Examinations Into The Calcium Hypothesis Of Alzheimer's Disease

172 Pages·2014·14.61 MB·English

by  Dustin J. Shilling

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Publicly Accessible Penn Dissertations
2013
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Dustin J. Shilling
University of Pennsylvania, [email protected]
Follow this and additional works at: https://repository.upenn.edu/edissertations
Part of the Neuroscience and Neurobiology Commons
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Shilling, Dustin J., "Examinations into the Calcium Hypothesis of Alzheimer's Disease" (2013). Publicly
Accessible Penn Dissertations. 697.
https://repository.upenn.edu/edissertations/697
This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/697
For more information, please contact [email protected].
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Alzheimer's disease (AD) is devastating to the patient, their family and friends, and represents a
significant fiscal burden to our society. Currently available therapeutics provide only mild symptomatic
relief and do not alter the course of the disease. Developing the next generation of disease modifying
therapies requires an understanding of the early cellular changes responsible for AD. A hindrance to
progress is the fact that most patients develop AD sporadically. However, mutations in the presenilin (PS)
homologs cause dominantly inherited, early-onset AD. These mutations provide an important tool for
understanding the cellular changes that cause AD. One consequence of PS mutations is exaggerated
intracellular Ca2+ ([Ca2+]i) signaling. However, the mechanisms underlying this phenomenon remain
controversial and its role in AD pathogenesis is unknown. Presented here are data indicating that
exaggerated [Ca2+]i signaling is dependent upon the inositol 1,4,5-trisphosphate receptor (InsP3R) and
contributes to AD pathogenesis in vivo. We began our studies by testing multiple proposed mechanisms
for exaggerated [Ca2+]i signaling. To do this we employed multiple Ca2+ imaging protocols and Ca2+
indicators to directly measure ER Ca2+ dynamics in several cell systems. We found that decreasing
InsP3R protein level rescues exaggerated [Ca2+]i signaling in primary cortical neurons and hippocampal
slices from mutant PS1 expressing mice. We then determined the contribution of exaggerated [Ca2+]i
signaling to AD pathogenesis. Using a combination of genetic, biochemical, electrophysiological and
behavioral techniques, we found that rescue of exaggerated [Ca2+]i signaling dramatically attenuates
mild cognitive impairment and AD phenotypes in AD mouse models. Reduction of InsP3R1 protein level in
PS1M146V-KIN mice rescued enhanced hippocampal ryanodine receptor protein level, enhanced
hippocampal synaptic potentiation, and constitutive activation of the CaMKIV-CREB transcriptional
pathway. In 3xTg mice, reduced InsP3R1 protein level attenuated Aβ and phospho-tau accumulation and
hippocampal electrophysiology and memory impairments. Together, these results reveal that mutant PS-
associated exaggerated [Ca2+]i signaling is InsP3R1-dependent, a proximal event, and contributes to the
development of AD in vivo. These findings advance our understanding of the pathological role of
exaggerated [Ca2+]i signaling in AD and identify several novel targets for the development of disease
modifying therapeutics.
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Dissertation
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Doctor of Philosophy (PhD)
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Neuroscience
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J. K. Foskett
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Neuroscience and Neurobiology
This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/697
EXAMINATIONS INTO THE CALCIUM
HYPOTHESIS OF ALZHEIMER’S DISEASE

Dustin J. Shilling

A DISSERTATION
in
Neuroscience
Presented to the Faculties of the University of Pennsylvania
in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
2013

Supervisor of Dissertation
Signature __________________________________
Dr. J. Kevin Foskett, Professor of Physiology, Perelman School of Medicine

Graduate Group Chairperson
Signature __________________________________
Dr. Joshua I. Gold, Associate Professor, Department of Neuroscience, Perelman School of
Medicine

Dissertation Committee
Dr. Douglas Coulter (Chair), Professor of Pediatrics, Children’s Hospital of Philadelphia
Dr. Steven Arnold, Professor of Psychiatry, Perelman School of Medicine
Dr. Ted Abel, Professor of Biology, School of Arts and Sciences
Dr. Frank LaFerla, Professor of Neurobiology, School of Biological Sciences
ACKNOWLEDGMENTS

