Noradrenergic Deficits Contribute to Impairment in the TgCRND8 Mouse Model of Alzheimer's ...

229 Pages·2013·21.09 MB·English

by  Beverly Francis

Noradrenergic Deficits Contribute to Impairment in the
TgCRND8 Mouse Model of Alzheimer’s Disease
by
Beverly M. Francis
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Department of Physiology
University of Toronto
© Copyright by Beverly M. Francis (2013)
Noradrenergic Deficits Contribute to Impairment in the TgCRND8
Mouse Model of Alzheimer’s Disease
Beverly M. Francis
Doctor of Philosophy
Department of Physiology
University of Toronto
2013
Abstract
Autosomal-dominant mutations in the amyloid precursor protein (APP) gene increase
the production and aggregation of toxic amyloid-β (Aβ) peptides and cause early-onset
Alzheimer’s disease (AD). Noradrenergic cell loss is well documented in AD and has
been posited to play a role in cognitive symptoms as well as disease progression. We
investigated memory and affect, tissue levels of catecholamines, brain-derived
neurotrophic factor (BDNF) mRNA and bioenergetic homeostasis in TgCRND8 mice
that express a double mutant (K670N/M671L + V717F) human APP transgene. We
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found that TgCRND8 mice develop object memory impairment and behavioural despair,
as well as reductions in noradrenaline and BDNF expression in the hippocampus and
cortex, before the appearance of Aβ plaques. Animals with more advanced Aβ
pathology exhibit disruptions in energetic status, along with diminished complex I+III
activity in the electron transport chain. To test whether the AD-like phenotypes of
TgCRND8 mice might be due to altered noradrenergic tone, pre-plaque mice were
treated with dexefaroxan, an antagonist of presynaptic inhibitory α -adrenoceptors that
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are highly expressed on both noradrenergic and cholinergic terminals. Effects of
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dexefaroxan were compared to those of rivastigmine, a cholinesterase inhibitor. Both
dexefaroxan and rivastigmine improved behavioural phenotypes and BDNF expression
without affecting tissue Aβ load. Drug treatments also restored complex I+III
mitochondrial activity and increased ATP levels. Reductions in noradrenergic tone
appear to underlie Aβ-induced functional impairment in TgCRND8 mice, in addition to
BDNF deficits and bioenergetic stress. These studies suggest that α -adrenoceptor
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targeting may warrant consideration as a therapeutic strategy in AD.
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To Leo and Moira,
with love.
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Acknowledgments

I extend my deepest gratitude to my supervisor, Dr. Howard Mount, for guiding me both
professionally and personally. Howard is a wonderful role model, teacher and mentor.
His advice set me on the right path and helped me achieve my best.
I would also like to thank my thesis committee members, Dr. JoAnne McLaurin and Dr.
Linda Mills, whose support and recommendations were crucial to the completion of this
work. I also thank Dr. Paul Frankland, Dr. James Eubanks, Dr. Sheena Josselyn, Dr.
Jason Lerch and Dr. Douglas Feinstein for evaluating my thesis. I am grateful to our
collaborators, Dr. Margaret Fahnestock, Dr. Brian Robinson, Dr. Yeni Yücel and Dr.
Richard Bazinet, for sharing their technical expertise and passion for neuroscience.
It has been a pleasure working in the Mount lab, due in large part to Dr. Jennifer Griffin,
Keith Ho, Emily Lam, Jimmy Yang, Elizabeth Cumyn, Valeriya Larskova and Zohar
Weinbrand, who have provided me with assistance, encouragement and laughter. I
also thank Dr. Janice Robertson, Dr. Cheryl D’Souza, Dr. Jesse McLean, Dr. Vivian Ng,
Dr. Christopher Bohm, Dr. John Sévalle, Robert Chen and Rosemary Ahrens, for their
comradeship and advice. I treasure these friendships formed at the CRND. To my dear
friends outside of the lab, particularly Anna Stasienko and Sunil Rao, thank-you for
giving me much needed perspective, coffee and companionship during stressful times.
I also gratefully acknowledge support from the Ontario Graduate Scholarship (OGS), the
Peterborough K.M. Hunter Alzheimer’s Graduate Studentship and the Ontario Graduate
Scholarship in Science and Technology (OGSST) programs.
I am indebted to my loving family for their unwavering support. This thesis is dedicated
to my beloved father Leo (Nov. 25th 1953 – April 22nd, 2012), who would have read
every word in it and been all the more proud, and mother Moira, who refuses to read it
but has given me all I need to accomplish it. I thank my brother Dalton, for leading me
to the finish line and teaching me more than I could ever teach him. Finally, I thank my
fiancé Ian Aranjo, for enduring the worst of me and inspiring the best in me. I love you.
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Contributions

This thesis consists of 4 original research studies. Chapter 3 was published in the
journal Neurobiology of Aging (Francis et al., 2012a) and Chapter 4 was published in
the journal Neuropyschopharmacology (Francis et al., 2012b). Research described in
Chapters 5, 6 and 8 has yet to be published.

