Cell- and Stage-specific Impact of TNF-alpha Receptor Signaling in Alzheimer's Disease By Sara L ...

226 Pages·2013·1.81 MB·English

Cell- and Stage-specific Impact of TNF-alpha Receptor Signaling

in Alzheimer’s Disease

By
Sara L. Montgomery

Submitted in Partial Fulfillment of the
Requirements for the degree
Doctor of Philosophy

Supervised by
Professor M. Kerry O’Banion
and
Professor William J. Bowers

Department of Pathology
School of Medicine and Dentistry

University of Rochester
Rochester, New York

2013
i

Cell- and Stage-specific Impact of TNF-alpha Receptor Signaling

in Alzheimer’s Disease

By
Sara L. Montgomery

Submitted in Partial Fulfillment of the
Requirements for the degree
Doctor of Philosophy

Supervised by
Professor M. Kerry O’Banion
and
Professor William J. Bowers

Department of Pathology
School of Medicine and Dentistry

University of Rochester
Rochester, New York

2013
ii

Dedication

This thesis work is dedicated to my family who has provided me with constant
encouragement and inspiration. To my mother, Mona, who has given me
unconditional love and instilled a sense of dedication, self-discipline, and hard work
in order to make my goals become reality. To my grandfather, Richard, who has
guided me in spirit, and to my brother and sisters, Nick, Toni, and Susie who have
kept me humble and grateful.
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Curriculum Vitae
Sara Lynn Montgomery was born in Cortland, NY on April 26, 1985. She graduated
from Groton High School in 2004. She attended Rochester Institute of Technology
from 2004-2008 and graduated with a Bachelor of Science in Biotechnology. Sara
entered graduate school at the University of Rochester School of Medicine and
Dentistry in the fall of 2008 in the Department of Pathology. She trained in the
laboratory of Dr. William J. Bowers from 2009-2011, and then in the laboratories of
Drs. M. Kerry O’Banion and John Olshowka from 2011-2013 where she continued
her studies on the role of TNF-α in Alzheimer’s disease. In 2010, she received travel
awards from the Department of Pathology and Graduate Student Society poster
sessions. That same year she was a recipient of a Ruth L. Kirchstein National
Research Service Award (NRSA) from the National Institutes of Health. She was
awarded a travel award from the Department of Neurobiology and Anatomy in 2012.
Sara earned her Masters of Science degree from the Department of Pathology in
October 2010.
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Publications
In Print

Montgomery, S.L., Narrow, W.C., Mastrangelo, M.A., Olschowka, J.A., O’Banion,
M.K., Bowers, W.J. (2013) “Chronic Neuron- and Age-selective Downregulation of
TNF Receptor Expression in Triple-transgenic Alzheimer’s Disease Mice Leads to
Significant Modulation of Amyloid and Tau-related Pathologies.” Am J Pathol

Wu, M.D., Montgomery, S.L., Olschowka, J.A., O’Banion, M.K. (2013) “Sustained
IL-1β Expression Impairs Adult Hippocampal Neurogenesis Independent of IL-1
Signaling in Nestin+ Neural Precursor Cells.” Brain Behav Immun

Montgomery, S. L. and W. J. Bowers (2012). "Tumor Necrosis Factor-alpha and the Roles it
Plays in Homeostatic and Degenerative Processes Within the Central Nervous
System." J Neuroimmune Pharmacol 7(1):42-59.

Montgomery, S. L., M. A. Mastrangelo, et al. (2011). "Ablation of TNF-RI/RII Expression
in Alzheimer's Disease Mice Leads to an Unexpected Enhancement of Pathology:
Implications for Chronic Pan-TNF-alpha Suppressive Therapeutic Strategies in the
Brain." Am J Pathol 179(4): 2053-2070.

Dolan K, Montgomery S, Buchheit B, Didone L, Wellington M, Krysan DJ (2009).
“Antifungal Activity of Tamoxifen: In Vitro and In Vivo Activities and Mechanistic
Characterization.” Antimicrob Agents Chemother 53:3337-3346.
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Meeting Abstracts
Montgomery SL, Mastrangelo MA, Narrow WC, Knowlden SA, Wright TW, Bowers
WJ (2011) “Ablated TNF-alpha Receptor Expression Leads to Enhanced Alzheimer’s
Disease Pathology in 3xTg-AD Mice” Keystone Symposia: The Molecular and
Cellular Basis for Neurodegeneration (Poster)

