The Role of Calcium in Alpha-Synuclein
Aggregation: A Potential Mechanism of
Neurodegeneration
Jacob J Goodwin
Bachelor of Science with Honours
School of Medical Science
Griffith Health Institute
Griffith University
A Thesis submitted in fulfilment of the requirements of the degree of
Doctor of Philosophy
January 2014
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Abstract
Abnormal protein aggregation has been implicated in the pathogenesis of many
neurological disorders. This study focuses on the protein alpha-synuclein (α-syn), a presynaptic protein that is involved in a number of diseases collectively termed alphasynucleinopathies, which include Parkinson’s disease (PD) and Multiple System Atrophy
(MSA). α-syn aggregation and microscopically-visible α-syn-positive intracellular inclusion
bodies are common features of these diseases, occurring in multiple cell types and
localisation throughout the central nervous system. Although gene mutations in a variety of
molecular pathways have been identified in rare familial forms, the majority of αsynucleinopathy cases are sporadic in origin and have a late onset (>60 years) and therefore it
is important to study age related changes in neurochemistry and how these changes may be
responsible for the neurodegeneration associated with α-syn aggregation.
With aging, tightly regulated cellular processes start to lose the capacity to maintain
homeostasis. It is known that there is increased level of oxidative stress in aged compared to
young brains, and that intracellular free calcium (Ca2+) is increased at both the resting level
and upon neuronal activation. This research is focused on these two processes: firstly the
increase of intracellular free Ca2+; and secondly, the increase in oxidative stress.
This thesis demonstrates that calcium addition in vitro to α-syn promotes aggregation of
the protein. Incubation of 10µM purified human recombinant α-syn with 100µM CaCl2
resulted in the formation of 10-20nm globular structures making up plaques of up to 1µm, as
well as larger annular rings of α-syn approximately 70-90nm in diameter measured by
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Scanning electron microscopy (SEM). This aggregation of α-syn was shown not to be
stabilised using fast protein liquid chromatography (FPLC). CaCl2 addition resulted in a left
shift indicating an increase in particle size, however no resolved peak was observed
indicating that oligomeric proteins were unstable and were disaggregating as they moved
down the column.
It was demonstrated that calcium induced α-syn aggregation is a dose dependent event.
Fluorescently labelled α-syn was incubated on glass coverslips, exposed to different CaCl2
concentrations (0.1mM to 1mM) resulting in surface aggregates of 1.5+/- 0.7µm2, with a half
maximal calcium concentration of 80µM. Using a higher concentration of α-syn (75µM) the
half maximal calcium concentration was increased to 180µM.
This phenomenon was mirrored in an in vivo cell culture model system whereby free
intracellular calcium was increased via either intra or extracellular calcium sources leading to
increased cytoplasmic α-syn aggregates. Human glial 1321N1 cells, transiently transfected
with an α-syn-GFP construct were incubated with Thapsigargin (TG) (10µM) or Calcium
Ionophore A23187 (CI) (1µM) to induce calcium influx into the cytoplasm. Aggregate
formation was monitored using confocal microscopy and the percentage of cells containing
aggregates was counted at 6, 12 and 24 hours. It was shown that at 6 and 12 hours α-syn
aggregates were significantly increased in both treatments. The increase in α-syn aggregates
was confirmed to be calcium dependent as the co-incubation with 1,2-bis (o-aminophenoxy)
ethane-N,N,N',N'-tetraacetic acid (BAPTA), a calcium chelator, reduced aggregate formation.
To asses a more physiological stimuli, K+ was used as a trigger of N-type calcium
channels for calcium influx of the human neuronal cell types SHSY-5Y and HEK293T.
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Trimethadione (TMO) treatment was able to reduce calcium induced α-syn aggregation. The
formation of 2-3µm α-syn aggregates were observed in HEK293T cells 72 hours post K+
treatment, and <2µm aggregates 48 hours post K+ treatment in SHSY-5Y cells. Pre-treatment
of these cell types with TMO abolished the transient cytosolic calcium influx and
subsequently the α-syn aggregate formation. Similarly, BAPTA significantly decreased both
small and large aggregate formation in HEK293T cells.