The author performed all experiments described in the thesis, except as noted below:
Mr. Jimao Yang – HPLC analyses of monoamines and high-energy tissue
phosphates in mouse brain tissue
Dr. Margaret Fahnestock – guidance in RT-PCR measurement of BDNF mRNA
in mouse brain tissue (performed jointly with Ms. Bernadeta Michalski)
Dr. JoAnne McLaurin – assessment of spatial memory in mice (performed by Dr.
Meredith Barakat) and guidance with western blot analyses of holoAPP and β-
CTF (assistance provided by Ms. Mary Brown)
Dr. Brian Robinson – guidance in planning and analyses of mitochondrial
enzymatic activities and tissue expression in mouse brain (assistance provided
by Dr. Mary Maj)
Dr. Yeni Yücel – assessment of visual function in mice (performed by Dr.
Stephan Fraenkl and Mr. John Kim in the Mount laboratory)
Dr. Richard Bazinet – TLC measurement of cardiolipin in mouse brain (performed
by Mr. Chris Song)
Ms. Enid Hajderi – artwork for the microwave dissection procedure

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Table of Contents
ACKNOWLEDGMENTS ……………………………………………………………………... V
CONTRIBUTIONS …………………………………………………………………………….VI
TABLE OF CONTENTS ……………………………………………………………………..VII
LIST OF TABLES ……………………………………………………………………………XIII
LIST OF FIGURES …………………………………………………………………………. XIV
LIST OF ABBREVIATIONS ………………………………………………………………..XVI
PREFACE …………………………………………………………………………………...…. 1
CHAPTER 1 LITERATURE REVIEW …………………………………………………... 2
1.1 Alzheimer’s Disease ……………………………………………………………........…3
1.1.1 Clinical Presentation ……………………………………………………………3
1.1.1.1 Cognition ………………………………………………………………4
1.1.1.2 Affect …………………………………………………………....……..5
1.1.2 Neuropathology …………………………………………………………………5
1.1.2.1 Amyloid Plaques ……………………………………………………...6
1.1.2.2 Neurofibrillary Tangles ……………………………………………….6
1.1.3 Neurotransmitter System Involvement ……………………………………….7
1.1.3.1 The Cholinergic Hypothesis …………………………………………8
1.1.3.2 Damage to the Locus Coeruleus Noradrenergic System .…….....9
1.1.4 Neurotrophic Mechanisms ……………………………………………….......10
1.1.5 Bioenergetic Factors in Disease Progression ……………………………...12
1.1.5.1 The Electron Transport Chain and ATP Homeostasis ……….....12
1.1.5.2 Oxidative Stress and Mitochondrial Dysfunction …………..........14
1.1.5.3 Decreased Metabolic Activity ……………………………………...16
1.2 The Amyloid Cascade Hypothesis ………………………………………………….18
1.2.1 Early-Onset Familial Mutations ……………………………………………...18
1.2.2 APP Processing, Aβ Production and Fibrillogenesis ……………………...19
1.2.3 Toxicity of Aβ ……………………………….………………………….......….22
1.2.3.1 Distal Axonopathy and Synaptic Dysfunction …………………....22
1.2.3.2 Aβ Downregulation of BDNF Signaling ………...………………...23
1.2.3.3 Aβ Interactions with Mitochondria ……...…………………...........25
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1.3 Noradrenergic Mechanisms in Alzheimer’s Disease …………………………... 26
1.3.1 Noradrenaline in the Brain ……………………………………………….......27
1.3.1.1 Anatomical Locus …………………………………………………...27
1.3.1.2 Synthesis and Metabolism …………………………………………29
1.3.1.3 Receptors and Signal Transduction Pathways ……………....….30
1.3.1.4 Functions …………………………………………………………….32
1.3.2 Noradrenergic Depletion Potentiates Amyloid Toxicity …………………...33
1.3.2.1 Decreased Clearance of Aβ and Increased Inflammation ....…..33
1.3.2.2 Decreased Cholinergic Transmission ………………………........35
1.3.2.3 Decreased Trophic Support …………………………………....….38
1.3.2.4 Increased Oxidative and Energetic Stress ……………….......….38
1.4 APP-Transgenic Mouse Models of Alzheimer’s Disease ……………………….40
1.4.1 Mouse Models of Aβ Deposition ………………………………...….......…..41
1.4.2 Limitations in Modeling Late-Stages of Disease ………………………..... 43
1.4.3 Noradrenergic Involvement in APP-Tg mice ……………………………….45
1.4.3.1 Noradrenaline Depletion Studies ………………………………….46
1.4.3.2 Noradrenaline Enhancement Studies …………………………….48
1.4.4 The TgCRND8 Mouse ………………………………………………………..49
1.4.4.1 Neuropathological and Neurochemical Changes …………….....50
1.4.4.2 Behavioral Profile …………………………………………………...53
1.4.4.3 Validity as a Model of Alzheimer’s Disease ……………………...54
CHAPTER 2 HYPOTHESES AND EXPERIMENTAL APPROACH ………………..56
2.1 Thesis Aims ………………………………………………………………………....….57
2.2 Hypotheses ……………………………………………………………………………..57
