Evaluation of antioxidant and neuroprotective therapies in a mouse model of amyotrophic lateral ...

261 Pages·2014·9.35 MB·English

by  Lewis, Kate

Evaluation of antioxidant and neuroprotective
therapies in a mouse model of amyotrophic
lateral sclerosis

Katherine Elizabeth Amy Lewis (BBiotech Hons)
School of Medicine

Submitted in fulfilment of the requirements for the
Doctor of Philosophy

University of Tasmania, October 2014

____________________________________________________________Declarations
______________________________________________________________________
Declaration of originality
This thesis contains no material which has been accepted for a degree or diploma by the
University or any other institution, except by way of background information and duly
acknowledged in the thesis, and to the best of the my knowledge and belief no material
previously published or written by another person except where due acknowledgement
is made in the text of the thesis, nor does the thesis contain any material that infringes
copyright.
Katherine Lewis …………………. October 2014

Authority of access
This thesis may be made available for loan and limited copying and communication in
accordance with the Copyright Act 1968.
Katherine Lewis …………………. October 2014

Statement of ethical conduct
The research associated with this thesis abides by the international and Australian codes
on human and animal experimentation, the guidelines by the Australian Government's
Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and
Institutional Biosafety Committees of the University.
Katherine Lewis …………………. October 2014

Statement regarding published work contained in thesis
Data in this thesis has given rise to one publication in Journal of Neuroinflammation.
The authors retain copyright for that article, and grant any third party, in advance and in
perpetuity, the right to use, reproduce or disseminate the article, according to the
BioMed Central copyright and license agreement. The remaining non-published content
of the thesis may be made available for loan and limited copying and communication in
accordance with the Copyright Act 1968.
Katherine Lewis …………………. October 2014
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____________________________________________________________Declarations
______________________________________________________________________
Statement of co-authorship
The following people and institutions contributed to the publication of work undertaken as part
of this thesis:
Katherine E Lewis, School of Medicine, University of Tasmania
Roger Chung, Australian School of Advanced Medicine, Macquarie University
Meng Inn Chuah, School of Medicine, University of Tasmania
Paper 1:
Microglia and motor neurons during disease progression in the SOD1G93A mouse model of
amyotrophic lateral sclerosis: Changes in arginase1 and inducible nitric oxide synthase
Katherine E Lewis, Anna L Rasmussen, William Bennett, Anna King,
Adrian K West, Roger S Chung, Meng Inn Chuah
Journal of Neuroinflammation, accepted for publication 7 March 2014
Data used for publication is located within Chapter 2 of this thesis.
Katherine Lewis (Candidate) conducted most of the experiments (about 60%) and all analyses;
she was also the primary author. In this publication, Anna Rasmussen contributed to some of the
data presented in Figures 1, 2, 3 and 6 while Anna King contributed to Figure 7. Authors West,
Chung and Chuah contributed to the idea, design and development of the project. Author
Bennett collected tissue samples and offered general laboratory assistance.

We the undersigned agree with the above stated “proportion of work undertaken” for each of the
above published (or submitted) peer-reviewed manuscripts contributing to this thesis:
Signed: ___________________
A/Prof MI Chuah Prof J Walls
Supervisor Head of School
School Of Medicine School of Medicine
University of Tasmania University of Tasmania

