The Protective Effect of Calbindin-D28K on a-Synuclein Aggregation in #- Synucleinopathies

Author Rcom-H'cheo-Gauthier, Alexandre Nay

Published 2016

Thesis Type Thesis (PhD Doctorate)

School School of Medical Science

DOI https://doi.org/10.25904/1912/3816

Copyright Statement The author owns the copyright in this thesis, unless stated otherwise.

Downloaded from http://hdl.handle.net/10072/368168

Griffith Research Online https://research-repository.griffith.edu.au

School of Medical Science, Griffith University, Gold Coast

Submitted in fulfilment of the requirements of the degree of Doctor of

Philosophy

The protective effect of Calbindin-D28K on α-synuclein aggregation in α- synucleinopathies

August 2016

Name: Alexandre Rcom-H’cheo-Gauthier, BSc Biomedical Science, M. Med. Res. (Biomedical Science)

Student Number: 2627404

Principal Supervisor: Dr Dean L. Pountney

Associate Supervisor: Dr Adrian C. B. Meedeniya

Abstract

Neurodegeneration in Dementia with Lewy Bodies (DLB) and Parkinson’s disease

(PD) is associated with the formation of neuronal inclusion bodies composed mainly of aggregated α-synuclein (α-syn) protein. Aggregation may be associated with disturbed

Ca2+ homeostasis and oxidative stress. Post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca2+ buffering protein, Calbindin-D28k (CB).

CB has been shown to be induced by the hormonal form of vitamin D, Calcitriol, and could be induced by other vitamin D analogue such as Calcipotriol (Cp). Furthermore, recent cell culture and in vitro studies have shown that α-syn aggregation can be induced by potassium depolarization, hence we hypothesized that Cp may suppress their formation.

We investigated the interplay between α-syn aggregation, expression of the calbindin-D28k (CB) Ca2+-buffering protein and oxidative stress in neurons by comparing DLB and “healthy” human brain tissue and examining a unilateral oxidative stress lesion model of -syn disease (rotenone mouse), using the combination of immunofluorescence double labelling and Western blot (WB) analysis. DLB cases showed a greater proportion of CB-positive (CB+) neurons in affected brain regions compared to normal cases. Lewy bodies were present predominantly in CB-negative

(CB-) neurons and were virtually undetected in CB+ neurons. The unilateral rotenone- lesioned mouse model showed a greater proportion of CB+ cells and α-syn aggregates within the lesioned hemisphere than the control hemisphere, and α-syn inclusions occurred primarily in CB- cells and were almost absent in CB+ cells. WB showed the total CB level was 20% higher in the lesioned hemisphere compared to the control

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 2 hemisphere. In addition, aged animals which are more sensitive to lesion had a 25% higher level in CB total expression compared to young mice in lesioned hemisphere.

After inducing CB with a vitamin-D analogue in a cell model, we observed through immunofluorescence exclusion of α-syn aggregates in cells expressing high levels of

CB. Taken together, the findings show α-syn aggregation is excluded from CB+ neurons, although the increased sensitivity of aged animals to lesion may not be directly related to differential CB expression.

A previously developed PD cell culture model was used in the following study. SH-

SY5Y human neuroblastoma cells containing endogenous levels of α-syn were treated with KCl or KCl/H2O2 to induce intracellular α-syn aggregation. These cells with a “PD phenotype” were treated with Cp. Upon immunofluorescence and WB analysis, we observed that Cp induced CB in a dose dependent manner. Moreover, cells treated with

Cp showed a significant decrease in the frequency of α-syn positive cells treated with

KCl, KCl/H2O2 and rotenone. Immunofluorescence also showed that CB-siRNA treated cells showed higher α-syn aggregation levels than untreated cells.

Taken together, the findings show that α-syn aggregation is excluded from CB+ neurons and in turn, CB is protective against -syn aggregation in neurons, although the increased sensitivity of aged animals to lesion was not related to differential CB expression. Successfully targeting raised intracellular free Ca2+ in the brain by promoting the expression of CB at the transcriptional level by using inducers, such as calcitriol or Cp may be used as potential therapeutics to prevent α-syn aggregation in

DLB or PD.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 3

Statement of Originality

This work has not previously been submitted for a degree or diploma in any university.

To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself.

______

Alexandre Rcom-H’cheo-Gauthier

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 4 Table of Content

Abstract ...... 2 Table of Content ...... 5 List of figures ...... 7 List of tables ...... 8 Abbreviations ...... 9 Acknowledgment: ...... 13 Chapter 1: Introduction ...... 16 1.1 Parkinson’s disease ...... 17 1.2 Parkinson’s disease and environmental factors ...... 19 1.3 Treatment of Parkinson’s disease ...... 19 1.4 Dementia with Lewy Bodies ...... 21 1.5 Clinical diagnosis of DLB ...... 22 1.6 Treatment of Dementia with Lewy Bodies...... 23 1.7 Lewy Bodies ...... 24 1.8 α-Synuclein ...... 28 1.9 α-Synuclein in neurodegeneration ...... 30 1.10 α-Synuclein oligomerization and cytotoxicity...... 33 1.11 α-Synuclein Post-Translational Modifications ...... 35 1.12 Neuronal spread of α-synuclein ...... 36 1.13 Oxidative stress and the Ageing Brain ...... 39 1.14 The Role of Ca2+ in the Neuron and Age Mediated Changes ...... 41 1.15 Increased intracellular Ca2+ induces α-synuclein oligomerisation ...... 42 1.16 Oxidative stress and α-synuclein oligomerization ...... 44 1.17 α-Synuclein oligomerization induces raised Ca2+ and oxidative stress ...... 46 1.18 Synergistic effect of Ca2+ and oxidative stress ...... 49 1.19 Voltage-Gated Ca2+ Channels in the Brain ...... 50 1.20 Ca2+ buffering proteins in the brain ...... 51 1.21 Calbindin D28K ...... 53 1.22 Vitamin D and the brain in Parkinson’s disease ...... 55 1.23 Challenges for future therapeutics ...... 57 1.24 Aims ...... 58 1.24.1 Aim one: To investigate the proportion of CB and α-syn aggregates positive neurons in DLB and healthy, non-DLB tissue...... 59 1.24.2 Aim two: To investigate the proportion of CB and neurons positive for α- syn aggregates in a mouse model of α-synucleoinpathy...... 60

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 5

1.24.3 Aim three: To investigate the correlation between the overexpression of CB and α-syn aggregation in SH-SY5Y cells...... 60 1.25 Hypotheses ...... 61 1.25.1 Aim one: Investigation of the proportion of CB and α-syn aggregates positive neurons in DLB and healthy, non-DLB tissue ...... 61 1.25.2 Aim two: Investigation of the proportion of CB and neurons positive for α- syn aggregates in a mouse model of α-synucleinopathy ...... 62 1.25.3 Aim three: Investigation of the correlation between the overexpression of CB and α-syn aggregation in SH-SY5Y cells ...... 63 1.26 Significance ...... 64 Chapter 2: Interactions between Calcium and Alpha-Synuclein in Neurodegeneration ...... 66 Chapter 3: Methods ...... 85 3.1 Research Plan ...... 86 3.2 Resources & health and safety ...... 90 3.3 Overview of ethics requirements ...... 91 3.4 Methods and Materials ...... 91 3.4.1 Human Model ...... 91 3.4.2 Animal Model ...... 96 3.4.3 Cell culture ...... 110 Chapter 4: Alpha-Synuclein aggregates are excluded from Calbindin-D28k-positive neurons in dementia with Lewy bodies and a unilateral rotenone mouse model ...... 114 4.1 Introduction ...... 117 4.2 PDF of paper ...... 118 Chapter 5: Calcipotriol inhibits α-synuclein aggregation in SH-SY5Y neuroblastoma cells by a Calbindin-D28K-dependent mechanism ...... 157 5.1 Introduction ...... 158 5.2 PDF of paper ...... 159 Chapter 6: General Discussion ...... 188 References ...... 196 Appendices ...... 216