Montgomery SL, Mastrangelo MA, Narrow WC, Wright TW, O’Banion MK, Bowers
WJ (2012) “Cell- and Stage-specific Impact of TNF-alpha Receptor Signaling in
Alzheimer’s Disease” Society for Neuroscience (Nanosymposium Platform Talk)
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Acknowledgements
There are a number of people who have been instrumental in my thesis work and
success. First, I’d like to thank my advisors, Dr. William J. Bowers and Dr. M. Kerry
O’Banion. I am appreciative of Dr. Bowers’ outstanding guidance and support over
the years, especially from afar, and for the many opportunities he has given me. Dr.
Bowers has been a true mentor. Under his guidance I have learned how to
confidently and independently form and test a hypothesis. He has provided
invaluable direction in strengthening my writing and public communication skills,
and importantly, grantsmanship, all of which will be useful as my career progresses.
Dr. Bowers is incredibly kind and has led by example in showing me the importance
of hard work and dedication. Furthermore, he has been a motivating force, always
letting me know his appreciation for my efforts. I am truly thankful for Dr. Kerry
O’Banion’s guidance and encouragement and for letting me join his lab later in my
graduate career. He is very compassionate and established an inviting, flexible and
nurturing environment for my graduate studies. Dr. O’Banion has also been an
outstanding mentor by challenging my scientific conceptualization and by providing
invaluable career advice. I’m also grateful for Dr. John Olschowka, of whom the “O
Labs” would not be complete. He was vital in introducing me to the lab space and
was always available for my many questions. Dr. Olschowka also provided a sense
of serenity by putting the given situation in perspective. I am grateful for his
enthusiasm for science and for his inspiration. Dr. Olschowka is highly generous and
hosted many wonderful lab celebrations. I am confident that the mentorship of Drs.
Bowers, O’Banion, and Olschowka will continue influencing my scientific career
throughout my lifetime.
I’d also like to thank my thesis committee members, Dr. Gail Johnson-Voll,
Dr. Sanjay Maggirwar, and Dr. Terry Wright for their exceptionally helpful and
motivating comments through my PhD training. I am fortunate to have such a
supportive and student-oriented committee and am sincerely thankful for their interest
in my project. I extend my thanks to Dr. Fran Lund for playing an early instrumental
role during my qualifying exam.
I owe many thanks to past and current members of the Bowers and
O’Banion/Olshowka (O Labs) laboratories for their scientific encouragement,
criticisms and discussion. I also thank them for their generous friendships and
providing an environment where I am excited to come in each day. Michael
Mastrangelo devoted a significant portion of his time in training me in the many
techniques that I have mastered over the years. I thank Mr. Mastrangelo for his
efforts in generating the 3xTg-ADxTNF-RI/RII KO mice utilized in Chapter 1 of this
thesis and for teaching me efficiency in the laboratory. Wade Narrow was
fundamental for the data generated in Chapter 2. He provided rAAV expertise and
aided in the design, construction, and viral packaging of the siRNA. Wade also
strengthened my in vitro culturing skills. Moreover, he provided many wonderful
homemade hard cider tastings to help us decompress. Special thanks should be given
to Rita Giuliano who spent countless hours in the tissue culture room with me and
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was critical in teaching me the ins and outs of culturing primary microglia. I also
acknowledge Lou Lotta for his hard work in maintaining the animal colonies. Clark
Burris and Marilyn Elliot deserve many thanks for their organization and prompt
ordering of reagents and supplies. Previous graduate students of the Bowers
laboratory, Drs. Keigan Park, Debbie Ryan, Suresh DeSilva, and Maya Desai
provided a source of encouragement and are examples of stellar students. I owe Dr.
Diala Habib for her time in optimizing the in vivo LTP experiments that are presented
in this thesis and am appreciative of her expertise in hippocampal circuitry and
synapse function. I thank Wenli Wang for her insightful and challenging questions
and being a respectful bench mate. I express unexplainable gratitude to Sara
Knowlden who is a dear friend and an exceptional immunologist. She challenged my
scientific thinking with an immune perspective and provided boundless support both
scientifically and personally. Sara is and will be a true lifetime friend.
I am fortunate to have worked with not only one wonderful group of people in
lab, but two. Lee Trojanczyk, Mallory Olshowka, and Jack Walter deserve credit for
maintaining the day-to-day organization in the O Labs and for keeping lab members
up-to-date on safety trainings, maintaining animal colonies, being available for aiding
in tissue mounting and technical questions, and for making the lab environment fun
and cheerful. I am lucky to have worked with a diverse and hard-working group of
graduate student, all of whom enriched my scientific conceptualization and mentoring
skills. Dr. Mike Wu provided useful insights for the stressful FACs experiments that