Finally it was determined that oxidative stress and increased intracellular calcium work
additively or synergistically in the generation of α-syn aggregates. Interestingly in vitro, the
aggregated α-syn species promoted by calcium addition is not stabilised and exchanges
between monomeric and oligomeric forms of the protein. On addition of hydrogen peroxide
(H2O2) as an oxidizing agent, α-syn oligomeric species become stabilised as seen in FPLC
chromatography. The addition of H2O2 (1mM) to α-syn (10µM) resulted in a discrete second
peak representing a larger stabilised species, co-incubation with H2O2 and calcium resulted in
even larger, stable species.
H2O2 (10mM) treatment of 1321N1 cells transiently transfected with α-syn-GFP, resulted
in a significantly increased percentage of cells containing α-syn aggregates as well as with
TG/CI treatments. This increase in aggregate formation was additive. However when cells
were co-treated with H2O2 and TG/CI, there was a dramatic increase in the number of
cytoplasmic inclusions per cell compared to control and to raised calcium alone. In H2O2/CI
and H2O2/TG treatments 80 and 90% of cells contained three or more α-syn positive
aggregates respectively, this is compared to less than 10% in CI/TG treatment, or 40% in
H2O2 single treatment.
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This work shows that there is a direct link between α-syn aggregation and the normal age
related changes in neurochemistry, increased intracellular free calcium and increased
oxidative stress. These findings will lead towards new avenues of research into therapeutic
treatment of α-synucleinopathies, which traditionally respond poorly, or transiently to current
treatment options.
These treatments may include chemical antioxidant and chelation
therapies, or biological therapies to increase the activity of calcium binding proteins, such as
calbindin.
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Acknowledgments
First and foremost, I would like to thank my supervisor Dr Dean Pountney for giving me
this opportunity to undertake my PhD in his Laboratory. I am truly grateful for your support,
guidance and patience over the years.
I would like to thank Joe Tiralongo, for his help with the protein purification side of my
work, and for his support early on in my PhD.
Also a thank you Mathew Wong, Jorden Follet, and Bonnie Darlow for your help during
your time in the lab, and just for being there, making the Lab a fun place to work.
A special thank to Professor Lyn Griffiths for her support, especially early on, and for
allowing me to use her facility to undertake my work, and to all of the GRC members who
were very welcoming.
I would like to thank two of my greatest friends Dr Marina Stantic, and Dr Diego Chacon
who were always there when I needed them.
Your support and encouragement meant
everything, and will never be forgotten.
I would like to thank all of my friends who have come and gone through Griffith over the
years, Particularly Dr Carlos Aya, Dr Emily Camilleri, Dr Anna Butcher, Rebecca Grealy, Dr
Javed Fowder and Melissa Kuwahata. Thank you guys so much, and to everyone else, too
numerous to mention for making the experience here a memorable one.
Finally, my greatest thanks go to my Family, without your support and sacrifice over the
years not only during my PhD but always. To my mother, Roslyn Goodwin, for your
unconditional love and support, and to my father, Daryl Goodwin for teaching me the value
of common sense. To my sister and brother, Heidi and Dan, thank you for always being
there.
