Temporal and regional changes in glial markers in post-mortem Alzheimer's disease brain

308 Pages·2017·7.84 MB·English

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Investigating the contribution of astrocytes and neuroinflammation to pathological tau
changes in Alzheimer's disease

Phillips, Emma Claire

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King's College London

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D ownload date: 06. Nov. 2017
Investigating the contribution of astrocytes
and neuroinflammation to pathological tau
changes in Alzheimer's disease

Emma Phillips

Thesis submitted in fulfilment of the degree of Doctor of
Philosophy

Department of Basic and Clinical Neuroscience
Institute of Psychiatry, Psychology and Neuroscience
King’s College London

September 2016
1
Declaration
I hereby declare that all of the work presented in this thesis is my own, with the exception of
the following:
 Electron microscopy of synthetic Aß was carried out by Covance (Princeton, NJ, US).
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 Immunohistochemical staining of post-mortem brains was carried out by the London
Neurodegenerative Diseases Brain Bank.

Emma Phillips
September 2016

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Acknowledgements
Firstly, many thanks to Dr Wendy Noble for her continual guidance and support. Wendy was
always able to provide encouragement and advice when most needed. Thank you also to Prof
Diane Hanger for her invaluable additional input and guidance. I also appreciate the perspective
gained by being mentored by two strong women so early in my career.
Thanks also to Dr Michael O’Neill and everyone at Eli Lilly for being so helpful and making me
feel so welcome. In particular, thank you to Dr Joanna Wolak and Dr Daniel Ursu. Also, Emma,
Sarah, Yugesh, Elena, Jasmeet and Silvia, without your help I would have been completely lost.
Thanks to Fiona for helping me with the CHO cells. A huge thank you to Team Tau, particularly
the Noble group. Also, especially to Dawn and Teresa for being lovely right from the start. Lizzie,
for early morning neuron prep gossips and Charlotte for wanting to throw yourself over
obstacles just as much as me. Also to Martina, for the chocolate and Mount Everest chat!
Thank you to my friends who supported me along the way: my ice skating family, GoodGym
friends and the Wadhamites. Particularly thanks to Sam, Dan, Ella and Ella for the pub and tube
walks, Abi and Emily for the girl’s nights, and to Becca, without whom I wouldn’t have got
through the last four years - thank you for the wine, feminism and Meryl Streep films!
A special thanks goes to my family who have always been there, even when I’m a crying mess
ruining our Canadian holiday! Thank you to my parents who support and believe in me no matter
what. To my sisters, Sal, Soph and Grace (adopted sister!), who always know how to lighten the
mood and remind me what life is actually about – fun! Most importantly, thanks to Ed, literally
my other half, without you this thesis would not have happened. Thank you for caring about
Alzheimer’s and tau even though it’s really not your thing!

Finally, this thesis is dedicated to my Gransha, Mac Norgrove, whose love, laughter and dignity
in the face of neurodegeneration sparked this whole journey off.

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Abstract
Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterised by
accumulation of ß-amyloid in extracellular plaques, intracellular neurofibrillary tangles
composed of abnormally phosphorylated and aggregated tau, and widespread synaptic
dysfunction and neuron loss that underlie the clinical symptoms of AD. Glial activation and a
neuroinflammatory immune response is also a key aspect of the pathological progression of AD.
The activation of astrocytes appears to be particularly associated with pathological changes in
tau. This thesis aims to investigate the association between astrocyte activation and abnormal
tau processing using primary cell culture and human post-mortem brain. Furthermore, it aims
to explore possible regional differences in this role of astrocytes, and the molecular signalling
pathways by which astrocytes exert their effects on tau.
Experiments in primary astrocyte and neuron co-cultures demonstrated that astrocytes were
involved in accelerating Aß-induced neurotoxicity in hippocampal cultures, but not cortical
cultures although the differences were quite subtle. Interestingly, astrocytes were important for
the neuronal release of tau from cortical neurons under basal conditions, suggesting that
astrocytes may be important for pathological tau spread in AD. Analysis of human post-mortem
brain showed differences in astrocytic changes in hippocampus and cortex as AD progresses. In
addition, these experiments also suggested regional differences in mechanisms related to
synaptic dysfunction and loss as disease progresses. These data suggest that different
mechanisms may underlie the neurodegenerative effects of ß-amyloid and/or activated
astrocytes in distinct brain regions; an important consideration when considering therapeutic
strategies for AD.
In addition, the potential benefits for tauopathy of repurposing an already licenced drug with
anti-inflammatory action were investigated. Despite showing significant modulation of tau
phosphorylation in primary cultures, dimethyl fumarate had little influence on disease-
associated tau species when tested in vivo in a mouse model of tauopathy.
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Overall, the findings of this thesis suggest that there are regional differences in astrocyte
activation during the development of AD, that are somewhat associated with AD-relevant
changes in tau. This work also supports a role for astrocytes in physiological tau release. Further
elucidating these differences will increase understanding of neurodegenerative mechanisms.
Moreover, these data suggest that regional involvement at different disease stages could be an
important consideration when targeting specific mechanisms for therapeutic development.
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Contents