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 6

List of figures

Figure 1: Schematic diagram of the basal ganglia pathways ((VA) Ventral Anterior

Nucleus, (VL) Ventral Lateral Nucleus) [9]...... 18

Figure 2: LBs stained with anti-α-synuclein (α-syn) [56]...... 25

Figure 3: α-Syn protein domain structure...... 30

Figure 4: Hypothetical diagram relating Ca2+ dysfunction and oxidative stress to cytotoxicity...... 38

Figure 5: Raised intracellular Ca2+ promotes α-syn aggregation [153]...... 43

Figure 6: Mechanism of α-syn aggregation induced by raised Ca2+ and oxidative stress.

(Store-operated channels (SOC), Receptor operated channels (ROC), Na+-Ca2+ exchanger (NCX)) ...... 48

Figure 7: Synergistic effect of α-syn aggregation, Ca2+ regulation and oxidative stress

(positive feedback loop)...... 50

Figure 8: NMR solution superimposition of the ten lowest-energy structures of Ca2+- loaded Calbindin-D28K, (CB) showing representative pair-pair interactions between

EF1 (Red)-EF2 (Orange) and EF3 (Yellow)-EF4 (green) [195] ...... 53

Figure 9: Vitamin D3 metabolizing pathway ...... 55

Figure 10: PET scan from control and patient subject showing high uptake of 18- fluoro-dopa in the striatum [226]...... 57

Figure 11: Overview of methods used to accomplish aims ...... 88

Figure 12: Target for stereotaxic surgery ((MFB) medial forbrain bundle)...... 98

Figure 13: Location of the sutures bregma and lambda in mouse ...... 99

Figure 14: Area of the brain analysed showing motor cortex (blue), somatosensory cortex (green) and striatum (red)...... 106

Figure 15: Protective effect of CB in the mechanism of α-syn aggregation...... 192

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 7

List of tables

Table 1: Molecular components of LBs [66]...... 28

Table 2: Dementia with Lewy Body and control Post-Mortem Tissue ...... 92

Table 3: Antibodies used for immunofluorescence labelling ...... 95

Table 4: Animal use for Western analysis ...... 96

Table 5: Animal use for immunohistochemistry ...... 97

Table 6: Chemical anaesthetic (For euthanasia Ketamine/xylazine combinations was utilized at 4 times the anaesthetic dose by the normal route) ...... 100

Table 7: Analgesic Temgesic...... 100

Table 8: Antibiotic Enrofloxacin ...... 100

Table 9: Modified Zamboni’s fixative ...... 102

Table 10: 12% SDS-PAGE (quantity for 1 gel) ...... 103

Table 11: Solutions used in Western blot analysis ...... 104

Table 12: Antibodies used for immunofluorescence labelling ...... 109

Table 13: Antibodies used for immunofluorescence labelling ...... 113

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 8

Abbreviations

α-syn – α-synuclein

β-syn - β-synuclein

Aβ - Amyloid-β aa - Amino Acid

AD – Alzheimer’s Disease

AF - Alexafluor

BAPTA - 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

BiFC - Bimolecular Fluorescence Complementation

CB – Calbindin D28K

CBP - Calcium Buffering Protein

CHei – Cholinergic Inhibitor

CNS- Central Nervous System

Cp- Calcipotriol

CR- Calretinin

DCX - Doublecortin

DLB – Dementia with Lewy Body

ER - Endoplasmic Reticulum

FRET - Förster resonance energy transfer

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 9

GFP – Green Fluorescent Protein

GPN - Using glycyl-L-phenylalanine-beta-naphthylamide

HRP - Horseradish peroxidase

JNK - c-Jun N-terminal kinase

L-DOPA – L-3,4-dihydroxyphenylalanine

LB – Lewy Body

MALDI-TOF - Matrix-Assisted Laser Desorption/ionization Time Of Flight

MFB – medial forebrain bundle

MPP+ - 1-methyl-4-phenylpyridinium

MSA – Multiple System Atrophy

NCX - Na+-Ca2+ Exchanger

NHS - Normal Horse Serum

NMDA - N-methyl-D-aspartate

NMDAR - N-methyl-D-aspartate Receptor

NO – Nitrous Oxide

OCT - Optimal Cutting Temperature compound

PCA- Protein fragment Complementation Assay

PD – Parkinson’s Disease

PDD - Parkinson’s Disease Dementia

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 10

PNS - Peripheral Nervous System

PV - Parvalbumin

REM – Rapid Eye Movement

ROC - Receptor Operated Channels

ROS – Reactive Oxygen Species

SN – Substantia Nigra

SNAP - Soluble NSF Attachment protein

SNARE - Soluble NSF Attachment protein Receptor

SnPc – Substantia Nigra Pars Compacta

SOC - Store-Operated Channels

SOD - Superoxide dismutase

TBS - Tris-buffered saline

TH – Tyrosine Hydroxylase

TMO - Trimethadione

UPS – Ubiquitin Proteosome System

VA - Ventral Anterior Nucleus

VDCC - voltage-dependent Ca2+ channel

VL - Ventral Lateral Nucleus

WT – Wild Type

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 11

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 12

Acknowledgments:

Firstly, I would like to express my sincere gratitude to my supervisor, Dr Dean

Pountney for his continuous support throughout my PhD study and related research and for his patience and motivation. His guidance has helped me in the entirety of my research and thesis I could not have imagined having a better advisor and mentor for my

PhD study.

I would also like to acknowledge my associate supervisor Dr. Adrian Meedeniya for his help and support throughout my PhD.

I would also like to thank all the Griffith University staff who have provided their help during my three years, especially the animal facility staff and G12 building technical staff.

I thank my fellow DLP labmates over the years, especially Bruno, Shamini,

Fleur and Dario for the stimulating discussions, for the times we worked together, and for all the fun we have had in the last three years. Also, I thank my friends in the School of Medical Science and other fellow students with whom I shared the lab. Especially to

Avi, Sora, Reem, Luqman and Mushfiq-thank you for your advice when I was having trouble with my experiments and giving me reagents when I was running out of them at the worst time.

To all my friends and to those who believe in me when I struggled- you are all precious and I am so grateful for each one of you. To my friends from New Caledonia:

JS, Amaury, Bouboune, Nico, Toto, Jojo, Tony, Rod, Anso, Sarah, Ju, Lolo and Elo, more than 15 years of friendship in the making.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 13

I would like to thank my girlfriend Junna Hayashi for her insightful comments and encouragement. Without her precious support, I would have been unable to conduct this project until the end.

