αα-Synuclein-Sy-Synucleinnuclein phosphorylationphosphorylation andand re relatedlated kinaseskinases inin ParkinsonParkinson’s diseasedisease
Jin-XiaJin-Xia ZhouZhou
A thesis submitted in fulfillment of the requirement of the degree of
Doctor of Philosophy
School of Medical Sciences, Faculty of Medicine
and
Neuroscience Research Australia
November 2013
I PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: Zhou
First name: Jin-Xia Other name/s:
I Abbreviation for degree as given in the University calendar: PhD
School: School of Medical Sciences Faculty: Medicine
lation and related kinases in Parkinson's disease
Abstract 350 words maximum: (PLEASE TYPE) ' Parkinson's disease (PO) is the most common neurodegenerative movement disorder pathologically identified by degeneration of the nigrostriatal system and the presence of Lcwy bodies (LBs) and neurites. structuTal pathologies largely made from insoluble a-synuclein phosphorylated at serine 129 (S 129P). Several kinases have been suggested to facilitate a-synuciein phosphorylation in PD, but without
significant human data the changes that precipitate such pathology remain conjecture. The major aims of this pr~ject were to assess the
dynamic changes of a -synuclein phosphorylation and related kinases in the progression of PD and in animal models of PD. and to determine whether Tenuigenin (TEN), a Chinese medicinal herb, can prevent cc-synucleln-induc.?d toxicity in a cell model.
The levels of non-phosphorylated a-synuclein decreased over the course ofPD, becoming increasingly phosphorylated and insoluble. There was a dramatic increase in phosphorylated a-synuclein that preceded LB formation. Importantly, three a-synuc!ein-relatec
ki nases [polo-like kinase 2 {PLK2), lcuc.:inc- rich repeat kinase 2 (LRRK2l and cyclin G-~tssoc i ated kinase (GAK)] were found to be
involved at different times in the evolution of LB formation in P.O. A naly.~is of a subacute \IIPTP model of PO neurodegeneration revealed similar increases in S 129P a-synuclein and PLK2/3 levels occurred immediately after the toxic insult, revealing this in 1•ivo model as suitable to assess this molecular mechanism. Assessment of a-sy11uclein over-expressing cell models of PO cytotoxicity 1 revealed increased S l29P a-synuclein but no increase in PLK2/3 levels, however treatment of these cells with the Chinese herbal ~ extract TEN was effective in reducing the increased S 129P a-synuclein and cell toxicity in association with reducing levels of PLK3. 1 This suggests that TEN may be an effective treatment for S129P' a-synuclein induced cytotoxicity as observed in PD. I In summary, these studies indicate that an increase in S 129P a-synuclein occurs early in response to cell damage in both PD and toxin induced PD models and that the levels of PLK2/3 increase in concert. Human data suggests that different kinases play a precipitating role in LB formation and compaction. Importantly TEN treatment appears to reduce PLK3 levels and ameliorateS 129P a-synuclein toxicity, identifying this herbal extract as a potential therapeutic drug for this PO relevant mechanism.
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Q'-¥1 II / )lOt) Date AcknowledgmentsAcknowledgments
Foremost, I would like to express the deepest appreciation to my primary supervisor
Professor Glenda Halliday. Thank you so much for the guidance and continuous support of my PhD study. Your extreme enthusiasm on science has inspired me to continue on this field and your patience and immense knowledge on neuroscience make this project special. You are the first-class supervisor and I feel so lucky to be your PhD student.
I would like also thank my co-supervisor Dr. Yue Huang for all the help, support and encouragement for my studying and living during the PhD period. You have taught me scientific thinking and problem solving skills. And also your patience and consideration made my life easier in Sydney. I feel like you are not only a supervisor but also a friend.
My sincere thanks also go to my conjoint-supervisor Professor Xiao Min Wang from
China Capital Medical University, Beijing, China. Thank you for all your generous support, encouragement and consideration for my PhD project. Your excellent organization and management have provided me a valuable and fruitful exchange experience during my PhD study. Without your support, this project would not have gone so well.
Thank you to all my colleagues in Halliday group in Neuroscience Research Australia.
You all have made my working environment exciting, motivating and fun. A special thanks to Ms Heidi Cartwright, Ms Heather McCann, Ms Shelley Forrest, Ms Danielle
Small, Ms Germaine Chua and Ms Amanda Gysbers for technical assistance. This thesis would not have been possible without your help. III Also thank you to all the colleagues in the Xiao Min Group in China Capital Medical
University. Your friendly and easy going characters made the laboratory environment so comfortable and fun. A special thanks to Ms Hao Bo Zhang and Dr. E Lv for the experimental cooperation. Your selfless contribution has made this project more comprehensive.
I would also like to thank all my friends at Neuroscience Research Australia. Your pleasant company has colored my life during my PhD. A special thanks to Ms Bonnie
Lam, Ms Puika Yeung, Ms Jac Kee Low, Dr. Claire Stevens, Dr. Andy Liang and Dr. Jie
Zhang. Thank you for leaving me such beautiful memories. The friendship from you all is a treasure to me.
Very special and sincere thanks go to all my family. Thank you all for supporting me spiritually throughout my life. Without you, I could not have gone so far. Words can not express my gratitude to you.
Finally, thanks to the Australian Government, University of New South Wales (UNSW) and Neuroscience Research Australia (NeuRA) for supporting me financially with the
International Postgraduate Research Scholarship (IRPS) and NeuRA PhD Scholarship.
It has been a great honor receiving these scholarships, and I will continue research in the future.
The PhD journey was very challenging for me and I wish to thank a lot of other people.
Although I can not state all the names here, I have a deep appreciation for everyone in my heart. Thank you.
IV AbstractAbstract
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder pathologically identified by degeneration of the nigrostriatal system and the presence of
Lewy bodies (LBs) and neurites, structural pathologies largely made from insoluble α- synuclein phosphorylated at serine 129 (S129P). Several kinases have been suggested to facilitate α-synuclein phosphorylation in PD, but without significant human data the changes that precipitate such pathology remain conjecture. The major aims of this project were to assess the dynamic changes of α-synuclein phosphorylation and related kinases in the progression of PD and in animal models of PD, and to determine whether
Tenuigenin (TEN), a Chinese medicinal herb, can prevent α-synuclein-induced toxicity in a cell model.
The levels of non-phosphorylated α-synuclein decreased over the course of PD, becoming increasingly phosphorylated and insoluble. There was a dramatic increase in phosphorylated α-synuclein that preceded LB formation. Importantly, three α-synuclein- related kinases [polo-like kinase 2 (PLK2), leucine-rich repeat kinase 2 (LRRK2) and cyclin G-associated kinase (GAK)] were found to be involved at different times in the evolution of LB formation in PD. Analysis of a subacute MPTP model of PD neurodegeneration revealed similar increases in S129P α-synuclein and PLK2/3 levels occurred immediately after the toxic insult, revealing this in vivo model as suitable to assess this molecular mechanism. Assessment of α-synuclein over-expressing cell models of PD cytotoxicity revealed increased S129P α-synuclein but no increase in
PLK2/3 levels, however treatment of these cells with the Chinese herbal extract TEN
V was effective in reducing the increased S129P α-synuclein and cell toxicity in association with reducing levels of PLK3. This suggests that TEN may be an effective treatment for S129P α-synuclein induced cytotoxicity as observed in PD.
In summary, these studies indicate that an increase in S129P α-synuclein occurs early in response to cell damage in both PD and toxin-induced PD models and that the levels of
PLK2/3 increase in concert. Human data suggests that different kinases play a precipitating role in LB formation and compaction. Importantly TEN treatment appears to reduce PLK3 levels and ameliorate S129P α-synuclein toxicity, identifying this herbal extract as a potential therapeutic drug for this PD relevant mechanism.
VI PublicationsPublications ari arisingsing fromfrom thisthis thesisthesis
Jinxia ZhouZhZhou,ou, Melissa Broe, Yue Huang, John P. Anderson, Wei-Ping Gai, Elizabeth A.
Milward, Michelle Porritt, David Howells, Andrew J. Hughes, Xiaomin Wang, Glenda
M. Halliday. (2011). Changes in the solubility and phosphorylation of α-synuclein over the course of Parkinson's disease." Acta Neuropathol 121(6): 695-704.
JinxiaJinxia ZhouZhou, Haobo Zhang, Yue Huang, Yi He, Yan Zheng, John P. Anderson, Wei-
Ping Gai, Zhigang Liang, Yong Wang, Xinmiao Ren, Qi Wang, Xiaoli Gong, Jian Yang,
Xuan Wang, Glenda Halliday, Xiaomin Wang.(2013) Tenuigenin attenuates α- synuclein-induced cytotoxicity bydown-regulating polo-like kinase 3.CNS
Neuroscience & Therapeutics, 19(9):688-94
JinxiaJinxia Zhou,Zhou, E Lv, Yizheng Wang, Haobo Zhang, Jun Jia, Yue Huang, John P.
Anderson, Yan Yu, Jiahui Deng, Yi He, Yan Zheng, Xuan Wang, Glenda M. Halliday,
Xiaomin Wang. α-Synuclein phosphorylation and related kinases in a subacute MPTP mouse model of Parkinson’s disease. [submitted to PLoS ONE]
JinxiaJinxia Zhou,Zhou, Shelley Forrest, Yue Huang, John P. Anderson, Nicholas Dzamko, Glenda
M. Halliday. α-Synuclein-related kinases and Lewy pathology in Parkinson’s disease.
[submitted to Acta Neuropathol]
VII ConferenceConference presentationspresentations
Oral
JinxiaJinxia Zhou,Zhou, Melissa Broe, Yue Huang, John P. Anderson, Wei-Ping Gai, Elizabeth A.
Milward, Michelle Porritt, David Howells, Andrew J. Hughes, Glenda M. Halliday.
(2010). Changes in the solubility and phosphorylation of α-synuclein over the course of
Parkinson’s disease. TOW research awards meeting, Prince of Wales hospital, Sydney,
Australia
PosterPoster
JinxiaJinxia Zhou,Zhou, Yue Huang, Weiping Gai, Xiaomin Wang, Glenda M. Halliday. (2010).
Accumulation and phosphorylation of α-synuclein in the progression of Parkinson’s disease. Brain Sciences UNSW Symposium, Sydney, Australia
JinxiaJinxia Zhou,Zhou, Yue Huang, John P Anderson, Wei Ping Gai, Xiaomin Wang, Glenda M.
Halliday. (2011). Changes in the solubility and phosphorylation of α-synuclein over the course of Parkinson's disease. The 15th International Congress of Parkinson's Disease and Movement Disorders, Toronto, Canada
JinxiaJinxia Zhou,Zhou, E Lv, Yizheng Wang, Haobo Zhang, Yue Huang, Glenda M. Halliday,
Xiaomin Wang. (2013). Increased α-synuclein phosphorylation in mice with transient
VIII MPTP toxicity correlates with increased levels of polo-like kinases. Australian
Neuroscience Society Annual Meeting, Melbourne, Australia
JinxiaJinxia Zhou,Zhou, Yue Huang, Glenda M. Halliday. (2013). Changes in α-synuclein phosphorylation and associated kinases in Parkinson’s disease. The International
Conference On α-synuclein in Parkinson’s Disease & Related Neurodegenerative
Diseases, Dubai, UAE
JinxiaJinxia Zhou,Zhou, Yue Huang, Glenda M. Halliday. (2013). Increasing α-synuclein Ser129 phosphorylation in Parkinson’s disease is associated with increasing kinase levels. The
11th International Conference On Alzheimer’s & Parkinson’s Disease, Florence, Italy
JinxiaJinxia Zhou,Zhou, Haobo Zhang, Yue Huang, Glenda Halliday, Xiaomin Wang. (2013).
Tenuigenin attenuates over-expressing α-synuclein induced cytotoxicity via down- regulating polo-like kinase 3. The 17th International Congress of Parkinson's Disease and Movement Disorders, Sydney, Australia
IX AwardsAwards
International Postgraduate Research Scholarship, 2010-2013
NeuRA Supplementary Scholarship, 2010-2013
Movement Disorder Society Travel Grant, 2011
Chinese Government Award For Outstanding Self-financed Students Abroad, 2012
Postgraduate Research Student Scholarship Travel Grant from UNSW, 2013
School of Medical Sciences Research Travel Grant from UNSW, 2013
Movement Disorder Society Travel Grant, 2013
X TableTable ofof ContentsContents
α-Synuclein phosphorylation and related kinases in Parkinson’s disease...... I
Originality Statement...... II
Acknowledgements...... III
Abstract...... V
Publications arising from this thesis...... VII
Conference presentations...... VIII
Awards...... X
Table of Contents...... XI
Abbreviations...... XVII
List of Figures...... XIX
List of Figures continued...... XX
List of Tables...... XXI
ChapterChapter 1: IntroductionInIntroduction...... troduction...... 1
1.1 Parkinson’s disease...... 1
1.1.1 The clinical symptoms of Parkinson’s disease...... 1
1.1.2 Treatments for Parkinson’s disease...... 2
1.1.3 Parkinson’s disease pathology...... 5
1.1.4 Animal models of Parkinson’s disease...... 7
1.1.4.1 Toxin-based models of Parkinson’s disease...... 7
1.1.4.2 Gene-based models of Parkinson’s disease...... 9
1.2 α-Synuclein...... 10
1.2.1 α-Synuclein phosphorylation in Parkinson’s disease...... 12
1.3 α-Synuclein phosphorylation related kinases in Parkinson’s disease...... 14 XI 1.3.1 Polo-like kinases (PLKs)...... 14
1.3.1.1 PLKs in the central nervous system...... 15
1.3.1.2 PLKs and α-synuclein...... 17
1.3.2 Casein kinases (CKs)...... 18
1.3.2.1 CKs in neurodegenerative disease...... 19
1.3.2.2 CKs and α-synuclein...... 20
1.3.3 G protein-coupled receptor kinases (GRKs)...... 21
1.3.3.1 GRKs in neurodegenerative diseases...... 22
1.3.3.2 GRKs and α-synuclein...... 22
1.3.4 Leucine-rich repeat kinase 2 (LRRK2)...... 23
1.3.4.1 LRRK2 and α-synuclein...... 24
1.3.5 Adenosine monophosphate activated protein kinase (AMPK)...... 27
1.3.6 Cyclin G-associated kinase (GAK)...... 28
1.3.6.1 GAK and Parkinson’s disease...... 28
1.3.6.2 GAK and α-synuclein...... 28
1.4 Conclusion...... 29
1.5 Hypothesis...... 29
1.6 Aims...... 30
ChapterChapter 2: ChangesChanges inin αα-synuclein-s-synucleinynuclein solubilitysolubility andand S129S129 phosphorylationphosphorylation duringduring thethe
coursecourse of ParkinsonParkinson’s diseasedidisease...... sease...... 31
2.1 Introduction...... 31
2.2 Aim and Hypothesis...... 32
2.3 Materials and Methods...... 32
2.3.1 Overall study design...... 32
2.3.2 Cases...... 32
2.3.3 Tissue sampling and fractionation...... 34
XII 2.3.4 Quantitative Western immunoblotting for α-synuclein proteins...... 34
2.3.5 Statistical analyses...... 35
2.4 Results...... 36
2.4.1 Forms of α-synuclein expressed in controls...... 36
2.4.2 Changes observed in Parkinson’s disease...... 39
2.4.3 Changes over the disease course...... 45
2.5 Discussion...... 47
2.6 Strengths and weaknesses of this study...... 53
2.7 Conclusion...... 53
ChapterChapter 3: αα-Synuclein-related-S-Synuclein-relatedynuclein-related kinaseskinases andand LewyLewy bodybody formationformation inin ParkinsonParkinson’s
diseasedidisease...... sease...... 55
3.1 Introduction...... 55
3.2 Aim and Hypothesis...... 56
3.3 Materials and Methods...... 56
3.3.1 Overall study design...... 56
3.3.2 Cases...... 57
3.3.3 Tissue sampling and processing...... 57
3.3.4 Semi-quantitative Western blotting...... 58
3.3.5 Single-labelling immunoperoxidase for protein localization...... 59
3.3.6 Double-labelling immunofluorescence for protein localization...... 60
3.3.7 Assessment of kinase immunoreactivity in LBs and LB containing neurons...... 61
3.3.8 Statistical plan and analyses...... 62
3.4 Results...... 62
3.4.1 PLKs and CK2β...... 62
3.4.2 LRRK2 and GAK...... 67
3.4.3 Correlation between kinase levels and S129 phosphorylation of α-synuclein..... 71
XIII 3.4.4 Changes in kinases over the course of the disease and the development and
maturation of LBs...... 73
3.5 Discussion...... 77
3.5.1 Relationship between the levels of S129P α-synuclein and kinases known to
phosphorylate α-synuclein...... 78
3.5.2 Relationship between the levels of S129P α-synuclein and other related
kinases...... 79
3.5.3 Kinases associated with the development and maturation of Lewy pathologies..81
3.6 Strengths and weaknesses of this study...... 81
3.7 Conclusion...... 83
ChapterChapter 4: AssessmentAssessment of acuteacute changeschanges inin αα-synuclein-s-synucleinynuclein phosphorylationphosphorylation andand
relatedrelated kinaseskinases inin an MPTPMPTP mousemouse modelmodel of ParkinsonParkinson’s diseasedidisease...... sease...... 84
4.1 Introduction...... 84
4.2 Aim and Hypothesis...... 85
4.3 Materials and Methods...... 85
4.3.1 Overall study design...... 85
4.3.2 Animals...... 86
4.3.3 MPTP administration and tissue preparation...... 86
4.3.4 Immunohistochemistry for tyrosine hydroxylase (TH)...... 87
4.3.5 Dopaminergic neuronal counts in the substantia nigra...... 87
4.3.6 Western blotting for tyrosine hydroxylase, α-synuclein and related kinases...... 88
4.3.7 Statistics...... 89
4.4 Results...... 89
4.4.1 Characterizing the subacute MPTP mouse model...... 89
4.4.2 α-Synuclein S129 phosphorylation in the subacute MPTP mouse model...... 92
4.4.3 Related kinases in the subacute MPTP mouse model...... 95
XIV 4.4.4 Correlations between the changing levels of α-synuclein phosphorylation and
its related kinases...... 98
4.5 Discussion...... 99
4.6 Strengths and weaknesses of this study...... 102
4.7 Conclusion...... 102
ChapterChapter 5: EffectsEffects of a traditionaltraditional ChineseChinese herbherb on polo-likepolo-like kinasekinase levelslevels inin an αα--
synuclein-inducedsynuclein-induced cellcell modelmodel of ParkinsonParkinson’s diseasedisease cytotoxicitycytcytotoxicity...... otoxicity...... 104104
5.1 Introduction...... 104
5.2 Aim and Hypothesis...... 105
5.3 Materials and Methods...... 105
5.3.1 Overall study design...... 105
5.3.2 Cell culture model...... 106
5.3.3 Drug treatment regimen...... 107
5.3.4 Outcome measures and analyses...... 108
5.3.4.1 Cell viability (proliferation and apoptosis) assays...... 108
5.3.4.2 Western blotting of cellular proteins...... 108
5.3.4.3 Statistical analyses...... 110
5.4 Results...... 110
5.4.1 α-Synuclein over-expressing SH-SY5Y cells have increased phosphorylation
of α-synuclein at S129 and reduced cell viability...... 110
5.4.2 α-Synuclein over-expressing SH-SY5Y cells do not have increased kinase
expression...... 113
5.4.3 Identification of the most effective non-toxic TEN concentration for
treatment...... 113
5.4.4 TEN treatment alleviates α-synuclein-induced cytotoxicity...... 114
5.4.5 TEN treatment down-regulates S129 phosphorylation of α-synuclein...... 116
XV 5.4.6 TEN treatment down-regulates PLK3 levels in SH-SY5Y cells...... 118
5.5 Discussion...... 120
5.6 Strengths and weaknesses of this study...... 123
5.7 Conclusion...... 124
ChapterChapter 6: GeneralGeneral discussiondidiscussion...... scussion...... 125
6.1 S129 α-synuclein phosphorylation precedes Lewy pathology formation in PD...... 125
6.2 PLK2, LRRK2 and GAK are involved in the evolution of S129 α-synuclein phosphorylation in relation to LB formation in PD...... 127
6.3 Identification that similar pathways are involved in a subacute MPTP mouse model of PD...... 129
6.4 TEN attenuates over-expressing α-synuclein induced toxicity and reduces PLK3 and
S129P α-synuclein levels...... 130
6.5 Future directions...... 131
References...... 133
XVI AbbreviationsAbbreviations
6-OHDA 6-hydroxydopamine Aβ beta-amyloid AD Alzheimer's disease AMP adenosine monophosphate AMPK adenosine monophosphate activated protein kinase ATP adenosine triphosphate BDGF brain-derived growth factor CKs casein kinases COMT-I catechol-o-methyltransferase-inhibitor COR C-terminal of Roc CP cerebral peduncle CSP cysteine-string protein CTSD cathepsin D DA dopamine agonists DBS deep brain stimulation DLB dementia with Lewy bodies DMEM/F12 Dulbecco’s Modified Eagle’s Medium/F12 DMSO dimethyl sulfoxide ERK extracellular signal-regulated kinase FBS fetal bovine serum GAK cyclin G-associated kinase GFP green fluorescent protein GPCRs G protein-coupled receptors GRKs G protein-coupled receptor kinases GWAS genome-wide association studies HEK293 Human Embryonic Kidney 293 IHC Immunohistochemistry IP interpeduclar nucleus LBs Lewy bodies
XVII AbbreviationsAbbreviations continuedcontinued L-DOPA 3,4-dihydroxyphenyl-L-alanine LNs Lewy neurites LPS lipopolysaccharides LRRK2 Leucine-rich repeat kinase 2 MAO-B monoamine oxidase B MAP1B microtubule-associated protein 1B MPP+ 1-methyl-4-phenylpyridinium MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MSA multiple system atrophy MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2Htetrazolium NGF neuronal growth factor PBD polo-box domain PBS phosphate-buffered saline PD Parkinson’s disease PLKs polo-like kinases RapGAP Rap guanosine triphophatase activating protein S129 Serine 129 S129P Serine 129 phosphorylated SN substantia nigra SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor SNpc substantia nigra par compact SNR substantai nigra pars reticulata TBS Tris-buffered saline TBST Tris-buffered saline Tween-20 TEN Tenuigenin TH tyrosine hydroxylase VTA ventral tegmental area
XVIII ListList of of FiguresFigures
Chapter 1
Figure 1.1: German guideline for treatment of PD 2012...... 4
Figure 1.2: Chemical structure of Tenuigenin (TEN)...... 5
Figure 1.3: Braak PD staging system...... 7
ChapterChapter 2
Figure 2.1: α-synuclein and S129P α-synuclein in controls...... 38
Figure 2.2: Relative amounts of α-synuclein and S129P α-synuclein in controls and PD cases...... 42
Figure 2.3: Relationships between α-synuclein and S129P α-synuclein amounts between the different protein fractions and over the disease course in the PD cases...... 44
Figure 2.4: Relative amounts of α-synuclein and S129P α-synuclein in the frontal cortex from controls and PD cases at different disease stages...... 46
ChapterChapter 3
Figure 3.1: Representative Western blots of PLK1, PLK2 and CK2 in human brain.....63
Figure 3.2: PLK1, PLK2 and CK2β in controls...... 64
Figure 3.3: changes in PLK1, PLK2 and CK2β in the nigrostriatum and frontal cortex of
PD compared with controls...... 66
Figure 3.4: LRRK2 and GAK in controls...... 68
Figure 3.5: changes in LRRK2 and GAK in the nigrostriatum and frontal cortex of PD compared with controls...... 70
XIX ListList of FiguresFigures continuedcontinued Figure 3.6: Relationships between kinase levels and S129P α-synuclein amounts in the putamen of PD cases...... 72
Figure 3.7: Relationships between kinases amounts and Lewy pathologies development
...... 74
Figure 3.8: Double immunofluorescence staining for kinases and S129P α-synuclein in anterior cingulate neurons...... 77
ChapterChapter 4
Figure 4.1: Dynamic changes in TH expression in the nigrostriatum of subacute MPTP mice over the experiment period...... 91
Figure 4.2: Dynamic changes in α-synuclein expression in nigrostriatum of subacute
MPTP mice over the experiment period...... 94
Figure 4.3: Dynamic changes in kinases expression in nigrostriatum of subacute MPTP mice over the experiment period...... 97
Figure 4: Correlations between α-synuclein Ser129 phosphorylation and PLK2 and 3 in the striatum and SN...... 99
ChapterChapter 5
Figure 5.1: Characterization of the α-synuclein over-expressing SH-SY5Y cell models
...... 112
Figure 5.2: Effects of TEN treatment on SH-SY5Y cell proliferation and apoptosis... 115
Figure 5.3: Effects of 10μM TEN treatment on S129α-synuclein phosphorylation..... 117
Figure 5.4: Effects of 10μM TEN treatment on PLK3 expression in SH-SY5Y cells..119
Figure 5.5: Representative Western blots of PLK1-2 and CK1-2...... 120
XX ListList of of Ta Tablesbles
Table 2.1: Case demographic details...... 33
XXI ChapterChapter 1: 1: Int Introductionroduction
1.11.1 ParkinsonParkinson’s diseasedisease
Parkinson’s disease (PD) is the most common progressive neurodegenerative movement disorder, eventually leading to considerable motor disability which profoundly affects quality of life for patients and caregivers (de Lau & Breteler, 2006). PD affects approximately 1% of patients over the age of 60, with a rising prevalence to around 2% in the population over 80 years of age (Mutch, et al., 1986). With the increasing lifespan of the population, the number of PD cases is likely to double worldwide by 2030
(Dorsey, et al., 2007) imposing an increasing social and economic burden (de Lau &
Breteler, 2006).