2.2.1 Object Memory Impairment is an Early Endophenotype ……………....…57
2.2.2 Noradrenaline Content is Reduced in Major Terminals and Antagonism of
α -Adrenoceptors Mitigates Functional Impairment ……………………….57
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2.2.3 Bioenergetic Homeostasis is Disrupted by Mitochondrial Dysfunction ….58
2.2.4 Blockade of α -Adrenoceptors Alleviates Mitochondrial Impairment ....…58
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CHAPTER 3 OBJECT RECOGNITION MEMORY AND BDNF EXPRESSION
ARE REDUCED IN YOUNG TgCRND8 MICE ………………………..59
3.1 Abstract ……………………………………………………………………………….…60
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3.2 Introduction ……………………………………………………………………………..61
3.3 Methods ………………………………………………………………………………….63
3.3.1 Mice ………………………………………………………………………….....63
3.3.2 Object Recognition ……………………………………………………………63
3.3.3 Visual Function ………………………………………………………………..65
3.3.4 Spatial Memory ………………………………………………………………..66
3.3.5 Measurement of BDNF mRNA ………………………………………………66
3.3.6 Statistical Analysis …………………………………………………………….67
3.4 Results ……....…………………………………………………………………………..68
3.4.1 Progressive Object Recognition Deficits Precede Amyloid Plaque
Accumulation ………………………………………………………………..…68
3.4.2 Pre-plaque TgCRND8 Mice are Not Impaired in the Morris Water
Maze Task …………………………………………………………………......70
3.4.3 BDNF mRNA is Reduced in the Hippocampus and Frontal Cortex of
Young TgCRND8 Mice ……………………………………………………….71
3.5 Discussion ………………………………………………………………………………73
CHAPTER 4 REDUCED TISSUE LEVELS OF NORADRENALINE ARE
ASSOCIATED WITH BEHAVIORAL PHENOTYPES OF THE
TgCRND8 MOUSE MODEL OF ALZHEIMER’S DISEASE …………77
4.1 Abstract …………………………………………………………………........……..…..78
4.2 Introduction …………………………………………………………………………..…79
4.3 Materials and Methods ………………………………………………………………..81
4.3.1 Transgenic Mice ……………………………………………………....………81
4.3.2 Brain Fixation and Microdissection ………………………………………….81
4.3.3 Tissue Catecholamines ………………………………………………....……83
4.3.4 Tail Suspension Test …………………………………………………....…....84
4.3.5 Assessing Forelimb Coordination with the Tape Removal Task ….....….84
4.3.6 Drug Treatments, Behavioral Testing and Tissue Processing ……..........84
4.3.7 Aβ Immunoassays …………………………………………………………….86
4.3.8 Western Blotting ……………………………………………………………....86
4.3.9 Measurement of BDNF mRNA ………………………………………………87
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4.3.10 Statistical Analysis …………………………………………………………….88
4.4. Results ………………………………………………………………………………......89
4.4.1 TgCRND8 Mice Exhibit Early Reductions in Noradrenaline Content
Within Major Terminal Fields ………………………………………………..89
4.4.2 TgCRND8 Mice Display Behavioral Despair in the Tail Suspension
Test ……………………………………………………………………………..90
4.4.3 Desipramine Alleviates Behavioral Despair ……………………………......91
4.4.4 TgCRND8 Mice are Not Impaired in a Test of Forelimb Coordination …..92
4.4.5 Dexefaroxan and Rivastigmine Reduce Object Memory Impairment
and Behavioral Despair ……………………………………………………....93
4.4.6 Treatments Do Not Affect Amyloid Burden …………………………….…..95
4.4.7 Dexefaroxan and Rivastigmine Increase BDNF mRNA …………………..97
4.4.8 Dexefaroxan and Rivastigmine Differentially Affect Noradrenaline
Turnover in the Hippocampus …………………………………………….....98
4.5 Discussion ………………………………………………………………………………99
CHAPTER 5 SELECTIVE TARGETING OF MITOCHONDRIAL COMPLEX I
IN AGED APP-TRANSGENIC TgCRND8 MICE ……………….....…105
5.1 Abstract ……………………………………………………………………………......106
5.2 Introduction …………………………………………………………………………...107
5.3 Methods ………………………………………………………………………………..109
5.3.1 Transgenic Mice ……………………………………………………………..109
5.3.2 Metabolic Enzyme Activities ………………………………………………..109
5.3.3 Western Blot Analysis ……………………………………………………….112
5.3.4 Heat-Inactivated Tissue Preparation ………………………………………112
5.3.5 Measurement of Cardiolipin ……………………………………………......113
5.3.6 High-Energy Phosphate Donors …………………………………………...113
5.3.7 Statistical Analysis …………………………………………………………..114
5.4 Results …………………………………………………………………………….…...115
5.4.1 Linked Complex I+III Activity and NDUFB8 Complex I Subunit
Expression are Reduced in Aged TgCRND8 Mice ………………………115
5.4.2 Cardiolipin is Unaltered in TgCRND8 Mice ………………………............118
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