I would like to thank Parkinson’s Disease Queensland for financial support.

Last but not the least; I would like to thank my family: my parents, my brother and sister for supporting me throughout writing this thesis and my life in general. Thank you for cultivating my interest in science and encouraging me to be the best I can.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 14

Included in this thesis are papers in Chapters 2, 3 and 5 for which I am the first author. My contribution to the co-authored papers is outlined at the front of the relevant chapter. The bibliographic details for this paper including all authors are:

The bibliographic details submitted for publication for this paper is:

Chapter 2: Interactions between Calcium and Alpha-Synuclein in Neurodegeneration

Rcom-H'cheo-Gauthier, A.; Goodwin, J.; Pountney, D.L. Interactions between Calcium and Alpha-Synuclein in Neurodegeneration.Biomolecules 2014, 4, 795-811 doi:10.3390/biom4030795.

Chapter 4: Alpha-Synuclein aggregates are excluded from Calbindin-D28k-positive neurons in dementia with Lewy bodies and a unilateral rotenone mouse model

Rcom-H’Cheo-Gauthier, A., Davis, A., Meedeniya, A., Pountney, D.L. Alpha- Synuclein aggregates are excluded from Calbindin-D28k-positive neurons in dementia with Lewy bodies and a unilateral rotenone mouse model. Molecular and Cellular Neuroscience, submitted June 17, 2015, accepted Fri, Sep 11, 2015

Chapter 5: Calcipotriol inhibits α-synuclein aggregation in SH-SY5Y neuroblastoma cells by a Calbindin-D28K-dependent mechanism, Molecular and Neuroscience,

Rcom-H’Cheo-Gauthier, A., Meedeniya, A., Pountney, D.L. Cacipotriol inhibits α- synuclein aggregation in SH-SY5Y neuroblastoma cells by a Calbindin-D28K- dependent mechanism. Journal of Neurochemistry, Submitted August 12 2016.

(Signed) ______(Date)______

Alexandre Rcom-H’cheo-Gauthier

(Countersigned)_ ____ (Date)_16/08/2016__

Supervisor: Dr Dean Pountney

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 15 Chapter 1: Introduction

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 16 1.1 Parkinson’s disease

Neurodegenerative diseases are caused by a gradual loss of neurons leading to central nervous system (CNS) dysfunction. The onset of these disorders is often later in life, hence they are termed “diseases of the elderly.” Currently, our population is dramatically shifting towards an increase in the aged population. Concomitantly, this will result in an increase in the prevalence of these disorders. One of these diseases is

PD which was named after James Parkinson who first described it in 1817 in ‘An essay on the Shaking Palsy’[1]. It is the second most prevalent neurodegenerative disorder, with a worldwide incidence of 2% in those over the age of 65 years old. The prevalence is predicted to double within the next 20 years.

Although there are many speculations on the causes of PD, most of the aetiologies are unknown. however it is widely accepted that it can be either genetically inherited or sporadic [2]. Researchers theorise the underlying cause of sporadic PD, such as exposure to pesticides [3], but to this day there is no unifying theory or established mechanisms.

PD has three major symptoms: motor dysfunction such as resting tremor- bradykinesia, rigidity and postural instability [4]. These symptoms are a consequence of the progressive loss of dopamine secreting cells in the substantia nigra (SN), located in the mid brain. Unfortunately, symptoms start to arise when 50 to 70% of neurons had been lost therefore diagnosis is only possible at an advanced stage of the disease [5].

Late diagnosis severely limits treatments. Surgery and deep brain stimulation [6], or use of drugs such as L-dopamine agonists, anti-cholinergic drugs are currently the only treatments available to treat PD [7]. These treatments do not prevent disease progression and dopaminergic neurons continue to degenerate. This observation highlights the

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 17 importance of the dopaminergic neurons of the nigrostrial pathway in the substantia nigra pars compacta (SnPc) in PD.

Voluntary motion is controlled from the motor and premotor cortex, through the basal ganglia network to the supplementary motor cortex [8]. In PD as seen in figure 1, death of dopaminergic neurons in the SN leads to a decrease in dopamine secretion and in turn a decreased inhibition of the striatum. The decrease inhibition of the subthalamic nucleus is due to the increase inhibition of the caudate/putamen on the globus pallidus, external segment. This leads to an increased stimulation on the globus pallidus, internal segment and more tonic inhibition on the ventral anterior nucleus/ventral lateral nucleus

(VA/VL) complex of thalamus. This results in less stimulation of the motor complex.

Figure 1: Schematic diagram of the basal ganglia pathways ((VA) Ventral Anterior Nucleus, (VL) Ventral Lateral Nucleus) [9].

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 18 1.2 Parkinson’s disease and environmental factors

Environmental factors, such as exposure to pesticides and insecticides, have long been thought to play a role in the pathogenesis of idiopathic PD. Rotenone, 1-methyl-4- phenylpyridinium (MPP+) and paraquat have all been linked to PD [10-12]. Rotenone has been utilised as a pesticide and insecticide while MPP+ and paraquat are both herbicides. These chemicals generate oxidative stress in the mitochondria through the inhibition of complex I of the electron transport chain (rotenone, MPP+) or by acting as a general inducer or reactive oxygen species (ROS) (paraquat). Although only a few cases of PD have been attributed to these chemicals, their mechanism of action points towards a common feature of mitochondrial dysfunction and oxidative stress that may help to explain the pathogenesis of this disease. On the other hand, consumption of caffeine and tobacco smoking has been identified as protective behaviours against PD

[13]. Although the reasons for such protection is unclear, it has been proposed that caloric restriction and the subsequent decrease in metabolic oxidative stress may be responsible. While PD has been shown to have genetic (discussed in section 1.8) and environmental risk factors, the vast majority of cases are late onset and idiopathic in nature. This indicates that age-related factors could be closely related to PD.

1.3 Treatment of Parkinson’s disease

Degeneration of cholinergic, serotonergic, noradrenergic, peptidergic and dopaminergic brainstem nuclei are in involved in the pathology of PD together with the presence of Lewy bodies (LBs), proteinaceous intra-neuronal inclusions in the soma or dendrites of neurons in the central, autonomic and enteric nervous system [14-20]. The main neurological symptoms of the disease are due to the loss of nigrostriatal dopamine neurons and consequential reduction in the level of dopamine in the striatum. Dopamine

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 19 replacement therapy such as the dopamine precursor L-3,4-dihydroxyphenylalanine (L-

DOPA) used in conjunction with a peripheral dopa-decarboxylase inhibitor or by treatment with direct acting dopamine receptor agonists can restore motor function [21,

22]. However, chronic treatment of PD with L-DOPA and direct acting dopamine receptor agonists causes serious adverse motor and psychiatric effects which limits their use [23, 24]. Blandini et al. (2009) showed that Ca2+ entry through the cell membrane into peripheral blood lymphocytes was impaired in PD patients with L-DOPA-induced dyskinesia, indicating that altered voltage-gated calcium channel (Cav) function may have a role in the adverse effects of anti-parkinsonian drug therapies. There is a pre- symptomatic period of 5 to 10 years during which the motor impairments of PD are masked by compensatory mechanisms within the basal ganglia, such that clinical signs do not appear until striatal dopamine levels have been reduced by 70% and approximately 50% of nigral dopaminergic neurons have degenerated [14, 26]. The pre- symptomatic striatal dopamine depletion and nigral degeneration may therefore have occurred in parallel with other pathological changes (e.g. degeneration of hindbrain nuclei) that may underlie some prodromal non-motor symptoms of PD, rather than in sequence after them. Since a definitive diagnosis of PD requires post-mortem examination, it is difficult to know the functional status of midbrain dopaminergic neurons during the pre-symptomatic stage of the disease. That is, dopamine neurons projecting to the striatum from the SnPc could be functionally compromised in the absence of overt pathological changes, namely, noticeable dopaminergic cell loss or the presence of LB or neurites. This view is supported by the observations of Hirsh et al.

