Infection in Alzheimer's disease

231 Pages·2017·6.12 MB·English

Infection in Alzheimer’s disease

A thesis submitted to the University of Manchester for the degree of
Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2016
REBECCA C MONTACUTE

School of Biological Sciences

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Contents

Abbreviations 10
Abstract of thesis 14
Declaration 15
Copyright statement 15
Acknowledgements 16
1. Introduction 17
1.1 Alzheimer’s disease 19
1.1.1 How AD pathology is driven: the amyloid cascade hypothesis 21
1.2 Neuroinflammation in AD 23
1.2.1 Microglia in AD 24
1.2.2 The role of astrocytes in AD 27
1.2.3 Cytokines in AD 27
1.2.4 Complement in AD 28
1.3 Peripheral immune responses in AD 28
1.3.1 Immunosenescence in AD 31
1.4 Infections in AD 32
1.4.1 Increased susceptibility to infection in AD 32
1.4.2 The effects of infection on AD pathology and symptoms 32
1.4.3 Infection as a risk factor for AD 33
1.5 Toxoplasma gondii 35
1.5.1 Life cycle and transmission 35
1.5.2 Immune responses to T. gondii infection 38
1.5.3 Chronic T. gondii infection in the brain 41
1.5.4 T. gondii and Alzheimer’s disease 42
1.6 Trichuris muris 45
1.6.1 Life cycle of T. muris 45
1.6.2 Immune responses to T. muris 45
1.7 Aims 49
2 Methods 51
2.1 Animals 52
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2.2 Infections 54
2.2.1 Toxoplasma gondii 54
2.2.2 Trichuris muris 54
2.2.3 Tissue collection 54
2.3 Behavioural testing 55
2.3.1 Y maze 55
2.3.2 Novel Object 56
2.3.3 Novel Smell 57
2.3.4 Open Field 58
2.3.5 Urine smell preference test 59
2.4 Tissue processing 61
2.5 Histology 61
2.6 Immunohistochemistry 62
2.6.1 Colon 62
2.6.2 Brain 63
2.7 Cell culture 64
2.8 Flow cytometry 65
2.9 Quantitative PCR 66
2.9.1 RNA isolation 66
2.9.2 CDNA conversion 67
2.9.3 PCR 67
2.10 Statistics 68
2.10.1 Power calculations 68
3. Systemic immune responses to infection in transgenic AD mouse models 69
3.1 Introduction 70
3.1.1 Aims 71
3.1.2 Experimental design 71
3.1.2.1 T. gondii infection 71
3.1.2.2 T. muris infection 72
3.2 Results 73
3.2.1 Increased inflammation in 3xTg-AD and APP PS1 mice following
infection with T. gondii 73
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3.2.2 3xTg-AD but not APP PS1 mice have an increased pro-inflammatory
response following infection with T. gondii 82
3.2.3 Elevated pro-inflammatory responses in AD mouse models after
T. muris infection 88
3.3 Summary of results 94
3.4 Discussion 95
4. The effects of infection on neuroinflammation, AD pathology and behaviour in
AD mouse models 101
4.1 Introduction 102
4.1.1 Aims 104
4.1.2 Experimental Design 105
4.1.2.1 T. gondii infection 105
4.1.2.2 T. muris infection 106
4.2 Results 106
4.2.1 Increased microglia activation in 3xTg-AD mice following infection
with T. gondii and T. muris 106
4.2.2 Microglia activation in APP PS1 mice following infection with
T. gondii and T. muris 115
4.2.3 Behavioural testing in 3xTg-AD mice following infection with T. gondii. 124
4.2.4 Behavioural testing in APP PS1 mice following infection with T. gondii
and T. muris. 133
4.3 Summary of results 139
4.4 Discussion 139
5. Chronic T. gondii infection in the TgF344-AD transgenic AD rat 148
5.1 Introduction 149
5.1.1 Aims 152
5.1.2 Experimental design 152
5.1.2.1 Chronic infection with T. gondii in 3xTg-AD mice 152
5.1.2.2 Chronic infection with T. gondii in TgF344-AD rats 158
5.2 Results 153
5.2.1 Attempted chronic T. gondii infection in the 3xTg-AD mouse model of AD 153
5.2.2 Chronic T. gondii infection in the TgF344-AD rat model 158
5.2.3 Systemic immune responses in naïve TgF344-AD rats 167
5.3 Summary of results 169
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5.4 Discussion 169

