Cameron, Ryan T. (2014) The role of Hsp20 in Alzheimer's disease.

192 Pages·2014·6.92 MB·English

by  Ryan Cameron

Cameron, Ryan T. (2014) The role of Hsp20 in Alzheimer's disease.
PhD thesis.

http://theses.gla.ac.uk/5128/

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The Role of Hsp20 in Alzheimer’s Disease

By
Ryan Thomas Cameron

A thesis submitted in fulfilment of the requirements for the Degree of

Doctor of Philosophy
In
Molecular & Cellular Biology

Institute of Cardiovascular & Medical Sciences
College of Medical, Veterinary & Life Sciences
University of Glasgow
Scotland
September 2013
This thesis is dedicated to
my Wife & Son...
Table of Contents

Abstract .................................................................................................................................. i
Author’s Declaration ............................................................................................................ iv
Acknowledgements ................................................................................................................ v
List of Figures ...................................................................................................................... vi
List of Tables....................................................................................................................... vii
Abbreviations ..................................................................................................................... viii
Publications ............................................................................................................................ x
Conferences ............................................................................................................................ x
1 Introduction .................................................................................................................... 1
1.1 Alzheimer’s disease ................................................................................................. 1
1.2 Discovery of Alzheimer’s disease ........................................................................... 1
1.2.1 Amyloid Precursor Protein ............................................................................... 3
1.2.2 The Amyloid Hypothesis ................................................................................. 6
1.3 Small Heat Shock Proteins .................................................................................... 10
1.3.1 Discovery ....................................................................................................... 10
1.3.2 sHSP expression profile ................................................................................. 13
1.3.3 Structure & Function of small Heat Shock Proteins ...................................... 15
1.3.4 sHSPs & Amyloids ........................................................................................ 18
1.3.5 sHSPs and Aβ ................................................................................................. 19
1.4 Second Messenger Signal transduction in Cognition & Disease .......................... 22
1.4.1 Cyclic Adenosine Monophosphate (cAMP) .................................................. 22
1.4.2 Cyclic Guanosine Monophosphate (cGMP) .................................................. 25
1.4.3 PKA & PKG ................................................................................................... 26
1.4.4 Phosphodiesterases and Cognition ................................................................. 30
1.5 Thesis Aims & Hypothesis .................................................................................... 38
2 Materials & Methods ................................................................................................... 41
2.1 Materials ................................................................................................................ 41
2.2 Preparation of Aβ .................................................................................................. 41
2.3 Plasmid DNA ........................................................................................................ 42
2.3.1 Site directed mutagenesis ............................................................................... 42
2.3.2 Transformation ............................................................................................... 42
2.3.3 Amplification & Purification ......................................................................... 43
2.3.4 Quantification ................................................................................................. 43
2.3.5 Plasmid Storage .............................................................................................. 44
2.4 Expression and Purification of Recombinant Proteins .......................................... 44
2.4.1 Histidine purification ..................................................................................... 44
2.4.2 Protein concentration ..................................................................................... 45
2.5 Protein-Protein Interaction Studies ....................................................................... 45
2.5.1 Peptide Arrays ................................................................................................ 45
2.5.2 In vitro pull-down assay ................................................................................. 48
2.6 Protein Analysis .................................................................................................... 48
2.6.1 SDS-PAGE ..................................................................................................... 48
2.6.2 Coomassie Staining ........................................................................................ 48
2.6.3 Western blotting ............................................................................................. 49
2.6.4 Ponceau Staining ............................................................................................ 49
2.7 Amyloid Aggregation Analysis ............................................................................. 51
2.7.1 NMR ............................................................................................................... 51
2.7.2 Fluorescence quenching Aggregation Protocols ............................................ 51
2.8 Mammalian Cell Culture ....................................................................................... 53
2.8.1 Transfection of plasmid DNA ........................................................................ 54
2.8.2 Stable cell-lines .............................................................................................. 54
2.9 Preparation of Whole Cell Lysates ........................................................................ 54
2.10 Cell-Based Experiments ........................................................................................ 55