1.1.1 The clinical sy symptomsmptoms of Parkins Parkinsonon’s disease
The classic symptoms of PD are well known as bradykinesia, resting tremor, rigidity and abnormal posture (Gelb, et al., 1999). These motor symptoms are attributed to considerable loss of dopaminergic neurons in the substantia nigra par compact (SNpc)
(Braak & Del Tredici, 2008). Besides these cardinal motor symptoms, non-motor symptoms of anosmia (the loss of sense of smell)(Kertelge, et al., 2010), sleep disturbances (Ferrer, et al., 2011), constipation (Kim, et al., 2009), emotional apathy
(Ravina, et al., 2009), depression (Felicio, et al., 2010) and physical pain (Beiske, et al.,
2009) are also found in patients with PD many years before the onset of motor symptoms. Thus, it has become increasingly clear that PD is not a disease only affecting the SNpc but is a multisystem disease affecting many brain regions and peripheral nervous system.
1 1.1.2 Tr Treatmentseatments for ParkinParkinsonson’s disease
To date there has been no cure for PD and all the current treatments can only provide relief from the symptoms. The dopamine precursor 3,4-dihydroxyphenyl-L-alanine (L-
DOPA) in combination with a dopa decarboxylase inhibitor such as carbidopa (Sinemet) or benserazide (Madopa) is the most effective drug in controlling motor symptoms
(Goetz, et al., 2005). However, long-term use of Madopa or Sinemet will result in a number of side effects, including motor fluctuations and dyskinesias (Fahn, et al., 2004).
Dopamine agonists (DA) that bind to dopaminergic postsynaptic receptors in the brain have similar effects to L-DOPA. These were initially used for individuals experiencing on-off fluctuations and dyskinesias as a complementary therapy to L-DOPA, but now are mainly used on their own as an initial therapy for motor symptoms with the aim of delaying motor complications (Goldenberg, 2008). In addition, monoamine oxidase B
(MAO-B) inhibitors are also applied to inhibit the metabolism of dopamine, while catechol-o-methyltransferase inhibitors (COMT-I) are used to decrease L-DOPA breakdown, resulting an increasing dopamine levels (Muller, 2012). Unfortunately, all these treatments can only alleviate motor symptoms, but have no effect on preventing neuronal loss.
For the patients with PD who suffer from motor fluctuations and tremor that are inadequately controlled by medication or for those who can not tolerate the medication, functional surgery is recommended (Bronstein, et al., 2011). Deep brain stimulation (DBS) is the most commonly used surgical treatment and the target areas for DBS include the subthalamic nucleus, globus pallidus, and thalamus (Pedrosa &
Timmermann, 2013). The safety and efficacy of DBS in PD has been proven based on the clinical experience in thousands of patients. However, there are still several open
2 and general questions concerning DBS, such as how many targets should be operated on and when is the best moment for the operation (Pedrosa & Timmermann, 2013).
In addition to pharmaceutical treatments and surgery, there are some other additional therapeutic options for PD, including physical, speech and/or occupational therapies
(Pedrosa & Timmermann, 2013). These treatments are used as complementary therapies in certain circumstances. For a practical approach, the current German guidelines for
PD therapy are referred to (Pedrosa & Timmermann, 2013) (Figure 1.1).
In China and some other Asian countries, traditional Chinese herbs have also been widely prescribed for PD-like symptoms for thousands of years, especially for the non- motor symptoms. Over recent decades, extensive efforts have been made to investigate the role of herbal and plant extracts in PD (Chen, et al., 2007; Li, et al., 2013) and dozens of active compounds extracted from herbal medicines have been indicated to have effects on PD molecular models, including Tenuigenin (TEN) (Liang, et al., 2011),
Quercetin (Karuppagounder, et al., 2013), Polyphenolic catechins (Guo, et al., 2007;
Levites, et al., 2001), Ginkgo (Rojas, et al., 2008) and others. TEN[ (2β,3β,4α,12α)-12-
(Chloromethyl)-2,3-dihydroxy-27-norolean-13-ene-23,28-dioic acid, molecular formula
C30H45ClO6, average molecular weight 537.14kDa, Figure 1.2] is a natural extract from the Polygala tenuifolia root, a traditional Chinese herb that has been widely prescribed in traditional Chinese medicine for amnesia, neurasthenia, palpitation, insomnia and cognitive dysfunction. The recent finding that TEN alleviates both toxin- [1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP+), 6- hydroxydopamine (6-OHDA)] and inflammation-induced [lipopolysaccharides (LPS)] models of dopaminergic neuronal injury (Choi, et al., 2011; Liang, et al., 2011; Lv, et al., 3 2009; Naito & Tohda, 2006; Yuan, et al., 2012) raises the possibility of its therapeutic benefit for patients with PD. But the underlying mechanisms remain unclear.
Figure 1.1: GermanGerman guidelineguideline forfor treatmenttreatment of PDPD 2012
4 Figure 1.2: ChemicalChemical structurestructure of TenuigeninTenuigenin (TEN).(TEN).
1.1.3 ParkinsonParkinson’s disease patholpathologyogy
The pathology of PD is characterized by the loss of dopaminergic neurons in the SNpc and the presence of Lewy bodies (LBs) and Lewy neurites (LNs) (Spillantini, et al.,
1998; Spillantini, et al., 1997; Wakabayashi, et al., 1997). LNs and LBs are intracytoplasmic inclusions mainly consisting of filaments of phosphorylated α- synuclein (Galvin, et al., 2001; Spillantini, et al., 1998). LBs are thought to form in two ways - a ‘compaction model’ from punctate α-synuclein material in cell bodies
(Kuusisto, et al., 2003) and a ‘growth model’ (Kanazawa, et al., 2008) from loosely packed α-synuclein filaments that mature to LNs (Kanazawa, et al., 2012) then aggregate and transform into LBs over time (Kanazawa, et al., 2008). A formal staging scheme has been proposed (Wakabayashi, et al., 2007) where Stage 1 is diffuse, pale cytoplasmic staining of α-synuclein, Stage 2 is irregularly shaped, uneven α-synuclein staining of moderate intensity, Stage 3 is a discrete, often peripheral α-synuclein staining of pale bodies or neurites, and Stage 4 is ring-like staining of a typical LB with a central core and a surrounding halo. In the early stages of PD, pale bodies outnumber typical LBs (Wakabayashi, et al., 2007).
5 LNs and LBs are found in many brain regions spreading from their initial sites in the medulla oblongata and olfactory bulb through the brainstem and forebrain to the neocortex in PD (Braak, et al., 2003). Based on the semi-quantitative assessment of the distribution of LBs and LNs, Braak and colleagues proposed a PD staging system to indicate a predictable sequence of pathological lesions (Braak, et al., 2003; Braak, et al.,
2004)(Figure 1.3). According to this classification system, PD pathology can be divided into six successive stages. The earliest lesions are seen in the medulla oblongata and olfactory bulb (stage 1), then both medulla oblongata and pontine tegmentum become involved (stage 2) with the upper brainstem involved by stage 3, but without substantial loss of the SNpc. These three stages are suggested to be preclinical. The SNpc becomes denuded of cells and α-synuclein pathology continues rostrally to affect the anteromedial temporal mesocortex in stage 4, the stage when the motor symptoms of
PD become clinically evident. Neocortical areas are infiltrated in stage 5 and stage 6.
The critical difference between stages 5 and 6 is the severity of involvement of the neocortex which is more severe in stage 6. These two stages frequently associate with cognitive impairment (Jellinger, 2009). It should be noted that the mechanism underlying this anatomical progression of PD pathology remains unclear.
6 Figure 1.3: BraakBraak PDPD stagingstaging systemsystem
Figure adapted from (Braak, et al., 2004). (A)A) The presymptomatic phase is marked by the appearance of
LNs/LBs in the brains of asymptomatic persons. In the symptomatic phase, the individual neuropathological threshold is exceeded (black arrow). The severity of the pathology is indicated by darker degrees of shading in the colored arrow left. ((B)B)B) Diagram showing the ascending pathological process (white arrows). The shading intensity of the colored are as corresponds to that in (A).
1.1.4 Animal models of Parkinson ’s disease
Animal models are an important approach to study the pathogenesis and therapeutic intervention strategies of human diseases. Based on etiology, PD animal models can be generally classified into toxin- and gene-based models.
1.1.4.11.1.4.1 Toxin-basedToxin-based modelsmodels ofof Pa Parkinsonrkinson’s diseasedisease
Several neurotoxins, including 6-OHDA, MPTP, paraquate and rotenone, have been used to induce PD-like dopaminergic neurodegeneration in rodents and primates (Dauer
& Przedborski, 2003). Importantly, only MPTP is clearly linked to a form of human parkinsonism, and it is thus the most widely studied model (Dauer & Przedborski,
2003).
7 MPTP was discovered to be a dopaminergic neurotoxin in the early 1980s after venous injection of synthetic heroin contaminated with the substance was used recreationally by several young Californian drug users (Langston, et al., 1983). These addicts displayed the cardinal features of idiopathic PD including bradykinesia, rigidity, and in some cases, resting tremor (Ballard, et al., 1985). Post-mortem analysis demonstrated that there was neuronal cell loss relatively specific for cells in the SNpc (Ballard, et al.,
1985). MPTP is a highly lipophilic mitochondrial toxin which rapidly crosses the blood- brain barrier (Przedborski, et al., 2004). Once in the brain, the pro-toxin MPTP is converted into the neurotoxic metabolite MPP+ by MAO-B (Przedborski & Vila, 2001) and released where it can enter dopaminergic neurons via membrane located dopamine transporters. Once inside dopaminergic neurons, MPP+ inhibits enzymes in the mitochondrial electron transport chain, resulting in ATP deficits and increased
“leakage” of superoxide from the respiratory chain, contributing to a cascade that leads to cell death (Dauer & Przedborski, 2003; Przedborski & Vila, 2001). MPTP is known to cause parkinsonism in humans, monkeys and mice (Bezard & Przedborski, 2011).
For both technical and economical reasons, MPTP treated mice are still the most common model to study PD-like SNpc degeneration (Przedborski, et al., 2001).
According to the schedule of MPTP administration, at least three regimens of MPTP mouse models have been reported: acute treatment, subacute treatment and chronic treatment (Jackson-Lewis & Przedborski, 2007; Luchtman, et al., 2009). These models are used to provide a better understanding of distinct phases of neuronal death such as the pre-symptomatic, the immediate onset, the progressive and final stage of PD
(Schmidt & Ferger, 2001). However, it is worth noting that all these MPTP models use
8 end points in the range of days or weeks that are far away from modeling the much more slowly progressive neurodegenerative processes observed in patients with PD, a disease that develop over decades.
6-OHDA is a neurotoxin specific for catecholaminergic cells including dopaminergic cells. It was first used in the late 1960s to target dopaminergic cells (Ungerstedt, 1968).
The neurotoxic effect of 6-OHDA is mediated by its structural similarity to catecholamines facilitating its uptake into catecholaminergic neurons via acting as a
“false transmitter” (Schwarting & Huston, 1996). In dopaminergic neurons, 6-OHDA is taken up by the dopamine transporter and once in the cell, it is rapidly oxidised resulting in metabolites which include free radicals and hydrogen peroxides (Lotharius, et al., 1999). 6-OHDA has conventionally been used to produce dopaminergic lesions in rodents rather than monkeys. Because 6-OHDA does not cross the blood brain barrier efficiently, it must be injected stereotaxically in order to produce depletion of dopaminergic neurons (Tolwani, et al., 1999). In rodents it is most commonly injected unilaterally into the nigrostriatal bundle. This results in the near complete reduction of dopaminergic cells in the SNpc, with acute onset and cell death being complete by 3–4 days after injection (Schwarting & Huston, 1996). This model therefore represents the end stage of the idiopathic PD, similar to that seen after acute bilateral MPTP treatment.
1.1.4.21.1.4.2 Gene-basedGene-based mo modelsdels ofof Pa Parkinsonrkinson’s diseasedisease
Gene mutations cause PD in people providing a direct mechanistic link between these models and human disease (Shulman, et al., 2011). Causal genetic mutations for PD are rare but several seem to be relevant to the more frequent sporadic forms of PD. Notably,
9 multiplication of the SNCA gene cause rare familial PD (Chartier-Harlin, et al., 2004;
Fuchs, et al., 2007; Ibanez, et al., 2004; Nishioka, et al., 2006), with α-synuclein accumulating in LBs in neurons of these patients, in a similar way to that seen in patients with sporadic PD (Galvin, et al., 2001; Spillantini, et al., 1998). Hence, selective cerebral transgenic over-expression of α-synuclein is used in animals as a model of PD (Shulman, et al., 2011). Links between other mutations and sporadic PD are increasingly recognized (Shulman, et al., 2011) and a selection of different genetic mouse models is now available (Chesselet & Richter, 2011) with each model having different advantages and disadvantages. However, the prevalent belief is that none of these other genetic models have reproduced many of the key features of PD (Chesselet
& Richter, 2011).
1.21.2 αα-Synuclein-Sy-Synucleinnuclein
α-Synuclein is a small (140 amino acid residues) presynaptic cytosolic phosphoprotein
(Jakes, et al., 1994; Tong, et al., 2010a) encoded by the SNCA gene (Spillantini, et al.,
1995). The SNCA gene (also known as the PARK1 and PARK4 gene) is a PD causative gene whose point mutations (A53T, A30P, E46K and G51D )(Kiely, et al., 2013; Kruger, et al., 1998; Polymeropoulos, et al., 1997; Zarranz, et al., 2004) and multiplications
(Chartier-Harlin, et al., 2004; Fuchs, et al., 2007; Ibanez, et al., 2004; Nishioka, et al.,
2006) are associated with familial autosomal-dominant inherited PD.
In physical conditions, α-synuclein is soluble with no structure, but under certain pathological conditions, it can aggregate and fibrillize resulting in the formation of intracellular inclusions (known as LBs and LNs) and neurodegeneration (Beyer, 2006;
10 Uversky, et al., 2001; Weinreb, et al., 1996). The physiological function of α-synuclein in normal brain is not fully understood. It is proposed to be a molecular chaperone capable of binding to other intracellular proteins, such as soluble N-ethylmaleimide- sensitive factor attachment protein receptor (SNARE) and cysteine-string protein-α
(CSPα) (Burre, et al., 2010; Chandra, et al., 2005; Ostrerova, et al., 1999), having roles in neuronal differentiation and cell viability, in regulating dopamine release and uptake, and in modulating synaptic transmission and vesicle recycling (Abeliovich, et al., 2000;
Lucking & Brice, 2000; Uversky, 2007).
α-Synuclein exists in at least two different structural forms, one of which is a membrane-bound form and the other an essentially disordered free cytosolic form
(Bussell, et al., 2005; Eliezer, et al., 2001). The function of membrane-bound α- synuclein is still hotly debated. Some studies show binding to membranes promotes stabilization of α-synuclein secondary structure (Davidson, et al., 1998; Uversky &
Eliezer, 2009; Vamvaca, et al., 2009) and inhibits fibril formation (Zhu & Fink, 2003) while others suggest that membrane-bound α-synuclein has the property of initiating and promoting self aggregation of the cytosolic form (Lee, et al., 2002). These pre- fibrillar, oligomeric intermediates, referred to as “protofibrils”, may further oligomerize into fibrillar structures that ultimately organize into LBs (Sen & West, 2009). Although the process of α-synuclein fibrillization is acknowledged to be toxic to cells, which structural forms of α-synuclein in this process are the critical toxic species remains unclear. LBs are made of protease resistant insoluble filaments, but their toxicity is questioned (Obeso, et al., 2010). The recent identification of LBs in grafted fetal neurons in patients with PD has suggested the propagation of toxicity through the
11 extrusion and uptake of small α-synuclein aggregates in a prion-like process (Angot &
Brundin, 2009). The strong finding that genetic multiplications cause PD has many believing that toxicity is caused by simply increasing the cellular monomeric form of α- synuclein (Fuchs, et al., 2007; Singleton, et al., 2003), but evidence for such an increase in idiopathic PD is limited and not without challenge (Devi, et al., 2008; Xu, et al.,
2002). Recent studies suggest that soluble oligomers underlie neuronal death (Brown,
2010), while others suggest that the increased binding of oligomers to membranes are necessary for toxicity (Sulzer, 2010; van Rooijen, et al., 2010). The concept that such membrane-associated forms of α-synuclein play a large role in idiopathic PD is directly challenged by a recent study (Tong, et al., 2010a). This study comparing membrane- associated α-synuclein across synucleinopathies showed a massive increase largely restricted to the pathologically affected basal ganglia regions in MSA, while widespread changes in PD were not observed (Tong, et al., 2010a). Surprisingly, in PD there was no increase in membrane-associated α-synuclein in either the putamen (which has an early loss of dopamine terminals) or frontal cortex (which accumulates LBs and LNs in the late stages of the disease), highlighting that idiopathic PD is not a simple, global over- expression of the protein in the regions affected by the disease process (Tong, et al.,
2010a). Importantly, α-synuclein can be phosphorylated at serine 129 (S129), and most
α-synuclein in LBs is phosphorylated at this site (Anderson, et al., 2006; Fujiwara, et al.,
2002), suggesting its phosphorylation state has an important pathogenic role.
1.2.1 αα-Synuclein-Sy-Synucleinnuclein phosphorylationphosphorylation in ParkinParkinsonson’s disease
Phosphorylation is the main post-translational modification of α-synuclein in PD
(Oueslati, et al., 2010). Under physiological conditions, only 4% of total α-synuclein is
12 phosphorylated in the brain, while in PD more than 90% of α-synuclein deposited in
LBs is phosphorylated (Fujiwara, et al., 2002). At least five α-synuclein phosphorylation sites have been reported so far, including S129, S87, Y125, Y133, and
Y136 (Oueslati, et al., 2010). Among them, only the phosphorylation of S129 has been identified as the dominant pathological modification and most α-synuclein in LBs is phosphorylated at this site (Anderson, et al., 2006; Fujiwara, et al., 2002). The low levels of S129 phosphorylated (S129P) α-synuclein under normal physiological conditions are considered protective (Sato, et al., 2011), while the function of hyperphosphorylated α-synuclein at S129 is still controversial. It has been reported that
S129 phosphorylation in vitro disrupts the intra-molecular long-rang interaction of wild type α-synuclein (Paleologou, et al., 2008) and increased levels of S129P α-synuclein in vivo exacerbate α-synuclein toxicity (Chen & Feany, 2005b; Sato, et al., 2011).
However, this toxic effect is challenged by other studies (Azeredo da Silveira, et al.,
2009; Gorbatyuk, et al., 2008; McFarland, et al., 2009). Over-expression of α-synuclein phosphorylation-prevented mutant S129A enhances the loss of dopaminergic neurons in the SNpc and reduces dopamine and tyrosine hydroxylase (TH) levels in the striatum
(Gorbatyuk, et al., 2008), whereas over-expression of the phosphorylation-mimicking mutant S129D variant does not accelerate α-synuclein toxicity (Azeredo da Silveira, et al., 2009; Gorbatyuk, et al., 2008; McFarland, et al., 2009).
The levels of phosphorylated α-synuclein are regulated by the interplay of the amount of total α-synuclein produced and degraded, and the up-stream kinases and down- stream phosphatases that interact with the molecule. Investigation of the up-stream kinases delivers new insight for understanding the role of phosphorylated α-synuclein in the pathogenesis of PD and may provide novel therapeutic targets for PD
13 (Vancraenenbroeck, et al., 2011a).
1.31.3 αα-Synuclein-Sy-Synucleinnuclein phosphorylationphosphorylation re relatedlated kinaseskinases inin Pa Parkinsonrkinson’s diseasedisease
1.3.1 Polo-likePolo-like kinaseskinases (PLKs)
PLKs are a serine/threonine kinase family containing an N-terminal serine/threonine kinase catalytic domain and a C-terminal polo-box domain (PBD) involved in substrate binding and regulation of kinase activity. Five mammalian PLK family members from three subfamilies have been identified so far, including the PLK1 subfamily, the PLK4 subfamily, and the PLK2 subfamily (containingPLK2, PLK3 and PLK5)(de Carcer, et al., 2011b). Studies on PLKs have been primarily focused on their critical role in the cell cycle (Winkles & Alberts, 2005), but recent studies suggest that PLKs also have important roles in terminally differentiated cells of the nervous system (Seeburg, et al.,
2005b).
PLK1 is expressed at the highest levels in tissues with actively proliferating cell populations (Winkles & Alberts, 2005). It localizes in the cytoplasm and centrosomes during interphase and concentrates in the kinetochores and the cytokinetic bridge during cell division, controlling a number of processes throughout the cell cycle (Hood, et al.,
2012; Liu, et al., 2012a). Early observations on the over-expression of PLK1 in human tumors initiated a series of studies that hold great hope for PLK1 being a potential target for the treatment of cancer (Lens, et al., 2010).
PLK2, 3 and 5 belong to the PLK2 subfamily, which are found only in some bilaterian animals (de Carcer, et al., 2011b). Whereas PLK2 and PLK3 transcripts are widely expressed in distinct proliferative and non-proliferative tissues (de Carcer, et al., 2011b;
14 Winkles & Alberts, 2005), PLK5 is only expressed in a few non-proliferative tissues, such as the brain and eye (de Carcer, et al., 2011a). PLK2 is concentrated in centrosomes and may participate in centrosome biology and S-phase checkpoints;
PLK3 localizes to the nucleolus in interphase and may function in S-phase entry; PLK5 does not seem to have a role in cell cycle progression. Although the biological roles of the PLK2 subfamily has not been fully resolved, all of them have been suggested as possible tumor suppressors and stress-response proteins (Strebhardt, 2010). The PLK2 and PLK3 mediated stress response depends on the p53 pathway, while the PLK5 related stress response seems to be independent of p53 (Strebhardt, 2010).
From a structural point of view, PLK4 is the most divergent PLK family member
(Strebhardt, 2010). Similar to PLK1, human PLK4 is also highly expressed in all embryonic tissues and in adults is predominantly found in proliferative tissues (Winkles
& Alberts, 2005). Both gain-of-function and loss-of-function approaches clearly show that the presence of PLK4 is crucial for centriole duplication in Drosophila and mammals (Bettencourt-Dias, et al., 2005; Habedanck, et al., 2005).
1.3.1.11.3.1.1 PLKsPLKs in in the centrcentralal nervousnervous sy systemstem
Although the polo gene was first described two decades ago (Llamazares, et al., 1991), the role of PLKs in the nervous system has been explored only recently. The central nervous system has relatively high expression of PLK2, 3 and 5, low expression of
PLK1 and apparently lacks PLK4 expression (de Carcer, et al., 2011a; de Carcer, et al.,
2011b; Winkles & Alberts, 2005). While PLK2 and PLK3 are expressed in most regions of the brain, there is surprisingly almost no expression of either PLK2 or PLK3
15 in the cerebellum (de Carcer, et al., 2011b; Winkles & Alberts, 2005). PLK2 and PLK3 levels are highly up-regulated by synaptic activity and their activation influences the modulation of synaptic inputs to both proximal and distal dendrites (Seeburg, et al.,
2005b). The discovery of specific interactions between a postsynaptic Rap guanosine triphophatase activating protein (RapGAP) known as spine-associated RapGAP and
PLK2/3 brings more understanding of how PLK2 and PLK3 remodel synapses (Pak &
Sheng, 2003a) as spine-associated RapGAP promotes the growth of dendritic spines, the postsynaptic compartment for excitatory synapses (Pak, et al., 2001). PLK2, and possibly PLK3, phosphorylates spine-associated RapGAP, leading to the degradation of spine-associated RapGAP and depletion of a core postsynaptic molecular scaffold that finally causes the loss of mature dendritic spines and synapses (Pak & Sheng, 2003a).
In addition, PLK2 is also found to be essential for neuron differentiation driven by neuronal growth factor (NGF) (Draghetti, et al., 2009). Elevated neural PLK2 has been found in the brains of patients with Alzheimer's disease (AD) or dementia with Lewy bodies (DLB), indicating its involvement in neurodegeneration (Mbefo, et al., 2010).
Intriguingly, the other PLK2 subfamily member, PLK5, is highly expressed in cortical neurons and glia cells, and in the granular layer of the cerebellum (de Carcer, et al.,
2011a) where PLK2 and PLK3 are absent (de Carcer, et al., 2011b; Winkles & Alberts,
2005). This suggests that PLK5, but not PLK2/3, may participate in functions specific to these cells. A recent study shows that PLK5 modulates neurite formation on stimulation of NGF/brain-derived growth factor (BDGF)-Ras pathway (de Carcer, et al.,
2011a).
16 Although PLK1 is poorly expressed in normal brain, neuronal PLK1 is up-regulated in the hippocampus and cortex of patients with AD (Harris, et al., 2000); moreover, inhibition of PLK1 reduces beta-amyloid (Aβ)-induced neuron death in a cell model
(Song, et al., 2011), indicating the involvement of PLK1 in neurodegeneration.
1.3.1.21.3.1.2 PLKsPLKs and and α α-synuclein-s-synucleinynuclein
PLK1-4 have been identified to be capable of phosphorylating several substrates, while human PLK5 lacks a functional kinase domain (de Carcer, et al., 2011a; de Carcer, et al., 2011b). Comparative studies on kinases phosphorylating synucleins demonstrate that PLK1-3 directly phosphorylate α-synuclein at S129 in vitro, while PLK4 seems unable to phosphorylate any synuclein (Inglis, et al., 2009; Mbefo, et al., 2010). The low kinase activity of PLK4 against α-synuclein and other substrates is partially explained by its unique structure with only a single polo-box, resulting in a much reduced electropositive environment in its substrate binding site (Mbefo, et al., 2010).
Among PLK1-3, PLK2 and PLK3 are more efficient than PLK1 in phosphorylating α- synuclein at S129 in vitro (Inglis, et al., 2009; Mbefo, et al., 2010), and are specifically co-localized with S129P α-synuclein in various subcellular compartments in cell culture systems and transgenic mouse brain (Mbefo, et al., 2010); while PLK1 is mainly involved in phosphorylating aggregated α-synuclein at S129 (Waxman & Giasson,
2011). Moreover, increases in PLK2 or PLK3 levels significantly up-regulate S129P α- synuclein (Mbefo, et al., 2010; Waxman & Giasson, 2011), while their inhibition or reduction remarkably decreases α-synuclein phosphorylation in both cell and animal models (Inglis, et al., 2009; Waxman & Giasson, 2011). Combined, these data in concert with the findings of up-regulated PLK1 levels and/or PLK2 immunoreactivity
17 in AD and DLB (Harris, et al., 2000; Mbefo, et al., 2010) suggest that PLKs increase their expression levels or/and kinase activity under pathological conditions, phosphorylating more α-synuclein.