(1988) who found that 17% of neuromelanin containing (i.e. dopaminergic) neurons in

PD were not tyrosine hydroxylase (TH) positive. In addition, a recent study by Good et al. (2011) showed that impaired dopamine release from striatal slices and altered

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 20 electrophysiological characteristics of SN dopamine neurons occurred before the appearance of motor symptoms in a genetic mitochondrial respiratory chain deficit model of PD called the MitoPark mouse [29, 30].

1.4 Dementia with Lewy Bodies

Dementia with Lewy Bodies (DLB) is the second most frequent cause of dementia in the elderly after Alzheimer disease (AD) [31]. Primary symptoms include progressive dementia which is often accompanied by Parkinsonism and psychiatric symptoms [32]. DLB sufferers display an inability to plan or a loss of analytical or abstract thinking and show markedly fluctuating cognition. Wakefulness, alertness and short-term memory vary randomly daily. Early symptoms such as recurring visual hallucinations with vivid and detailed pictures can be use as diagnostics. Anatomically

DLB is characterized by the presence of LBs, clumps of α-syn and ubiquitin protein in neurons, detectable in post mortem brain histology [32].

DLB unlike PD shows a widespread distribution of LBs in almost every brain area, with the frontal cortex, pigmented midbrain and brainstem nuclei, dorsal efferent nucleus of the vagus basal forebrain nuclei, and limbic cortical regions expressing a greater number of LBs than other brain regions [33].

No genetic marker has been found to be associated with sporadic DLB.

However, four mutations, the E46K mutation on SNCA gene [34], the I93M mutation on the UCH-L1 gene [35] and two β-synuclein (β-syn) mutations (V70M and P123H)

[36] have been described in DLB pedigrees.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 21

1.5 Clinical diagnosis of DLB

As with all dementias, accurate clinical diagnosis can only be made after a thorough clinical assessment including a detailed history and full mental-state, cognitive and physical examinations. There should be particular emphasis on examining the core diagnostic features of fluctuating cognitive impairment, parkinsonism and recurrent visual hallucinations and the supportive features of falls, depression, other hallucinations and rapid eye movement (REM) sleep disorder [37]. Diagnosis is made on the basis of the consensus diagnostic criteria for DLB, which are the most widely accepted and have been the best validated by autopsy. The main differential diagnoses are AD, vascular dementia, Parkinson’s disease dementia (PDD), atypical parkinsonian syndromes (such as progressive supra-nuclear palsy, multiple system atrophy (MSA) and cortico-basal degeneration), and Creutzfeldt-Jakob disease [32].

In addition to LB related pathology, most DLB cases revealed AD characteristic symptoms [38-40], where higher Braak stages (method used to diagnose PD and AD) of

AD-type pathology result in a misdiagnosis of AD rather than DLB [41-43]. In this regard, the misdiagnosis of DLB increases with increasing Braak stages of AD associated pathology [44].

Consistent application and greater reliability of the consensus criteria is facilitated by more detailed definitions of the quality, frequency, and severity of core and supportive features [39, 45]. The assessment of fluctuating cognitive impairment poses substantial difficulty to many clinicians, and newly proposed rating scales could be particularly helpful in this regard [46]. The best way to take advantage of supportive diagnostic features in improving diagnostic accuracy also needs to be identified. Repeated falls, syncope, transient loss of consciousness, and depression are common in older people

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 22 with cognitive impairment and can serve as red flags to a possible diagnosis of DLB. By contrast, neuroleptic sensitivity and REM sleep behaviour disorder can be highly predictive of DLB, but their detection depends on having a high index of suspicion and asking appropriate screening questions.

Since PDD is common and typically shares the features of DLB [47], there is much debate about the relation between the two disorders. There are at present no specific operational clinical criteria to diagnose PDD. The arbitrary 1-year rule used to separate

DLB from PDD is helpful in individual case diagnosis but is increasingly hard to justify from a neurobiological point of view. Therefore current DLB criteria need to be revisited with respect to their relation to PDD; this process could be facilitated by improved operational criteria for PDD. The need for a collaborative research effort by specialists in movement disorders and dementia is apparent and is already being addressed by interdisciplinary task groups [48].

1.6 Treatment of Dementia with Lewy Bodies

When dealing with the management of DLB patient a list of cognitive, psychiatric, and motor disabilities is established. Then highest priority drug is administrated to treat the most disabling or distressing symptoms. Before any drugs are prescribed, physicians must check that the treatment gains in target symptoms may not be associated with worsening of symptoms such as neuroleptic sensitivity reactions which are a risk factor in all cases [49].

Nonpharmacological strategies for cognitive symptoms, including education, orientation and memory prompts, and targeted behavioural interventions, are an essential part in dealing with DLB. Pharmacological treatments are most successful when prescribed as part of a comprehensive management approach [50].

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 23

Levodopa is used to treat motor symptoms in DLB; however it is less effective than in PD. Treatment refractoriness may be linked to intrinsic striatal degeneration in

DLB. Low effective dose of levodopa monotherapy are administrated as higher doses or other antiparkinsonian drugs, are usually associated with increased confusion and hallucinations [51].

Evidence is accumulating that cholinesterase inhibitor (ChEI) drugs are effective and relatively safe in the treatment of neuropsychiatry and cognitive symptoms in DLB, but the number of patients studied was relatively small [52, 53]. In addition to the usual gastrointestinal side effects associated with this class of drugs, increased cholinergic activity in DLB patients may cause hyper-salivation, rhinorrhoea, lacrimation, and exacerbate postural hypotension and falls. Improvements are generally reported as greater than those achieved when treating AD [54].

1.7 Lewy Bodies

LBs are intraneuronal inclusion bodies which were first discovered by Friedrich

Lewy in 1912. LBs are composed of proteins such as α-syn, ubiquitin, neurofilaments protein, and α-crystallin B. Tau proteins may also be present, and LBs may occasionally be surrounded by neurofilaments tangles [55]. LBs are found in the SN of many patients with idiopathic PD. Cortical LBs are a distinguishing feature for DLB also seen in cases of MSA.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 24 Lewy Body

Figure 2: LBs stained with anti-α-synuclein (α-syn) [56].

Inclusion bodies usually form when a cell cannot degrade misfolded or damaged proteins at a sufficient rate, leading to an accumulation of protein that cannot be degraded by the ubiquitin proteasome system. Reactive oxygen and nitrogen species contribute to protein misfolding and damage. The current literature demonstrates the importance of inclusion bodies in cell survival [57]. They could be a protective response to toxic intrusion in cells sequestering cytotoxic aggregates and a precursor of autophagic clearance. It has been demonstrated that cells with inclusion bodies show a greater rate of survival than cells that does not possess this defence mechanism [58, 59].