2.10.1 Cell Viability Assays ..................................................................................... 55
2.10.2 Real-Time Cell Monitoring (xCELLigence) .................................................. 55
2.10.3 Hsp20 phosphorylation assays ....................................................................... 58
2.11 Phosphodiesterase Activity Assay ......................................................................... 58
2.12 Microscopic Analysis ............................................................................................ 59
2.12.1 FRET Imaging ................................................................................................ 59
2.12.2 Immunocytochemical staining of SH-SY5Y cells ......................................... 60
2.13 Statistical Analysis ................................................................................................ 60
3 Hsp20 phosphorylation modulates its binding to Aβ and promotes neuroprotection .. 61
3.1 Introduction ........................................................................................................... 61
3.1.1 Experimental Aims......................................................................................... 62
3.1.2 Experimental Procedure ................................................................................. 62
3.2 Results ................................................................................................................... 63
3.2.1 Mapping the interaction between Hsp20 and Aβ using Peptide Array ..... 63
1-42
3.2.2 In vitro pull-down of Aβ with His-Hsp20 ................................................. 66
1-42
3.2.3 Hsp20 phosphorylation alters Aβ aggregation dynamics .............................. 69
3.2.4 Hsp20 overexpression attenuates Aβ induced cytotoxicity ...................... 83
1-42
3.3 Discussion ............................................................................................................. 90
3.3.1 The interaction of Hsp20 with Aβ is modulated via PKA/G
1-42
phosphorylation ............................................................................................................ 90
3.3.2 Hsp20 interacts with Aβ to maintain it in a non-toxic conformation ............. 91
3.3.3 Hsp20 attenuates two morphologically distinct Aβ aggregation pathways ... 93
3.3.4 Increased intracellular expression of Hsp20 is protective against Aβ
1-42
induced cytotoxicity ..................................................................................................... 95
4 PDE Inhibition Promotes Neuroprotection Via Hsp20 Phosphorylation ..................... 99
4.1 Introduction ........................................................................................................... 99
4.1.1 Experimental Aims....................................................................................... 104
4.1.2 Experimental Procedure ............................................................................... 105
4.2 Results ................................................................................................................. 105
4.2.1 PDE9 is expressed in human neuronal-like SH-SY5Y cells ........................ 105
4.2.2 PDE9 inhibition promotes Hsp20 Phosphorylation ..................................... 106
4.2.3 PDE inhibition attenuates Aβ induced cytotoxicity ................................ 108
1-42
4.3 Discussion ........................................................................................................... 118
4.3.1 PDE9 inhibition promotes Hsp20 Phosphorylation ..................................... 118
4.3.2 PDE inhibition attenuates Aβ induced cytotoxicity ................................ 120
1-42
5 The Development of Novel PDE4 Inhibitors to Induce Hsp20 Phosphorylation ...... 123
5.1 Introduction ......................................................................................................... 123
5.1.1 Experimental Aims....................................................................................... 125
5.1.2 Experimental Procedure ............................................................................... 125
5.2 Results ................................................................................................................. 126
5.2.1 Chemical informatics and docking studies identify moexipril as a candidate
PDE4 inhibitor. .......................................................................................................... 126
5.2.2 Model of moexipril and its analogues bound to catalytic domain of PDE4 127
5.2.3 Biochemical determination of moexipril potency as a PDE4 inhibitor ....... 129
5.2.4 Moexipril increases in intracellular cAMP .................................................. 131
5.2.5 Moexipril treatment promotes PKA-mediated phosphorylation of Hsp20 .. 133
5.2.6 Moexipril attenuates Aβ induced cytotoxicity ........................................ 135
1-42
5.3 Discussion ........................................................................................................... 136
6 Final Discussion ......................................................................................................... 142
6.1 Hsp20 Inhibits Aβ Aggregation .......................................................................... 143
6.2 Hsp20 attenuates Aβ toxicity .............................................................................. 145
6.3 Targeting Hsp20 phosphorylation as a therapeutic strategy for treating AD ...... 147
6.4 Final Conclusions ................................................................................................ 149
6.5 Limitations & Future Directions ......................................................................... 152
7 Appendices ................................................................................................................. 155
8 References .................................................................................................................. 158
Abstract
Alzheimer’s disease is the most common of the degenerative brain diseases and is
characterised by impairment of cognitive function. Patients with this disorder lose the
ability to encode new memories. Eventually, both declarative and non-declarative memory
is significantly impaired, resulting in the capacity for reasoning, abstraction and language
becoming progressively reduced. Alzheimer’s disease and other dementias have
devastating effects on families and caregivers, and is an increasing burden in an ageing
society. It is estimated that 36 million people worldwide are living with dementia and this
figure is expected to double every 20 years. The worldwide costs of dementia in 2010 were
estimated to be $604 billion, an exorbitant figure that represents 1% of global GDP (World
Alzheimer Report 2011).
Alzheimer’s disease is the fourth leading cause of death in industrialised nations, preceded
by cardiovascular disease, cancer and stroke. As yet there are currently no disease-
modifying drugs approved to treat Alzheimer’s disease. The therapeutics that are available