Although previous studies have suggested associations between PLKs and α-synuclein phosphorylation, some questions still remain. Even though PLK2/3 is co-localized with
α-synuclein aggregates in cell and animal models of PD (Mbefo, et al., 2010), they are not co-localized with α-synuclein inclusions in synucleinopathies(Mbefo, et al., 2010).
In addition, although PLK1 is involved in phosphorylating α-synuclein aggregates in cell models, it fails to be co-localized with α-synuclein aggregates in the same system
(Waxman & Giasson, 2011). Therefore, more studies are needed to determine whether it is necessary for PLKs to co-localize with α-synuclein to phosphorylate it; and to determine whether the interaction between PLK2/3 and S129P α-synuclein under pathological conditions is different from that under normal physiological conditions.
1.3.2 Casein kinasekinasess (CKs)
CKs are a serine/threonine kinase family ubiquitously expressed in eukaryotic organisms (Peters, et al., 1999). They were first characterized in the 1970s as actively phosphorylating casein in vitro in a cyclic adenosine monophosphate (AMP)- independent manner. CKs contain two members, CK1 and CK2, which are quite different from each other in structure, localization, and function (Perez, et al., 2011).
CK1 consists of a small N-terminal lobe and a large C-terminal lobe, and a catalytic cleft where adenosine triphosphate (ATP) and substrates bind (Cheong & Virshup,
2011). To date at least seven CK1 isoforms (α, β, γ1-3, δ, and ε) and their various splice 18 variants have been identified in different organisms. These isoforms range from 22 to
55 kDa and localize in membranes, nucleus, and cytoplasm of eukaryote cells and additionally in the mitotic spindle in mammalian cells (Fish, et al., 1995). All CK1 isoforms are highly homologous in their kinase domains (Knippschild, et al., 2005), presenting a strong preference for “primed”, pre-phosphorylated substrate; however, they also phosphorylate related unprimed site under certain conditions (Cheong &
Virshup, 2011). CK1 is involved in diverse cellular processes ranging from circadian rhythms to cell proliferation, playing a crucial role in human diseases like cancer and sleep disorders (Cheong & Virshup, 2011; Perez, et al., 2011).
CK2 is a tetrameric enzyme composed by the assembly of two catalytic subunits (CK2α and CK2α’) and a regulatory subunit (CK2β dimer). The two catalytic subunits α and α’ share 90% sequence homology in their N-terminal region, but the regulatory subunit β does not have any similarity to the other two subunits. CK2 is found in many organisms and tissues and nearly every subcellular compartment. It can phosphorylate more than
300 substrate proteins (Meggio & Pinna, 2003), which are involved in diverse cell processes including cell division, proliferation, apoptosis, and DNA repair. Among the many cell processes regulated by this protein kinase, the central task of CK2 is the promotion of cell growth and cell viability (St-Denis & Litchfield, 2009).
1.3.2.11.3.2.1 CKsCKs inin neur neurodegenerativeodegenerative di diseasesease
Recent studies indicate that CKs are involved in neurodegenerative disease, especially in AD and PD. It has been shown that CKs are co-localized in neurofibrillary lesions and granulovacuolar degeneration bodies in AD and that the levels of CK1 mRNA are
19 elevated in the pathological regions of AD brains (Perez, et al., 2011). Moreover, both
CK1 and CK2 are found to associate with the pathological phosphorylation and/or accumulation of tau and Aβ in AD (Perez, et al., 2011). Similar findings in PD show that the CKs are associated with α-synuclein phosphorylation.
1.3.2.21.3.2.2 CKsCKs andand α α-synuclein-s-synucleinynuclein
Although previous studies have shown that both CK1 and CK2 can constitutively phosphorylate α-synuclein at S129 in vitro (Okochi, et al., 2000; Waxman & Giasson,
2008) and that inhibition of CK1 or CK2 reduces α-synuclein S129 phosphorylation in vivo (Ishii, et al., 2007; Okochi, et al., 2000; Waxman & Giasson, 2008), in cells the inhibition of CK2 is more efficient in reducing S129P α-synuclein levels than that of
CK1 (Ishii, et al., 2007). Furthermore, CK2 rather than CK1 co-localizes with synucleinopathologies (Ryu, et al., 2008; Wakamatsu, et al., 2007). This data suggests that CK2 is more involved in phosphorylating α-synuclein in synucleinopathies. In addition, CK1 has been reported to directly phosphorylate another PD-associated protein parkin (Kitada, et al., 1998), whose over-expression decreases α-synuclein phosphorylation (Khandelwal, et al., 2010) and attenuates cell death (Khandelwal, et al.,
2010; Petrucelli, et al., 2002) while CK2 is responsible for the phosphorylation of synphilin-1 (Lee, et al., 2004), a protein that is present in LBs and binds to α-synuclein
(Buttner, et al., 2010; Engelender, et al., 1999; Eyal, et al., 2006). The interaction between synphilin-1 and α-synuclein is remarkably dependent on phosphorylation and is blocked by CK2 inhibition (Lee, et al., 2004). Combined, these data suggest that CKs regulate α-synuclein interactions and its potential toxicity via both direct phosphorylation of α-synuclein, or via phosphorylation of an interacting partner.
20 It is worth noting that although previous studies have suggested that an interaction between CKs and α-synuclein, there has been no assessment of either kinase activity or protein levels of CKs in PD and related synucleinopathies. In addition, a recent study failed to find an effect of CK1 inhibition on α-synuclein S129 phosphorylation in a cell model (Waxman & Giasson, 2011), challenging the association between CK1 and α- synuclein phosphorylation. This discrepancy could result from, at least partially, the specificity of current CK1 inhibitors. Hence, more studies are needed to determine the relationship between CKs and α-synuclein in both physiological and pathological conditions.
1.3.3 G protein-coupledprotein-coupled re receptorceptor kinases ((GRKs)GRKs)
GRKs are a serine/threonine kinase family which regulate the activity of G protein- coupled receptors (GPCRs) by phosphorylating their intracellular domains after their associated G protein has been released and activated (Gurevich, et al., 2012).
Structurally, GRKs contain a central catalytic domain flanked by an N-terminus containing a regulator of G protein signaling homology domain and a variable length C- terminal end. Based on sequence homology and tissue expression, GRKs are further classified into three subfamilies: the rhodopsin kinase or visual GRK subfamily (GRK1 and GRK7), the β-adrenergic receptor kinase subfamily (GRK2 and GRK3), and the
GRK4 subfamily (GRK4, GRK5, and GRK6)(Gurevich, et al., 2012; Kamal, et al.,
2012). GRK2, 3, 5 and 6 are ubiquitously expressed in mammalian tissue, whereas
GRK1 and 7 are confined to retinal rods and cones, respectively, and GRK4 is mainly present in testis, cerebellum and kidney (Kamal, et al., 2012). GRKs primarily phosphorylate activated GPCRs, followed by binding arrestin proteins, which prevent 21 receptors from downstream heterotrimeric G protein pathways while allowing activation of arrestin-dependent signaling pathways (Premont & Gainetdinov, 2007). In addition, GRKs are also capable of phosphorylating non-GPCRs and may play a role in the process of cell growth and proliferation, cell death and motility (Gurevich, et al.,
2012).
1.3.3.11.3.3.1 GRKsGRKs inin neurodegenerativeneurodegenerative di diseasesseases
Four out of five non-visual GRK isoforms, GRK2, 3, 5 and 6, are highly expressed in the brain. GRK2 and 5 are the main isoforms in the primate brain with GRK3 and 6 at lower levels. GRK3 is least abundant in rodent and primate subcortical brain areas, but its expression in cortex is comparable to that of other isoforms. GRK2 is required for normal chemosensation in C. elegans and circadian odorant responses in Drosophila, and mammalian GRK3 mediates desensitization of odorant receptors in the olfactory epithelium. Dysfunction of GRK isoforms have been implicated in neurodegenerative disorders, especially AD and PD. It has been reported that GRK2 is up-regulated in AD patients and in a rat model (Obrenovich, et al., 2006) with elevated GRK2 protein and mRNA levels correlated with the degree of cognitive impairment (Leosco, et al., 2007); moreover, GRK2 is associated with neurofibrillary tangles in AD (Takahashi, et al.,
2006). GRK5 deficiency accelerates accumulation of Aβ (Cheng, et al., 2010) and increases both microgliosis and astrogliosis (Li, et al., 2008).
1.3.3.21.3.3.2 GRKsGRKs andand αα-synuclein-s-synucleinynuclein
Extensive evidence from both in vitro and cell model experiments indicates that GRKs may be responsible for α-synuclein phosphorylation. An early study reported that all
22 synuclein (α, β, γ and synoretin) are substrates of GRK, with GRK2 preferentially phosphorylating α and β synucleins while GRK5 prefers α-synuclein as a substrate
(Pronin, et al., 2000a). GRK5 has been confirmed to phosphorylate α-synuclein in vitro, and also to promote the oligomerization of α-synuclein and is localization in LBs in vivo (Arawaka, et al., 2006). These findings support the concept that GRK2 and 5 are capable of phosphorylating α-synuclein and the GRK5 may be the major GRK isoform for this effect in synucleinopathies. However, this hypothesis is questioned by later studies which have failed to reproduce the co-localization of GRK5 in LBs (Takahashi, et al., 2006) and failed to diminish the phosphorylation of α-synuclein by knockdown of either GRK5 or GRK2 in cell models (Liu, et al., 2010; Sakamoto, et al., 2009). Instead, knockdown of GRK3 or GRK6 significantly decreased S129P α-synuclein levels
(Sakamoto, et al., 2009). The expression of GRKs tends to decrease in PD brains
(Bychkov, et al., 2008); however, because chronic L-DOPA treatment suppresses GRK expression in animal models (Bezard, et al., 2005) and PD patients are routinely treated with L-DOPA, it is difficult to differentiate the effects of PD itself from those of L-
DOPA treatment for these kinases.
1.3.4 Leucine-richLeucine-rich repeat repeat kinase 2 (LRRK2(LRRK2))
LRRK2 is a newly identified kinase encoded by the LRRK2 gene (also known as
PARK8 gene), and found to be responsible for a proportion of autosomal-dominant inherited PD. It is an extremely large protein of approximate 280kDa and is comprised of five predicted functional domains: a leucine-rich repeat domain, a Roc GTPase domain, followed by its associated C-terminal of Roc (COR) domain, a MAPKKK domain and a WD40 domain (Mata, et al., 2006). LRRK2 is widely expressed in normal human brain, with highest level in striatum and cortex but a relatively low abundance in 23 the SN (Higashi, et al., 2007). In addition, it displays particularly high expression in the kidney and appears to increase in expression during organogenesis and cell maturation
(Biskup, et al., 2007; Westerlund, et al., 2008a). LRRK2 is localized in the cytoplasm of neurons (Miklossy, et al., 2006) as well as associated with membranous structures including the mitochondrial outer membrane, lysosomal vesicles and punctuate structures within the perikarya dendrites and axons (Biskup, et al., 2006; Hatano, et al.,
2007). The physiological function of LRRK2 remains unclear. It may play a role in regulating dopamine neurogenesis, vesicle endocytosis, cytoskeleton dynamics and autophagy in immune cells (Gomez-Suaga, et al., 2012; Yue, 2012). At least 20 LRRK2 gene mutations have been linked to autosomal-dominant PD so far, with the G2019S the most common mutant responsible for not only familiar but also sporadic PD (Gasser,
2009). Extensive studies have shown that increased kinase activity is associated with some pathogenic LRRK2 mutations, like G2019S (Cookson, et al., 2007; West, et al.,
2005). G2019S mutants have significantly increased autophosphorylation and substrate phosphorylation compared to wild type LRRK2 (West, et al., 2005). Reduction in the kinase activity of the G2019S mutant reduces its neurotoxicity (Greggio, et al., 2006;
Smith, et al., 2006; West, et al., 2007). These results suggest that deregulation of
LRRK2 kinase activity and modulation of subsequent phosphorylation might be the pathogenic mechanism for LRRK2 related PD.
1.3.4.11.3.4.1 LRRK2LRRK2 and and α α-synuclein-s-synucleinynuclein
Both LRRK2 and α-synuclein are dominant PD-linked gene products, and α-synuclein aggregation is the major pathology associated with LRRK2 mutations (Gomez & Ferrer,
2010; Zimprich, et al., 2004). Additionally, LRRK2 immunoreactivity occurs in some
24 LBs (Alegre-Abarrategui, et al., 2008; Higashi, et al., 2007; Perry, et al., 2008; Zhu, et al., 2006a; Zhu, et al., 2006b). This data suggests that LRRK2 and α-synuclein may have common processes relevant to PD pathogenesis.
The coincidence in PD patients of increased kinase activity from LRRK2 mutations
(Cookson, et al., 2007; West, et al., 2005) and hyperphosphorylation of α-synuclein
(Anderson, et al., 2006; Fujiwara, et al., 2002) raises the possibility that LRRK2 may directly phosphorylate α-synuclein in the process of PD. Qing and collaborators report in in vitro experiments that LRRK2 could phosphorylate α-synuclein at S129 under certain conditions(Qing, et al., 2009a) and that LRRK2 and α-synuclein co- immunoprecipitated with each other from pathological DLB tissue and from Human
Embryonic Kidney 293 (HEK293) cells exposed to oxidative stress (Qing, et al., 2009b).
Furthermore, immunostaining shows co-localization of LRRK2 and α-synuclein in some neurons and LBs in PD and DLB cases (Guerreiro, et al., 2012; Perry, et al., 2008;
Qing, et al., 2009b).
However, the hypothesis of direct phosphorylation of α-synuclein by LRRK2 is challenged by other studies. Cell culture and animal studies show that over-expressing either wild-type or PD-related G2019S LRRK2 fails to increase α-synuclein phosphorylation at S129 (Herzig, et al., 2012; Lin, et al., 2009). Moreover, co- expression of LRRK2 with A53T mutant α-synuclein in a double-transgenic mouse model dramatically accelerates the neurodegenerative process in a dose dependent manner and independently from the LRRK2 genotype, suggesting that the kinase activity is not important for the observed phenotype (Lin, et al., 2009). Hence, there is another possibility that LRRK2 interplays with α-synuclein via different mechanisms
25 rather than direct phosphorylation. The loss of LRRK2 causes accumulation of endogenous α-synuclein in mouse kidney (Tong, et al., 2010b); increased α-synuclein levels induce increased LRRK2 mRNA levels in rat striatum (Westerlund, et al., 2008b); and over-expression of LRRK2 up-regulates α-synuclein expression in a cell model via the extracellular signal-regulated kinase (ERK) pathway (Carballo-Carbajal, et al.,
2010). These data suggest that LRRK2 and α-synuclein may interact with each other by co-regulating their expression levels. In addition, the findings that both LRRK2 and α- synuclein are associated with the 14-3-3 family of proteins, cytoskeleton proteins, mitochondria, and the proteasome and autophagy pathways support the concept that
LRRK2 and α-synuclein may indirectly interact with each other via these common pathways (Liu, et al., 2012b).
While these studies indicate a potential pathophysiological interplay between α- synuclein and LRRK2, there are still many questions. First, although α-synuclein positive LBs are the major pathology of PD cases with LRRK2 mutations, there are also appreciable LRRK2 mutant cases which lack LB pathology (Cookson, et al., 2008).
Secondly, LRRK2 phosphorylates α-synuclein only at high temperature and with long incubations in vitro (Qing, et al., 2009a) but not under physiological conditions (West, et al., 2005). Thirdly, some studies failed to observe co-localization of LRRK2 in synucleinopathologies (Higashi, et al., 2007). Fourthly, results from recent transgenic studies do not support an interaction between LRRK2 and α-synuclein in either over- expressing or knock-out transgenic mice (Daher, et al., 2012; Herzig, et al., 2012). Last but not least, even the reported pathways in which LRRK2 and α-synuclein act together in cell (Carballo-Carbajal, et al., 2010) and transgenic (Lin, et al., 2009) models are discrepant. Therefore, more studies are needed to determine whether there is a
26 relationship between α-synuclein and LRRK2 under both physiological and pathological conditions.
1.3.5 Adenos Adenosineine mo monophosphatenophosphate activated prproteinotein kinase (AMPK)
Different from above kinases, AMPK has been reported to phosphorylate α-synuclein only in a very recent study. AMPK is a serine/threonine kinase protein complex that consists of a catalytic α subunit and regulatory β and γ subunits (Hardie, 2011). It exists in all eukaryotes as heterotrimeric complexes and acts as a key regulator of cellular energy metabolism (Hardie, 2011). AMPK had been the focus of research on obesity, diabetes and other metabolic disorders for decades while its role in the brain, the most energy-consuming organ in our body, has only recently been studied and appreciated
(Amato & Man, 2011). It is suggested that AMPK signaling is intimately implicated in multiple aspects of brain development and function including neuronal proliferation, migration, morphogenesis and synaptic communication, as well as in pathological conditions such as neuronal cell death, energy depletion and neurodegenerative disorders (Amato & Man, 2011). Recently, Jiang and colleagues found that AMPK is capable of phosphorylating α-synuclein both in vitro and in vivo (Jiang, et al., 2012). In vitro, AMPK phosphorylates α-synuclein at S129, and enhanced AMPK activity facilitates α-synuclein accumulation and phosphorylation in neuronal culture; in addition, immunoprecipitation with cell lysates indicates an interaction between AMPK and α-synuclein (Jiang, et al., 2012). This is the first report about an association between AMPK and α-synuclein phosphorylation. More studies both in animal models and postmortem human brains are required to further identify its role in synucleinopathies.
27 1.3.6 CyclinCyclin G-associatedG-associated kinase (GAK)
GAK is a serine/threonine kinase encoded by the GAK gene (Kimura, et al., 1997). It is a ubiquitously expressed 160kD protein containing a highly conserved N-terminal serine/threonine kinase domain, a C-terminal J-domain, a clathrin-binding domain and a tension-like N-terminal domain (Kanaoka, et al., 1997; Kimura, et al., 1997; Zhang, et al., 2005). GAK is localized in the cytoplasm perinuclear area and trans-Golgi network
(Sato, et al., 2009), participating in regulating membrane trafficking, maintaining centrosome maturation and mitotic chromosome congression (Korolchuk & Banting,
2002; Shimizu, et al., 2009).
1.3.6.11.3.6.1 GAKGAK and and ParkinsonParkinson’s diseasedisease
Gene expression profiling shows that GAK is differentially expressed in the SNpc of
PD patients compared with controls (Grunblatt, et al., 2004), indicating the possible involvement of GAK in PD. Recent Genome-wide association studies (GWAS) further identify an association between a locus in the GAK gene and increased PD risk (Nalls, et al., 2011; Pankratz, et al., 2009; Sharma, et al., 2012). Hence, the GAK gene is defined as PARK17 for PD by some researchers (Mata, et al., 2011).
1.3.6.21.3.6.2 GAKGAK and and αα-synuclein-s-synucleinynuclein
There is a reported interaction between GAK and α-synuclein where polymorphism in the GAK locus (rs1564282) can result in higher α-synuclein expression in postmortem
PD frontal cortex compared with controls (Dumitriu, et al., 2011); in addition, knockdown of GAK in an α-synuclein based PD cell model results in increased α- synuclein levels and enhanced cytotoxicity (Dumitriu, et al., 2011). This interaction
28 between GAK and α-synuclein is thought to be medicated by pre-cathepsin D (CTSD), which binds to the clathrin-binding domain of GAK (Kametaka, et al., 2007). Cathepsin
D is the main lysosomal enzyme in α-synuclein degradation (Cullen, et al., 2009;
Sevlever, et al., 2008). However, there are limited details on how α-synuclein and GAK interact under normal or pathological conditions.
1.41.4 ConclusionConclusion
Changing the dynamics of S129 α-synuclein phosphorylation through related kinases could provide promising therapeutic targets for PD and related synucleinopathies
(Braithwaite, et al., 2012a; Vancraenenbroeck, et al., 2011b; Wang, et al., 2012).
However, it remains unclear which kinases are responsible for the hyperphosphoryaltion of α-synuclein in PD and related synucleinopathies. While in vitro studies provide useful information for screening possible kinases, results from these studies should be judged carefully when relating them directly to PD, as in vitro studies have extremely different conditions from that occurring in vivo. A related problem is the specificity and activity of inhibitors or agonists for these kinases. Data from the assessment of kinase expression or activity experiments in postmortem tissue from PD and related synucleinopathies are necessary as they would provide a basis for determining the kinases most related to α-synuclein phosphorylation and deposition in
PD.
1.51.5 HypothesisHypothesis
I hypothesize that increasing α-synuclein S129 phosphorylation and accumulation in disease affected brain regions will correlate with the progression of PD. Furthermore,
29 this PD related α-synuclein modification will relate to changes in up-stream kinases in the pathogenesis of PD. Inhibiting upstream kinases will reduce α-synuclein-induced cytotoxicity and pathology in model systems.
1.61.6 AimsAims
The overall objective of this thesis is to assess the role of α-synuclein phosphorylation and related kinases in the progression of PD. There are four specific aims:
Aim 1: To determine changes in the amount of α-synuclein phosphorylation over the
course of PD (Chapter 2).
Aim 2: To determine changes in the levels of α-synuclein related kinases in PD and
identify any relationships to the changes observed in α-synuclein
phosphorylation over the disease course (Chapter 3).
Aim 3: To confirm that similar α-synuclein phosphorylation and related kinase changes
to those observed in patients with PD also occur in a commonly used animal
model of PD (Chapter 4).
Aim 4: To determine whether a Chinese herbal treatment can reduce α-synuclein
phosphorylation and toxicity by inhibiting the relevant kinases identified in
patients with PD (Chapter 5).
30 ChapterChapter 2:2: ChangesChanges in α-s--synucleinsynucleinynuclein so solubilitylubility and and S1 S12929 phosphorylationphosphorylation during tthehe coursecourse ofof ParkinsonParkinson’s dis diseaseease
2.12.1 IntroductionIntroduction
As stated in Chapter 1, α-synuclein is a soluble protein making up 1% of all cytosolic proteins found in neurons (Iwai, et al., 1995) with approximately 15% transiently membrane bound (Lee, et al., 2002). It forms two types of pathologic inclusions in neurodegenerative disorders; protease-resistant insoluble α-synuclein fibrils in neuronal
LBs and LNs in PD and DLB, and less protease-resistant α-synuclein fibrils in oligodendroglial inclusions in multiple system atrophy (MSA)(Campbell, et al., 2001;
Tong, et al., 2010a). In MSA there is a massive increase in the membrane associated form of α-synuclein largely restricted to pathologically affected regions, which surprisingly is not seen in PD (Tong, et al., 2010a). This shows that idiopathic PD is not a simple, global over-expression of the protein in the regions affected by the disease process (Tong, et al., 2010a). As phosphorylation is the main post-translational modification of α-synuclein in PD and most α-synuclein in LBs is phosphorylated at
S129 (Anderson, et al., 2006; Fujiwara, et al., 2002), α-synuclein phosphorylation in
PD may be more important. Previous studies have assessed only a single form of α- synuclein (membrane associated α-synuclein), and did not assess the levels of phosphorylation of the protein in PD. They also did not examine the dynamics of any change over the course of PD. It therefore still remains unclear which forms of α- synuclein may play a role in the slow propagation of LBs and LNs that characterize PD.
31 2.22.2 AimAim an andd Hy Hypothesispothesis
The studies described in this chapter will determine changes in the amount of α- synuclein phosphorylation over the course of PD and assess any changes associated with its solubility and LB formation. It is hypothesized that there will be increased phosphorylation of α-synuclein prior to changes in its solubility and LB formation.
2.32.3 MaterialsMaterials and and Metho Methodsds
2.3.1 Overall study design
To determine changes in the solubility and phosphorylation of α -synuclein over the course of PD, tissue from cases at the three progressive stages of PD (stages IV, V and
VI) who have no other neurodegenerative conditions will be used and compared to tissue from neurological and neuropathological controls. Tissue proteins will be serially extracted to examine proteins of different solubility. Two brain regions affected at different times in patients with PD will be examined, the putamen which is always affected due to the early loss of dopamine terminals with progression to even more severe loss over the disease stages, and the superior frontal cortex which only has LB pathology by stage VI of PD. In this second region, the evaluation of early changes that result in LB formation over the disease course can be assessed. The use of different antibodies to α -synuclein that identify all forms of α -synuclein or only S129P α - synuclein will be compared to determine which forms relate most to the disease and to regional LB formation.
2.3.2 Cases
Approval was obtained to assess brain tissue from 33 PD cases and 13 controls 32 collected with consent through the Australian Network of Brain Banks. Controls had no significant neuropathology and no evidence of neurological or psychiatric disease. PD cases fulfilled the UK Parkinson’s Disease Society Brain Bank Diagnostic Criteria
(Hughes, et al., 1992) and were levodopa-responsive and medicated throughout their disease course. The PD cases were pathologically classified into three groups according to simplified diagnostic criteria (Harding & Halliday, 1998); brainstem or stage IV disease (N=14), limbic or stage V disease (N=9) and neocortical or stage VI disease
(N=10). No case had brain injury due to non-degenerative mechanisms (such as head trauma, infarction or sepsis) or other significant neuropathological changes.
Demographic details of the groups are given in Table 2.1.