My graduate career has provided an extremely rewarding learning experience, and
I have many people to thank for that. To start, I would like to thank my mentor, Kevin
Foskett, for his expertise, advice and guidance. His willingness to invest his time,
resources, and to let me explore my interests was invaluable to my development as a
scientist. For that I am extremely grateful.
I would like to thank the members of the Foskett laboratory who provided
important technical training, advice, and were always willing to discuss my data. Many
thanks to Marioly Muller, Adam Siebert, Daniel Mak, King-Ho Cheung, Rob Lee, Cesar
Cardenas, Jung Yang, Horia Vais, Zhongming Ma, and Akiyuki Taruno.
I am extremely grateful to Ted Abel and Doug Coulter who were willing to
collaborate with me on my thesis work. The expertise, training and access to
experimental hardware they provided were critical in accomplishing this work. I am
indebted to all those in their laboratories who were so willing to selfishness allow me to
work on their rigs. Special thanks to Hajime Takano, Morgan Bridi, and Alan Park, all of
whom spent an enormous amount of time providing technical support and training. I
would also like to thank the University of Pennsylvania Behavioral Core, specifically
Tim O’Brien for providing training in behavioral assays.
Thank you to Doug Coulter for Chairing my thesis committee and the other
members, Ted Abel, Steven Arnold, Robert Neumar and Frank LaFerla. These
individuals have dedicated time to support my growth as a scientist, and their comments
have guided my project and allowed for its success. I would also like to thank the

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Neuroscience Graduate Group and the countless faculty and staff members that
contributed to my training during my graduate career. In combination their efforts have
provided me with a strong foundation in neuroscience.
Lastly, I would like to thank my family and friends for their support and
understanding during my time in graduate school. Their encouragement and distraction
allowed me to make it through the roughest times during my training. Special thanks to
Sheena Baratono for her willingness to counsel, sympathize, and celebrate with me
through the ups-and-downs of graduate school.

My thesis training was supported in part by a National Institutes of Health Grant
AG038240.

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ABSTRACT

EXAMINATIONS INTO THE CALCIUM HYPOTHESIS OF
ALZHEIMER’S DISEASE

Dustin J. Shilling
J. Kevin Foskett, Ph.D.

Alzheimer’s disease (AD) is devastating to the patient, their family and friends,
and represents a significant fiscal burden to our society. Currently available therapeutics
provide only mild symptomatic relief and do not alter the course of the disease.
Developing the next generation of disease modifying therapies requires an understanding
of the early cellular changes responsible for AD. A hindrance to progress is the fact that
most patients develop AD sporadically. However, mutations in the presenilin (PS)
homologs cause dominantly inherited, early-onset AD. These mutations provide an
important tool for understanding the cellular changes that cause AD. One consequence of
PS mutations is exaggerated intracellular Ca2+ ([Ca2+]) signaling. However, the
i
mechanisms underlying this phenomenon remain controversial and its role in AD
pathogenesis is unknown. Presented here are data indicating that exaggerated [Ca2+]
i
signaling is dependent upon the inositol 1,4,5-trisphosphate receptor (InsP R) and
3
contributes to AD pathogenesis in vivo. We began our studies by testing multiple
proposed mechanisms for exaggerated [Ca2+] signaling. To do this we employed multiple
i
Ca2+ imaging protocols and Ca2+ indicators to directly measure ER Ca2+ dynamics in

  - iv -
several cell systems. We found that decreasing InsP R protein levels rescues exaggerated
3
[Ca2+] signaling in primary cortical neurons and hippocampal slices from mice
i
expressing mutant PS1. We then determined the contribution of exaggerated [Ca2+]
i
signaling to AD pathogenesis. Using a combination of genetic, biochemical,
electrophysiological and behavioral techniques, we found that rescue of exaggerated
[Ca2+] signaling attenuates mild cognitive impairment and AD-like phenotypes in mouse
i
models. Reduction of InsP R1 protein level in the PS1M146V knock-in AD mouse
3
rescued enhanced hippocampal ryanodine receptor protein level, enhanced hippocampal
synaptic potentiation, and constitutive activation of the CaMKIV-CREB transcriptional
pathway. In 3xTg AD mice, reduced InsP R1 protein level attenuated Aβ and phospho-
3
tau accumulation and hippocampal electrophysiology and memory impairments.
Together, these results reveal that mutant PS-associated exaggerated [Ca2+] signaling is
i
InsP R1-dependent, a proximal event, and contributes to the development of AD in vivo.
3
These findings advance our understanding of the pathological role of exaggerated [Ca2+]
i
signaling in AD and identify several novel targets for the development of disease
modifying therapeutics.