Date: _____________________
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________________________________________________________________Abstract
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Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised
by dysfunction and degeneration of motor neurons innervating skeletal muscle. ALS
patients experience progressive muscle weakness and atrophy, leading to paralysis and
death within 3-5 years of diagnosis. The mechanisms underlying neurodegeneration in
ALS are unknown; studies of patient tissue and of transgenic mouse models of ALS
have implicated oxidative stress, neuroinflammation, aberrant RNA metabolism,
excitotoxicity, protein misfolding, autophagy and proteasome dysfunction, and
intracellular transport deficits in disease processes.
Current ALS therapeutics can only extend lifespan by a matter of months, so it is vital
that novel therapeutic targets and therapeutic molecules are identified. The many
putative “triggers” of ALS are predicted to converge upon common mechanisms of
degeneration, with oxidative stress being identified as one of the major pathological
hallmarks of ALS. Therapeutics capable of modulating oxidative stress and preventing
neuronal death may be of value in treating human ALS.
In this thesis, the temporal correlations between microglial activation, development of
pathological alterations in the spinal cord, and functional decline, were explored in the
transgenic SOD1 mouse model of ALS (carrying the ALS-linked mutant human Cu,Zn-
superoxide dismutase gene SOD1G93A), with non-transgenic (WT) mice used as
controls. The ability of three putative therapeutic compounds for ALS – Gemals,
metallothionein-2 protein, and Emtin peptides – to increase survival time in SOD1 mice
was also examined.
Pathological alterations in motor neurons preceded an increase in microglial numbers,
suggesting microglial activation occurs as a reactive response to neuronal degeneration
or dysfunction. Microglial activation occurred concurrently with disease onset at 14
weeks of age, but preceded the development of overt functional deficits around 18
weeks of age. Interestingly, microglial activation was associated with an increase in the
number of microglia expressing the M2-like, putative neuroprotective, marker arginase1
(Arg1), and to a lesser extent with an increase in the number of microglia expressing the
M1-like, putative neurotoxic, marker inducible nitric oxide synthase (iNOS). These data
suggest the concurrent presence of ongoing neuroprotective and neuroinflammatory
processes in the spinal cord of SOD1 mice; microglial activation may not be a primary
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________________________________________________________________Abstract
______________________________________________________________________
cause of neurodegeneration, but may drive disease progression after onset. Additionally,
the expression of the antioxidant protein metallothionein-1/2 (MT-1/2) increased from
18 weeks of age, possibly in response to oxidative stress or neuronal degeneration.
Gemals, an antioxidant and anti-inflammatory combination therapy, has been previously
shown to extend the lifespan of an ALS rat model, but has not been tested in ALS mice.
Here, Gemals was administered subcutaneously to SOD1 mice from the age of
symptom onset through to disease endpoint. No significant changes in survival time
were identified in Gemals-treated SOD1 mice compared to controls, indicating that
Gemals treatment may be less effective when administered after symptom onset.
MT-1/2 protein has previously shown both antioxidant and neuroprotective properties,
and its ablation in SOD1 mice has been shown to accelerate disease progression. In this
study, SOD1 mice were treated with MT2 injections, and/or with treadmill running
exercise to upregulate endogenous MT-1/2. MT2 treatment slightly but significantly
delayed disease onset, and tended to increase survival time, in SOD1 mice, whereas
treadmill running exercise had little effect. However, the mechanism of action for MT2
is as yet unknown – preliminary data suggest that MT2 treatment did not substantially
prevent spinal cord motor neuron degeneration or muscle endplate denervation.
Peptide derivatives of MT-1/2, termed Emtins, have previously displayed similar
neuroprotective properties to their parent MT-1/2 protein in vitro and in vivo, and
additionally can readily cross the blood-brain barrier. Emtins were administered
subcutaneously to SOD1 mice from the onset of disease symptoms, resulting in
increased survival time compared to control mice, although this result was not
significant due to a smaller number of animals used during this trial. These data indicate
that both MT2 and Emtins have pro-survival effects in the SOD1 mice. Emtin peptides
are thought to have limited metal-binding and antioxidant properties; however, both
MT-1/2 and Emtins are known to interact with low-density lipoprotein receptor-related
proteins (LRPs) and activate the Akt pathway, leading to increased cellular survival. It
is possible that the pro-survival effects of MT2 and Emtins seen in these studies were
mediated through LRP binding and activation of downstream pathways. MT2 and
Emtins show potential as therapeutic molecules for ALS, but more work is required to
elucidate the mechanism of action.
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_______________________________________________________Acknowledgements
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Acknowledgements
First and foremost, I would like to thank my supervisors Associate Professor Meng Inn
Chuah, School of Medicine, University of Tasmania, and Professor Roger Chung,
Australian School of Advanced Medicine, Macquarie University, for the endless
support, advice, and guidance they have given me throughout my candidature.
The work described in this thesis has been supported by funding from the Motor Neuron
Disease Research Institute of Australia (MNDRIA) and the National Health and
Medical Research Council (NHMRC).
I would like to acknowledge past student Ms Anna Rasmussen for her contribution to
the microglial protein expression data in Chapter 2, Dr Bill Bennett for his preparation
of the spinal cord tissue library in Chapter 2 and his assistance in the pre-clinical trial in
Chapter 3, Ms Emma Eaton for preparing biotinylated Emtin in Chapter 5, and
Associate Professor Leigh Blizzard for his patient explanations of statistical procedures
involved in constructing linear mixed models.
Thanks also to past and present animal services staff – Murray Plaister, Angela Maher,
Keri Playford, and Peta Lawrie – and to animal welfare officer Barrie Wells, for their
expert and diligent care of animals, and advice in all aspects of animal trials. I would
also like to thank Professor Peep Palumaa and his laboratory, especially Dr Julia
Smirnova, Dr Kairit Zovo, Andra Noormägi, and Merlin Friedemann, for their support
and assistance during my visit to the Tallinn University of Technology in 2012.
I would like to thank my fellow MT lab students for being the best bunch of people to
work with – to Emma Eaton, Ros Herbert, Dr Jackie Leung, and Lila Landowski, thank
you for all of your friendship, advice, problem-solving, support, and cake. A huge
thankyou goes to Dr Bill Bennett, for being an infinite source of knowledge and
support. I would also like to thank Professor Adrian West, and all past members of the
MT and MBU groups, for sharing their considerable collective wisdom and insight with
me across the years.
To my dear friends and fellow students who have shared this PhD journey with me –
thank you so much for the friendship, camaraderie, laughs, commiserations, cups of tea,
Friday drinks, and epic ASMR dinners we have shared over the past four years.
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_______________________________________________________Acknowledgements
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Thanks also go to my family – my father David, mother Krystyna, and brothers Andrew
and Alex, for always being supportive and interested in what I was doing.
Finally, to my incredible partner Anthony, who has endured the worst of my late nights
in the lab and my bad moods when experiments failed; I simply could not have finished
my PhD without your unwavering love and support.