6. General Discussion 173
6.1 Introduction 174
6.2 Is the immune response to infection altered in AD? 175
6.3 Does age affect immune responses in AD? 177
6.4 Does infection alter neuroinflammation in AD? 179
6.5 Does infection alter AD pathology and behaviour? 181
6.6 Could infections be prevented in AD patients? 182
6.7 Does chronic infection with T. gondii alter AD pathology and behaviour? 185
6.8 Are infections important in other CNS diseases? 187
6.9 Are mice and rats good models for AD? 189
6.10 Further Research 193
6.10.1 What are the mechanisms behind the altered immune response to
infection in AD? 193
6.10.2 Are activated microglia pro- or anti-inflammatory in AD following
infection? 195
6.10.3 How do other immune cells in the brain respond to infection in AD? 198
6.10.4 What could be learnt about chronic T. gondii infection in AD by using
the TgF344-AD rat model? 198
6.11 Conclusions 199
7. Appendix 201
7.1 General linear model repeated measures, weight throughout infection 201
8. References 208

Final word count: 43,631

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List of figures and tables

Figures

Chapter 1 - Introduction
Figure 1.1: APP processing in Alzheimer’s disease 21
Figure 1.2: Life cycle and transmission of T. gondii 36
Figure 1.3: Immune response following acute infection with T. gondii 39
Figure 1.4: Overview of the immune response to T. muris infection 46
Figure 1.5: Comparison of immune responses to T. gondii and T. muris 48

Chapter 2 - Methods
Figure 2.1: Timelines of AD pathology in 3xTg-AD, APP PS1 and TgF344-AD models 53
Figure 2.2: Y maze apparatus 56
Figure 2.3: Novel Object test apparatus 57
Figure 2.4: Novel Smell test apparatus 58
Figure 2.5: Open Field apparatus 59
Figure 2.6: Urine smell preference arena, set-up 1 60
Figure 2.7: Urine smell preference arena, set-up 2 61

Chapter 3 - Systemic immune responses to infection in transgenic AD mouse models
Figure 3.1: Inflammatory cell infiltration in the liver following T. gondii infection in
3xTg-AD mice. 75
Figure 3.2: Small intestine villi length following T. gondii infection in 3xTg-AD mice 76
Figure 3.3: Weight change in 3xTg-AD mice following infection with T. gondii 77
Figure 3.4: Inflammatory cell infiltration in the spleen following T. gondii infection in
3xTg-AD mice. 78
Figure 3.5 Inflammatory cell infiltration in the liver following T. gondii infection in
APP PS1 mice. 80
Figure 3.6: Small intestine villi length following T. gondii infection in APP PS1 mice 81
Figure 3.7: Weight change in APP PS1 mice following infection with T. gondii 82
Figure 3.8: Cytokine and chemokine expression after STAg stimulation of splenocytes
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following infection with T. gondii in 3xTg-AD mice. 84
Figure 3.9: Cytokine expression after STAg stimulation of splenocytes following infection
with T. gondii in 3xTg-AD mice. 85
Figure 3.10: Cytokine expression after STAg stimulation of splenocytes following
infection with T. gondii in APP PS1 mice. 85
Figure 3.11: Immune cell numbers and cytokine production following T cell stimulation
of splenocytes post infection with T. gondii in 3xTg-AD mice. 87
Figure 3.12: Crypt length in the colon in 3xTg-AD mice following infection with T. muris 89
Figure 3.13: CD45+ and F4/80+ cells following infection with T. muris in 3xTg-AD mice 90
Figure 3.14: CD11c+ cell responses in 3xTg-AD mice to infection with the nematode
parasite T. muris 91
Figure 3.15: T cell Responses in 3xTg-AD mice to infection with the nematode parasite
T. muris 93
Figure 3.16: T. muris worm burden in 3xTg-AD and APP PS1 mice following infection 94