TableTable 2.1: CaseCase demographicdemographic detailsdetails (given as means ± their standard deviation)
GroupGroup ControlsControls StageStage IVIV PDPD StageStage V PDPD StageStage VIVI PDPD
N 13 14 9 10
Male:female 6:7 6:8 7:2 7:3
Age at onset (y) - 72±6 62±8* 71±8
Age at death (y) 81±9 80±5 77±5 80±5
Duration (y) - 8±5 15±9 9±6
Postmortem delay 31±17 28±26 28±15 34±30
(h)
PD=Parkinson’s disease
*ANOVA p<0.05, different from other groups on posthoc testing
33 2.3.3 Tissue Tissue sampli samplingng and fractionatiofractionationn
Half the hemisphere was freshly sectioned and frozen blocks stored at -80°C prior to fractionation. One gram of the posterior putamen and 1g of the mid-superior frontal cortex from each case was homogenized in 4ml of homogenization buffer [0.32M sucrose, 20mM Tris-HCl pH7.4, 5mM EDTA, 1X protein inhibitor cocktail (EDTA free;
Roche, NSW, Australia), 1X phosphatase inhibitor (Roche, NSW, Australia)], sonicated
2x10s on ice and then cleared by centrifugation at 16,000g for 10min at 4°C. The supernatant was labeled the Tris-buffered saline (TBS)-soluble extract (S1). The pellet was washed twice with homogenization buffer, resuspended in 2ml homogenization buffer with 5% SDS, sonicated for 2x10s and centrifuged at 100,000g for 30min at
24°C. The supernatant was labeled the SDS-soluble fraction (S2). The pellet was rinsed twice with homogenization buffer with 5%SDS, then resuspended in 400ul 8%SDS/8M urea for 2h at room temperature and termed the urea-soluble/SDS-insoluble fraction
(S3). All protein concentrations were analyzed using a BCA kit (Thermo scientific, IL,
USA).
2.3.4 QuantitativeQuantitative We Westernstern immun immunoblottingoblotting for α-sy-synucleinnuclein pr proteinsoteins
For each gel, a previously tested sample of known antibody reactivity (20μg/well) was run against the unknown samples as an internal control. Aliquots containing equal amount of protein samples (TBS-soluble, 20μg; SDS-soluble and urea-soluble, 10μg respectively) were dissolved in an equal volume aliquot of 2×Laemmli buffer (4% SDS,
20% glycerol, 0.004% bromphenol blue, 0.125M Tris-HCl pH6.8, 5% mercaptoethanol), boiled at 95°C for 5min, loaded onto a 10% SDS-polyacrylamide gel and then subjected to electrophoresis with constant voltage of 100V. After separation, protein was
34 transferred by electrophoresis to a 0.22mm nitrocellulose filter membrane (Bio-Rad,
CA, USA) by the application of 100V for 1h. Blots were blocked with 5% milk in Tris- buffered saline Tween-20 (TBST) buffer (10mM Tris-HCl, pH7.5, 150mM NaCl,
0.05%Tween-20) and probed by using the primary antibodies suspended in 5% milk in
TBST buffer at 4°C overnight. Mouse monoclonal anti-α-synuclein antibodies were applied [syn-1, 42/α-synuclein, BD Transduction Laboratories, CA, USA, 1:1000; anti-
S129P-α-synuclein from Elan Pharmaceuticals, South San Francisco, CA, USA,
1:12,500] at 4°C overnight. Membranes were washed three times with TBST buffer and probed with goat anti-mouse horseradish peroxidase-conjugated secondary antibodies
(Bio-Rad, CA, USA, 1:7000) suspended in 5% milk in TBST buffer, applied for 1h at room temperature. After washing, chemiluminescence was produced using an ECL kit
(Amersham Biosciences, NJ, USA). Films were scanned and the intensity of each band was quantified with Quantity One software (Bio-Rad, CA, USA) and expressed as arbitrary units relative to the standard for comparison across groups. For α-synuclein qualification, the intensity of 18kD and 25kD bands were quantified in the TBS-soluble and SDS-soluble fractions, while in the Urea-soluble fraction the intensity of the whole lane from 18kD to the top loading well was qualified.
2.3.5 StatisticalStatistical analysesanalyses
SPSS 18 (IBM, Chicago, USA) was used for all analyses and significance established when p<0.05. There were no correlations between the levels of the different α- synuclein species in the different regions analysed and postmortem delay (P>0.05).
Wilcoxon Signed Rank and Median tests were used to describe the relationships between different forms of α-synuclein in different control regions. Mann Whitney U
35 tests were used to describe any overall difference in the levels of the different forms of
α-synuclein between PD and controls. Stepwise multiple regression analyses were used to determine any relationships to disease stage and between the different forms of α- synuclein changing over the disease course, covarying for age at onset and disease duration which differed for the cases in the different PD stages (see Table 2.1).
2.42.4 ResultsResults
2.4.1 Forms of α-synuclein ex expressedpressed in contrcontrolsols
In controls, monomeric α-synuclein was the dominant species in all tissue fractions
(Figure 2.1). In both the putamen and frontal cortex, there was more TBS-soluble α- synuclein (55-65% of relative α-synuclein amounts) compared with SDS-soluble α- synuclein (30-37% of relative α-synuclein amounts, Median tests p=0.03) and very low levels of urea-soluble α-synuclein (<10% of relative α-synuclein amounts, Figure 2.1
A-C). There was no difference between the distribution of the forms of α-synuclein between the regions examined (Wilcoxon Signed Rank test p=0.6). In controls, S129 phosphorylation was observed in a small proportion of both the TBS-soluble (Figure 2.1
D, 3-8% of relative α-synuclein amounts) and SDS-soluble (5-10% of relative α- synuclein amounts) protein fractions. Because of the low relative levels of S129P α- synuclein, both monomeric as well as a 25kD species was revealed (Figure 2.1 D). This higher molecular weight species has been shown previously to be a mono-ubiquitinated
α-synuclein species (Anderson, et al., 2006; Hejjaoui, et al., 2011) which must be in low overall abundance compared with the total amount of monomeric α-synuclein, but is a larger fraction of monomeric S129P α-synuclein (Figure 2.1 C&D). No S129P α-
36 synuclein was observed in the urea-soluble fractions in controls. There was a striking regional variation in the distribution of the forms of phosphorylated α-synuclein at
S129 in controls (Wilcoxon Signed Rank test p=0.03), with much greater phosphorylation of the TBS-soluble compared with SDS-soluble protein in the putamen
(65% versus 35% of relative putamen S129P) than in frontal cortex (both fractions 49-
51% of relative frontal S129P). This was largely due to greater phosphorylation (and monoubiquitination) of the TBS-soluble α-synuclein in the putamen compared with the frontal cortex (3.3x more S129P α-synuclein, Wilcoxon Signed Rank test p=0.02).
37 Figure 2.1: α-synuclein-synuclein andand S129PS129P α-synuclein-synuclein inin controlscontrols
(A)(A) Some monomeric 18kD urea-soluble (insoluble) α-synuclein was observed in both the frontal cortex
38 and putamen in control samples using the Syn-1 antibody to assess total α-synuclein. No oligomeric species were observed, and the proportion of insoluble α-synuclein as a fraction of the standardized total found in all tissue fractions for each region was similar (<10%, see Figure 2.2 B).
(B)(B) Only monomeric 18kD SDS-soluble (membrane associated) α-synuclein was observed in both the frontal cortex and putamen in control samples using the Syn-1 antibody to assess total α-synuclein, with between 30-37% of the standardized total amount of Syn-1 α-synuclein identified in these regions (see
Figure2.3 D).
(C)(C) Mainly monomeric 18kD and some 14kD TBS-soluble (cytosolic) α-synuclein were observed in both the frontal cortex and putamen in control samples using the Syn-1 antibody to assess total α-synuclein.
Only the 18kD monomer was quantified and represented between 55-65% of the standardized total amount of Syn-1 α-synuclein identified in these regions (see Figure 2.3 H).
(D)(D) Both monomeric 18kD and a 25kD species of TBS-soluble (cytosolic) S129P α-synuclein were observed in both the frontal cortex and putamen in control samples using the S129 antibody to assess phosphorylated α-synuclein. The 25kD band has been identified previously as monoubiquitinated monomeric α-synuclein (Anderson, et al., 2006; Hejjaoui, et al., 2011) and was therefore quantified in addition to the 18kD monomeric band. Significantly more S129P α-synuclein was identified in the putamen compared with the frontal cortex in controls (see Figure 2.3 H).
2.4.2 ChangesChanges observed in ParkinParkinsonson’s disease
As may have been expected, the most striking change observed in PD is the amount of higher molecular weight urea-soluble species of α-synuclein in both regions analysed
(Figure 2.2 A) and the overall increase in α-synuclein in the urea-soluble fraction
(Figure 2.2 B, 7.0-8.6x increase from control levels, Mann-Whitney U tests p<0.001).
Surprisingly, this large increase in urea-soluble α-synuclein was not reflected by an increase in its phosphorylation at S129, with only a few cases having comparatively weak immunoreactive blots (data not shown). Significantly more urea-soluble α-
39 synuclein was observed in the frontal cortex compared with the putamen in PD (Figure
2.2 B, 67% versus 44% of relative control α-synuclein amounts, Wilcoxon Signed Rank test p=0.035), with the amount of insoluble frontal α-synuclein being highly variable between cases (Figure 2.2 B).
There was no increase in either the TBS-soluble or SDS-soluble α-synuclein levels in the PD putamen samples compared with controls (Figure 2.2 D&H, Mann-Whitney U tests p>0.14). There was a small but significant increase in frontal TBS-soluble α- synuclein levels (Figure 2.2 G&H, 1.14±0.12x increase, Mann-Whitney U test p=0.047), but no difference between the levels in the frontal cortex versus the putamen due to the large variation in amounts (Wilcoxon Signed Rank test p=0.64). There was a larger and less variable increase in the SDS-soluble α-synuclein in the frontal cortex in PD (Figure
2.2 C&D, 1.4±0.1x increase, Mann-Whitney U test p<0.001) differentiating this region from the putamen (1.8±0.6x difference in SDS-soluble α-synuclein between the frontal cortex versus the putamen, Wilcoxon Signed Rank test p=0.001).
There was a striking increase in the levels of S129P α-synuclein in both the TBS- soluble and SDS-soluble fractions in the putamen (Figure 2.2 F&J, 5.8-18x, Mann-
Whitney U tests p<0.001) and frontal cortex (Figure 2.2 E, F, I&J, 21-28x, Mann-
Whitney U tests p<0.001) with significantly more S129P α-synuclein in the frontal cortex compared with the putamen in PD (Figure 2.2 F&J, 1.7-3.6x, Wilcoxon Signed
Rank text p<0.001).
40 41 FigureFigure 2.2: RelativeRelative amountsamounts of α-synuclein-synuclein andand S129PS129P α-synuclein-synuclein inin controlscontrols andand
PDPD casescases
(A,B)(A,B) There was a substantial overall increase in the amount of urea-soluble (insoluble) α-synuclein in both the frontal cortex and putamen in the PD samples, with the majority of the protein occurring in oligomeric species. There was more insoluble α-synuclein in the frontal cortex compared with the putamen in the cases with PD (double asterisk in B).
(C,D)(C,D) SDS-soluble (membrane associated) α-synuclein was predominantly monomeric and selectively increased in the frontal cortex in the PD samples, with no change over control levels observed in the putamen.
(E,F)(E,F) SDS-soluble (membrane associated) S129P α-synuclein was substantially increased in both the frontal cortex and putamen in the PD samples, with the monomeric species dominating but increasing oligomerization also observed. More membrane associated S129P α-synuclein occurred in the frontal cortex compared with the putamen in the cases with PD (double asterisk in F).
(G,H)(G,H) The amounts of TBS-soluble (cytosolic) α-synuclein mirrored the data observed for the SDS- soluble fraction, being predominantly monomeric and selectively increased in the frontal cortex in the PD samples.
(I,J)(I,J) The amounts of TBS-soluble (cytosolic) S129P α-synuclein also mirrored the data observed for the
SDS-soluble fraction and substantially increased in both the frontal cortex and putamen in the PD samples, with the monomeric species dominating but increasing oligomerization also observed. In PD, there was more TBS-soluble S129P α-synuclein in the frontal cortex compared with the putamen, whereas the reverse was true for controls (double asterisks in J).
To determine the relationships between the different α-synuclein species in patients with PD, stepwise multiple regression analyses were performed using either the urea- soluble frontal syn-1 α-synuclein (high levels in PD, see above) or the TBS-soluble
S129P putamen α-synuclein (high levels found in controls, see above), covarying for age at onset and disease duration. To identify changes most associated with LBs (made
42 of insoluble α-synuclein), the species relating most to changes in the levels of urea- soluble α-synuclein in the frontal cortex was determined to be frontal TBS-soluble phosphorylated S129 α-synuclein and frontal SDS-soluble α-synuclein (Figure 2.3 B, p<0.001, β coefficients for TBS-soluble S129P=0.78 and SDS-soluble syn-1=0.33). In addition, disease duration affected this relationship (more frontal urea-soluble α- synuclein with increasing disease duration, β coefficient=0.27). In PD, the species relating most to changes in the levels of TBS-soluble phosphorylated S129 α-synuclein in the putamen were SDS-soluble phosphorylated S129 α-synuclein in the putamen and
TBS-soluble phosphorylated S129 α-synuclein in the frontal cortex (Figure 2.3 A, p<0.001, β coefficients for putamen SDS-soluble=0.51 and frontal TBS-soluble=0.49).
43 Figure 2.3: RelationshipsRelationships betweenbetween α-synuclein-synuclein andand S129PS129P α-synuclein-synuclein amountsamounts betweenbetween thethe differentdifferent proteinprotein fractionsfractions andand overover thethe diseasedisease coursecourse inin thethe PDPD casescases 44 (A)(A) The α-synuclein species relating most to the levels of TBS-soluble (cytosolic) S129P α-synuclein in the putamen were the SDS-soluble (membrane associated) S129P α-synuclein in the putamen and TBS- soluble (cytosolic) S129P α-synuclein in the frontal cortex.
(B)(B) The α-synuclein species relating most to the levels of urea-soluble (insoluble) α-synuclein in the frontal cortex were the TBS-soluble (cytosolic) S129P α-synuclein and SDS-soluble (membrane associated) α-synuclein in the frontal cortex.
(C)(C) The α-synuclein species relating most to disease stage were a decrease in the levels of TBS-soluble
(cytosolic) and an increase in the SDS-soluble (membrane associated) α-synuclein in the frontal cortex, with increasing membrane levels of α-synuclein relating to LB formation (B). Grey bars stand for the range of control samples (Mean±SEM).
2.4.3 ChangesChanges over the disease course
As disease duration does not directly reflect disease stages (see Table 2.1), the amount of α-synuclein in the different fractions was assessed by disease stage. The urea-soluble and SDS-soluble syn-1 and TBS-soluble and SDS-soluble S129P in PD appeared to increase with disease stage (Figure 2.4). To determine the regional changes relating most to the different stages of PD, stepwise multiple regression analysis was performed in the PD cases, covarying for age at onset and disease duration. The factors relating most to disease stage were a decrease in the levels of TBS-soluble and an increase in the
SDS-soluble syn-1 in the frontal cortex (Figure 2.3 C, p<0.001, β coefficients for TBS- soluble=-0.74 and SDS-soluble=0.29, 36% reduction in TBS-soluble and 24% increase in SDS-soluble), with increasing levels of SDS-soluble syn-1 relating to LB formation
(see above).
45 Figure 2.4: RelativeRelative amountsamounts of α-synuclein-synuclein andand S129PS129P α-synuclein-synuclein inin thethe frontalfrontal
46 cortexcortex fromfrom controlscontrols andand PDPD casescases at differentdifferent diseasedisease stagesstages
(A)(A) There was an increase in the amount of urea-soluble (insoluble) α-synuclein with increasing disease stage, as reflected in the greater variation observed between cases (see PD amounts in Figure 2.2 B).
(B)(B) There was an increase in the amount of SDS-soluble (membrane associated) α-synuclein with increasing disease stage (see Figure 2.3 C).
(C)(C) There was an increase in the amount of SDS-soluble (membrane associated) S129P α-synuclein with increasing disease stage, as reflected in the greater variation observed between cases (see PD amounts in
Figure 2.2 F).
(D)(D) There was a decrease in the amount of TBS-soluble (cytosolic) α-synuclein with increasing disease stage (see Figure 2.3 C).
(E)(E) There was an increase in the amount of TBS-soluble (cytosolic) S129P α-synuclein with increasing disease stage, as reflected in the greater variation observed between cases (see PD amounts in Figure 2.2
J).
2.52.5 DiscussionDiscussion
This was the first study assessing the changes in α-synuclein phosphorylation and solubility with increasing pathological stage of PD, although previous studies have observed significant general increases in S129P α-synuclein in PD (Anderson, et al.,
2006; Fujiwara, et al., 2002). The findings from this chapter confirm the observations by Tong and colleagues (Tong, et al., 2010a) that there is only a small increase over control levels in the TBS-soluble and SDS-soluble α-synuclein levels in PD, although we have found that variation in these measures occurs with greater pathological severity and with increasing disease duration. In the frontal cortex where the deposition of LBs and LNs is a later pathological event, the initially ~15% higher cytosolic (TBS-soluble) levels of α-synuclein that precedes LB formation normalises to control levels as the disease progresses. This transient increase in the cytosolic levels of α-synuclein in PD
47 appears to precipitate a shift in the location of intracellular α-synuclein increasing its membrane (SDS) association by around 40% and changing the ratio of cytosolic:membrane associated α-synuclein from around 2:1 to 1:1 by late disease stages. Importantly, this intracellular shift and increased accumulation of membrane associated α-synuclein over time is directly related to both increased cytosolic S129 phosphorylation of the protein and to its increased insolubility (in the urea-soluble fraction), as would be expected for LB formation. The amount of cytosolic α-synuclein that is phosphorylated at S129 progressively rises from its basal level of around 5% to between 30-100% depending on the region and severity of pathology in the patients with PD. These data suggest that the factors driving LB formation in sporadic PD are an increase in both the cytosolic phosphorylation of α-synuclein and its membrane associations rather than an increase in the soluble cytosolic amount of the protein, as previously hypothesised (Cookson, 2009; McCormack & Di Monte, 2009; Shtilerman, et al., 2002). Previous pathological studies using immunohistochemistry have identified that S129P α-synuclein accumulates in LBs (Anderson, et al., 2006), although this was not reflected in the data from the urea-soluble extracts but was observed in the SDS- soluble extracts. SDS-soluble α-synuclein is generally considered a membrane- associated species (Tong, et al., 2010a), but it may also reflect the formation of less soluble oligomeric intermediates that perhaps aggregate in the superficial layers rather than the insoluble cores of LB. This suggests that dephosphorylation of S129P α- synuclein is likely to occur during the further fibrillation of α-synuclein and LB maturation.
The findings of no substantial increase in cytosolic α-synuclein protein levels in brain
48 regions affected by LB formation in patients with sporadic PD in this chapter confirm findings from recent studies using a variety of techniques [see Table 1 in (Tong, et al.,
2010a) and (Wills, et al., 2010)]. This contrasts with the slow increase in the SDS membrane associated levels of α-synuclein and has important implications for the pervasive concept that an increase in cytosolic α-synuclein concentration is the main pathogenic mechanism for PD (Cookson, 2009; McCormack & Di Monte, 2009;
Shtilerman, et al., 2002). The relatively stable amount of soluble α-synuclein measured in the present and previous PD studies is surprising, and perhaps suggests that regulatory mechanisms are required to maintain such normal α-synuclein cytosolic levels and protein turnover when insoluble intracellular pathogenic depositions of the protein are forming. Opposing changes in the expression levels of α-synuclein mRNA have been documented in sporadic PD (Grundemann, et al., 2008; Simunovic, et al.,
2009) and the amount of cytosolic α-synuclein may also be held relatively constant through micro-RNA regulation (Doxakis, 2010) or methylation (Jowaed, et al., 2010) or mechanisms involved in the membrane localization of the protein. Understanding the cellular regulation of the gene and any additional non-translational mechanisms regulating the levels of the major cytosolic α-synuclein species may assist further with understanding disease pathogenesis.
Despite no substantive change in the relative amount of soluble cytosolic levels of α- synuclein, the small changes noted over time were directly related to an increase in the amount of SDS membrane associated protein that occurred with increasing Braak stage of LB formation. This intracellular shift in protein location not only related to disease onset and progression in the frontal cortex, but directly related to the large increase in
49 insoluble α-synuclein observed in the PD tissue fractions over time, suggesting that membrane localization is a key event in LB formation. Membrane-associated α- synuclein is documented to play a critical role in its secondary structure (Davidson, et al., 1998; Uversky & Eliezer, 2009; Vamvaca, et al., 2009), protein aggregation and cellular degeneration (Auluck, et al., 2010; Jo, et al., 2000; Lee, et al., 2002). In vitro studies show that membrane-associated α-synuclein accelerates fibril formation (Jo, et al., 2000) and seeds the aggregation of cytosolic α-synuclein (Lee, et al., 2002), indicating it can initiate and precipitate inclusion formation (Auluck, et al., 2010). α-
Synuclein translocates to mitochondrial membranes with increasing intracellular acidification (Cole, et al., 2008) and patients with PD have significantly increased accumulation of mitochondrial α-synuclein and decreased complex 1 activity compared with controls (Devi, et al., 2008). Such cellular deficits impact on α-synuclein’s interaction with synaptic vesicles to decrease neurotransmitter release through lowering the recycling pool of synaptic vesicles (Nemani, et al., 2010). Overall the increase in membrane associated α-synuclein observed would significantly impact on cell function, in addition to increasing its aggregation propensity.
Importantly, this intracellular shift and increase in membrane associated α-synuclein over time is directly related to increased cytosolic S129 phosphorylation of the protein which also directly related to the amount of insoluble protein found in the tissue. This chapter shows a dramatic rise in the relative proportion of cytosolic S129P α-synuclein over the disease course, which suggests that such cytosolic phosphorylation is important for its membrane association and subsequent accumulation into the protease resistant fibrillar forms found in PD (Lue, et al., 2012). This is consistent with the diffuse non- fibrillar neuronal cytoplasmic staining using S129 specific immunohistochemsitry in 50 regions with LB formation (Saito, et al., 2003). It is well known that α-synuclein is extensively phosphorylated at S129 in PD (Anderson, et al., 2006; Fujiwara, et al., 2002;
Okochi, et al., 2000) and that such phosphorylation makes the protein more acidic, possibly increasing its capacity for membrane translocation (Cole, et al., 2008). A proportion of the cytosolic S129P α-synuclein appears to be monoubiquitinated according to its molecular weight (Anderson, et al., 2006; Hejjaoui, et al., 2011), although the extent of S129 α-synuclein phosphorylation is not influenced by its monoubiquitination, rather the monoubitquitination of α-synuclein has been shown to increase the stability of the monomer (Hejjaoui, et al., 2011). Pulldown assays show that S129P α-synuclein binds to different cellular proteins compared to non- phosphorylated α-synuclein (McFarland, et al., 2008). The phosphorylated form of the protein selectively binds to enzymes and signaling proteins involved in serine/threonine phosphorylation (McFarland, et al., 2008), although its role in such signaling remains unexplored. It also selectively binds to clathrin heavy chain and subunits involved in clathrin-mediated endocytosis of vesicles destined for the recycling pool (McFarland, et al., 2008), an association likely to impact on neurotransmitter release (Nemani, et al.,
2010). In addition, it binds to cytosolic proteins that form the presynaptic web required for synapse stability (McFarland, et al., 2008). Proteins of the presynaptic web which bind phosphorylated α-synuclein include spectrins and spectrin-interacting proteins, cytoplasmic actins, non-muscle myosins as well as microtubule-associated protein 1B
(MAP1B) and neurofilament L (McFarland, et al., 2008), with many of these proteins found in LBs (Leverenz, et al., 2007). The presynaptic web is essential for stability during vesicle exocytosis and membrane retrieval, dissolving at high basic pHs and being more stable at lower acidic pHs, bridging between the synaptic cell adhesion and the microtubule cytoskeleton (Phillips, et al., 2001; Pielage, et al., 2005). While 51 phosphorylated α-synuclein immunoreactive dot-like structures were identified some time ago in cases with LB formation (Saito, et al., 2003), it has only recently been recognized that these structures are synaptic accumulations of the phosphorylated protein (Schulz-Schaeffer, 2010; Tanji, et al., 2010). These data suggest that increased phosphorylation of cytosolic α-synuclein may increase its signaling capacity as well as synaptic and membrane stability.
The substantial LB-related increase in cytosolic phosphorylation of α-synuclein in the frontal cortex was directly related to the amount of S129P α-synuclein in the earlier affected putamen in the same cases, a region we have found to contain a significantly higher basal control level of S129P α-synuclein compared with the frontal cortex. These data support the concept that α-synuclein pathology may propagate between different brain regions (Angot, et al., 2010; Braak, et al., 2003), but suggests that the species involved may be the S129P form of the protein rather than insoluble α-synuclein. The regional difference between the degree of α-synuclein phosphorylation in controls also suggests that its signaling and synaptic functions following S129 phosphorylation
(McFarland, et al., 2008) are more necessary in the basal ganglia compared with the cortex. Whether other regions predisposed to α-synuclein pathology also have high
S129 phosphorylation levels remains to be determined. Synaptic damage in the putamen occurs preclinically in PD (Booij & Knol, 2007) with limited LB formation found in this region (Braak, et al., 2003) despite extensive α-synuclein synaptic and neuritic pathology (Duda, et al., 2002). This higher level of α-synuclein phosphorylation in the putamen may predispose this region to the synaptic degeneration that precedes the motor signs of PD.
52 2.62.6 StrengthsStrengths and and wea weaknessesknesses ofof thisthis study study
The present pathological study has used cross-sectional data from different PD cases with different disease severities to interpret the progression of pathological changes over the disease course and is not a longitudinal study of individuals. However, this paradigm has provided important information previously. The sample size at each stage is relatively small but also fairly homogeneous, as significant coexisting and age-related neuropathologies were excluded in all cases. The homogeneity of the cases was a strength of this study. All cases had typical PD using the UK Parkinson’s Disease
Society Brain Bank Diagnostic Criteria (Hughes, et al., 1992) and were levodopa- responsive with average disease durations of 8 years or more. As may have been expected, cases with stage IV disease had shorter disease durations than stage V, although this did not hold true for cases with stage VI disease. No case had clinical
DLB, or had the more rapid clinical phenotype often seen in such cases (Williams, et al.,
2006). In addition to case homogeneity, the same extraction methods were used in all cases, and the relative α-synuclein levels directly compared using the same techniques.