These seem to protect the cell by the uptake of misfolded and dysfunctional proteins

[60, 61]. However, it has been suggested that the accumulation of larger LB may interfere with normal cell function and metabolism and cause neurodegeneration.

Recasens et al. (2014) showed that intra-nigral or intrastriatal inoculations of PD- derived LB extracts resulted in progressive nigrostriatal neurodegeneration starting at striatal dopaminergic terminals.

Morphologically, LBs can be divided into two categories: nigral and cortical.

Nigral LBs are spherical, intraneuronal cytoplasmic inclusions, characterized by hyaline eosinophilic cores, concentric lamellar bands, narrow pale halos, and are

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 25 immunoreactive for α-syn and ubiquitin and present in the brainstem nuclei and diencephalon [63]. On the other hand, cortical LBs are found in the cerebral limbic cortex and amygdala and do not possess a halo [64]. α-Syn immunolabeling of PD brains have revealed that α-syn immunostaining can detected approximately 64% of nigral LBs and 31% of cortical LBs [65].

More than 70 components have been observed in LBs so far [66]. The main component of inclusion bodies is α-syn and after its initial aggregation, it is thought that other proteins are attached and aggregate to the LB. Numerous proteins have been identified as being present in LBs, but many of them have not had their precise biochemical roles determined [67]. Those proteins belong into classes, including structural elements, α-syn-binding proteins, components of the ubiquitin proteosome system (UPS), proteins involved in cellular responses, proteins linked with phosphorylation and signal transduction, cytoskelatal, synphilin-1-binding, cell cycle and cytosolic proteins [66].

+, positive; +/-, partially or weakly positive; –, negative; ND, not described. Brainstem-type LBs Cortical LBs Advanced glycation end-products + + Agrin + ND ab-crystallin - +/- a2-macroglobulin + ND α-synuclein + + Amyloid precursor protein + +/- ATPase of the 26S proteasome ND +/- Basic fibroblast growth factor +/- - b-TrCP + + C terminus of Hsp70-interacting protein + ND Calcium/calmodulin-dependent protein + + kinase II Calbindin-D + ND Centrosome/aggresome-related proteins + + Chondroitin sulfate proteoglycans + + Chromogranin A + + Clusterin/apolipoprotein J +/- + Complement proteins (C3d, C4d, C7 and +/- +/-

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 26

C9) Cullin-1 + + Cyclin B + ND Cyclin-dependent kinase 5 + + Cytochrome c + - DJ-1 +/- - Dorfin + + Extracellular signal-regulated kinases + - 14-3-3 protein + + G-protein-coupled receptor kinase 5 + - Gelsolin-related amyloid protein Finnish + + type Glyceraldehyde-3-phosphate +/- ND dehydrogenase Heat-shock proteins (Hsp 27, 40, 60, 70, 90 + + and 110) Heme oxygenase-1 + + Histone deacetylase 4 + + Immunoglobulin (IgG) + ND IkBa + + Leucine-rich repeat kinase 2 + + Lipids + + Microtubule-associated protein 1 + - Microtubule-associated protein 1B (MAB + + 5) Microtubule-associated protein 2 + +/- Multicatalytic proteinase +/- + MxA protein +/- ND NEDD8 + + Neurofilaments + + NFkB + + NUB1 + + Omi/HtrA2 + ND Pael-R + ND Parkin + + Phospholipase C-d - +/- p35 + + p38 + ND p62/sequestosome 1 + ND PINK1 +/- - Prolyl-isomerase Pin 1 + ND Proteasome + + Proteasome activators (PA700, PA28) + + Retinoblastoma protein + - ROC1 + + Sept4/H5 + + SIAH-1 + ND Superoxide dismutase 1 (Cu/Zn + ND

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 27 superoxide dismutase) Superoxide dismutase 2 (Mn superoxide +/- ND dismutase) Synphilin-1 + + Synaptophysin +/- +/- Synaptotagmin XI + ND Tau +/- +/- Tissue transglutaminase + ND TorsinA + + Tropomyosin - + Tubulin + ND Tubulin polymerization promoting + + protein/p25 Tyrosine hydroxylase + ND Ubiquitin + + Ubiquitin activating enzyme (E1) + + Ubiquitin conjugating enzyme UbcH7 + ND (E2) Ubiquitin C-terminal hydrolase ND + Vesicular monoamine transporter 2 + -

Table 1: Molecular components of LBs [66].

1.8 α-Synuclein

α-Syn is a 14kDa protein that is encoded by the SNCA gene and highly conserved in vertebrate species. Although the exact role of α-syn still remains unclear, the protein is primarily expressed in the olfactory bulb, frontal cortex, striatum and the hippocampus with lower expression levels also observed in hypothalamus, thalamus midbrain, cerebellum, and the pons/medulla oblongata [68] where it localises to the presynaptic terminals of dopaminergic neurons [69, 70]. It is believed to be involved in vesicle transport and dopamine neurotransmission through interaction with SNAP

(Soluble NSF Attachment protein) Receptor (SNARE) [71]. Three putative domains of the α-syn protein have been identified. The N-terminal domain is made up of seven repeating 11 amino acid (aa) imperfect repeat sequences, which have been predicted to form aliphatic helices allowing α-syn to associate with lipids. The second motif contains

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 28 acidic residues in the C-terminus which have been identified as a Ca2+ binding site, where it was determined that Ca2+ binds in a 0.5M Ca2+: 1M α-syn stoichiometry and can rapidly induce oligomerization of α-syn [72]. The third domain is a region of hydrophobic amino acids which are important for aggregation [73]. The conformation of α-syn is highly dependent on environmental conditions. In the aqueous cellular environment, α-syn adopts a random coiled structure, but adopts a helical conformation upon binding to acidic phospholipid vesicles [74, 75]. In vitro model studies suggest that seeding of NAC amyloid formation leads to an accumulation of ordered NAC [76], pointing out the importance of the NAC domain in aggregate formation. α-Syn is also prone to nucleation dependent aggregation [77] through the N-terminus [78] and this aggregation transforms α-syn from the random coiled formation to beta-pleated sheets, which is inhibited upon membrane binding [79]. Circular dichroism (CD) spectroscopy has also been used to confirm this conformational change [80].

α-Syn has a high sequence homology with β-syn which like α-syn is localised to presynaptic vesicles, however unlike α-syn, it does not form the fibrillar structures [81].

Comparison of the aa sequence of these two proteins revealed two divergent regions. A hydrophobic region of 12 aa was found in the central region of α-syn but not in β-syn. It was also found that the C-terminus was also highly divergent. To ascertain which diverged region was responsible for protein aggregation, several recombinant synuclein proteins were generated. α/β-syn (containing the first 97 aa of α-syn (including the 12 aa hydrophobic region) and the C-terminal 48 aa of β-syn) was found to have similar aggregation properties to that of α-syn. Deletion of the hydrophobic 12 aa region from

α-syn, however resulted in the loss of α-syn aggregation dynamics. It was also found that the hydrophobic 12 aa region alone was sufficient to form aggregates [73]. Under normal conditions, α-syn has been shown to interact with membranes, McLean et al.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 29

(2000) showed, by using Förster resonance energy transfer (FRET) on primary cortical neurons, that both the N- and C-terminus of the protein can be bound to membranes. In addition, both ends of the protein have some degree of interaction with each other. This membrane interaction and its subsequent function as a SNARE related protein is mediated by Rab3a (a protein involved in Ca2+ exocytosis in neurons) [83].