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Contents
The Role of Calcium in Alpha-Synuclein Aggregation: A Potential Mechanism of
Neurodegeneration .................................................................................................................................. 1
Abstract ........................................................................................................................................... iii
Acknowledgments .......................................................................................................................... vii
Statement of Originality ................................................................................................................ xiii
Acknowledgement of published papers included in this thesis ...................................................... xv
List of Publications ........................................................................................................................xvii
Journals......................................................................................................................................xvii
Conferences .............................................................................................................................. xviii
List of figures .................................................................................................................................xix
List of Tables:.................................................................................................................................xxi
Abbreviations ............................................................................................................................... xxiii
Chapter 1: Introduction .................................................................................................................... 1
1.1 Protein Aggregation and Neurodegeneration ......................................................................... 1
1.2 Alpha-synuclein in Neurodegeneration .................................................................................. 2
1.2.1 Alpha-synuclein in Parkinson ’s disease. ............................................................................ 3
1.2.1.1 Genetics and Familial Parkinson’s disease ......................................................................... 4
1.2.1.2 Parkinson’s Disease and Environment ................................................................................ 7
1.2.1.3
Idiopathic Parkinson’s disease ........................................................................................ 7
1.2.1.4 Pathology of Parkinson’s disease ........................................................................................ 8
1.2.2 Multiple System Atrophy .................................................................................................... 9
1.3 Alpha-synuclein.................................................................................................................... 11
1.3.1 Expression of Alpha-synuclein ............................................................................................ 14
1.3.2 Alpha-synuclein oligomerisation and toxicity. .................................................................... 16
1.3.3 Alpha-synuclein Post-translational modifications ............................................................... 18
1.3.4 Exosomes: Cell to cell spread of alpha-synuclein................................................................ 20
1.4 The Role of Calcium in the Neuron and Age Mediated Changes ........................................ 23
1.5 Oxidative stress and the Ageing Brain ................................................................................. 27
Chapter 2: Aims and Significance .................................................................................................. 29
2.1 Aims ..................................................................................................................................... 29
2.2 Significance .......................................................................................................................... 31
Chapter 3: Raised calcium promotes α-synuclein aggregate formation ......................................... 33
Abstract ...................................................................................................................................... 37
Introduction ................................................................................................................................ 37
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Results ........................................................................................................................................ 38
Scanning electron microscopy reveals calcium-dependent α-syn plaques.................................... 38
Analysis of fluorescent-labelled α-synuclein demonstrates calcium dose-dependent aggregation
...................................................................................................................................................... 38
Formation of calcium-induced surface aggregates requires surface binding sites ..................... 41
Calcium dependent α-synuclein aggregation in solution is concentration dependent ................ 41
Increased intracellular free calcium yields α-synuclein aggregates in α-synuclein-GFPtransfected 1321N1 cells ................................................................................................................... 42
Discussion .................................................................................................................................. 43
Experimental Methods ............................................................................................................... 44
Acknowledgments ...................................................................................................................... 47
References .................................................................................................................................. 47
Chapter 4: Potassium Depolarization and raised calcium induces α-synuclein aggregates ........... 49
Abstract ......................................................................................................................................... 53
Introduction ................................................................................................................................... 53
Material and methods .................................................................................................................... 55
Results ........................................................................................................................................... 56
Raised calcium induc3ed by K+ depolarization causes α-synuclein aggregation ........................ 60
BAPTA-AM buffers intracellular free Ca2+ and suppresses α-synuclein aggregation ............. 61
Discussion...................................................................................................................................... 64
Acknowledgments......................................................................................................................... 65
References ..................................................................................................................................... 65
Chapter 5: Raised calcium and oxidative stress cooperatively promote α-synuclein aggregate
formation ............................................................................................................................................... 69
Abstract ...................................................................................................................................... 73
Introduction ................................................................................................................................ 73
Material and methods ................................................................................................................. 74
Results ........................................................................................................................................ 74
Increased intracellular free calcium and hydrogen peroxide cooperatively promote α-syn
aggregates in α-SYN-GFP-transfected 1321N1 cells ................................................................... 75
Size exclusion chromatography shows an increase in α-syn oligomers with combined
calcium/hydrogen peroxide treatment in vitro .............................................................................. 75
Scanning electron microscopy reveals calcium/hydrogen peroxide-dependent spherical and
annular α-syn oligomers ............................................................................................................... 76
Discussion ..................................................................................................................................... 79
Acknowledgments ...................................................................................................................... 80
References .................................................................................................................................. 80
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Chapter 6: Discussion ..................................................................................................................... 83
6.1 Increased intracellular calcium induces alpha-synuclein oligomers..................................... 84
6.2 Oxidative stress and alpha-synuclein oligomerisation ......................................................... 86
6.3 Synergistic effect of calcium and oxidative stress................................................................ 88
6.4 Future Directions .................................................................................................................. 89
References ...................................................................................................................................... 92
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Statement of Originality
This work has not previously been submitted for a degree or diploma in any university. To the
best of my knowledge and belief, the thesis contains no material previously published or written by
another person except where due reference is made in the thesis itself.