The methods and results used are similar to those recently published by Tong et al.
(Tong, et al., 2010a) where they showed that the most substantial changes occurred in the nigrostriatal pathway in the early disease cases they assessed. By using such methods, confirmation of previous results has been achieved and gives strength to the novel results identified in this study.
2.72.7 ConclusionConclusion
As previously stated, there were no studies assessing the changes in the relative α-
53 synuclein levels or phosphorylation status over the different stages of PD prior to the present study, although subsequent studies have identified similar changes in the phosphorylation of α-synuclein over the course of PD (Lue, et al., 2012). By using the same methods across the different stages of PD, this study shows that soluble non- phosphorylated α-synuclein decreases in PD vulnerable brain regions, becoming increasingly phosphorylated and insoluble over the course of PD. These relationships have not been previously described and provide important key information on the key protein change underlying PD pathology. In addition, the levels of S129P α-synuclein in the nigrostriatal pathway relates to the pathogenic forms of α-synuclein in frontal brain regions, suggest a propagating role of putamenal S129P α-synuclein in PD pathogenesis.
54 ChapterChapter 3: 3: α α-Synuclein-related-Sy-Synuclein-relatednuclein-related kina kinasesses andand LewyLewy bodybody formationformation in PPaParkinsonarkinsonrkinson’s diseasedisease
3.13.1 IntroductionIntroduction
As mentioned in Chapter 1, LBs and LNs are intraneuronal inclusions largely made of filamentous and hyperphosphorylated α-synuclein (Anderson, et al., 2006; Galvin, et al.,
2001). LBs are thought to form in two different way (Kuusisto, et al., 2003) via a progressive process and a formal staging scheme has been proposed (Wakabayashi, et al., 2007). The data from Chapter 2 shows that a substantive increase in the phosphorylation of cytosolic α-synuclein at S129 precedes Lewy pathology formation, confirming its phosphorylation state has an important pathogenic role (Braithwaite, et al., 2012a; Oueslati, et al., 2010).
As described in Chapter 1, several kinases are capable of phosphorylating α-synuclein at S129 in vitro and in cell models, including PLKs (Mbefo, et al., 2010), CKs
(Waxman & Giasson, 2008), and GRKs (Pronin, et al., 2000a). Human PLKs consist of five different members (PLK1-5) but only PLK1, 2 and 3 are able to phosphorylate α- synuclein in vitro (de Carcer, et al., 2011a; Lowery, et al., 2005; Mbefo, et al., 2010), and there is little information about PLK3 levels in human brain tissue due to the lack of specific antibodies (Mbefo, et al., 2010). Both CK1 and CK2 have been identified as capable of phosphorylating α-synuclein in vitro (Okochi, et al., 2000; Waxman &
Giasson, 2008), although only CK2 is localized in Lewy pathologies. There are seven different GRK members (GRK1-7) and at least five of them (GRK1, 2, 3, 5 and 6) have
55 been reported to phosphorylate α-synuclein (Pronin, et al., 2000a; Sakamoto, et al.,
2009), but the major isoform(s) responsible for this effect are contentious (Pronin, et al.,
2000a; Sakamoto, et al., 2009). The expression of GRKs tends to decrease in PD brains
(Bychkov, et al., 2008); however, because chronic L-DOPA treatment suppresses GRKs expression in animal models (Bezard, et al., 2005) and PD patients are routinely treated with L-DOPA, it is difficult to differentiate the effects of PD itself from those of L-
DOPA treatment for this kinase. In addition to these kinases, other kinases have been indicated to interact with α-synuclein in the pathogenesis of PD, such as LRRK2 (Liu, et al., 2012b) and GAK (Dumitriu, et al., 2011). Overall there is little information on the levels of most of these kinases in relation to the changes observed in α-synuclein phosphorylation in PD or the kinases associated with the different stages of LB formation.
3.23.2 AimAim an andd Hy Hypothesispothesis
The studies described in this chapter will determine changes in the levels of α-synuclein related kinases in PD and identify any relationships to the changes observed in α- synuclein phosphorylation over the disease course and during LB formation. It is hypothesized that there will be increased levels of α-synuclein related kinases that are associated with the increased levels of α-synuclein phosphorylation, and that α- synuclein-related kinases will be associated with the different stages of LB formation.
3.33.3 MaterialsMaterials and and Metho Methodsds
3.3.1 Overall study design
To determine changes in the levels of α-synuclein related kinases in PD, five S129 α- 56 synuclein phosphorylation-related kinase candidates [PLK1, PLK2, CK2β, LRRK2 and
GAK] will be assessed in the same human brain tissue fractions used to assess the levels of α-synuclein in chapter 2. The same two brain regions affected at different times in patients with PD will be examined, the early affected putamen and the only late affected superior frontal cortex. To assess the different types of LB, assessment of the dopamine neuronal cell bodies in the SN rather than their terminals will be used, and the anterior cingulate region of the frontal lobe will be assessed, as this region is known to have the greatest accumulation of S129P α-synuclein in LBs compared with other cortical regions (Lue, et al., 2012; Walker, et al., 2013). It should be noted that cortical LBs in early disease cases are often considered immature compared with their counterparts in the SN (Goedert, et al., 2013; Wakabayashi, et al., 2007). Therefore any differences between the LBs in these regions would also be assessed to determine any influence on
LB maturation. This experimental design allows for the examination of both end-stage disease effects and changes associated with the early and subsequent development of
Lewy pathologies in different brain regions at different disease stages.
3.3.2 Cases
The same cases were used as in Chapter 2 (stated in section 2.3.2).
3.3.3 Tissue Tissue sampli samplingng and prprocessingocessing
For all cases, half the hemisphere was freshly sectioned and frozen blocks stored at -
80°C prior to fractionation. Frozen putamen and frozen superior frontal cortex were fractionated as described in section 2.3.3 and TBS-soluble and SDS-soluble fractions were used for western blotting in this Chapter.
57 The other hemisphere was fixed in formalin for two weeks prior to tissue preparation for neuropathological examination and screening, as previously described (Harding &
Halliday, 1998). Tissues blocks from SN and anterior cingulate for immunohistochemistry were embedded in wax and sectioned at 10µm thick on a microtome, mounted on 3-aminopropyltriethoxysilane-coated slides, and de- paraffinized for the following staining for this study.
3.3.4 Semi-quantitativeSemi-quantitative WesternWestern blotting
For each gel, a previously tested sample of known antibody reactivity was run against the unknown samples as an internal control and β-actin was used as a control for sample loading. Aliquots containing equal amount of protein samples (40μg/well) were dissolved in an equal volume aliquot of 4x sample buffer (Life Technologies, CA, USA) with 10x reducing buffer (Life Technologies, CA, USA) and boiled at 70°C for 10min.
For LRRK2 and GAK detection, samples were loaded onto a 3-8% gradient Tris-acetate pre-cast gel (Life Technologies, CA, USA) and then subjected to electrophoresis in 1x
NuPAGE Tris-acetate running buffer (Life Technologies, CA, USA) with constant voltage of 150V. For PLK1, PLK2 and CK2β detection, samples were loaded onto a
10% SDS-polyacrylamide gel and then subjected to electrophoresis in 1x Tris-glycine
SDS running buffer (pH=8.9) with constant voltage of 100V. After separation, protein was transferred by electrophoresis to a 0.22mm nitrocellulose filter membrane (Bio-Rad,
CA, USA) by the application of 100V for 1-2h. Blots were blocked with 5% milk in
TBST buffer and probed by using the primary antibodies [anti-LRRK2 (Epitomics, CA,
USA; 1:1000); anti-GAK (Sigma, CA, USA; 1:2000); anti-PLK1 (Santa Cruz
58 Biotechnology, TX, USA; 1:300); anti-PLK2 (Santa Cruz Biotechnology, TX, USA;
1:500); anti-CK2β (Santa Cruz Biotechnology, TX, USA; 1:500); or anti-β-actin
(Abcam, Cambridge, UK; 1:25,000)] suspended in 5% milk in TBST buffer at 4°C overnight. Membranes were washed three times with TBST buffer and probed with goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies
(Bio-Rad, CA, USA; 1:7000) suspended in 5% milk in TBST buffer, applied for 1h at room temperature. After washing, chemiluminescence was produced using home-made
ECL [fresh mixture of buffer A (0.1M Tris pH8.5, 2.5mM luminol, 0.396mM p- coumaric acid) and buffer B (0.1M Tris pH8.5, 0.0192% hydrogen peroxide)]. Films were scanned and the intensity of each band was quantified with Image J software
(http://rsbweb.nih.gov/ij/) and expressed as arbitrary units relative to the standard for comparison across groups.
3.3.5 Single-labellingSingle-labelling im immunoperoxidasemunoperoxidase for pr proteinotein localizat localizationion
10µm thick formalin-fixed paraffin-embedded tissue sections of the SN and anterior cingulate cortex were dewaxed and hydrated followed by antigen retrieval with 0.1 mol/L citrate buffer (3 min in the microwave then cooled to room temperature). After washing, sections were blocked by in 50% ethanol containing 1% H202 for 30 min and in 10% normal horse serum in Tris buffer for 20 min at room temperature. Sections were incubated in primary antibodies [anti-PLK1 (Santa Cruz Biotechnology, TX, USA; anti-rabbit, 1:50); anti-PLK2 (Santa Cruz Biotechnology, TX, USA; anti-rabbit, 1:20); anti-CK2β (Santa Cruz Biotechnology, TX, USA; anti-rabbit, 1:200); anti-LRRK2
(Epitomics, CA, USA; anti-rabbit, 1:500) or anti-GAK (Sigma, CA, USA; anti-rabbit,
1:2000)] prepared in Tris buffer overnight at 4°C, followed by an avidin–biotin-
59 peroxidase detection system (Vector Laboratories, Burlingame, CA, USA). Western blotting was used to confirm antibody specificity with the expected size band(s) detected and omission of the primary antibody was used to confirm specificity of ligand binding.
3.3.6 Double-labellingDouble-labelling immunofluorescenceimmunofluorescence for prproteinotein localizat localizationion
10µm thick formalin-fixed paraffin-embedded tissue sections of the SN and anterior cingulate cortex were prepared for double-labelling immunofluorescence as previously described (Guerreiro, et al., 2013). Briefly, sections were dewaxed and hydrated followed by antigen retrieval by formic acid (90% formic acid for 3 min at room temperature) and citrate buffer (microwaved for 3 min). After washing, sections were blocked in 50% ethanol containing 1% H2O2 for 30 min and then blocked in 10% normal horse serum for 20 min. Sections were then incubated overnight at 4°C in a combination of primary antibodies [anti-S129P-α-synuclein (Elan Pharmaceuticals,
South San Francisco, CA, USA; anti-mouse, 1:5000) with kinases of anti-PLK2 (Santa
Cruz Biotechnology, TX, USA; anti-rabbit, 1:20), anti-LRRK2 (Epitomics, CA, USA; anti-rabbit, 1:200) or anti-GAK (Sigma, CA, USA; anti-rabbit, 1:2000)] prepared in Tris buffer. Appropriate secondary antibodies [goat anti-rabbit AF568 (Life Technologies,
CA, USA; 1:200) and goat anti-mouse AF488 (Life Technologies, CA, USA; 1:200)] were applied for 2 hr at room temperature. Slides were washed and coverslipped with
Vectashield fluorescent mounting medium (Vector Laboratories, CA, USA). Western blotting confirmed antibody specificity and omission of either one or both primary antibodies was used to confirm specificity of ligand binding.
60 3.3.7 Assessme Assessmentnt of kinase immunimmunoreactivityoreactivity in LBs and LB containcontaininging neuronsneurons
Immunofluorescent sections were viewed under a Zeiss Imager.M1 microscope (Zeiss;
Munchen-Hallbergmoos, Germany) and images captured using a Zeiss AxioCam HRc camera and Axiovision 4.7 software. Neuronal counts and intensity analysis was performed on 8-bit unsaturated monochrome images (1388 x 1040 or 1300 x 1030 pixels) that were captured under a 40x objective using Image J software
(http://rsbweb.nih.gov/ij/). All images, counts and intensity analysis were performed with the observed blind to case type.
For kinase co-localization analysis, a maximum of 15 neurons containing S129P α- synuclein-immunoreactive inclusions were imaged for each kinase antibody used
(LRRK2, GAK or PLK2) in each region of each PD case, and each neuronal inclusion assessed as either kinase positive or kinase negative. To assess the relative amount of kinase proteins in neurons, the intensity of immunofluorescence in neurons labelled in the same sections was performed. For this intensity analysis, 50 kinase-immunoreactive neurons for each kinase antibody used in each region in each case were assessed and the staining intensity (pixel grey level) compared within a circular region of interest (6 μm diameter) that was placed in the neuronal cytoplasm using Image J software
(http://rsbweb.nih.gov/ij/). Every kinase-positive neuron that contained a S129P α- synuclein-immunoreactive inclusion was included so that the two groups (kinase neuron with and without pathological inclusions) could be compared. The staining intensity assessments were performed blinding by two investigators with only 7.9% variability.
61 3.3.8 StatisticalStatistical plan and analyanalysesses
SPSS 18 (IBM, Chicago, USA) was used for all analyses and significance established when p<0.05. There were no correlations between the different protein levels or intensity in the different regions analysed and any demographic variable (see Table 2.1), including age and postmortem delay (p>0.05), and therefore these variables will not be considered further.
To determine any differences in kinase levels in controls between different fractions or between control and PD, Wilcoxon Signed Rank test and Mann White U tests were used, respectively. When this quantitative method identified any change in PD, the intensity of the kinase immunoflourescence between control and PD cases in each region was also evaluated using Mann Whitney U tests and the kinase intensity in histological immunofluorescence between neurons with or without S129P α-synuclein- immunoreactive inclusions in PD cases was evaluated using Wilcoxon Signed Rank tests. To determine any relationships between the levels of kinases and S129P α- synuclein, or any relationships between kinase levels in the different frontal tissue fractions and disease stage, stepwise multiple regression analyses covarying for onset age and duration were used.
3.43.4 ResultsResults
3.4.1 PLKs and CK2CK2ββ
As described previously, PLKs and CKs have been identified as capable of phosphorylating α-synuclein at S129. To determine whether the levels of these kinases are increased in PD, the levels of PLK1, PLK2 and CK2β in the frontal cortex and
62 nigrostriatal pathway were analysed. Normal levels were established in controls (Figure
3.1), with most PLK1 in the SDS-soluble fraction in both regions analysed (Figure 3.2
A&B), while PLK2 differed by region in controls. There was more PLK2 in the TBS- than the SDS-soluble fraction in the putamen (Figure 3.2 A&B, 0.7x more in putamen,
Wilcoxon Signed Rank test p=0.018). CK2β was mostly observed in the TBS-soluble fraction in both regions (Figure 3.2 A&B). Immunohistochemistry for all three kinases showed neuronal cytoplasmic localization, with PLK2-immunoreactivity found more localized to small neuronal cytoplasmic punctuate structures (Figure 3.2 C), while
PLK1 and CK2β immunoreactivities were throughout the entire neuronal cytoplasm
(Figure 3.2 D&E).
Figure 3.1: RepresentativeRepresentative WesternWestern blotsblots of PLK1,PLK1, PLK2PLK2 andand CK2CK2 inin humanhuman brain.brain.
Around 45kD and 66kD bands were observed in the blots of PLK1 and PLK2 in human brain; In addition, a 20kD band was also detected in PLK1. The 66kD bands in PLK1 and PLK2 (in red boxes ) are full length of PLKs while the 45kD and 20kD bands could be truncated PLKs or cross-reaction of primary 63 antibodies as they were undetectable with primary antibodies omitted(data not shown). A 26kD band, which is the right size for full length of CK2, was observed in TBS-soluble fraction; in addition, this band immigrated to 28kD in SDS-soluble fraction. Series of high molecular bands were also observed in CK2 blots and they could be cross-reaction of primary antibody.
Figure 3.2: PLK1,PLK1, PLK2PLK2 andand CK2CKCK2β2β inin controlscontrols 64 (A,B)(A,B) Representative Western blots(A) and related graphs(B) show PLK2 differed by region in control with more PLK2 in the TBS-soluble than the SDS-soluble fraction in putamen but no significant difference in frontal; most PLK1 was in the SDS-soluble fraction while most CK2β was in the TBS- soluble fraction in both regions analysed of controls.
(C-E)(C-E) Immunohistochemistry shows neuronal cytoplasmic localization of PLK2(C), PLK1(D) and
CK2 β (E), with PLK2-immunoreactivity more localized to small neuronal cytoplasmic punctuate structures(C), while PLK1(D) and CK2β(E) immunoreactivities were throughout the entire neuronal cytoplasm.
Regional comparisons in cases with PD revealed significant changes in PLK2 levels compared to controls. In the region demonstrating limited or less mature Lewy pathologies (frontal cortex), there was a significant increase in the levels of TBS- soluble PLK2 compared with controls (Figure 3.3 A&B, 1.6±0.4x increase, Mann-
Whitney U test p=0.005) but no significant change in the levels of SDS-soluble PLK2
(Figure 3.3 B, Mann-Whitney U test p=0.30). There was also no significant change in the intensity of neuronal PLK2 immunoreactivity in PD frontal cortex (Figure 3.3 B,
Mann-Whitney U test p=0.11), indicating that this staining is more reflective of the
SDS-soluble than TBS-soluble fraction. This is consistent with the extraction of many soluble proteins during tissue fixation with formalin and some lipid associated proteins during paraffin infiltration with xylene and chloroform (Halliday & McCann, 2008) regions demonstrating end-stage pathologies (nigrostriatal pathways), there was no change in the levels of PLK2 in either fraction analysed (Figure 3.3 B, Mann-Whitney
U test p>0.16), although there was a measurable decrease in the intensity of PLK2 immunoreactivity in pigmented SN neurons in PD cases compared with controls (Figure
3.3 B, 85±4.5% of mean control values, Mann-Whitney U test p=0.001). This suggests that the protein was in more soluble forms that were extracted during tissue processing.
65 In contrast, assessment of PLK1 and CK2β in cases with PD revealed no significant changes in levels compared to controls (Figure 3.3 C&D, Mann-Whitney U test p>0.12).
Figure 3.3: changeschanges inin PLK1,PLK1, PLK2PLK2 andand CK2CKCK2β2β inin thethe nigrostriatumnigrostriatum andand frontalfrontal cortexcortex of PDPD comparedcompared withwith controlscontrols 66 (A)(A) Representative Western blots show a significant increase in the levels of TBS-soluble PLK2 in PD frontal compared with controls.
(B)(B) There was a significant increase in the levels of TBS-soluble PLK2 in PD frontal compared with controls but no significant change in the levels of other PLK2 species in either regions analysed or the intensity of neuronal PLK2 immunoreactivity in frontal cortex compared with controls( *p<0.05).
(C,D)(C,D) There was no significant change in levels of either PLK1(C) or CK2β (D) in either regions analysed compared to controls.
3.4.2 LRRK2 and GAK
LRRK2 and GAK are PD-linked genes encoding serine/threonine kinases (Kimura, et al., 1997; Paisan-Ruiz, et al., 2004). As detailed previously, both LRRK2 and GAK have been reported to interact with α-synuclein. To determine whether there is any change in the levels of these kinases in PD, the same analysis as described above for these kinases was performed. Normal levels were established in controls, with no regional differences in the levels of GAK in different tissue fractions (Figure 3.4 A&B,
Wilcoxon Signed Rank test p>0.13), while there was more SDS-soluble than TBS- soluble LRRK2 in both regions assessed in controls (Figure 3.4 A&B, 2.7x more of putamental LRRK2, Wilcoxon Signed Rank test p=0.018; 0.5x more of frontal LRRK2,
Wilcoxon Signed Rank test p=0.018). LRRK2 immunoreactivity was mainly localized to small neuronal cytoplasmic punctuate structures (Figure 3.4 C), while GAK immunoreactivity was somewhat more diffuse within the cytoplasm (Figure 3.4 D).
67 Figure 3.4: LRRK2LRRK2 andand GAKGAK inin controlscontrols
(A,B)(A,B) Representative Western blots(A) and related graphs(B) show in controls, there was more SDS- soluble than TBS-soluble LRRK2 in both regions assessed while no fractional differences in GAK levels in either regions analyzed.
(C,D)(C,D) Immunohistochemistry shows LRRK2 was mainly localized to small neuronal cytoplasmic punctuate structures (C), while GAK immunoreactivity was more diffuse within the cytoplasm (D). 68 Significant changes were seen in both kinases in patients with PD. In the region demonstrating early and less mature pathology (frontal cortex), there was a decrease in
TBS-soluble LRRK2 compared with controls (Figure 3.5 A&B, 66±34% of mean control levels, Mann-Whitney U test p=0.033 for LRRK2), and a trend for decreased
SDS-soluble LRRK2 levels (Figure 3.5 B, 71±30% of mean control levels, Mann-
Whitney U test p=0.085). Despite this reduction, there was no measureable change in the intensity of neuronal LRRK2 immunoreactivity in PD frontal cortex (Figure 3.5 B,
Mann-Whitney U Test p=0.68), suggesting that the remaining LRRK2 was associated with fixed cellular structures. In contrast, there was a significant increase of the intensity of neuronal GAK immunoreactivity in PD frontal cortex compared with controls (Figure 3.5 C, 1.4±0.5x increase, Mann-Whitney U Test p<0.001) but no significant change in the relatively low total GAK levels compared with controls
(Figure 3.5 C, Mann-Whitney U Test p>0.11). This suggests that in PD GAK is more associated with fixed cellular structures.
In regions demonstrating end-stage pathologies (nigrostriatal pathways), there was a significant decrease in SDS-soluble LRRK2 in the PD cases (Figure 3.5 A&B, 52±37% of mean control levels, Mann-Whitney U test p=0.008) but no significant change in
TBS-soluble LRRK2 (Mann-Whitney U test p=0.51). There was also a decrease in the intensity of LRRK2 immunoreactivity in pigmented SN neurons compared with controls (Figure 3.5 B, 83±15% of mean control levels, Mann-Whitney U Test p<0.001) suggesting that there is a reduction in the LRRK2 associated structures in these cells.
There was a small but significant decrease of pigmented neuronal GAK intensity in PD nigra (Figure 3.5 C, 92±6% of control, Mann-Whitney U Test p=0.032) but no significant change in the relatively low total GAK levels compared to controls (Figure
69 3.5 C, Mann-Whitney U Test p>0.11). This also suggests there is a reduction in GAK associated structures in these cells.
Figure 3.5: changeschanges inin LRRK2LRRK2 andand GAKGAK inin thethe nigrostriatumnigrostriatum andand frontalfrontal cortexcortex of PDPD comparedcompared withwith controlscontrols
70 (A)(A) Representative Western blots showed dramatic decreases in the levels of frontal TBS-soluble LRRK2 and nigrostriatal SDS-soluble LRRK2 in PD compared with controls.
(B)(B) There were significant decreases in the levels of frontal TBS-soluble LRRK2 and nigrostriatal SDS- soluble LRRK2 in PD compared with controls.but no significant change in the levels of other LRRK2 species in either regions analysed or the intensity of neuronal LRRK2 immunoreactivity in frontal cortex
(*p<0.05).
(C)(C) There was no significant change in the relatively low total GAK levels compared with controls in either regions analysed (*p<0.05).
3.4.3 CorrelationCorrelation betw betweeneen kinase levels and S129 phosp phosphorylationhorylation of α- synucleinsynuclein
Stepwise multiple regression analysis was performed in the PD cases, covarying for onset age and duration, to determine if the changes in the levels of any of these kinases predicted the levels of S129P α-synuclein in PD (data from Chapter 2, section 2.4.2).
No kinase levels predicted the levels of S129P α-synuclein in PD frontal cortex, although increasing levels of S129P α-synuclein in PD putamen related to the increasing levels of SDS-soluble PLK2 (Figure 3.6 A&B, for putamental TBS-soluble
S129P p=0.09, β coefficients =0.60; for putamental SDS-soluble S129P p=0.10, β coefficients =0.60).
71 Figure 3.6: RelationshipsRelationships betweenbetween kinasekinase levelslevels andand S129PS129P α-synuclein-synuclein amountsamounts inin thethe putamenputamen of PDPD casescases
The kinases analysed relating most to the levels of TBS-soluble S129P α-synuclein (A) and SDS-soluble
S129P α-synuclein (B) in putamen was the putamental SDS-soluble PLK2.
3.4.4 ChangesChanges in kinases over the course of the disease and the developdevelopmentment
72 and maturatmaturationion of LBs
Stepwise multiple regression analysis was performed in the PD cases, covarying for onset age and duration, to determine if the changes in the levels of any of these kinases predicted the stage of pathological progression of PD. Increasing pathological stage of
PD was predicted by decreasing levels of TBS-soluble PLK2 and increasing levels of
TBS-soluble LRRK2 in PD frontal cortex (Figure 3.7 A, p=0.002, β coefficients for
PLK2=-0.37 and LRRK2=0.29, 14% reduction in TBS-soluble PLK2 over the stages and 42.5% increase in TBS-soluble LRRK2 over the stages).
73 FigureFigure 3.7:
RelationshiRelationshi
psps betweenbetween
kinaseskinases
amountsamounts
andand LewyLewy
pathologiespathologies
developmedevelopme
ntnt
(A)(A) The
kinases
analyzed
relating most
to disease
pathological
stages were a
decrease in the
levels of TBS-
soluble PLK2
and an
increase in the
levels of TBS-
soluble
LRRK2 in the frontal cortex. Grey bars stand for the range of control samples (Mean±S.D).
(B)(B) PLK2 and LRRK2 immunoreactivity were localized in LBs in both SN (containing more mature 74 LBs)and anterior cingulate cortex(containing less mature LBs) while GAK was localized more in nigral
LBs.
(C)(C) There was a significant increase in the intensity of GAK immunoreactivity in nigral LBs compared with the intensity of GAK immunoreactivity in non-LB containing pigmented nigral neurons, but no difference in the GAK immunoreactivity in the cortical neurons containing LBs compared with those without LBs. There was no difference in the intensity of PLK2 or LRRK2 immunoreactivity between neurons containing LBs compared with those without LB in either regions analysed.