Figure 3: α-Syn protein domain structure. α-Syn contains three putative domains. The N-terminus involved in lipid interaction, the hydrophobic Non-amyloid-β-component (NAC) domain important for aggregation and the C-terminal Ca2+ binding site can increase the rate of oligomerization. The Parkinson’s disease (PD)-linked point mutations are indicated within the neurotransmitter vesicle binding domain.

1.9 α-Synuclein in neurodegeneration

Under normal cellular conditions, proteins have a stable biological conformation that is necessary to perform their biological function. Misfolded proteins are normally degraded by the UPS or via lysosomal degradation. However, in disease, misfolded proteins may be resistant to degradation and can form aggregates. Aggregation of a protein can result from instability of a protein's native structure and may be caused by

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 30 gene mutations or by environmental stressors, such as pH or metal ion concentration

(Ca2+, Mn2+, Cu+, ect …) changes within the local environment. Abnormal protein aggregation has been implicated in a number of neurological diseases including PD. The protein α-syn has been implicated in a number of neurodegenerative disorders characterised by either nuclear or cytoplasmic aggregation in multiple cell types throughout the CNS. These diseases are collectively termed α-synucleinopathies and include PD and atypical PD such as MSA and DLB [84-86].

PD can be classified into two main groups. Idiopathic PD accounts for roughly

85-90% of all PD cases, and as the name suggests, has no known cause. However, there is some evidence that PD may be a result of environmental factors. Familial PD accounts for the remaining 10-15% of cases and has primarily been shown to be involved in early onset Parkinsonism, due to mutations in a number of genes. The dysfunction of these genes may provide clues to important pathways that give rise to idiopathic PD. Although approximately 90% of PD cases are not attributed to genetic abnormality, there are a number of specific genetic mutations or genetic regions that have been implicated in familial PD. To date, six mutations in PARK1/4, the gene responsible for the expression of α-syn, have been identified. They include the A30P

[87], A53T [88], E46K [34], H50Q [89], A53E [90] and G51D [91] aa substitutions.

The A53T and A30P mutations also alter neuronal cytotoxicity in response to hydrogen peroxide and MPP+ treatment. Expression of these mutant isoforms significantly increases cytotoxicity in comparison to cells expressing wild-type (WT) α-syn, which was similar to control cells [92]. Gene duplication [93] and triplication of α-syn [94] have also been shown to be involved in the development of familial PD. It has been shown that there is a difference in the fibrillation dynamics between the mutations,

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 31 where the oligomerization rate is increased compared to WT. Both A30P and A53T mutations showed increased fibril formation compared to WT [95-97].

Gene triplication of SNCA was found previously to give rise to an autosomal dominant form of early onset PD, outlining the importance of α-syn concentration to aggregation [98]. Interestingly, mice studies have shown that there is an age dependent decline in both α-syn protein and mRNA levels. A study showed that 10 month old mice had only 25% mRNA levels compared to two month old mice in both the SN and hippocampal regions and that this decrease was not associated with a reduction in tyrosine hydroxylase (TH) positive neurons [99]. This was confirmed in a study looking at mRNA levels in rats that also showed an age related loss in α-syn expression. In a study that looked at levels in PD patients, they found that there was no significant difference in the mRNA levels in the cerebral cortex [100]. This data suggests that there is no difference in expression and that there must be another important element in LB formation. Real-time polymerase chain reaction analysis of single dopaminergic neurons from post mortem SN tissue of both PD affected individuals and controls revealed that there was a significant three fold increase in α-syn mRNA levels [101].

This suggests that inhibition of protein degradation pathways may not account for accumulated α-syn in PD affected neurons. Another case control study that looked at the expression of α-syn found that there was no significant difference between cases and controls [102]. This conflicting data may be explained by the loss of neurons from the

SN during different stages of the disease.

α-Syn species contained in PD-derived LBs are pathogenic and have the capacity to initiate a PD-like pathological process, including intracellular and

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 32 presynaptic accumulations of pathological α-syn in different brain areas and slow progressive axon-initiated dopaminergic nigrostriatal neurodegeneration [103].

1.10 α-Synuclein oligomerization and cytotoxicity.

Abnormal oligomeric α-syn species have been implicated in the pathogenesis of

α-synucleinopathies. Mechanisms that increase the rate of oligomerization or clearance could be important focal points on research into pathophysiology. Danzer et al. (2007) examined α-syn oligomers that were generated in different manners (with or without

FeCl3, long or short incubation with stirring or spinning), therefore had different size and morphologies, and how they affected SH-SY5Y human neuroblastoma cells culture looking specifically at cytosolic Ca2+ levels and their ability to seed intracellular aggregation. Small, annular α-syn species but not monomeric proteins were able to increase cytosolic Ca2+ levels in SH-SY5Y cells, shown via fluorescent microscopy.

This increase in cytosolic Ca2+ was rapid, reaching a plateau around 200 seconds. To support this, annular species were also shown to cause membrane depolarisation. In an effort to find where the Ca2+ came from, the cells were incubated in Ca2+ free media. No influx of Ca2+ was observed, indicating that the Ca2+ was coming from extracellular sources indicating a pore forming ability of α-syn oligomeric species. Treatment with these oligomers resulted in an increased level of cleaved active caspase 3 directly indicating α-syn oligomers induced apoptosis. These observations were replicated in cells to generate oligomeric species. Outeiro et al. (2008) used Protein fragment

Complementation Assay (PCA) and Bimolecular Fluorescence Complementation

(BiFC) assay to monitor α-syn monomer interactions in vitro. A number of α-syn constructs were fused to partial green fluorescent protein (GFP) fragments.

Fluorescence only occurred when a full GFP complement was achieved. It was

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 33 determined that the most favourable α-syn interaction was anti-parallel α-syn formation.

Toxicity was then tested by measuring the release of adenylate kinase from damaged cells and was establish to be increased against other constructs. Previously HSP70 (a cytoplasmic heat shock protein and chaperone) was shown to be protective against α- syn cytotoxicity in these GFP constructs. Co-transfection with the α-syn constructs with

HSP70 lead to a decrease in α-syn oligomerization. The link between these provides further evidence that the oligomeric forms of α-syn are cytotoxic.

Oligomeric species of α-syn were shown to induce ROS, and this may be one of the inducers of apoptosis. Using PD cytoplasmic hybrids, which have relatively high levels of oxidative stress and an enhancement in oligomer formation, treatment with

CoQ10 and GSH antioxidants resulted in a decrease in oligomer formation. A possible mechanism for the increase rate of oligomerization is an increase in the monomer/polymerised tubulin ratio caused by mitochondrial mediated reactive oxygen species (ROS) production. This ratio increased when treated with CoQ10 and increased levels of ROS. [106].