(Signed)_____________________________
Jacob J Goodwin
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Acknowledgement of
included in this thesis
published
papers
Included in this thesis are published papers in Chapters 3, 4 and 5 which are co-authored with
other researchers. My contribution to the co-authored papers is outlined at the front of the relevant
chapter. The bibliographic details for these papers are:
Chapter 3: *Nath, S., *Goodwin, J.,Engelborghs, Y., Pountney, D.L. (2011) Raised calcium
promotes α-synuclein aggregate formation. Molecular and cellular Neuroscience. 46: 516-526
Chapter 4: Follet, J., Darlow, B., Wong, M., Goodwin, J., Pountney, D.L. (2012) Potassium
depolarization and raised calcium induces α-synuclein aggregates. Neurotoxicity Research. 23(4):37892
Chapter 5: Goodwin, J., Nath, S., Engelborghs, Y., Pountney, D.L. (2012) Raised calcium and
oxidative stress cooperatively promote alpha-synuclein aggregate formation. Neurochemistry
International. 62(5):703-11
Appropriate acknowledgements of those who contributed to the research but did not qualify as
authors are included in each published paper.
(Signed) _________________________________
Jacob J Goodwin
(Countersigned) ___________________________
Supervisor: Dr Dean L Pountney
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List of Publications
Journals
Goodwin, J., Nath, S., Engelborghs, Y., Pountney, D.L. (2011) Raised calcium promotes αsynuclein aggreagate formation. Molecular and cellular Neuroscience. 46: 516-526
Goodwin, J., Nath, S., Engelborghs, Y., Pountney, D.L. (2012) Raised calcium and oxidative
stress cooperatively promote alpha-synuclein aggregate formation. Neurochemistry International.
62(5):703-11
Follet, J., Darlow, B., Wong, M., Goodwin, J., Pountney, D.L. (2012) Potassium depolarization
and raised calcium induces α-synuclein aggregates. Neurotoxicity Research. 23(4):378-92
Wong, M.B., Goodwin, J., Norazit, A., Meedeniya, A.C.B., Richter-Lansberg, C., Gai, W.P.,
Pountney, D.L. (2012) SUMO-1 is associated with a subset of lysosomes in glial protein aggregate
diseases. Neurotoxicity Research. 23(1):1-21
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Conferences
Goodwin, J.J., Gai, W.P., Voelcker N.H., Pountney, D.L. (2006) Molecular architecture and
Cytotoxic Action of Alpha-Synuclein Oligomers. ASBMB Combio : Brisbane, September 24-28.
Goodwin, J.J., Gai, W.P., Voelcker N.H., Pountney, D.L. (2006) Molecular architecture and
Cytotoxic Action of Alpha-Synuclein Oligomers. Griffith University Health and Medical Research
Conference : Gold Coast,December 14-15.
Goodwin, J., Pountney, D.L. (2007) Development of a Method to Study the Interaction of Alphasynuclein and Calcium in Live Cells. Griffith University Health and Medical Research Conference :
Gold Coast, December 6-7.
Goodwin, J., Pountney, D.L. (2008) Role of Calcium in the Formation of Potentially Cytotoxic
Alpha-Synuclein Species: Possible Role in Neurodegeneration. Griffith University Health and
Medical Research Conference : Gold Coast, December 4-5.
Goodwin, J., Pountney, D.L. (2009) Increased Cytosolic Calcium Levels and Oxidative Stress
Promote Alpha-synuclein aggregation in vivo. 13th International Congress of Parkinson’s Disease and
Movement Disorders: Paris, June 7-11.