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________________________________________________________Table of contents
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Table of contents
Declaration of originality ................................................................................................. i
Authority of access ........................................................................................................... i
Statement of ethical conduct ........................................................................................... i
Statement regarding published work contained in thesis ............................................ i
Statement of co-authorship ............................................................................................ ii
Abstract ........................................................................................................................... iii
Acknowledgements .......................................................................................................... v
Table of contents ........................................................................................................... vii
List of figures ................................................................................................................... x
List of tables ................................................................................................................... xii
Abbreviations ............................................................................................................... xiii
Chapter 1 Literature Review Molecular pathology leading to motor neuron
degeneration in amyotrophic lateral sclerosis .............................................................. 1
1.1 Motor neuron disease ......................................................................................... 2
1.1.1 An overview of the motor system ............................................................... 2
1.1.2 Types of motor neuron disease ................................................................... 2
1.2 Amyotrophic lateral sclerosis ............................................................................. 4
1.2.1 Clinical features and disease course ............................................................ 4
1.2.2 Diagnosis ..................................................................................................... 4
1.2.3 Prevalence and risk factors.......................................................................... 5
1.2.4 Prognosis and treatment strategies .............................................................. 5
1.2.5 Pathological features of ALS ...................................................................... 6
1.2.6 Mouse models of ALS ................................................................................ 6
1.3 Etiology of ALS ................................................................................................. 8
1.3.1 Genetic evidence for molecular pathologies in ALS .................................. 8
1.3.2 Oxidative stress ......................................................................................... 11
1.3.3 Glial activation and neuroinflammation.................................................... 16
1.3.4 Mitochondrial dysfunction ........................................................................ 20
1.3.5 RNA processing dysfunction .................................................................... 22
1.3.6 Excitotoxicity ............................................................................................ 24
1.3.7 Protein misfolding and protein degradation pathways .............................. 27
1.3.8 Intracellular transport deficits ................................................................... 29
1.3.9 Convergent pathological mechanisms in ALS aetiology .......................... 32
1.4 Therapeutic strategies to combat ALS ............................................................. 37
1.4.1 Gemals....................................................................................................... 37
1.4.2 Metallothionein and Emtins ...................................................................... 37
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________________________________________________________Table of contents
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1.5 Research questions and aims ............................................................................ 38
Chapter 2 Characterisation of neuroinflammatory and functional changes over
time in SOD1 mice......................................................................................................... 40
2.1 Background ...................................................................................................... 41
2.1.1 Microglial activation status in ALS .......................................................... 41
2.1.2 Measuring disease progression in SOD1 mice ......................................... 42
2.1.3 Aims and hypothesis ................................................................................. 43
2.2 Methods ............................................................................................................ 44
2.2.1 Animal ethics ............................................................................................ 44
2.2.2 Maintenance and genotyping of SOD1 mice ............................................ 44
2.2.3 Spinal cord changes between SOD1 and WT mice .................................. 46
2.2.4 Functional characterisation of SOD1 and WT mice ................................. 52
2.3 Results .............................................................................................................. 56
2.3.1 Cellular changes over time in SOD1 mouse spinal cord .......................... 56
2.3.2 Functional changes over time in SOD1 mice............................................ 70
2.4 Discussion ........................................................................................................ 80
2.4.1 The relationship between microglial activation and disease progression . 80
2.4.2 Microglial phenotype in SOD1 mice ........................................................ 81
2.4.3 Pathological changes in SOD1 motor neurons ......................................... 85
2.4.4 Increasing expression of the antioxidant response protein, MT-1/2 ......... 86
2.4.5 Functional decline ..................................................................................... 88
2.4.6 Summary and conclusions ........................................................................ 91
Chapter 3 The effects of Gemals compound in SOD1 mice .................................. 93
3.1 Background ...................................................................................................... 94
3.1.1 The need for multi-action therapies in ALS.............................................. 94
3.1.2 Endotherapia – a drug cocktail of small molecules .................................. 94
3.1.3 Aims and hypothesis ................................................................................. 98
3.2 Methods ............................................................................................................ 99
3.2.1 Animals ..................................................................................................... 99
3.2.2 Drug and dosage schedule ....................................................................... 100
3.2.3 Outcome measures .................................................................................. 100
3.3 Results ............................................................................................................ 103
3.3.1 Adverse effects ........................................................................................ 103
3.3.2 Survival ................................................................................................... 104
3.3.3 Body weights........................................................................................... 111
3.3.4 Rotarod performance ............................................................................... 111
3.3.5 Grip strength ........................................................................................... 115
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