α-Syn has been thought to exist as a natively unstructured protein. However, recently under non-denaturing conditions (native One dimensional polyacrylamide gel electrophoresis (PAGE)), α-syn has been shown to migrate at 45-50kDa in M17D,

HEK293, HeLa and COS-7 cells [107]. This was also observed with recombinant protein [12], confirmed to be approximately 56kDa by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy and α-syn tetramers had a predominantly helical structure and were resistant to aggregation. This indicates that an external stimulus is required for endogenous oligomeric species to become pathogenic. These normally occurring tetramers are proposed to be non-toxic,

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 34 with the abnormal, toxic oligomeric species produced via interconversion first to the monomer. However, it was suggested that traditional methods of α-syn purification resulted in the denaturation of this tetramer species. Though, [108] Coelho-Cerqueira et al. (2013) compared cell disruption and purification protocols. They found no great difference between the purification methods with a small proportion of tetrameric α-syn found, but predominantly monomeric and dimeric α-syn oligomers.

1.11 α-Synuclein Post-Translational Modifications

α-syn oxidation has been shown to be important in increasing the rate of aggregation and has also been shown to increase metal ion binding [110]. Oxidation leads to other common post-translational modifications, including nitrosylation, and it has been shown that oxidative stress can stabilise oligomeric α-syn species via the formation of di-tyrosine cross links [111, 112]. However, oxidation of recombinant human α-syn results primarily in the oxidation of methionine residues [113]. At physiological pH, this oxidation abolishes α-syn aggregation [114]. A striking characteristic of PD is the selective loss of dopaminergic neurons in the SN which has been mimicked in a Drosophila model system [115]. However, α-syn has been shown to play a cyto-protective role in dopaminergic cells. The N27 dopaminergic cell line transfected with human α-syn is protected against MPP+ induced apoptosis via inhibition of PKCδ cleavage. Moreover, the inhibition of Bcl-2-associated death promoter and the concentration of ROS is reduced, in comparison to non-transfected cells [116]. Dopamine has also been shown to inhibit the formation of α-syn fibrils via oxidative modification of the protein and enhances formation of protofibrillar α-syn species [117].

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 35 α-syn phosphorylation has also been revealed to be important in increasing the rate of aggregation [118] and has also been shown to increase metal ion binding [119,

120]. Truncation and proteolytic processing of α-syn have also been implicated in aggregate pathology [121]. Recent studies by Kleiknecht et al. (2016), describe how C- terminal Y133 is involved in α-syn aggregate clearance by supporting the protective

S129 phosphorylation for autophagy and by promoting proteasome clearance. Using yeast, they described how C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by α-syn di-tyrosine dimers [122].

1.12 Neuronal spread of α-synuclein

A major contribution to the toxicity of oligomeric α-syn species is cell to cell spread of α-syn [123]. α-Syn is suspected to spread three different ways, via exosomes, via directed secretion into the extracellular space or via direct cell to cell interaction.

Exosomes are membranous vesicles released from mammalian cells and have been shown to contain mRNA, microRNA and proteins. Their normal role is for the removal of unwanted proteins, signal transduction between cells. Alvarez-Erviti et al. (2011) demonstrated that α-syn+ exosomes isolated from a SH-SY5Y neuroblastoma α-syn overexpressing cell line were capable of transferring the protein to other SH-SY5Y cells. They also concluded that inhibition of the lysosomes that are involved in α-syn degradation resulted in an increase in α-syn exosome-mediated release into the culture media. Danzer et al. (2012) demonstrated in H4 cells and primary cortical neurons that

α-syn in exosomes was oligomeric. They also looked at the location of α-syn protein was in exosome enriched fractions; by trypsin treatment they deduced that α-syn was predominantly either outside or associated with the outer membrane of the exosome, but not totally excluded from the lumen. Microglial cells have also been shown to secrete α-

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 36 syn positive exosomes [126] using BV-2 mouse cells with treatment of the α-syn protein. Moreover, it was found in foetal tissue graft recipients that return of symptoms of a long term graft survivor correlated with classic PD markers such as α-syn and ubiquitin aggregation within the grafted region, replicated in patients who received foetal mesencephalic dopaminergic neurons [127, 128]. Furthermore, α-syn then taken up by other cells within the CNS after being secreted from the neurons through exosomes [129].

Prusiner et al. (2015) described how α-syn was likely to spread between neurons. It was reported that α-syn acted in a prion like manner and caused more neurodegeneration in MSA [130]. This ultimately leads to spread and further cell death.

Additionally, using a cell culture model which eliminates direct cell to cell contact,

Reyes et al. (2015) show that human dopaminergic neurons carrying a triplication of the

α-syn gene secrete α-syn and was then taken up by neighboring neurons.

Live cell imaging experiments by Reyes et al. (2015) showed direct transfer of α- syn-mCherry puncta between two GFP cells. Dynasore inhibited the GTPase activity of dynamin (implicated in endocytosis) and α-syn cell to cell transfer [131].

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 37 Figure 4: Hypothetical diagram relating Ca2+ dysfunction and oxidative stress to cytotoxicity. α-Syn can be induced to oligomerize via, interaction or modification by metal ions, such as Ca2+ or/and oxidative species. This oligomerization leads to protofilament formation, inhibition of the ubiquitin proteasome system and membrane permeabilisation, both of which lead to cell death. Furthermore, α-syn can spread through different mechanisms to other neurons.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 38 1.13 Oxidative stress and the Ageing Brain

Harman D (1956) first postulated a 'free-radical theory' of ageing where he linked oxidative stress mediated by normal metabolic processes, with rate of ageing. He hypothesised a process whereby metabolic oxidative stress affects tissue in a similar manner to oxidative stress resulting from pathological stimuli. Moreover, he postulated that decreasing oxidative stress could increase longevity [132]. Since then, studies have looked at the relationship between metabolic rate and lifespan in different species. In general, a low metabolic rate is indicative of a longer life span while a high metabolic rate coincides with a shorter life expectancy. There are however, a few exceptions in which species which do not follow this trend have been shown to have lower levels of

ROS [133]. In terms of ageing, there is evidence to show that the resulting products of

ROS such as protein carbonyls, protein bound 4-Hydroxynonenal and 3-nitrotyrosine are increased in young compared to old mice and in humans [134]. While there are many metabolic processes taking place in the cell, the electron transport chain of the mitochondria is responsible for the reduction of molecular oxygen in the cell and hence is the major source of cellular ROS. The high energy intermediates NADH/FADH2 produced from glycolysis and the citric acid cycle are fed into the oxidative phosphorylation chain at complex I and II respectively. Electrons from these intermediates are transferred to complex III and IV and at this stage molecular oxygen is reduced. The majority of oxidative stress in the cells comes from complex I and III of the electron transport chain in mitochondria [135]. The mitochondria is a primary source of radicals as illustrated by a greater level mitochondrial DNA damage compared to nuclear DNA damage [136].

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 39

Sims-Robinson et al. (2013) looked at the effect of superoxide dismutase (SOD)

(enzyme which catalyzes the dismutation of superoxide radicals) in aged neurons in

SOD1+/+ and SOD1-/- mice and assessed the level of nitrated proteins and lipid peroxidation. They showed a 1.7 fold increase in lipid peroxidation and a 2 fold increase in nitrated proteins in the SOD deficient mice at 20 months. There is evidence that oxidative stress is increased in the healthy aged brain however the level of oxidative stress is greatly increased in patients with neurodegenerative diseases such as PD.