Chapter 4: The effects of infection on neuroinflammation, AD pathology and behaviour
in AD mouse models
Figure 4.1: Microglia activation following T. gondii infection in 3xTg-AD mice 7 days PI 108
Figure 4.2: Microglia activation following T. gondii infection in 3xTg-AD mice 9 days PI 109
Figure 4.3: Parasite burden in the brain following T. gondii infection in 3xTg-AD mice 112
Figure 4.4: Cytokine levels in the brain at day 9 PI with T. gondii in 3xTg-AD mice 113
Figure 4.5: Microglia activation following T. muris infection in 3xTg-AD mice 114
Figure 4.6: Microglia activation following T. gondii infection in APP PS1 mice 117
Figure 4.7: Parasite burden in the brain following T. gondii infection in APP PS1 mice 118
Figure 4.8: Cytokine levels in the brain at day 9 PI with T. gondii in APP PS1 mice 120
Figure 4.9: Amyloid burden in the brain at day 7 PI with T. gondii in APP PS1 mice 121
Figure 4.10: Amyloid burden in the brain at day 9 PI with T. gondii in APP PS1 mice 122
Figure 4.11: Microglia activation following T. muris infection in APP PS1 mice 123
Figure 4.12: Behavioural testing in 3xTg-AD mice before infection with T. gondii 125
Figure 4.13: Weight change and splenocyte numbers following T. gondii infection
of 3xTg-AD mice 127
Figure 4.14: Y-maze behavioural testing of 3xTg-AD mice following infection with
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T. gondii 128
Figure 4.15: Open field behavioural testing of 3xTg-AD mice following infection
with T. gondii 129
Figure 4.16: Urine preference behavioural testing of 3xTg-AD mice following infection
with T. gondii 130
Figure 4.17: Urine preference behavioural testing of Naïve Non-Tg mice 131
Figure 4.18: Development of urine preference behavioural testing Non-Tg mice 132
Figure 4.19: Y-maze behavioural testing of APP PS1 mice following infection with
T. gondii 134
Figure 4.20: Novel Object behavioural testing of APP PS1 mice following infection with
T. gondii 135
Figure 4.21: Y-maze behavioural testing of APP PS1 mice following infection with
T. muris 136
Figure 4.22: Open Field behavioural testing of APP PS1 mice following infection with
T. muris 137
Figure: 4.23: Novel Object and Novel Smell behavioural testing of APP PS1 mice
following infection with T. muris 138

Chapter 5: Chronic T. gondii infection in the TgF344-AD transgenic AD rat
Figure 5.1: Weight change during attempted chronic infection with T. gondii in
3xTg-AD mice 155
Figure 5.2: Weight change during second attempted chronic infection with T. gondii
in 3xTg-AD mice 157
Figure 5.3: Figure 5.3: Weight change during acute infection with T. gondii in
Tg-F344-AD rats 159
Figure 5.4: Serum cytokine levels one week following infection with T. gondii in
Tg-F344-AD rats. 160
Figure 5.5: Serum cytokine levels one month following infection with T. gondii in
Tg-F344-AD rats 161
Figure 5.6: Serum cytokine levels two months following infection with T. gondii in
Tg-F344-AD rats 162
Figure 5.7: Serum cytokine levels three months following infection with T. gondii in
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Tg-F344-AD rats. 163
Figure 5.8: Amyloid burden in the brain at 3 months PI with T. gondii in Tg-F344-AD
rats 164
Figure 5.9: Cytokine expression in the brain at 3 months PI with T. gondii in
Tg-F344-AD rats 165
Figure 5.10: Cytokine level plotted against amyloid burden in the brain at 3 months PI
with T. gondii in Tg-F344-AD rats 166
Figure 5.11: Cytokine responses following ConA stimulation in TgF344-AD rats 168
Chapter 6: General discussion
Figure 6.1: Acute infection in transgenic AD mice 182

Tables
Table 1: Changes in blood and blood monocyte cytokine levels in AD patients 29
Table 2: Antibodies used for flow cytometry 66
Table 3: Group numbers of non-Tg and 3xTg-AD mice - T. gondii infection 72
Table 4: Group numbers of WT and APP PS1 mice - T. gondii infection 72

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Abbreviations

Aβ Amyloid beta
AD Alzheimer’s disease
AICD Amyloid intracellular domain
ANOVA Analysis of variance
APP Amyloid precursor protein
APOE Apolipoprotein E
Bordetella pertussis B. pertussis
BACE β-secretase 1
BBB Blood brain barrier
C. pneumoniae Chlamydia pneumoniae
CBA Cytometric bead array
CD Cluster of differentiation
cDNA Complementary DNA
CO Carbon dioxide
2
COX-2 Cyclooxygenase-2
CCR2 C-C chemokine receptor type 2
CCR5 C-C chemokine motif ligand 5
CCR6 C-C chemokine receptor 6
CNS Central nervous system
CSF Cerebrospinal fluid
CVO Circumventricular organs
CXCL-1 Chemokine C-X-C motif ligand 1
DAB 3,3-diaminobenzidine tetrahydrochloride
DG Dentate gyrus
DNA Deoxyribonucleic acid
E Edinburgh
E/S Excretory/secretory
EC Epithelial cell
FAD Familial Alzheimer’s disease
GABA γ-Aminobutyric acid
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