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List of figures
Chapter 1: Introduction
Figure 1. Signalling pathways and protein aggregation in Parkinson’s Disease…………………………… 8
Figure 2. Alpha-synuclein domain structure………………………………………………………………..12
Chapter 2: Aims and significance
Figure 1: Hypothetical schematic relating calcium dysfunction and oxidative stress to cytotoxicity .....….30
Chapter 3: Raised calcium promotes α-synuclein aggregate formation
Figure 1. Scanning electron microscopy of α-synuclein samples incubated with and without calcium pg.39
Figure 2. Fluorescence microscopy of fluorescently tagged α-synuclein precipitation on glass surfaceseffect of calcium.........………………………………………………………………………………………….. 40
Figure 3. Influence of surface modification on α-synuclein precipitation induced by calcium…….41
Figure 4. FCS monitoring of α-synuclein aggregation in solution in the presence of calcium …………….42
Figure 5. Influence of increased intracellular free calcium on α-synuclein aggregation in α-synuclein –GFP
transfected 1321N1 cells ………………………………………………………………………………………45
Figure 6. Raised intracellular calcium causes α-synuclein aggregation in α-synuclein –GFP transfected
1321N1 cells ……………………………………………………………………………………………………..46
Chapter 4: Potassium depolarization and raised calcium induces α-synuclein aggregates
Figure 1: HEK293T cells show Ca2+ transient after K+ depolarization that can be blocked by TMO and
causes α-synuclein aggregates …………………………………………………………………………………..57
Figure 2: SH-SY5Y cells develop small (<2 µm) and large (>2 µm) α-synuclein aggregates after K+
depolarization and raised Ca2+ ………………………………………………………………………………. 58
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Figure 3: SH-SY5Y cells develop small α-synuclein aggregates after K+ depolarization that can be
suppressed by TMO pre-treatment ………………………………………………………………………….. 59
Figure 4: 50 mM KCl evokes Ca2+ transient in HEK293T/α-synuclein cells …………………………...61
Figure 5: α-Synuclein aggregation following K+ depolarization in HEK293T/α-synuclein cells ……….62
Figure 6: Pre-loading with BAPTA-AM reduces intracellular Ca2+ transient and α-synuclein aggregates in
HEK293T/α-synuclein cells treated with 50mM KCl ………………………………………………………. 63
Chapter 5: Raised calcium and oxidative stress cooperatively promote α-synuclein aggregate
formation
Figure 1. Influence of increased intracellular calcium and hydrogen peroxide on α-syn aggregation in αsynuclein –GFP transfected 1321N1 cells ……………………………………………………………………..76
Figure 2: Raised intracellular free calcium and hydrogen peroxide cooperatively cause α-syn aggregation in
α-synuclein –GFP transfected 1321N1 cells ……………………………………………………………………77
Figure 3. Size exclusion chromatography reveals calcium/hydrogen peroxide co-treatment induces
increased high molecular weight α-syn species
…………………………………………............................................................…………………………………78
Figure 4. Scanning electron microscopy reveals α-synuclein incubated with hydrogen peroxide or
calcium/hydrogen peroxide forms both spherical and annular oligomeric species ……………………………. 78
Figure 5. Atomic force microscopy of hydrogen peroxide/calcium-treated α-syn ………………………... 79
Figure 6. FCS of α-syn subjected to calcium/hydrogen peroxide combined treatment ……………………79
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List of Tables:
Chapter 1: Introduction:
Table 1: Synucleinopathies ………………………………………………………………………2
Table 2: Genetics of Parkinson’s disease…………………………………………………………4
Chapter 3:
Table 1: FCS of 75µM α-synuclein samples incubated at 4°C with and without calcium ……. 41
Chapter 5:
Table 1: Mean particle diameter of annular alpha-synuclein oligomers by SEM ……………..79
Table 2: Mean particle height of alpha-synuclein oligomers by AFM ………………….……. 79
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Abbreviations
α-syn – Apha- synuclein
β- syn – Beta-synuclein
BAPTA - 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid
BBB - Blood brain barrier
CBP- Calcium binding protein
CD – Circular Dichroism
CI – Calcium Ionophore
DLB – Dementia with Lewy Bodies
ER- Endoplasmic Reticulum
GCI – Glial Cytoplasmic Inclusions
GSH- Glutathione
LB – Lewy Body
LRRK2 - Leucine Rich Repeating Kinase-1
MPP+ - 1-methyl-4-phenylpyridinium
MSA - Multiple System Atrophy
PD - Parkinson’s disease
PINK1 - PTEN-induced kinase 1
PMCA - Plasma membrane Ca2+ ATPase
PPS - Parkinson’s Plus Syndromes
ROS- Reactive oxygen species
SERCA- Sarcoplasmic endoplasmic reticulum calcium ATPase
Sn – Substantia Nigra
SNARE - SNAP (Soluble NSF Attachment protein) REceptor
SNP – Supra Nuclear Palsy
SUMO-1 – Small Ubiquitin-related modifier
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TG - Thapsigargin
UPDRS - Unified Parkinson’s disease rating scale
UCHL-1 - Ubiquitin C-terminal hydrolase-1
UPS – Ubiquitin Proteasome system
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