were performed and offered a voice of encouragement and support. Sage Begolly,
Jonathan Cherry, and Fatima Rivera-Escalera allowed me to be a part of their
qualifying exam journey by asking excellent questions and challenging my clarity,
patience, and guidance. I thank Dr. Tara Sweet for her career advice and for lending
her ear to discuss scientific and personal dilemmas. Sarah McConnell and Simi
Ghosh offered useful constructive criticisms that further strengthened my project.
Each person significantly impacted my graduate career and I am in their debt.
I acknowledge the support from the National Institute of Health for funding
my National Research Service Award entitled “Stage-specific Impact of TNF-alpha
Receptor Signaling in Alzheimer’s Disease” (F31-AG3800632). Contributions from
other investigators outside the University that helped make this thesis work possible
include Dr. Colin Combs (University of North Dakota) who shared his in vitro
phagocytosis assay protocol, Dr. William Klein (Northwestern University) for kindly
providing the Nu-4 antibody, Dr. Peter Davies (Albert Einstein School of Medicine)
for the PHF-1 antibody, and Dr. Takashi Matozaki (Gunma University, Gunma,
Japan) provided the SIRP-β1 antibody.
I am extremely thankful for the collaborative community at the University of
Rochester and would like to extend my appreciation to people that have worked
“behind the curtains.” I express my gratitude to Taimi Marple in the Pathology
Department for her diligence in scheduling rooms for the defense, organizing seminar
talks and student council meetings, completing my thesis paperwork and for her
supportive character. Special thanks to Dr. Jane Tolbert in CNDD for her
administrative efforts in keeping me on track with the NRSA grant.
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I am deeply indebted to my family and friends. I am appreciative of the many
friends that I have made at the University and would like to particularly mention
fellow graduate students of the Pathology Department, Mike Moses, Julie Smith,
Fiona Kuo and Laura Nguyen for their scientific advice and friendship. Life in
graduate school can sometimes be a roller coaster, and without support outside the lab
many challenges could have proven even more difficult. I give many thanks to
Michael and Chelsea Tolleson for their support, encouragement, and fun Friday and
Saturday night get-togethers. I extend my gratitude to my cherished friends, Lindsay
Schmidt, Jennifer Vuong, and Sara Knowlden for their continued friendship and
enlivenment. I thank Zack Knowlden for allowing me to steal his wife almost every
day for lunch. I’d like to thank my parents Mona Head and Ralph Montgomery, my
siblings and their spouses, Nick and Sophia, Toni and Matt, and Susie and Steve for
their love, support and wisdom. I am incredibly fortunate to be an aunt to my
amazing nephews and niece, Gannon, Lucas, Stephen, Mattison, and Lane of who let
me lose sense of responsibility, if only for a short amount of time. Finally, I give
immense thanks to my best friend and boyfriend, Trevor Smith. He has provided
continual support and love through my graduate training and all other endeavors. He
has been tremendously patient even when I was frustrated, and without his noble
character, undying sense of humor and thoughtfulness this work would have been
nearly impossible. Special thanks to Trevor’s family who consider me as their own
and were unbelievably supportive and interested in my graduate work. I am also
grateful for my canine companion, Chub-chubs who has given my laughter, joy, and
love.
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Abstract
Alzheimer’s disease (AD) is a progressive degenerative disorder characterized by
severe memory loss and cognitive impairment that elaborates in a temporal and
spatial manner. Neuropathological correlates include amyloid-beta (Aβ) deposition,
neurofibrillary tangles, synaptic loss, and neuronal cell death. AD is the most
common form of dementia and lacks truly disease ameliorating therapy. It is
estimated that 5.4 million Americans are currently afflicted by AD and as the
population ages, the incidence is estimated to increase dramatically. By 2050 it is
projected that between 11 and 16 million Americans will be living with the disorder,
making research in the diagnosis, treatment, and prevention of this devastating
malady a matter of great national importance.

Neuroinflammation drives disease AD pathogenesis through the production of pro-
inflammatory molecules and activated glia. Tumor Necrosis Factor- alpha (TNF-α), a
pleotropic pro-inflammatory cytokine, is produced in excess and implicated in Aβ-
induced inflammation and cognitive decline. While TNF-α has been well studied, its
purported function remains elusive, and surprisingly, little is known about the cell
type- and stage-specific roles of this signaling molecule in AD. These questions are
becoming ever more important since a growing body of research has been devoted to
preclinical and clinical testing of non-selective anti-TNF-α modulating drugs for the
treatment of AD. Our goal was to dissect the role of TNF-α and its associated
receptors to gain insight into the specific signaling requirements and outcomes of this