To further assess the involvement of these kinases in LB development, S129P α- synuclein was used to determine the type of LB stained and the intensity of any kinase immunofluorescence in both the SN and anterior cingulate cortex. This analysis revealed that in the region containing less mature LBs (frontal cortex), 91±14% of LBs co-localized PLK2 immunoreactivity, 70±12% co-localized LRRK2 immunoreactivity and 37±26 co-localized GAK immunoreactivity (Figure 3.7 B). PLK2 immunoreactivity
(Figure 3.8 A-F) and LRRK2 immunoreactivity (Figure 3.8 G-L) were identified in most LBs, including stage 4 LBs, while GAK immunoreactivity was only observed in mature stage 4 LBs (Figure 3.8 M-R). In stage 4 LBs, PLK2 immunoreactivity and
LRRK2 immunoreactivity was located in the outer halo surrounding the core of S129P
α-synuclein immunoreactivity (Figure 3.8 A-L). There was no difference in the intensity of any kinase immunoreactivity between cortical neurons containing LBs compared to those without LBs (Figure 3.7 C, Wilcoxon Signed Rank test p>0.12).
75 76 FigureFigure 3.8: DoubleDouble immunofluorescenceimmunofluorescence stainingstaining forfor kinaseskinases andand S129PS129P αα-- synucleinsynuclein inin anterioranterior cingulatecingulate neurons.neurons.
(A-C)(A-C) In S129P α-synuclein positive cortical LBs, PLK2 immunoreactivity was located in the outer halo surrounding the core of S129P α-synuclein.
(D-F)(D-F) In non-LB containing cortical neurons, PLK2 immunoreacitivity was located in the cytoplasm with punctuate staining.
(G-I)(G-I) In S129P α-synuclein positive cortical LBs, LRRK2 immunoreactivity was located in the outer halo surrounding the core of S129P α-synuclein.
(J-L)(J-L) In non-LB containing cortical neurons, LRRK2 immunoreacitivity was located in the whole cytoplasm.
(M-O)(M-O) In S129P α-synuclein positive cortical LBs, GAK immunoreactivity was mostly co-localized with
S129P α-synuclein.
(P-R)(P-R) In non-LB containing cortical neurons, GAK immunoreacitivity was located in the whole cytoplasm.
In the region containing mainly mature LBs (SN), 79±18% of LBs co-localized PLK2 immunoreactivity, 86±13% co-localized LRRK2 immunoreactivity and 91±12% co- localized GAK immunoreactivity (Figure 3.7 B). There was no difference in the intensity of either PLK2 immunoreactivity or LRRK2 immunorectivity between SN neurons containing LBs and those without (Figure 3.7 C, Wilcoxon Signed Rank test p>0.28), although the intensity of GAK immunoreactivity in SN LBs was increased compared with the intensity of GAK immunoreactivity in non-LB containing pigmented
SN neurons (Figure 3.7 C, 1.3±0.3x increase, Wilcoxon Signed Rank test p=0.024).
3.53.5 DiscussionDiscussion
This is the first systematic study of the relationship between α-synuclein
77 phosphorylation and α-synuclein-related kinases in human brain tissue. It was shown in this chapter that the protein levels and/or immunoreactivity of PLK2, LRRK2 and GAK are affected in PD, changing in association with either the amount of S129P α-synuclein
(PLK2), with disease stage (PLK2 and LRRK2) and with the maturation of Lewy pathology (GAK). This suggests that these kinases are involved at different times and in different ways in the evolution of α-synuclein phosphorylation in relation to LB formation in PD.
3.5.1 RelationshipRelationship betweenbetween the levels of S129P α-sy-synucleinnuclein sand kinasekinasess knownknown to phospphosphorylatehorylate α-synuclein
The experiments in Chapter 2 and studies from others in sporadic PD (Lue, et al., 2012) have shown substantial increases in S129P α-synuclein prior to LB formation and that the increased phosphorylation of α-synuclein at this time is not accompanied by a similar substantial increase in total α-synuclein levels in pathologically vulnerable regions. This indicates that the phosphorylation rather than global over-expression of α- synuclein is a key event prior to LB formation in PD. As discussed previously, several kinases are capable of phosphorylating α-synuclein at S129 in vitro, with PLK1, PLK2
(Mbefo, et al., 2010) and CK2 (Waxman & Giasson, 2008) being thought to be most associated with α-synuclein phosphorylation and subsequent LB formation. There has only been a single study on PLK2 levels previously in human brain tissue and no data on either of the other kinases known to phosphorylate α-synuclein in vivo. Data in this chapter shows that there were no changes in the levels of either PLK1 or CK2β in patients with PD, but there was an average 60% increase in the level of PLK2 in the same cases where it co-localized with S129P α-synuclein in most LBs. These data
78 suggest a more substantial role for PLK2 in the pathogenesis of PD than either of the other kinases. This increase in PLK2 levels in PD appears more substantial than the increase observed previously in the temporal cortex of patients with DLB (Mbefo, et al.,
2010), although less phosphorylated α-synuclein is detected in temporal compared with frontal cortices at all disease stages (Walker, et al., 2013). It was also found that TBS- soluble PLK2 increased prior to LB formation in PD tissue and that its levels correlated with increasing S129P α-synuclein. This strongly suggests that PLK2 is responsible for the change in phosphorylation of α-synuclein in human brain. This is in line with recent studies showing that PLK2 efficiently phosphorylates α-synuclein, interacting only with soluble α-synuclein (Inglis, et al., 2009; Waxman & Giasson, 2011). Interestingly, as the disease progresses and LB formation occurs in the tissue samples, there was a slight decrease in overall PLK2 levels, even though the levels of S129P α-synuclein do not decrease with disease progression (Walker, et al., 2013; Zhou, et al., 2011). Furthermore, within the vulnerable SN neurons, there was a decrease in the intensity of PLK2 immunoreactivity compared with controls. This suggests that as PD progresses other kinases are involved in the development of the insoluble Lewy pathologies.
3.5.2 RelationshipRelationship betweenbetween the levels of S129P α-sy-synucleinnuclein and other related related kinaseskinases
As described previously, in addition to those kinases identified as phosphorylating α- synuclein in vivo, both LRRK2 (Liu, et al., 2012b) and GAK (Dumitriu, et al., 2011) are associated with LB pathology in PD. However, how LRRK2 and GAK are involved in the pathogenesis of PD remains unclear, particularly for GAK. Enhanced kinase activity has been shown with certain pathogenic LRRK2 mutations (West, et al., 2005) but some
79 studies show that reduced LRRK2 can cause PD relevant changes (Tong, et al., 2010b).
A recent study from our lab shows LRRK2 directly interacts with α-synuclein in PD, co-localizing in Lewy pathologies (Guerreiro, et al., 2013). In this chapter, it was shown that there is an average 35% decrease in LRRK2 tissue levels prior to LB formation in
PD when there is a significant increase in α-synuclein phosphorylation, and no change in the very low levels of GAK. The reduction in LRRK2 protein levels is consistent with the widespread reduction of LRRK2 mRNA levels in PD brains (Sharma, et al.,
2011) and suggests that perhaps this kinase is downregulated at the initiation of PD in association with the upregulation of PLK2 and the increasing phosphorylation of α- synuclein. Interestingly, as the disease progresses and LB formation occurs in the tissue samples, there is an average 40% increase in LRRK2 with the levels returning to and surpassing average control levels as LBs form. This is consistent with the small ~20% increase in LRRK2 levels previously observed in the anterior cingulate cortex and amygdala (Guerreiro, et al., 2013) where significantly more mature Lewy pathologies are observed in PD, and suggests that LRRK2 may have a role in the formation of Lewy pathologies. The recent study from our lab shows that in areas with significant LB formation there is a positive correlation between the small increases in LRRK2 levels and the larger increases in S129P α-synuclein in the same regions (Guerreiro, et al.,
2013), suggesting that LRRK2 may play a role in the phosphorylation of α-synuclein during and/or after the initial formation of Lewy pathologies. This is consistent with the knock down experiments in a cell model where the loss of LRRK2 reduced the aggregation of α-synuclein (Guerreiro, et al., 2013).
3.5.3 KinasesKinases associat associateded with with the developdevelopmentment and maturatmaturationion of LewyLewy
80 pathologiespathologies
In this chapter, we found that three S129P α-synuclein-related kinases (PLK2, LRRK2 and GAK) occur in Lewy pathologies. As described in section3.4.4, PLK2 appears in the majority of LBs but is distributed to the periphery of the inclusions. The early trapping of PLK2 in Lewy pathologies may contribute to the reduction in the amount of soluble PLK2 protein in vulnerable PD regions. Increasing LRRK2 levels were associated with inclusion formation in this chapter, and a previous study shows that in areas with significant numbers of inclusions there is a correlation between small increases in the levels of LRRK2 and large increases in the levels of S129P α-synuclein
(Guerreiro, et al., 2013). In areas forming LBs, the majority co-localize LRRK2 where the kinase occurs in the periphery of the inclusion. As the disease progresses and more
LBs mature, less inclusions co-localize LRRK2 (Guerreiro, et al., 2013), suggesting that
LRRK2 is most associated with the formation of the inclusion. In contrast, GAK is co- localized only in mature Lewy pathologies where it occurs throughout the inclusion with increasing intensity. This is consistent with the increased GAK mRNA found in the
SN of PD (Grunblatt, et al., 2004). Combined these findings show a dynamic alternation of these kinases prior to and during the formation and maturation of Lewy pathologies in PD.
3.63.6 StrengthsStrengths and and wea weaknessesknesses ofof thisthis study study
A strength of this study is the use of the same cases as described in Chapter 2 to assess the changes in α-synuclein related kinases and their relationships to the previously observed increases of S129P α-synuclein in PD. The use of same cases in these two consecutive studies excluded difficulties of comparing different case groups. Although
81 it is not a longitudinal study, the application of homogenous cases at different pathological stages and the use of different brain regions which are affected at different stages made it possible to assess the dynamic role of kinases in the pathological development and progression of PD pathology. In this way these correlations are similar to those that would be performed in staged animal studies. Of course such observational studies can not assess the dynamic formation of Lewy bodies intracellular, studies which can only be performed in more isolated cell systems with external and perhaps artificial stimuli.
However, there are also other limitations in this study. Although PLK3 has been identified to phosphorylate α-synuclein in vitro and some cell models (Mbefo, et al.,
2010), we could not assess PLK3 in human brain tissue due to the lack of specific antibodies (Mbefo, et al., 2010). In addition, kinase function is controlled by protein expression level and activity. Protein kinase activity is usually analyzed by monitoring autophosphorylation in immunoprecipitates from cell lysates or phosphorylation of specific substrates with in vitro assays (Schlessinger, 2002). To date, the measurement of kinase activity in postmortem human brain tissue is still a big challenge due to the postmortem delay that can affect protein posttranslational modifications and preservation. For this reason in this study the expression and immunoreactivity rather than activity of kinases were analysed. Thus, further technical developments are needed to investigate kinase activity from proteins extracted from PD tissue in order to determine any relationship between kinase activity and protein levels.
82 3.73.7 ConclusionConclusion
In conclusion, the data in this chapter are consistent with different kinases associated with different pathogenic aspects of PD. They suggest PLK2 is involved in the early phosphorylation of α-synuclein prior to Lewy pathology formation, LRRK2 is involved in the initiation and formation of Lewy pathologies from phosphorylated α-synuclein, and GAK is involved in ensuring the final compaction and maturation of Lewy pathologies into the most insoluble form of α-synuclein. Further molecular and cell biology studies are needed to identify the dynamic process of kinase involvement in
Lewy pathology formation.
83 ChapterChapter 4:4: AssessmentAssessment of of acut acutee cha changesnges in α α--s-synucleinsynucleinynuclein phosphorylationphosphorylation and and re relatedlated kina kinasesses nin an an MPTPMPTP mo mouseuse modelmodel of of ParkinsonParkinson’s dis diseaseease
4.14.1 IntroductionIntroduction
As shown in Chapter 2 and 3, increasing levels of S129 phosphorylation of α-synuclein is correlated with increasing levels of PLK2 in PD cases, although whether such a change occurs dynamically requires longitudinal analyses. As discussed in Chapter 1, animal models allow the assessment of such changes over a reasonable time frame. The motor symptoms of PD are most rapidly replicated using environmental mitochondrial toxins which produce a PD-like loss of dopaminergic SNpc neurons and MPTP treated mice are still the most common model used to study PD-like SNpc degeneration (see
Chapter 1).
Up-regulation of α-synuclein levels has been found after MPTP administration in mice
(Vila, et al., 2000) and ablation of α-synuclein in mice prevents MPTP-induced SN neurodegeneration (Dauer, et al., 2002; Drolet, et al., 2004; Thomas, et al., 2011). These data indicate a potential role for α-synuclein in the pathogenesis of MPTP induced neurotoxicity. However, the underlying mechanism remains unclear. Phosphorylated α- synuclein has been observed in both MPTP treated mice and monkeys (McCormack, et al., 2008; Yasuda, et al., 2011), although the time course for this change and whether changes in related kinases occurs have not been evaluated. Determining whether the changes observed in PD can be replicated in an animal model will allow pharmacological testing of potential mechanistic therapies to ameliorate the changes
84 observed.
4.24.2 AimAim an andd Hy Hypothesispothesis
The studies described in this chapter will determine the progressive changes in the phosphorylation of α-synuclein following subacute MPTP neurotoxicity in mice and determine whether there are any associated changes in the levels of related kinases. It is hypothesized that similar changes to those observed in different patients with PD will occur progressively in the mouse model.
4.34.3 MaterialsMaterials and and Metho Methodsds
4.3.1 Overall study design
To determine the earliest dynamic changes in the levels of α-synuclein phosphorylation and α-synuclein related kinases in the nigrostriatal pathway, a subacute mouse model will be established which uses MPTP toxin to transiently damage dopaminergic neurons.
To establish that the mouse model damages dopaminergic SN neurons, measurements of reduced tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, in the SN and striatum will be measured and the reduction in the number of
SNpc neurons expressing TH identified. To determine changes in the levels of α- synuclein phosphorylation and α-synuclein related kinases in the mouse model, antibodies to α-synuclein, S129P α-synuclein, PLK1, PLK2, PLK3, CK1 and CK2 will be assessed.
85 4.3.2 Animal Animalss
This project was approved by the Ethics Committee on Animal Care and Usage (Beijing
Capital Medical University, China). 102 fourteen-week-old male C57/BL mice were obtained from the Laboratory Animal Center of Capital Medical University. Animals were housed in a controlled room (23±1°C) under 12-h light/12-h dark cycle with free access to food and water.
4.3.3 MPTP adminadministrationistration and tissue prpreparationeparation
Following one-week habituation, mice were randomly divided into MPTP groups (n=54) and control groups (n=48). MPTP mice received an intraperitoneal injection of MPTP-
HCl (30mg/kg/day in saline; 10ml/kg/day, Sigma, CA, USA) once a day for 5 consecutive days, while control mice received an equal volume of saline only. Both
MPTP and saline treated mice were sacrificed at six different time points after treatment with all efforts to minimize suffering [ie. at 1 day before ending treatment (labeled as -1 day) or at 1, 3, 7, 14 or 28 days after the last injection].
For tissue preparation, each group was randomly divided into two groups. The first group (n=36) were anaesthetized with 10% chloral hydrate and perfused via intracardiac infusion with saline (0.9%) followed by 4% paraformaldehyde in 0.1M phosphate- buffered saline (PBS), pH 7.4. After perfusion, brains were collected and fixed in the same 4% PFA for 24-h, then transferred to 20% sucrose and then 30% sucrose in 0.1M
PBS until the block sank. Tissue was cut and processed for immunohistochemistry as described below. The second group of mice (n=66) were sacrificed by head decapitation and the brains quickly removed, the SN and striatum dissected out (Paxinos & Franklin,
2004), snap-frozen on dry ice and stored at -80ºC for Western blotting. 86 4.3.4 ImmunohistochemistryImmunohistochemistry for tyr tyrosineosine hydroxylasehydroxylase (TH)
Serial coronal sections spanning the entire fixed midbrain and the mid striatum (Paxinos
& Franklin, 2004) were cut at 60μm on a cryostat and processed free floating. Sections were submerged in 0.3% hydrogen peroxide for 10-min to eliminate endogenous peroxidase activity. Then they were incubated with 0.3% TritonX-100 (Sigma, CA,
USA) for 30-min and 5% goat serum for 60-min at room temperature followed by the primary antibody (mouse anti-TH, Sigma, CA, USA) diluted 1:1000 in 0.1M PBS, pH
7.4, containing 1% BSA overnight at 4°C. After washing, the sections were incubated with biotinylated goat anti-mouse IgG (Bio-Rad, CA, USA) diluted 1:2000 in 0.1M
PBS for 60-min at room temperature then placed in a solution containing avidin-biotin peroxidase complex (ABC Elite kit; Zhongshan Golden-Bridge Biotechnology CO LTD,
Beijing, China). To test the specificity of the immunostaining, negative controls were processed in an identical manner but with the primary antibody omitted. All sections were finally mounted onto slides, air-dried, dehydrated in ascending grades of ethanol, cleared in xylene and coverslipped with natural mounting media (DPX, Sigma, USA).
4.3.5 DopaminergicDopaminergic neuronalneuronal counts in the substant substantiaia nigra
Stereological principles were applied to estimate TH-immunopositive neurons within the SN. Every fourth section through the SN was analyzed using Stereo investigator software (MicroBrightfield, VT, USA) attached to Leica DM4000B microscope (Leica
Microsystems, Wetzlar, Germany). The total number of TH-immunopositive neurons was calculated using the optical fractionator method (West, et al., 1991) and cell counting boxes measuring 40x40µm (optical frame size of 1600µm2) that were placed in a randomly-placed grid pattern 180x180µm to achieve a sampling of 15 disectors per
87 section (range from 11 to 23, average volume of 25600µm3). The average coefficient of error for sampling density/case was 0.12.
4.3.6 We Westernstern blottin blottingg for tytyrosinerosine hydroxylase,hydroxylase, α α-synuclein-sy-synucleinnuclein and rrelatedelated kinaseskinases
Frozen brain tissue of the SN and striatum was homogenised in RIPA buffer (Beyotime
Institute of Biotechnology, Shanghai, China) with 1×protease inhibitor cocktail (EDTA free; Roche, Shanghai, China) and 1× phosphatase inhibitor cocktail (Roche, Shanghai,
China). Protein concentration was determined using a BCA kit (Thermo Fisher
Scientific Inc, MA, USA). Semi-quantitative Western blotting was performed as stated in Section2.3.4. Briefly, 50µg of each protein sample were dissolved in an equal volume aliquot of 5×Laemmli buffer (Beyotime Institute of Biotechnology, Shanghai, China), boiled at 95°C for 5-min and loaded onto SDS-PAGE gels. To compare the results from different gels, a previously tested sample of known antibody reactivity (30µg/well) was loaded twice onto each gel as a standard reference. After separation, proteins were transferred onto 0.22µm nitrocellulose filter membranes (Bio-Rad, CA, USA) at 100V constant for 60-min. To ensure that the same amount of protein was loaded for each sample, mouse monoclonal β-actin (Sigma, CA, USA) was used as a loading control.
Blots were blocked with 5% milk in TBST buffer and probed using the primary antibodies antibodies [anti-TH (1:20,000, sigma, CA, USA); anti-α-synuclein (syn-1,
1:2,000, BD Transduction Laboratories, CA, USA); anti-S129P-α-synuclein (1:5000, a gift from Elan Pharmaceuticals, South San Francisco, CA, USA); anti-PLK1 (1:300,
Santa Cruz Biotechnology Inc, CA, USA); anti-PLK2 (1:500, Santa Cruz
Biotechnology Inc, CA, USA); anti-PLK3 (1:1,000, BD Transduction Laboratories, CA,
USA); anti-CK1 (1:500, Santa Cruz Biotechnology Inc, CA, USA); anti-CK2 (1:500, 88 Santa Cruz Biotechnology Inc, CA, USA); and anti-β-actin (1:15,000, Sigma, CA,
USA)] suspended in 5% milk powder in TBST buffer at 4°C overnight. Membranes were washed three times with TBST buffer and probed with fluorescently-labelled secondary antibodies (LI-COR Biosciences, NE, USA, diluted 1:10,000) suspended in
5% milk powder in TBST buffer, applied for 1 h at room temperature. After washing, membranes were scanned using the Odyssey infrared imaging system (LI-COR
Biosciences, NE, USA). Scanned images were exported in TIFF format and the intensity of each band was quantified with Quantity One software (Bio-Rad, CA, USA) and expressed as arbitrary units relative to the standard for comparison across groups.
4.3.7 StatisticsStatistics
SPSS 18 (IBM, Chicago, USA) was used for all analyses and significance established when P was < 0.05. General linear multivariate analyses with repeated measures were used to determine changes in dopaminergic neuronal counts and protein levels between control and MPTP groups and within MPTP groups over time. Stepwise linear regression modeling was used to identify which significant measures predicted α- synuclein phosphorylation.
4.44.4 ResultsResults
4.4.1 CharacterizingCharacterizing the subacute MPTP mouse mmomodelodeldel
The most striking change observed in the subacute MPTP mouse model was a significant decrease in TH levels in the striatum (41±3% of control, p<0.001, Figure 4.1
A&B). The acute decrease in TH levels in the striatum began during MPTP treatment
(34% of control at -1 day, p<0.001), peaking at 3 days post-treatment, and then 89 gradually recovering from 7 days onwards to still be only 56% of control levels at the end of the experiment (28 days, p=0.005, Figure 4.1 A&B). Stereological counts of TH- immunopositive SN neurons revealed a varied but significant decrease in neuron number in the MPTP group (36-70% of control, p<0.04) with recovery over a similar time course to that observed for striatal TH levels (Figure 4.1 A&C). Overall, these experiments show that subacute MPTP injury to dopaminergic neurons has two distinct phases (Figure 4.1 A); the first corresponding to a period of rapid decline in synaptic
TH levels and a small reduction in the number of TH-immunoreactive SN neurons during acute, ongoing injury (up to 3 days post-treatment); while the second period corresponds to a phase of gradual recovery from 7 days onward. These data are in line with previous descriptions of the subacute MPTP mouse model (Jacksonlewis, et al.,
1995; Schmidt & Ferger, 2001).
90 Figure 4.1: DynamicDynamic changeschanges inin THTH expressionexpression inin thethe nigrostriatumnigrostriatum of subacutesubacute
MPTPMPTP micemice overover thethe experimentexperiment periodperiod [at[at 1 dayday beforebefore en endingding treatmenttreatment (labeled(labeled as -1-1 day)day) or at 1, 3, 7, 14 or 28 daysdays afterafter thethe lastlast injection].injection].
91 IHC=immunohistochemistryIHC=immunohistochemistry
(A)(A) Graph shows that striatal TH levels and the number of SN TH-immunoreactive neurons were significantly decreased during MPTP treatment and for the 3 days post-treatment, gradually recovering from 7 days onwards but remained lower than control levels at the end of the experiment (28 days){*p<0.05). All data are expressed as a mean ± S.D. ratio compared to matched controls with n=5-
6/group.
(B)(B) Representative Western blots show obvious decreases in striatal TH at different time points.
(C)(C) Representative IHC shows decreases in TH-immunopositive SNC neurons in MPTP mice at different time points. cp=cerebral peduncle, IP=interpeduclar nucleus, SNR=substantai nigra pars reticulata,
VTA=ventral tegmental area.
4.4.2 αα-Synuclein-Sy-Synucleinnuclein S129 phosph phosphorylationorylation in thee subacut subacutee MPTP mouse model
There were no specific oligomeric forms of α-synuclein observed in our Western blotting condition, so quantification of the monomeric α-synuclein bands was performed. As predicted, the levels of nigrostriatal α-synuclein S129 phosphorylation were dynamically changed following MPTP treatment (Figure 4.2 A&B). The time course of change in S129P levels was significant but varied between the striatum and
SN (for striatal S129P p<0.001; for SN S129P p=0.004, Figure 4.2 A&B).
Striatal S129P levels were significantly reduced during treatment (33±8% of control, p=0.003), rebounding to higher than control levels during the acute, ongoing injury
(82±4% of control at 1 day and 138±8% of control at 3 days, p=0.03), before returning to normal levels from 7 days onwards (91±6% of control, p=0.002, Figure 4.2 A&B). A similar but not identical pattern was observed for total α-synuclein (syn-1) in the striatum (striatal syn-1 p= 0.005) with decreased levels during treatment that recovered to control levels (but not higher) during the ongoing injury phase (Figure 4.2 A&B). 92 The ratio of S129P to total α-synuclein (syn-1) in striatum significantly changed over these time points (p<0.001), suggesting hyperphosphorylation of α-synuclein during this period in the MPTP mice.
S129P α-synuclein in the SN were not reduced during the treatment period (117±12% of control, p=0.37), but dramatically increased over control levels during the acute, ongoing injury (129-174±11% of control, p<0.01), returning to normal levels from 7 days onwards (95±5.5% of control, Figure 4.2 A&B), paralleling the changes observed in total α-synuclein (syn-1) in this structure (SN syn-1 p=0.048, Figure 4.2 A&B).
Comparison of the average change overall showed a reduction of S129P α-synuclein levels in the more affected striatum reduced to 92±2% of control, p=0.034, Figure 4.2
C), while there was an increase in S129P levels in the SN (increased to 120±4% of control, p=0.002, Figure 4.2 C).
93 Figure 4.2: DynamicDynamic changeschanges inin αα-synuclein-s-synucleinynuclein expressionexpression inin nigrostriatumnigrostriatum of
94 subacutesubacute MPTPMPTP micemice overover thethe experimentexperiment period.period.
(A)(A) Graph show that striatal S129P levels were significantly reduced during MPTP treatment, rebounding to higher than control levels at 1-3 days post-treatment, before returning to normal levels from 7 days onwards. Syn-1 levels in the striatum were also decreased during treatment, and gradually recovered post-treatment to control levels (but not higher) by day 7. S129P and syn-1levels in the SN had a similar change over time; dramatically increasing over control levels, peaking at 3 days post-treatment, and returning to normal levels from 7 days onwards. All data are expressed as a mean ± S.D. ratio compared to matched controls with n=5-6/group.
