrin in Aβ oligomer-induced depletion of F-actin
and F-actin-associated synaptic proteins in primary hippocampal neurons ............... 41
Requirement of SSH1 in Aβ oligomer-induced cofilin translocation to mitochondria
and accumulation of mitochondrial cofilin in AD brains ........................................... 42

Aβ oligomer-induced ROS production, mitochondrial dysfunction, and apoptosis via
the β1-integrin–SSH1–Cofilin activation pathway .................................................... 43
Cofilin reduction rescues APP/Aβ42 oligomers-induced gliosis and loss of synaptic
protein, as well as LTP and contextual memory deficits in APP/PS1 mice ............... 44
Discussion ...................................................................................................................................... 46
Conclusion ..................................................................................................................................... 49

Chapter 3 RanBP9 at the intersection between Cofilin and Amyloid β pathologies .................................. 66
Background .................................................................................................................................... 66
Methods ......................................................................................................................................... 68
Cells, cDNA constructs, siRNA sequences, and transfection ........................................... 68
Antibodies, reagents, and Aβ oligomer preparation ......................................................... 69
Cell/tissue lysis and Immunoblotting ................................................................................ 70
Mice .................................................................................................................................. 70
Immunofluorescence ......................................................................................................... 70
Fear conditioning, open field, and rotarod behavior ......................................................... 71
Electrophysiology in mouse brain slices ........................................................................... 72
Results .......................................................................................................................................... 73
Endogenous RanBP9 mediates Aβ42 oligomer-induced translocation of cofilin to
mitochondria and promotes cofilin activation via positively regulating SSH1 .......... 73
Endogenous RanBP9 and SSH1 promote cofilin–actin rod formation and Aβ
oligomer-induced collapse of growth cones in primary hippocampal neurons .......... 75
Endogenous RanBP9 mediates the depletion of postsynaptic proteins induced by Aβ
oligomers in primary hippocampal neurons ............................................................... 76
Significant increase in endogenous RanBP9 in APP/PS1 mice brains ............................. 77
Rescue of neuroinflammation, synaptic damage, and Aβ accumulation by RanBP9
reduction in APP/PS1 mice ........................................................................................ 77
Accumulation of cofilin–actin rods in APP/PS1 mice and significant attenuation of
cofilin–actin rod formation by RanBP9 reduction ..................................................... 78
RanBP9 reduction rescues deficits in synaptic plasticity and contextual memory
associated with APP/PS1 mice ................................................................................... 79
Discussion ...................................................................................................................................... 80
Conclusion ..................................................................................................................................... 84

ii
Chapter 4 Discussion .................................................................................................................................. 95
Summary ........................................................................................................................................ 95
Conclusion ..................................................................................................................................... 96

References ................................................................................................................................................... 97

Appendix A IACUC Approval for Animal Research ............................................................................... 123

Appendix B Copyright Permissions .......................................................................................................... 124

Appendix C Copyright Permissions .......................................................................................................... 125

iii