Declaration .................................................................................................................................... 2
Acknowledgements ....................................................................................................................... 3
Abstract ......................................................................................................................................... 4
Contents ........................................................................................................................................ 6
List of Figures .............................................................................................................................. 12
List of Tables ............................................................................................................................... 16
Publications arising from this studentship .................................................................................. 17
Abbreviations .............................................................................................................................. 18
Chapter 1: Introduction ........................................................................................................ 24
1.1 Historical perspective .................................................................................................. 24
1.2 Neuropathology .......................................................................................................... 25
1.2.1 ß-amyloid plaques ............................................................................................... 26
1.2.2 Neurofibrillary tau tangles .................................................................................. 26
1.3 The genetics of AD ...................................................................................................... 28
1.4 Amyloid precursor protein and ß-amyloid .................................................................. 31
1.4.1 Amyloid precursor protein processing ................................................................ 31
1.5 Tau............................................................................................................................... 34
1.5.1 Protein structure ................................................................................................. 34
1.5.2 Post-translational modifications of tau .............................................................. 35
1.5.3 Tau cleavage ........................................................................................................ 37
1.5.4 Tau aggregation .................................................................................................. 38
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1.5.5 Tau functions in health and disease.................................................................... 39
1.5.6 Tau spread and seeding ...................................................................................... 41
1.6 The amyloid cascade hypothesis ................................................................................ 42
1.6.1 Modelling the amyloid cascade in vitro .............................................................. 45
1.7 Neuroinflammation ..................................................................................................... 47
1.7.1 Glia ...................................................................................................................... 48
1.7.2 Inflammatory mediators ..................................................................................... 60
1.7.3 Neuroinflammation as a therapeutic target in AD ............................................. 64
1.8 Aims and objectives of this thesis ............................................................................... 66
Chapter 2: Materials and Methods ....................................................................................... 67
2.1 General buffer solutions ............................................................................................. 67
2.2 Cell culture .................................................................................................................. 68
2.2.1 Rat primary neurons ........................................................................................... 68
2.2.2 Cell lines .............................................................................................................. 71
2.2.3 ß-amyloid characterisation ................................................................................. 73
2.2.4 Cell treatment ..................................................................................................... 78
2.2.5 Cell viability LIVE/DEAD assay ............................................................................. 81
2.3 Htau mice .................................................................................................................... 82
2.3.1 Genotyping and polymerase chain reaction (PCR) ............................................. 82
2.3.2 Treatment and testing of htau mice ................................................................... 85
2.4 Sample preparation and standardisatoin ................................................................... 89
2.4.1 Sample preparation ............................................................................................ 89
2.4.2 Protein standardisation....................................................................................... 94
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2.5 Immunostaining .......................................................................................................... 95
2.5.1 Immunocytochemistry (ICC) of primary rat cultures .......................................... 95
2.5.2 Immunohistochemical (IHC) staining of human post-mortem brain sections .... 97
2.6 Western blotting ......................................................................................................... 98
2.6.1 Materials ............................................................................................................. 98
2.6.2 Antibodies ......................................................................................................... 102
2.6.3 Methods ............................................................................................................ 104
2.7 Immunoassays........................................................................................................... 106
2.7.1 Standard tau ELISA ............................................................................................ 106
2.7.2 Tau sandwich ELISA ........................................................................................... 108
2.7.3 Alphascreen....................................................................................................... 110
2.8 Statistical analysis ..................................................................................................... 115
2.8.1 Protein analysis and cell death assays .............................................................. 115
2.8.2 Analysis of Alphascreen and MSD data ............................................................. 117
2.8.3 Behavioural analysis of htau mice .................................................................... 117
Chapter 3: Investigating regional differences in Aß-induced effects of astrocytes in primary
neuron cultures ......................................................................................................................... 118
3.1 Introduction .............................................................................................................. 118
3.2 Methods .................................................................................................................... 121
3.3 Results ....................................................................................................................... 123
3.3.1 Preliminary work ............................................................................................... 123
3.3.2 Aßcharacterisation ........................................................................................... 129
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3.3.3 Astrocytes do not affect Aß-induced changes in 14 DIV primary cortical cultures
141
3.3.4 Astrocytes may mediate some Aß-induced changes in primary hippocampal
cultures 152
3.3.5 Summary ........................................................................................................... 156
3.4 Discussion .................................................................................................................. 156
3.4.1 Aß characterisation ........................................................................................... 157
3.4.2 Aß-induced cell death and tau changes were not observed in 14 DIV primary
cortical cultures ................................................................................................................. 159
3.4.3 Astrocytes may regulate extracellular tau concentrations ............................... 161
3.4.4 Differences in Aß and minocycline effects in hippocampal cultures versus cortical
cultures 162
3.4.5 Conclusions ....................................................................................................... 163
Chapter 4: Exploring the potential of dimethyl fumarate repurposing for Alzheimer’s disease
165
4.1 Introduction .............................................................................................................. 165
4.2 Methods .................................................................................................................... 169
4.3 Results ....................................................................................................................... 171
4.3.1 Preliminary work ............................................................................................... 171
4.3.2 The effects of DMF on tau and astrocytes in mixed primary cortical cultures . 173
4.3.3 DMF treatment of htau mice ............................................................................ 175
4.3.4 Possible signalling pathways for DMF action .................................................... 186
4.3.5 Summary ........................................................................................................... 193
4.4 Discussion .................................................................................................................. 194
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