(B)(B) Representative Western blots show changes in S129P and syn-1 at1 day before ending treatment (-1d) and 3 days after treatment (3d) in the striatum and SN, separately.
(C)(C) Graph shows there were a small but significant decrease in striatal S129P levels and a moderate increase in SN S129P levels in MPTP-treated mice compared with control. Data are the mean ± S.D., n=5~6, *p<0.05, **p<0.01.
Overall, these data show a recovery of α-synuclein levels during acute, ongoing injury with significantly elevated levels of S129P in surviving nigrostriatal structures during this period in the MPTP treated mice. Again, the ratio of S129P to total α-synuclein
(syn-1) was significantly increased during the acute, ongoing injury (p<0.001), suggesting its hyperphosphorylation during the MPTP injury.
4.4.3 Related kinases in the subacusubacutete MPTP mouse model
To determine whether there were any similar changes in S129P-related kinases following MPTP treatment, the relative levels of five kinases (PLK1, 2 and 3, CK1 and
2) were assessed in the same tissue (Figure 4.3). Multivariate and repeated measures analyses showed that the SN levels of PLK2 and 3 were significantly increased during the acute, ongoing injury in the MPTP mice (for PLK2 p=0.015 and for PLK3 p=0.002,
Figure 4.3 A&B) with no significant changes in the levels of PLK1, CK1 or 2 in either 95 region (Figure 4.3 C), or in striatal PLK2 or 3. Nigral PLK3 levels peaked at an earlier time point (1 day, 134±18% of control, p=0.008, Figure 4.3 A&B) than PLK2 (3 days,
152±21% of control, p=0.02, Figure 4.3 A&B).
96 Figure 4.3: DynamicDynamic changeschanges inin kinaseskinases expressionexpression inin nigrostriatumnigrostriatum of subacutesubacute
MPTPMPTP micemice overover thethe experimentexperiment periodperiod
97 (A)(A) Graph shows that SN PLK2 and 3 levels were significantly increased during MPTP treatment and for the 3 days post-treatment, then gradually recovered to normal levels. All data are expressed as a mean ±
S.D. ratio compared to matched controls with n=5-6/group.
(B)(B) Representative Western blots show changes in SN PLK2 and PLK3 expression at 1 day (1d) and 3 days (3d) after treatment in subacute MPTP mice.
(C)(C) Representative Western blots show no change in PLK1, CK1 or 2 expression between MPTP and control mice brain.
4.4.4 CorrelationsCorrelations betw betweeneen the changing levels of αα-sy-sy-synucleinnuclein phosphorylationphosphorylation and its rrelatedelated kinase kinasess
To determine whether the MPTP-induced changes in S129P levels correlated with
PLK2 and 3 levels, stepwise multiple regression analyses were used with the α- synuclein phosphorylation ratios (S129P/syn-1) as the dependent variables. Increases in the phosphorylation ratio of striatal α-synuclein was related to both striatal PLK3 levels and SN PLK2 levels (P<0.001, β coefficient for striatal PLK3=0.32 and SN PLK2=0.51,
Figure 4.4 A), while an increased phosphorylation ratio of α-synuclein in the SN was related to SN PLK3 levels (P=0.041, β coefficient for PLK3=0.26, Figure 4.4 B).
98 Figure 4: CorrelationsCorrelations betweenbetween αα-synuclein-s-synucleinynuclein Ser129Ser129 phosphorylationphosphorylation andand PLK2PLK2 andand 3 inin thethe striatumstriatum andand SNSN
(A)(A) SN PLK2 and striatal PLK3 predicted α-synuclein Ser129 phosphorylation (ratio of S129P/syn-1) in the striatum, (B)(B) while SN PLK3 levels predicted α-synuclein Ser129 phosphorylation in SN.
4.54.5 DiscussionDiscussion
To our knowledge, this is the first systematic analysis of changes in the levels of S129P
α-synuclein and related kinases in the nigrostriatal system of subacute MPTP mouse 99 model of PD. The administration of MPTP in mice is used to mimic the PD-like dopaminergic SN loss (Jacksonlewis, et al., 1995; Schmidt & Ferger, 2001) and several dopaminergic deficits were successfully reproduced in the subacute MPTP mouse model in this chapter. Furthermore, these biochemical changes were found to be mainly confined to a one week period following MPTP cessation, which is consistent with previous studies (Jacksonlewis, et al., 1995; Schmidt & Ferger, 2001). During this time of MPTP neurotoxicity, there was significant hyperphosphorylation of α-synuclein at
S129, a finding related to increased PLK2 and 3 levels. These changes were only observed during the one week period of acute, ongoing injury and functional recovery in this mouse model. By 7 days post MPTP treatment, α-synuclein phosphorylation and
PLK2/3 levels had returned to baseline, although there was still an ongoing deficit in
TH levels until the end of the experiment at 28 days. These longer-term changes suggest synaptic plasticity and compensation occurs in the recovery period.
The observation in this chapter that nigrostriatal S129P increases during acute MPTP injury, suggests that hyperphosphorylation of α-synuclein at S129 is involved in MPTP induced neurotoxicity, similar to the hyperphosphorylation observed in patients with PD
(see Chapter 2). Low levels of S129P α-synuclein under normal physiological conditions is suggested to be protective (Sato, et al., 2011), although the effects of large amounts of S129P α-synuclein is still debated. It has been reported that increased S129 phosphorylation in vitro disrupts the intra-molecular long-rang interaction between wild type α-synuclein molecules (Paleologou, et al., 2008) and increased levels of S129P α- synuclein in vivo exacerbate α-synuclein toxicity (Chen & Feany, 2005b; Sato, et al.,
2011). However, this toxic effect is challenged by other studies (Azeredo da Silveira, et
100 al., 2009; Gorbatyuk, et al., 2008; McFarland, et al., 2009). Over-expression of mutant
S129A α-synuclein which can not be phosphorylated enhances the loss of dopaminergic neurons in the SNpc and reduces dopamine and tyrosine hydroxylase (TH) levels in the striatum (Gorbatyuk, et al., 2008), whereas over-expression of the phosphorylation- mimicking mutant S129D does not accelerate α-synuclein toxicity (Azeredo da Silveira, et al., 2009; Gorbatyuk, et al., 2008; McFarland, et al., 2009). In Chapter 3, PLK2 was identified as related to the increased levels of S129P α-synuclein in PD, with in vivo studies suggesting other kinases may also be involved (Mbefo, et al., 2010; Pronin, et al., 2000b; Waxman & Giasson, 2008). In this chapter, the levels of five of these kinases
(PLK1, 2 and 3, CK1 and 2) were assessed in the subacute MPTP mouse model and increases in both PLK2 and 3 levels were found during acute nigrostriatal toxicity. In addition, this increased expression of PLK2 and 3 was associated with nigrostriatal α- synuclein phosphorylation in vivo. Increased expression of neuronal PLK2 has been found in DLB (Mbefo, et al., 2010) and aged monkey nigra (McCormack, et al., 2012), while increased PLK2 and 3 is observed in mouse models of acute seizures (Pak &
Sheng, 2003b). These data may suggest that in primates PLK2 is more involved in this pathway, while both PLK2 and 3 are involved in mice. Alternatively, the data may suggest that PLK2 and 3 are initially involved in such neuronal damage, but that overtime the sustained response is driven by PLK2 and not PLK3. Further experiments are required to clarify this aspect. Overall the data suggests that an increase in nigrostriatal PLK2/3 phosphorylates a greater amount of α-synuclein at S129 and contributes to a cascade of injuries in vulnerable neurons.
101 4.64.6 StrengthsStrengths and and wea weaknessesknesses ofof thisthis study study
It is worth noting that the MPTP induced nigrostriatal injury in mice was reversible with treatment withdrawal (Jacksonlewis, et al., 1995; Schmidt & Ferger, 2001), rather than the permanent damage observed in PD. Therefore, there is another possibility that the alterations observed in the levels of phosphorylated α-synuclein and PLKs in these
MPTP mice are due to a successful synaptic plasticity response. Short-term synaptic alternations are mediated largely by post-translational modification of existing proteins
(Izquierdo, et al., 2002), while long-term synaptic changes require new protein synthesis (Bozon, et al., 2003). PLKs have been shown to mediate activity-dependent changes in the molecular composition and morphology of synapses, and up-regulation of PLK2 and 3 expression is required to maintain thresholds for synaptic activation
(Seeburg, et al., 2005a). Once induced by activity, PLK2 seems to negatively regulate its own expression at the protein level through autophosphorylation and subsequent degradation (Seeburg, et al., 2005a) . So it is possible that the MPTP induced injury signals are retrogradely transferred from synapses to cell bodies, resulting in a measureable increase in PLKs in the SN; this increase in PLKs make it possible to phosphorylate more α-synuclein to mediate increased excitatory synaptic strength during the acute injury period. After that, the PLKs down-regulate their own expression and, at least partially, contribute to the recovery of α-synuclein phosphorylation.
4.74.7 ConclusionConclusion
In summary, the findings in this chapter of increased nigrostriatal PLK2 and 3 expression in association with α-synuclein S129 phosphorylation during subacute
MPTP injury in a mouse model support the concept that these PLKs are involved in the
102 MPTP induced deleterious cascade causing the injury. This parallels the changes observed in damaged tissue from patients with PD, supporting the use of this model as a model for both the cell loss and α-synuclein changes observed in PD. The observed changes in the levels of these PLKs and S129P in this PD model, whether they are responsible for more permanent neuronal injury or in the longer-term recovery response, suggest they warrant more focused attention as potential modifiable targets for the treatment of PD.
103 ChapterChapter 5: 5: EffectsEffects ofof a tra traditionalditional Chines Chinesee nherb on on polo-likepolo-like kinasekinase levelslevels inn aann αα-synuclein-induced-s-synuclein-inducedynuclein-induced cell mmomodelodeldel of of ParkinsonParkinson’s diseasedisease cy cytotoxicitytotoxicity
5.15.1 IntroductionIntroduction
As discussed in Chapter 1, traditional Chinese herbs have also been widely prescribed for PD-like symptoms for thousands of years. Over recent decades, a large number of modern studies have been performed to investigate the role of herbal and plant extracts in PD (Chen, et al., 2007; Li, et al., 2013) and dozens of active compounds extracted from herbal medicines have been indicated to have effects on PD molecular models, including TEN (Liang, et al., 2011). TEN is a natural extract from a Chinese herb which has been widely prescribed in traditional Chinese medicine for amnesia, neurasthenia, palpitation, insomnia and cognitive dysfunction. The recent finding that TEN alleviates both toxin- (MPTP, MPP+ and 6-OHDA) and inflammation-induced (LPS) models of dopaminergic neuronal injury (Choi, et al., 2011; Liang, et al., 2011; Lv, et al., 2009;
Naito & Tohda, 2006; Yuan, et al., 2012) raises the possibility of its therapeutic benefit for patients with PD.
As highlighted in this thesis, initial increases in α-synuclein levels and phosphorylation characterize the earliest changes associated with PD neurodegeneration, and the initial testing of compounds to alleviate these changes are most commonly performed in cell models over-expressing α-synuclein (wild-type or mutant) (Chesselet, 2008; Lashuel &
Hirling, 2006; Ulusoy, et al., 2010). Such models recapitulate a known genetic cause of
PD in rare patients, that is multiplications (duplication and triplication) of or mutations
104 (A53T, A30P, E46K and G51D) in the α-synuclein gene (Chartier-Harlin, et al., 2004;
Kiely, et al., 2013; Singleton, et al., 2003; Zarranz, et al., 2004). While similar animal models take some time to have evident PD-like cytotoxicity, cell models have rapid cytotoxicity and provide capability to rapidly initially test treatments that may ameliorate α-synuclein-induced cytotoxicity. Whether there is an increase in PLK expression in such a model needs to be determined and also whether any treatments have an effect on PLK expression will be important for the development of mechanistic treatments for PD.
5.25.2 AimAim an andd Hy Hypothesispothesis
The studies described in this chapter will determine whether there is a similar increase in PLKs in a cell model that uses increased α -synuclein expression to induce cytotoxicity and also determine whether the Chinese herbal treatment TEN, that is known to alleviate toxin- and inflammation-induced cytotoxicity in dopaminergic neurons, can reduce PLK levels in this α-synuclein-induced model of cytotoxicity. It is hypothesized that increased PLK levels will be observed in such a cell model, and that
TEN will reduce PLK expression to alleviate the α-synuclein-induced cytotoxicity, providing initial evidence for a potential mechanistic treatment for this change.
5.35.3 MaterialsMaterials and and Metho Methodsds
5.3.1 Overall study design
It is first necessary to determine if there is an increase in the expression of PLKs in an
α-synuclein-induced dopaminergic cell model of cytotoxicity. The dopaminergic neuroblastoma SH-SY5Y cell line will be transiently transfected with pcDNA3.1 105 plasmids expressing either human wild type α-synuclein or the A53T mutation as the cell models for these experiments, with controls the same cell lines transfected with the pcDNA3.1 vector alone. α-Synuclein expression and phosphorylation will be checked using Western blotting, toxicity measured using assays of proliferation and apoptosis, and changes in the levels of PLK1, PLK2, PLK3, CK1 and CK2 following successful transfections assessed using Western blotting. Whether treatment with TEN can ameliorate the α-synuclein-induced cytotoxicity will then be assessed. To identify the most effective TEN treatment dose that the cells can tolerate, experiments using different doses of the purified herb will be performed and toxicity measured. The highest dose with the least toxicity will be used in further experiments to determine whether the maximal TEN treatment can reduce PLK levels and cytotoxicity.
5.3.2 Celle culturculturee model
Human neuroblastoma SH-SY5Y cells (Peking Union Medical College, Beijing, China) were maintained in Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12, GIBCO,
CA, USA) supplemented with 15% fetal bovine serum (FBS, GIBCO, CA, USA) at
37ºC in 5% CO2. The pcDNA3.1 plasmid expressing human wild type α-synuclein used for transfections has been previously described (Kragh, et al., 2009) and a pcDNA3.1 plasmid expressing A53T mutant was also constructed for transfections by introducing the G209A via site-directed mutagenesis kit (Promega, WI, USA). All constructs were confirmed by sequencing.
SH-SY5Y cells were plated and when 70-80% confluent transiently transfected with pcDNA3.1 plasmids expressing human wild-type or A53T mutant α-synuclein using
106 Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's protocol.
Plasmids encoding the pcDNA3.1 vector (Invitrogen, CA, USA) alone were transfected into cells as controls using the same protocol. Opti-medium (GIBCO, CA, USA) was changed 6h after transfection. To determine transfection efficiency, SH-SY5Y cells were also transfected with plasmids expressing green fluorescent protein(GFP)-tagged
α-synuclein (kind gifts from P.J. McLean, Massachusetts General Hospital East,
MA,USA ) using the same procedures, revealing that around 40-50% transfection efficiency was achieved.
5.3.3 Drug trtreatmenteatment regim regimenen
TEN powder (molecular formula C30H45ClO6, average molecular weight 537.14kDa,
Figure 1.2) was purchased from the Chinese National Institute for the Control of
Pharmaceutical and Biological Products (NICPBP, 111572-200702) with a purity of
98.7%. 20mg of TEN powder was dissolved in 500µl of dimethyl sulfoxide (DMSO) and filtered through a 0.22µm filter (Millipore, MA, USA) to prepare a 0.74M TEN stock solution. TEN working solutions were freshly prepared by diluting the stock solution in DMEM/F12 with 5% FBS. Control solutions were prepared by adding the same volume of DMSO to DMEM/F12 with 5% FBS. To determine the optimal dose of
TEN, SH-SY5Y cells were directly incubated with 0.1μM to 1mM TEN working solutions or related control solutions. For the drug treatment of the cell culture model,
Opti-medium was changed into the optimized TEN working solutions or related control solution 6h after transfection and the cells grown for another 24h prior to harvesting.
107 5.3.4 Outcome measures and analyanalysesses
5.3.4.15.3.4.1 CellCell vi viabilityability (proliferation(proliferation andand apo apoptosis)ptosis) as assayssays
Cell proliferation was assessed using a cell proliferation assay (CellTiter96 AQ,
Promega, WI, USA) according to the manufacture’s protocol. Briefly, SH-SY5Y cells were seeded at a density of 1.5×104 cells/well in 96 well plates. After transfection and drug treatment, previous medium was removed from cells and replaced by 100µl mixture of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2Htetrazolium (MTS) with phenazine methosulfate (20:1) diluted in culture medium (1:10). Cells were then incubated for 1-3h at 37°C with protection from light prior to measuring the 490nm light absorbance using a microplate reader
(PerkinElmer Inc, MA, USA).
Cell apoptosis was determined by using an FITC Annexin V Apoptosis Detection Kit
(BD Bioscience, CA, USA) according to the manufacture’s protocol. Briefly, 1x105 cells were resuspended in 1x Binging buffer and incubated with the mixture of 5µl of FITC
Annexin V and 5µl of propidium iodide for 15min at room temperature in the dark.
Detection and measurement of stained cells was performed by flow cytometry
(GE Healthcare, PA, USA). Early (Annexin V+, PI-) and late (Annexin V+, PI+) apoptotic cells were all counted.
5.3.4.25.3.4.2 WesternWestern bl blottingotting of of cellularcellular pr proteinsoteins
Cellular proteins from the SH-SY5Y cells were assessed whole or fractionated with
Triton X-100 into Triton-soluble and Triton-insoluble protein extracts using different lysis buffers and centrifuge speeds. For whole cell lysates preparation, cells were
108 harvested in RIPA lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 1% Triton X-100,
1% sodium deoxycholate, 0.1% SDS, 5mM EDTA, Beyotime Institute of Biotechnology,
Shanghai, China) with 1x protease inhibitor (Roche, Shanghai, China) and 1x phosphatase inhibitor b (Roche, Shanghai, China) with sonicating for 10s on ice, then centrifuging at 700g for 15min and the supernatant labeled as whole cell lysates.
For subcellular fractionation, detergent soluble proteins were separated from detergent insoluble fractions by lysing cells in TBST buffer with 1x protease inhibitor (Roche,
Shanghai, China) and 1x phosphatase inhibitor (Roche, Shanghai, China) by pipetting up and down for 10 times and incubating on ice for 30min. The lysate was centrifuged at 16,000g for 15 min at 4°C with the supernatant being the Triton-soluble fraction. The remaining pellet was washed in TBS buffer (50mM Tris pH 7.4, 150mM NaCl) and resuspended in SDS-Urea buffer (1% SDS, 6mM Urea, 5mM EDTA) with 1x protease inhibitor (Roche, Shanghai, China) and 1x phosphatase inhibitor (Roche, Shanghai,
China), centrifuged at 16,000g at room temperature with the supernatant being the
Triton-insoluble SDS-fraction. Protein concentrations were determined using a BCA kit
(Thermo Fisher Scientific Inc, MA, USA).
Semi-quantitative western blotting was performed as described in section 2.3.4. Briefly, equal amounts of protein sample were dissolved in an equal volume aliquot of
5×Laemmli buffer (Beyotime Institute of Biotechnology, Shanghai, China), boiled at
95°C for 5min and loaded onto SDS-PAGE gels. After separation, proteins were transferred onto 0.22µm nitrocellulose filter membranes (Bio-Rad, CA, USA). Blots were blocked with 5% milk in washing buffer (10mM Tris-HCl, pH7.5, 150mM NaCl,
0.05%Tween-20) and probed using the primary antibodies [anti-α-synuclein (syn-1, 109 1:2,000, BD Transduction Laboratories, CA, USA); anti-S129P-α-synuclein (1:5000, a gift from Elan Pharmaceuticals, South San Francisco, CA, USA); anti-PLK1 (1:300,
Santa Cruz Biotechnology Inc, CA, USA); anti-PLK2 (1:500, Santa Cruz
Biotechnology Inc, CA, USA); anti-PLK3 (1:1,000, BD Transduction Laboratories, CA,
USA); anti-CK1 (1:500, Santa Cruz Biotechnology Inc, CA, USA); anti-CK2 (1:500,
Santa Cruz Biotechnology Inc, CA, USA); and anti-β-actin (1:15,000, Sigma, CA,
USA)] suspended in 5% milk in washing buffer at 4°C overnight. The antibody to β- actin was used as an internal control to ensure that the same amount of protein was loaded for each sample. Membranes were washed three times with washing buffer and probed with the appropriate fluorescently-labeled secondary antibody (1:10,000, LI-
COR Biosciences, NE, USA) suspended in 5% milk in washing buffer, applied for 1h at room temperature. After washing, membranes were scanned using an Odyssey infrared imaging system (LI-COR Biosciences, NE, USA). Scanned images were exported in
TIFF format and the intensity of each band quantified with Quantity One software (Bio-
Rad, CA, USA) and related to the intensity of the β-actin band for that sample.
5.3.4.35.3.4.3 StatisticalStatistical ana analyseslyses
SPSS 18 (IBM, Chicago, USA) was used for all analyses and significance established when P was <0.05. Analysis of variance with Bonferroni posthoc protection was used to determine any differences between cell groups.
5.45.4 ResultsResults
5.4.1 αα-Synuclein-Sy-Synucleinnuclein over-expressingover-expressing SH-SY5YSH-SY5Y cells have incrincreasedeased phosphorylationphosphorylation of αα-synuclein-sy-synucleinnuclein at S129 and rreducededuced cell viability
As expected, cells over-expressing α-synuclein (either wild-type or A53T) had dramatic 110 increases in the levels of total α-synuclein compared with vector controls (4.2-4.5 fold increase, Figure 5.1 A-F). Intriguingly, while the levels of endogenous S129P α- synuclein in the vector controls were too low to detect, cells over-expressing α- synuclein (both wild type and A53T) had significant levels of S129P α-synuclein in all protein fractions extracted (Figure 5.1 A-F). These increases in the levels and phosphorylation of α-synuclein in SH-SY5Y cells were associated with decreased proliferation (by 18-23±4% compared with the vector controls, p<0.001; Figure 5.1 G) and increased apoptosis (by 21-33±5% compared with vector controls, p<0.003; Figure
5.1 H). No significant differences were observed between cells transfected with wild- type versus A53T mutant α-synuclein (p>0.57).
111 Figure 5.1: CharacterizationCharacterization of thethe αα-synuclein-synuclein over-expressing SH-SY5YSH-SY5Y cellcell modelsmodels
SH-SY5Y cells were transiently transfected with wild-type α-synuclein, A53T mutant α-synuclein or pcDNA3.1 vector control for 24h and harvested for semi-quantitative Western blotting (A-F) or cell viability measurements (G&H). (A-F) Representative Western blots of proteins prepared from whole cell lysates (A) or from serially extracted protein fractions (C&E) showing the increased expression of total α- synuclein (syn-1) and S129P α-synuclein relative to the loading control (β-actin) in the differently
112 transfected cells (A,C&E). The relative quantitation of the data showed that both wild-type and A53T mutant α-synuclein transfections produced over a 4-fold increase in total cellular α-synuclein levels compared with vector controls (B,D&F). Note that endogenous α-synuclein is low and primarily in the more soluble Triton-soluble fraction (vector in C compared with E and D compared with F) and has little
S129 phosphorylation. The relative increase in S129P α-synuclein is disproportionately higher than the increased level of total cellular α-synuclein, particularly in the more soluble fraction (D). (G&H) Over- expression of wild-type and A53T mutant α-synuclein in SH-SY5Y cells significantly decreased proliferation (G) and increased apoptosis (H) compared with vector groups. Data are the mean ± S.D, * p<0.05, ** p<0.01. Values represent the mean of three independent experiments preformed in triplicate.
5.4.2 αα-Synuclein-Sy-Synucleinnuclein over-expressingover-expressing SH-SY5YSH-SY5Y cells do not have incr increasedeased kinase expr expressionession
To determine whether the increase in S129P α-synuclein levels observed in the cell model is associated with increased expression of α-synuclein kinase candidates, the protein levels of PLK1-3 and CK1-2 were measured by semi-quantitative western blotting. The protein levels of these five kinases were not affected by α-synuclein transfection, as no significant differences were found between α-synuclein-expressing cells and their vector controls (Figure 5.4 A, C&E).
5.4.3 IdentificationIdentification of the most effective non-tox non-toxicic TEN concentra concentrationtion for trtreatmenteatment
Non-transfected SH-SY5Y cells were incubated with different concentrations of TEN working solutions from 0.1μM to 1mM for 24h. Cell viability assays showed that 1mM
TEN was toxic to SH-SY5Y cells, reducing cell proliferation by 53±2% (p<0.001,
Figure 5.2 A), 100μM TEN slightly affected cell proliferation but not significantly, and
0.1-10μM of TEN were not toxic to SH-SY5Ycells (Figure 5.2 A). 113 To identify the most effective non-toxic dose of TEN for further experiments, SH-SY5Y cells over-expressing α-synuclein (wild type or A53T mutant) were treated with 0.1-
10μM of TEN for 24h and cell proliferation assessed. 10μM TEN had the most protective effects on α-synuclein over-expressing cell models (increased cell proliferation by 12% with 10uM TEN, P<0.001; by 9-10 % with 1μM and 0.1μM TEN,
P<0.01) and will be used in all further experiments.
5.4.4 TEN tr treatmenteatment alleviate alleviatess α α-synuclein-induced-sy-synuclein-inducednuclein-induced cytotoxicitycytotoxicity
To verify that TEN ameliorates α-synuclein-related cytotoxicity, SH-SY5Y cells over- expressing α-synuclein (wild type or A53T mutant) were treated with 10μM and cell proliferation and apoptosis assessed. TEN treatment significantly increased cell proliferation by 12-20±3% (p<0.001, Figure 5.2 B) and decreased apoptosis by 22-
29±4.5% (p<0.031, Figure 5.2 C) in the α-synuclein over-expressing groups. No significant changes in either cell proliferation or apoptosis were observed with TEN treatment in the vector controls (Figure 5.2 B &C). These results indicate that 10µm
TEN treatment attenuates the cytotoxicity induced by the over-expression of α- synuclein without altering normal SH-SY5Y cell viability.