only temporarily alleviate symptoms of cognitive impairment, however, they do not halt
the inevitable progression of the disease. As such, major scientific efforts are underway in
order to develop drugs which can help stabilise the disease. The publication of the
“Amyloid Hypothesis” by Dennis Selkoe in 1991 helped to focus research efforts towards
a causative protein involved in the disease, the amyloid β protein (Aβ).
Aggregation and deposition of the Aβ protein is fundamental in the aetiology of
Alzheimer’s disease and its importance has been demonstrated by a number familial
heterogeneous mutations in the amyloid precursor protein that promote increased Aβ
deposition, resulting in early onset phenotypes. There are several other aspects involved in
disease progression such as neuroinflammation and aberrant neuronal signalling, however,
therapies targeting amyloid β aggregation have the potential to slow or even halt further
neurodegeneration and anti-Aβ therapies are regarded as a logical approach to treating
Alzheimer’s disease.
Several endogenous pathways exist to prevent protein misfolding and subsequent
aggregation following stressful cellular conditions. One pathway includes the amateur
chaperones of the small heat-shock protein family, which have recently garnered interest
due to their ability to inhibit the aggregation of amyloid-like proteins. In particular, Hsp20
was previously identified as having the ability to inhibit the aggregation of Aβ and could
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attenuate subsequent toxicity associated with Aβ peptides. Hsp20 was of particular interest
to the Baillie group as it has well established cardio-protective functions, which are
triggered by the phosphorylation of a serine residue (S16) at a consensus protein kinase
A/G site. Hsp20 “activity” can therefore be readily modulated via inhibition of second
messenger signal degradation by phosphodiesterases.
The first part of this thesis investigated the interaction between Hsp20 and Aβ using
Peptide Array technology. This technique allowed rapid characterisation of interacting
domains and pinpointed key residues that mediated the protein-protein interaction. Using
this approach, I demonstrated that the domain within Hsp20 that interacted with Aβ
included the consensus PKA phosphorylation site (R-R-X-S). Upon introduction of a
phospho-serine residue or a phospho-mimetic substitution, I was able to show that the
binding of Aβ was enhanced. Reciprocal peptide array experiments highlighted that Hsp20
bound to a domain within Aβ, which is key to the aggregation of the Aβ peptide and is
required to produce the higher order toxic Aβ species. The Peptide Array data was then
verified using full-length recombinant proteins and several Hsp20 mutants were developed
including a phospho-mimetic. The phospho-mimetic Hsp20 was shown to outperform the
wild-type variant in several assays such as, in vitro pull-down assays, Aβ aggregation
measured using nuclear magnetic resonance spectroscopy, and also a novel Aβ aggregation
assay which can differentiate between two distinct aggregation pathways, namely
fibrillisation and oligomerisation. These data demonstrated for the first time how the
interaction between Hsp20 and Aβ may be modulated by cell signalling cascades.
I then moved to investigate the cytotoxicity of Aβ in order to investigate whether
increasing Hsp20 expression in neuronal-like cells would confer protection against Aβ-
mediated toxicity. This was initially carried out using a standard MTT-based cell viability
assay, before utilising a real-time cell monitoring device to develop a novel Aβ toxicity
assay. In both assays, increasing Hsp20 expression was shown to be cytoprotective. The
wild-type variant of Hsp20 was found to be more effective in cell-based assays due to
increased levels of phosphorylated Hsp20. The real-time Aβ toxicity monitoring assay also
gave me a platform for testing agents with potential neuroprotective properties.
Given that increasing levels of phosphorylated Hsp20 could attenuate Aβ-mediated
cytotoxicity, I logically was drawn to study ways that this event could be targeted
therapeutically. Several drugs that target cAMP- and cGMP-dependent phosphodiesterases
have been shown to be effective in alleviating symptoms of Alzheimer’s disease in rodent
ii
models have also been studied here in cellular systems. These included the “blockbuster”
PDE5 inhibitor Viagra® (sildenafil), two novel compounds which selectively inhibit
PDE9, which were developed by the pharmaceutical company Lundbeck specifically as
Alzheimer’s treatments, and rolipram, a well established cognitive enhancer that was
developed originally as an anti-depressant. All of these compounds were shown to
“activate” endogenous Hsp20 to varying degrees in neuronal-like cells and the levels of
Hsp20 activation was found to correlate with both the level of induced Hsp20/Aβ co-
localisation, and subsequent attenuation of Aβ-mediated cytotoxicity. This suggests that
this endogenous protection pathway can be targeted by currently available therapeutics in
order to reduce the neurotoxic effects of Aβ.
Finally, we wanted to develop novel agents of our own that could promote Hsp20
phosphorylation. To do this, in silico docking of all FDA approved drugs against the
catalytic domain of PDE4 was undertaken in an attempt to find a novel compound with the
potential to reposition as an Alzheimer’s treatment. Using this methodology we discovered
an angiotensin converting enzyme inhibitor, moexipril to be a PDE4 inhibitor in the low
micro molar range. Unfortunately, moexipril works as an ACE inhibitor in the low nano
molar range making repositioning unviable. However, moexipril treatment was more
effective than rolipram in reversing Aβ toxicity and I speculate that this may be due to the
sub-family selective nature of its (moexipril) PDE4 inhibition. Furthermore, the lack of
emetic side effects associated with moexipril makes this compound an ideal starting point
for the development of isoform selective and/or non-emetic PDE4 inhibitor.
In summary, these studies describe a novel endogenous mechanism for combating the toxic
effects of the Aβ protein, which underpins the development and progression of
Alzheimer’s disease. Given that the interaction between Hsp20 and Aβ can be manipulated
via cAMP/cGMP signalling, the interaction could be targeted therapeutically. As there are
currently no effective drugs on the market for stabilising Alzheimer’s disease, I believe
that the data presented here opens up a potential new avenue that could lead to the
development of a new class of AD drugs.

iii
Author’s Declaration
I hereby declare that the work presented within this thesis has been conducted by myself,
unless otherwise cited or acknowledged. The work is entirely of my own composition and
has not been submitted, in whole or in part, for any other degree at the University of
Glasgow or any other institution.

Ryan Cameron
September 2013

iv