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TABLE OF CONTENTS
ACKNOWLEDGMENTS ……………………………………………..….. ii
ABSTRACT …………………………………………………………….… iv
TABLE OF CONTENTS …………………………………………………. vi
LIST OF FIGURES ……………………………………………………….. ix
CHAPTER 1 – INTRODUCTION …………………………………….….. 1
ALZHEIMER’S DISEASE ………………………………………..………. 1
ALZHEIMER’S DISEASE GENETICS ……………………..……….….…. 3
Sporadic Alzheimer’s disease genetics ……………………..…..………… 3
Familial Alzheimer’s disease genetics ……………………..………….….. 4
THE CELLULAR FUNCTIONS OF FAMILIAL ALZHEIMER’S
DISEASE PROTEINS ………………………………………..………….... 5
Amyloid precursor protein ………………………………..……….……. 5
Presenilins …………………………………………………...………... 7
HYPOTHESIZED MECHANISMS OF ALZHEIMER’S
DISEASE PATHOGENESIS ……………………………………..………... 8
CALCIUM HOMEOSTASIS …………………………………..………..… 9
DISRUPTION OF CALCIUM HOMEOSTASIS IN ALZHEIMER’S
DISEASE …………………………………………………………….… 10
Patient cell lines ……………………………...………………………. 11
Neuronal and non-neuronal cell lines ………………………………….... 11
Alzheimer’s disease mouse models ………………………………........... 12
CONTRIBUTIONS OF EXAGGERATED CALCIUM SIGNALING
TO ALZHEIMER’S DISEASE PATHOGENESIS ……....……………….… 13
Amyloid precursor protein processing and Aβ generation .……………...… 13
Tau hyperphosphorylation ………..……………………………………. 15
Neurodegeneration …………………...……………………………….. 17

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Synaptic deficits ………………………………………………..…..… 17
Targeting exaggerated [Ca2+] signaling ……………………...………..… 19
i
HYPOTHEISIZED MECHANISMS OF MUTANT
PRESENILIN EXAGGERATED CALCIUM SIGNALING ……………….... 20
RATIONALE …………………………………..……………………….. 24

CHAPTER 2 – Lack of Evidence for Presenilins as Endoplasmic Reticulum
Ca2+ Leak Channels …………………………………………..…………... 26
SUMMARY …………………………………………………………….. 27
INTRODUCTION ……………………………………………………..… 28
EXPERIMENTAL PROCEDURES ……………………………………….. 31
RESULTS ……………………………………………………..………... 37
DISCUSSION …………………………………………………..………. 48
CHAPTER 3 – Exaggerated Intracellular Calcium Signaling and Phenotypes
of Two AD-mouse Models are Attenuated by Decreasing Inositol 1,4,5-
trisphosphate Receptor Type 1 Protein Level …………….……………… 63
SUMMARY ………………………………………………..………….... 64
INTRODUCTION ……………………………………………………….. 65
EXPERIMENTAL PROCEDURES ……………………………………….. 69
RESULTS …………………………………………………………….… 81
DISCUSSION ………………………………..……….……………...... 106
CHAPTER 4 – DISCUSSION ……………………….…………….…… 129
EXAGGERATED CALCIUM SIGNALING IS INSP R1-DEPENDENT ....… 130
3
MECHANISMS OF CALCIUM-INDUCED ALZHEIMER’S
DISEASE PATHOGENESIS ………….……….……………….……..… 131
Amyloid precursor protein metabolism ………………….….…………. 131

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Tau hyperphosphorylation ……………………………………….....… 133
Synaptic deficits ………………………………...………………...… 135
IMPLICATIONS FOR THERAPEUTIC APPROACHES ………………..… 138
Current therapeutic approaches ……………………………….….…… 138
Future therapeutic approaches ………………………………..…….… 139
REFERENCES …………………………………………………..……… 141

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