114 Figure 5.2: EffectsEffects of TENTEN treatmenttreatment on SH-SY5YSH-SY5Y cellcell proliferationproliferation andand apoptosisapoptosis
(A) Non-transfected SH-SY5Y cells were incubated with different concentrations of TEN working solutions from 0.1μM to 1mM for 24h and cell viability assessed. 1mM TEN significantly decreased cell proliferation while 100 to 0.1 μM TEN did not alter the proliferation of SH-SY5Y cells. (B&C) SH-
SY5Y cells over-expressing α-synuclein (wild type or A53T mutant) were treated with 10μM TEN for
24h and cell viability assessed. 10μM TEN treatment increased proliferation (B) and decreased apoptosis
115 (C) only in cells over-expressing α-synuclein (wild type or A53T mutant) and not in the vector controls
(B&C). Data are the mean ± S.D, * p<0.05, ** p<0.01. Values represent the mean of three independent experiments preformed in triplicate.
5.4.5 TEN tr treatmenteatment dow down-regulatesn-regulates S129 phosp phosphorylationhorylation of αα-synuclein-synuclein
To determine whether TEN treatment attenuated α-synuclein-induced cytotoxicity by reducing α-synuclein S129 phosphorylation, SH-SY5Y cells over-expressing α- synuclein (wild type or A53T mutant) were treated with 10μM TEN for 24h and the levels of total and phosphorylated α-synuclein (syn-1 total and S129P) assessed using
Western blotting in the different protein extracts. As no specific oligomeric forms of α- synuclein were observed, only the monomeric α-synuclein bands were quantified.
In vector control lysates, S129P α-synuclein was undetectable and no changes were observed with TEN treatment (Figure 5.3 A&B). However, the increased S129P α- synuclein observed in the α-synuclein over-expressing cells (both wild type and A53T) was reduced by 30-35% with TEN treatment (Figure 5.3 A&B), representing a 25-29% decrease in the relative level of S129P to total α-synuclein in the whole lysates (Figure
5.3 B) rather than a reduction in the amount of α-synuclein over-expressed.
To determine whether the TEN effect occurred preferentially in the more soluble α- synuclein pool shown in previous chapters to initially increase in PD and the subacute
MPTP model of PD, the ratio of S129P to total α-synuclein proteins in the more soluble
Triton-soluble fraction and less soluble SDS fractions were assessed after TEN treatment. In the Triton-soluble protein fraction, TEN treatment reduced S129P α- synuclein levels by 18-30%, reducing the ratio of S129P to total α-synuclein by 15-24%
116 (Figure 5.3 C&D). In the SDS protein fraction, TEN treatments reduced S129P α- synuclein levels by 38-43%, reducing the ratio of S129P to total α-synuclein by 25-30%
(Figure 5.3 E&F). This data shows that TEN reduces the S129 phosphorylation of α- synuclein and that this effect is not confined to the more soluble forms of the protein.
Figure 5.3: EffectsEffects of 1010μμμMM TENTEN treatmenttreatment on S129SS129α129αα-synuclein-s-synucleinynuclein phosphorylationphosphorylation 117 SH-SY5Y cells over-expressing α-synuclein (wild type or A53T mutant) were treated with 10μM TEN for
24h and the levels of total (syn-1) and S129P α-synuclein assessed using Western blotting in the differently prepared protein fractions. Representative Western blots (A, C&E) and graphs of the relative quantitation of the change in levels of S129P α-synuclein and the ratio of S129P to total α-synuclein (B,
D&F). The data show that compared with their vector controls, 10μM TEN treatment of cells over- expressing wild-type or A53T α-synuclein reduced their levels of S129P α-synuclein and the ratio of
S129P to total α-synuclein in whole lysates (A, B) as well as in the separate protein fractions (C, D, E&F).
Data are the mean ± S.D. (ratio of S129P/syn-1). Values represent the mean of three independent experiments preformed in triplicate and are expressed as % change from the non-TEN treatment groups
5.4.6 TEN tr treatmenteatment dow down-regulatesn-regulates PLK3 levels in SH-SYSH-SY5Y5Y cells
Despite the α-synuclein-induced cell model having no increase in PLK levels, to determine whether TEN treatment reduced PLK levels to decrease the S129P α- synuclein levels in the model was explored. Remarkably, TEN treatment selectively decreased PLK3 levels in all cell groups regardless their α-synuclein-expressing status
(Figure 5.4 A-F). In contrast, TEN treatment had no effect on the levels of CK1, CK2,
PLK1 or PLK2 protein levels (Figure 5.5).
118 Figure 5.4: EffectsEffects of 1010μμμMM TENTEN treatmenttreatment on PLK3PLK3 expressionexpression inin SH-SY5YSH-SY5Y cellscells
SH-SY5Y cells over-expressing α-synuclein (wild type or A53T mutant) were treated with 10μM TEN for
24h and kinase levels assessed using semi-quantitative Western blotting in the differently prepared protein fractions. Representative Western blots (A, C&E) and graphs of the relative quantitation of the change in levels of PLK3 (B, D&F) showing that TEN treatment reduced endogenous PLK3 levels in all groups in all protein fractions. The reduction in PLK3 levels did not differ between the vector controls or cells over-expressing α-synuclein (wild or A53T) (A-F). Data are the mean ± S.D. Values represent the mean of three independent experiments preformed in triplicate and are expressed as % change of non-
TEN treatment groups.
119 Figure 5.5: RepresentativeRepresentative WesternWestern blotsblots of PLK1-2PLK1-2 andand CK1-2CK1-2
No significant changes of PLK1-2 or CK1-2 levels among α-synuclein over-expressing groups and vector groups, or between TEN treated groups and related control treated groups.
5.55.5 DiscussionDiscussion
The experiments in this chapter show that TEN attenuates α-synuclein-induced cytotoxicity, down-regulating excessive phosphorylation of α-synuclein at S129 probably by reducing the amount of PLK3 in this neuronal cell model. There have been limited previous studies assessing PLK levels in cell models, although knockdown or knockout of PLK proteins using genetic manipulations reduces S129P α-synuclein
(Inglis, et al., 2009; Mbefo, et al., 2010). Most previous studies have focused on the activity of PLKs using a non-specific inhibitor BI2536 (Inglis, et al., 2009; Waxman &
Giasson, 2011). It would appear that the reduction in PLK3 produced by TEN treatment holds considerable promise as a potential neuroprotective agent for the hyperphsophorylation of α-synuclein in PD and related synucleinopathies.
The over-expression of wild-type and A53T mutant α-synuclein in SH-SY5Y cells was successful in producing a PD model of cytotoxicity, with cells over-expressing 4x their normal levels of the protein and having decreased proliferation and increased apoptosis. 120 A53T mutant α-synuclein induced toxicity is widely accepted and considered to act through a gain-of-function mechanism (Bertoncini, et al., 2005; Chesselet, 2008;
Ulusoy, et al., 2010); however, the modes of toxicity of over-expressing wild-type α- synuclein in cells are hotly debated (Ko, et al., 2008; Sidhu, et al., 2004; Xu, et al.,
2002). This is perhaps explained by the utilization of different plasmids and transfection methods between studies. In this chapter the full length α-synuclein DNA cloned to the pc-DNA3.1 vector was used in a transient transfection system rather than with stable transfections. This technique potentially affects the biological characteristics of the cell colonies selected by their antibiotic resistance, as this over-expression system of either wild-type or A53T mutant α-synuclein was toxic to SH-SY5Y neurons. Such a finding is consistent with genetic observations in patients with PD (Chartier-Harlin, et al., 2004;
Singleton, et al., 2003; Zarranz, et al., 2004). Such over-expressing cell models have mostly been used to study the accumulation and aggregation of α-synuclein, and so the relatively high level of S129P α-synuclein in this cell model is a significant finding and suggests that, in addition to aggregation and toxicity, significant hyperphosphorylation occurs as a consequence of such increased α-synuclein expression in neurons. This greater increase in S129 phosphorylation is relevant to the findings in chapter 2 of increased S129 phosphorylation in sporadic PD patients without an increase in total α- synuclein protein level, and reveals a new characteristic of this cell model. The role of hyperphosphorylated α-synuclein in neurodegeneration is still not clear, with some studies showing S129 phosphorylation is harmful (Chen & Feany, 2005a; Sato, et al.,
2011) while others suggest the opposite (Azeredo da Silveira, et al., 2009; McFarland, et al., 2009). Some of these discrepancies can be attributed to the use of the phosphorylation-mimicking mutant S129D which may not truly reflect the biophysical and biochemical properties of authentic S129P α-synuclein (Sato, et al., 2011). Data in
121 this chapter show that high levels of S129P α-synuclein result from the over-expression of wild-type or A53T mutant α-synuclein in neurons, providing a simple model system to assess the effects of α-synuclein phosphorylation and pathways.
Importantly, TEN treatment protected SH-SY5Y cells from α-synuclein-induced cytotoxicity. These data suggest that TEN should be considered as a potential drug for the treatment of α-synuclein-related neurodegeneration. Previous studies have shown that TEN prevented MPTP/MPP+ and 6-OHDA-induced dopaminergic neuronal apoptosis and LPS-induced inflammation in cell and animal models of PD (Choi, et al.,
2011; Liang, et al., 2011; Yuan, et al., 2012). In these toxin-induced PD models the therapeutic effect of TEN was considered to be due to its anti-oxidative and anti- inflammatory properties (Choi, et al., 2011; Liang, et al., 2011; Yuan, et al., 2012). Such properties could indicate that TEN is effective in any condition where there is increased neuronal oxidation and inflammation. In the present study, TEN reduced the levels of
S129P α-synuclein and PLK3. It is unknown whether TEN affects protein kinase signaling pathways or alters function/s of cellular organelle/s (mitochondria, lysosome, proteasome, Golgi, etc). The data presented in this chapter suggest that the effect of
TEN is more specific to the PLK3 phosphorylation of α-synuclein, and so TEN may be especially beneficial early in the disease process when this effect is manifest (see chapter 4). The phosphorylation of α-synuclein precedes aggregation in patients with
PD (Lue, et al., 2012; Walker, et al., 2012; Zhou, et al., 2011), although increased levels of PLK3 are not observed in PD (chapter 3). In the present chapter, increased PLK levels were also not observed, but a reduction in PLK levels was associated with amelioration of α-synuclein-induced cytotoxicity. This suggests that both the levels and activity of PLKs appear to be involved in the cytotoxicity.
122 In the present study TEN treatment decreased S129P α-synuclein levels and increased cell viability, suggesting that these processes are linked. In addition, TEN treatment specifically reduced the levels of PLK3 in the SH-SY5Y cells, while the over- expression of α-synuclein did not alter the levels of PLK3 or the other kinases analyzed.
This indicates that PLK3 may be a specific target of TEN. As stated in Chapter 1, PLK3 is a member of PLKs, a serine/threonine kinase family containing two polo box regions, which bind to phosphoserine/threonine motifs. While five mammalian PLK family members have been identified so far (PLK1-5), the neuronal PLK2 and PLK3 have been shown to completely phosphorylate α-synuclein in vitro and specifically at S129
(Inglis, et al., 2009; Mbefo, et al., 2010; Waxman & Giasson, 2011). Intriguingly, in this study TEN treatment only reduced the levels of PLK3 to attenuate the α-synuclein- induced cytotoxic increase in S129 phosphorylation, consistent with studies showing that PLK3 is efficient in phosphorylating α-synuclein at S129 (Mbefo, et al., 2010).
5.65.6 StrengthsStrengths and and wea weaknessesknesses ofof thisthis study study
The data in this study shows high levels of S129P α-synuclein result from the over- expression of wild-type or A53T mutant α-synuclein in neurons, providing a simple model system to assess the effects of α-synuclein phosphorylation and the cellular pathways affecting its phosphorylation. The findings of significant improvement in cell viability and the specific down-regulation of PLK3 and S129P α-synuclein by TEN treatment indicate a potential therapeutic benefit of TEN in the early stages of PD.
However, the study is only correlational. Whether the observed reduction of S129P α- synuclein with TEN treatment is a consequence of a direct effect of TEN in reducing
123 PLK3 levels or whether TEN treatment works to reduce S129P α-synuclein through
PLK3-independent pathways remains conjecture. Further studies are required to test these different scenarios by inhibiting and/or knocking-out PLK3 using molecular techniques.
5.75.7 ConclusionConclusion
Overall the data in this chapter suggest that the phosphorylation of α-synuclein at S129 is toxic in this cell model of PD and that TEN treatment can reduce the levels of PLK3 and mitigate such toxicity. Further studies assessing the ability of TEN to reduce S129 phosphorylation and PLK levels in a variety of in vivo animal models of PD are now required, and if consistent with the theory that TEN can reduce PLK levels and S129P toxicity, it may be that TEN could be a potential mechanistic treatment for the early detrimental increases in the molecular levels of phosphorylated α-synuclein observed in
PD and related synucleinopathies.
124 ChapterChapter 6: 6: GeneralGeneral discussiondiscussion
α-Synuclein plays a key role in the pathogenesis of PD. In chapter 2 I have shown quite low levels of S129 phosphorylated α-synuclein in relevant brain regions of controls, while with dramatic increased S129 phosphorylated α-synuclein in PD, in line with previous data showing that most α-synuclein deposited in LBs is phosphorylated at
S129 (Anderson, et al., 2006; Fujiwara, et al., 2002). Since completing and publishing these data (Zhou, et al., 2011) , the importance of early phosphorylation of α-synuclein in patients with PD has been confirmed (Lue, et al., 2012). Overall, these data indicate that S129P α-synuclein has a pathogenic role in PD. As described in chapter 1, several kinases have been identified as capable of phosphorylating α-synuclein at S129 in vitro and in cell models, but the kinases associated with α-synuclein phosphorylation at S129 in patients with PD had previously been unknown. The studies in this thesis were designed to determine which α-synuclein related kinases were associated with the increase in S129 α-synuclein phosphorylation in PD and also in models of PD. By determining the changes in relevant kinases in patient tissues where LBs are forming, and by determining which models recapitulate these changes, mechanistic treatments to ameliorate these pathogenic changes can be trailed.
6.16.1 S129S129 α α-synuclein-s-synucleinynuclein pho phosphorylationsphorylation precedesprecedes Lew Lewyy pathologypathology formationformation in in PDPD
Previous studies had identified that LBs are largely made from insoluble, phosphorylated α-synuclein (Anderson, et al., 2006; Fujiwara, et al., 2002), but the earliest changes that precipitate such pathology were conjecture. In Chapter 2 of this 125 thesis [now published, (Zhou, et al., 2011)], different forms of α-synuclein species were assessed in human brain tissue from controls and cases with PD at different pathological stages. It was shown that the earliest changes in PD where a large increase in cytosolic and membrane-associated S129P α-synuclein without any significant increase in cytosolic total α-synuclein levels. These findings are consistent with previous studies showing a lack of increased total α-synuclein levels (Tong, et al., 2010a) and confirm that aggregation and phosphorylation rather than global over-expression are the major pathologic changes in α-synuclein observed in PD (Anderson, et al., 2006; Fujiwara, et al., 2002; Tong, et al., 2010a). Further analysis over the course of disease showed an increase in membrane-associated α-synuclein accompanied by a decrease in cytosolic α- synuclein as LBs form. All these data indicate that soluble non-phosphorylated α- synuclein decreases over the course of PD, becoming increasingly phosphorylated and insoluble in Lewy pathologies.
Recent studies revealed that α-synuclein could be phosphorylated at S129 in the fibrillar state (Mbefo, et al., 2010; Paleologou, et al., 2010; Waxman & Giasson, 2008) with the suggestion that S129 phosphorylation could have been a late event occurring after fibrillization (Paleologou, et al., 2010). However, the data in Chapter 2 of thesis showed a dramatic increase in S129P α-synuclein at early stages in regions lacking Lewy pathology. This shows that S129 α-synuclein phosphorylation precedes LB pathology, a finding more recently confirmed by others (Lue, et al., 2012). Importantly, the increased levels of S129P α-synuclein directly related to the amount of less soluble membrane- associated and insoluble α-synuclein found in tissue forming LBs, suggesting that
S129P α-synuclein is important for its membrane association and subsequent accumulation into the protease resistant fibrillar forms found in PD. 126 Pathological studies with immunohistochemistry staining have identified that abundant
S129P α-synuclein accumulates in human LBs (Anderson, et al., 2006). However, in
Chapter 2 we observed that the large increase in urea-soluble α-synuclein was not reflected by an expected increase in its phosphorylation at S129 in PD cortex, but was accompanied by a dramatic increase in SDS-soluble S129P α-synuclein in the Western blots. This discrepancy between pathology and biochemistry data may be explained by the differential distribution of S129P α-synuclein in different parts of the LB structure
(see Chapter 2 discussion). Further studies are required to confirm this conjecture.
6.26.2 PLK2,PLK2, LR LRRK2RK2 and and GA GAKK areare inv involvedolved inin the ev evolutionolution ofof S129S129 α- synucleinsynuclein phosphorylationphosphorylation in in relationrelation to LB foformationrmation in in PD PD
Several kinases have been suggested to facilitate α-synuclein phosphorylation in PD, including PLKs (Inglis, et al., 2009; Mbefo, et al., 2010), CKs (Ishii, et al., 2007;
Waxman & Giasson, 2008), GRKs (Arawaka, et al., 2006; Pronin, et al., 2000a),
LRRK2 (Liu, et al., 2012b) and GAK (Dumitriu, et al., 2011). However, little is known about the levels and locations of these kinases in relation to α-synuclein phosphorylation and Lewy pathology formation.
In Chapter 3 of this thesis, the same cases as described in Chapter 2 were utilized to assess the levels and immunoreactivity of five S129P α-synuclein related kinase candidates (PLK1, PLK2, CK2β, LRRK2 and GAK). The most striking finding was an early increase in PLK2, with increasing PLK2 levels correlating with increasing levels of phosphorylated α-synuclein in PD. This data highlights the role of PLK2 in the large, ongoing S129 α-synuclein phosphorylation in PD. Surprisingly the levels of LRRK2
127 were initially decreased in PD. As LB form the levels of PLK2 decreased while the levels of LRRK2 increased back to control levels and beyond. Most LBs co-localized
PLK2 and LRRK2 immunoreactivity in their periphery, with such localisation for PLK relating to decreased levels while such localisation for LRRK2 relates to its increasing levels. Only mature LBs co-localized GAK where this kinase is found throughout the inclusion. These data are consistent with different kinases being associated with different aspects of LB formation in PD. The early trapping of PLK2 in Lewy pathologies may contribute to the reduction in the amount of soluble PLK2 protein in vulnerable PD regions. Increasing LRRK2 levels were associated with inclusion formation in this thesis, and previous studies show that in areas with significant numbers of inclusions there is a correlation between small increases in the levels of
LRRK2 and large increases in the levels of S129P α-synuclein (Guerreiro, et al., 2013).
As the disease progresses and more LBs mature, less inclusions co-localize LRRK2
(Guerreiro, et al., 2013), suggesting that LRRK2 is most associated with the formation of the inclusion. In contrast, GAK was co-localized only in mature Lewy pathologies where it occurred throughout the inclusion with increasing intensity. This is consistent with the increased GAK mRNA found in the SN of PD (Grunblatt, et al., 2004). Overall, combined these findings suggest a dynamic alternation of these kinases prior to and during the formation and maturation of Lewy pathologies in PD. We speculate that
PLK2 is involved in the early phosphorylation of α-synuclein prior to Lewy pathology formation, that LRRK2 is involved in the initiation and formation of Lewy pathologies from phosphorylated α-synuclein, and that GAK is involved in ensuring the final compaction and maturation of Lewy pathologies into the most insoluble form of α- synuclein. Further molecular and cell biology studies are needed to test this speculation.
128 6.36.3 IdentificationIdentification thatthat similarsimilar pathwayspathways ar aree involvedinvolved in in a suba subacutecute MPTPMPTP mousemouse mo modeldel of of PDPD
To confirm the early dynamic changes α-synuclein phosphorylation and PLKs, an animal model is required where the initiation and subsequent sequence of changes can be strictly identified. Such animal models remain the most accepted tool to study pathogenesis and evaluate therapeutic strategies for disease, and MPTP treated mice are the most common model used to study PD-like SN degeneration (Przedborski, et al.,
2001). Phosphorylation of α-synuclein has been observed in this model (Yasuda, et al.,
2011), however the time course for this change has not been evaluated. In addition, there is no information about changes in S129P α-synuclein related kinases in this model. In Chapter 4 of this thesis, the levels of α-synuclein phosphorylation at S129 and related PLKs and CKs were assessed in a subacute MPTP mouse model followed for a further one month. An increase in the levels of S129P α-synuclein in both the SN and striatum was identified in this model, and a related increase in PLK2 and PLK3 in the
SN was shown, with these protein levels peaking in the week following MPTP treatment cessation and returning to normal during subsequent follow-up. The increase in PLK2 and PLK3 levels was selective, as no changes were observed in the levels of
PLK1, CK1 or CK2. These data suggest that PLK2 and PLK3 participate in phosphorylating α-synuclein following subacute nigrostriatal damage, and that such α- synuclein phosphorylation is reversible following the nigral reinnervation of the striatum. Hence, this subacute MPTP mouse model could be used not only to study the
PD-like dopaminergic neuronal damage (Przedborski, et al., 2001) but also to investigate α-synuclein phosphorylation and related PLKs associated pathophysiology, and particularly their treatment. Identifying compounds that can ameliorate such changes is now required.
129 6.46.4 TEN aatteattenuatesttenuatesnuates ov over-expressinger-expressing αα-synuclein-s-synucleinynuclein induc induceded toxicitytoxicity and and reducesreduces PLK3PLK3 and and S1 S129P29P α α-synuclein-s-synucleinynuclein lev levelsels
More rapid screening of potential compounds that may ameliorate the α-synuclein phosphorylation and related changes in PLKs is also required. In chapter 5 the most common cell model used for PD cytotoxicity was assessed for evidence of α-synuclein phosphorylation and related increases in PLK2 or PLK3 expression levels. While this model recapitulates genetic forms of PD cytotoxicity, there was no associated increase in PLKs, although an increase in α-synuclein phosphorylation was observed. This may suggest that there is an initial increase in PLK2 and PLK3 activity prior to any up- regulation of kinase levels. Alternatively the data may suggest that genetic forms of PD differ in their pathogenicity, as the data from Chapter 4 show that toxin-induced changes increase PLK2 and PLK3 levels as expected for PD. Further comparisons of such models are required to determine the most similar models to the findings observed in tissue from patients with PD.
As stated in Chapter 5, TEN is a Chinese herbal extract with anti-oxidative and anti- inflammatory effects on toxin-induced cell models of PD (Choi, et al., 2011; Liang, et al., 2011; Lv, et al., 2009; Naito & Tohda, 2006; Yuan, et al., 2012); however, its effects on α-synuclein toxicity based PD models were unknown. In Chapter 5 of this thesis,
TEN treatment was trialed in α-synuclein over-expressing cell models to determine whether TEN can alleviate α-synuclein-induced cellular toxicity and the levels of any of the related kinases. It was shown that over-expression of either wild-type or A53T mutant α-synuclein decreased cell viability and increased α-synuclein phosphorylation and that TEN treatment protected cells from this α-synuclein-induced toxicity. The data suggests that TEN non-specifically reduces PLK3 levels and α-synuclein
130 phosphorylation in this cell model. Whether the observed reduction of S129P α- synuclein with TEN treatment is a consequence of a direct effect of TEN in reducing
PLK3 levels or whether TEN treatment works to reduce S129P α-synuclein through
PLK3-independent pathways remains conjecture. Further studies are required to test these different scenarios by inhibiting and/or knocking-out PLK3 using molecular techniques. Identifying any similar product that can specifically reduce PLK2 levels would also by an important step forward.
6.56.5 FutureFuture directionsdirections
This thesis suggests that changes in S129 α-synuclein phosphorylation and related
PLKs, following by changes in LRRK2 levels and GAK localization all participate in the pathological progression of PD. Assessment of similar changes in relevant animal and cell models have been determined, revealing that similar early changes are observed in toxin-induced models. Importantly, in testing the effects of a promising Chinese herb,
TEN, on α-synuclein phosphorylation and related PLK increases found that TEN selectively reduced PLK3 levels, identifying TEN as a potential therapeutic target for the early treatment of PD. However, there are certainly more questions that warrant further exploration. Firstly, although PLK3 has been identified as efficient in phosphorylating α-synuclein in vitro (Mbefo, et al., 2010) and identified as a potential therapeutic target for PD in this thesis, we could not assess PLK3 in human brain tissue due to the lack of specific antibodies (Mbefo, et al., 2010). Hence, exploration of specific PLK3 antibodies to human tissues is necessary to understand better about
PLK3 in the progression of PD.
131 Secondly, kinase function is controlled by protein expression level and activity. The protein kinase activity is usually analyzed by monitoring autophosphorylation in immunoprecipitates from cell lysates or phosphorylation of specific substrates with in vitro assays (Schlessinger, 2002). To date, the measurement of kinase activity in postmortem human brain tissue is still a big challenge due to the postmortem delay and preservation. In this thesis, we analyzed the expression and immunoreactivity of kinases.
Thus, further studies are needed to investigate kinase activity in PD tissue and any relationship to kinase protein levels.
Thirdly, the levels of phosphorylated α-synuclein are regulated by the interplay of the amount of total α-synuclein produced and degraded, and the kinases (Wang, et al., 2012) and phosphatases (Braithwaite, et al., 2012b) that interact with the molecule. In this thesis, the levels of S129P α-synuclein, total α-synuclein and related kinases were assessed. Hence, further studies on the down-stream phosphatases are required to evaluate all processes affecting S129P α-synuclein production.
Last but not least, this thesis has determined that the same early dynamic changes in the levels of S129P α-synuclein and related PLK2 and 3 occur in a subacute MPTP mouse model. It will now be important to determine whether treatments can ameliorate these changes and reverse both the molecular and behavioral changes observed in this model.
Such data will be important in investigating potential modifying agents for the treatment of PD.
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