Interestingly, for the case of synucleiopathies, α-syn expression is increased in response to oxidative stress. Quilty et al. (2006) showed that when mouse primary neocortical cells are incubated in the absence of antioxidants, neurons exhibit a higher

α-syn expression. After 10 days in culture these α-syn-positive (α-syn+) neurons had a statistically significant decrease in condensed nuclei, a marker of apoptosis. This was not supported by Kanda et al. (2000) who showed that in human SH-SY5Y cells, there was no significant difference in the viability of cells with WT α-syn over expression.

However, they showed that the A53T and A30P mutations were more susceptible to

H2O2 insult. When discussing oxidative stress in the brain, the enzymatic anti-oxidant glutathione (GSH) deserves mention. This enzyme is highly efficient in scavenging

ROS such as superoxide, hydroxyl radicals and peroxynitrites. The levels of this enzyme have been shown to be slightly reduced in aged in comparison to young brain, but greatly reduced in patients with mild cognitive impairment and AD [139]. This may explain the increased levels of oxidative stress in neurodegeneration.

Recent studies have assessed the role that changes in normal physiological neurochemistry, increased oxidative stress and a de-regulation of Ca2+ homeostasis, which collectively may contribute in the pathogenesis of synucleinopathies. It was concluded that potential age related changes in neurochemistry are sufficient to produce

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 40 protein aggregation of α-syn similar to those seen in pathological tissue. The most significant finding was that increased intracellular Ca2+ and oxidative stress work synergistically to generate a greater number of α-syn aggregates. This finding gives rise to a number of therapeutic opportunities that may be used to help alleviate the protein aggregation within the cell, and, delay the onset and progression of symptoms.

1.14 The Role of Ca2+ in the Neuron and Age Mediated Changes

Ca2+ plays many important roles in normal cellular function and is thought to be one of the most important cellular signalling molecules. It is involved in many processes such as apoptosis, metabolism, signal transduction, gene expression, proliferation, maturation, binding of neurotransmitter vesicle binding plasmalemma and cell death

[140]. As Ca2+ is such an important molecule the regulation of intracellular concentrations must be tightly regulated in order for proper functioning of the cell

[141]. This homeostasis is not only important between the cytoplasm and intracellular

Ca2+ stores such as the endoplasmic reticulum (ER) [142], but between the intracellular and the extracellular environment which can be up to 20,000 times more concentrated outside the cell. Thus, a current focus of research is to look at a possible link between

Ca2+ and α-syn aggregation and the role that this might play in the development of late onset synucleinopathies such as PD [143].

It has been established that resting intracellular Ca2+ levels remain the same despite the age of the neuron. Studies confirm this notion in which they show no difference between intracellular Ca2+ levels of young and aged neurons. However, the return duration to resting levels after a stimulus is greatly increased in aged neurons.

This lag in Ca2+ uptake/removal can cause problems within the cells due to the activation of lipases, proteases, kinases along with a host of other proteins in the cell, all

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 41 of which when present at the wrong time can wreak havoc on the cell, even resulting in the activation of cell death [140, 141].

The major sources of intracellular Ca2+ include Ca2+ influx through ligand-gated glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptor (NMDAR) or various voltage-dependent Ca2+ channels (VDCCs), as well as the release of Ca2+ from intracellular stores [144-146]. Clearance of Ca2+ after depolarisation is achieved either by intracellular Ca2+ binding which facilitates its uptake into the endoplasmic reticulum

(ER) or mitochondria or upon pumping into the extracellular space via the plasma membrane. The key pump involved in this process, Ca2+ ATPase (PMCA), is impaired in aged neurons [147]. The Ca2+ buffering capacity of neurons is decreased in ageing neurons [148, 149]. Some studies have shown that after stimulation, the concentration of Ca2+ may be decreased but the recovery to resting levels is dramatically increased.

Aged neurons also exhibit a decreased capacity to recover from Ca2+ stimulus through uptake into the intracellular Ca2+ stores with a decline in SERCA (sarco/endoplasmic reticulum PMCA) function [150]. The mitochondria is a second intracellular Ca2+ store and like the ER, the ability of this organelle to act as a reservoir for Ca2+ is also decreased with age due to a decreased activity of the mitochondrial Ca2+ uniporter

[151].

1.15 Increased intracellular Ca2+ induces α-synuclein oligomerisation

Recent studies have shown that a transient increase in the intracellular free Ca2+ concentration induced in cultured 1321N1 glioma cells by thapsigargin or Ca2+ ionophore chemical treatments caused a significant increase in the proportion of cells bearing microscopically-visible α-syn aggregates. It was also demonstrated that chelating free Ca2+ with 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 42

(BAPTA), resulted in no significant difference in the number of α-syn inclusions between control and CI/BAPTA cells. This indicates that raised intracellular free Ca2+ directly induces α-syn aggregate formation. Moreover, in vitro studies using recombinant forms of α-syn indicated that direct binding of Ca2+ ion to α-syn promoted rapid oligomer formation [152]. More recently, Follett et al (2013) demonstrated that

KCl depolarization of the plasma membrane in HEK293T and SH-SY5Y human cell lines resulted in raised intracellular free Ca2+ and α-syn aggregate formation under more physiologically relevant cellular conditions [153]. Both raised free Ca2+ and α-syn aggregation were blocked by BAPTA chelation treatment. KCl depolarization was observed especially to trigger formation of frequent large, LB-like perinuclear α-syn inclusion bodies.

Figure 5: Raised intracellular Ca2+ promotes α-syn aggregation [153].

It is hence possible to conclude that Ca2+ is a major factor concerning α-syn aggregation, and potentially in the formation of the cytotoxic oligomeric species as seen

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 43 in disease. This provides a potential therapeutic target by using drugs that modulate the amount of free Ca2+ in the cell. Ca2+ channel blockers, such as those from the dihydropyridines family, may be used to lessen the increase in intracellular Ca2+ seen in aged neurons. A step towards replicating the complex architecture of the CNS and assessing Ca2+ blockade was performed by Chan et al (2007) who used brain slices prepared from a MPTP mouse model for PD. They found that by using blocking L-type

2+ Cav1.3 Ca channels with Isradipine, a common drug used to treat high blood pressure, they could recover dopaminergic neural activity. This supports the earlier data of

Yamada et al. (1990) who found that dopaminergic neurons of the SnPc that were high in the Ca2+ binding protein (CBP) Calbindin D28k (CB) were preferentially spared in control brain sections compared with PD patients. This indicates that increased Ca2+ may be a major factor in the pathogenesis of α-synucleinopathies. Also, in a mouse model with Parkinsonian like pathological features, the loss of dopaminergic neurons, it was found that neurons expressing CB were spared from this pathological loss [155].

Consistent with this, Trimethadione (TMO), a Ca2+ channel blocker with broad selectivity, commonly used as an anti-epileptic drug, blocked K+ depolarization induced

Ca2+ influx into the cell resulting in loss of α-syn+ aggregate formation post- depolarization [153].

1.16 Oxidative stress and α-synuclein oligomerization

Another potential target for therapeutics could be to target oxidative stress in the brain. Avramovic et al. (2012) showed that dietary supplementation with Omega 3 fatty acids was capable of significantly increasing the levels of SOD and decreasing malondialdehyde activity in the aged brains of male Wistar rats. A similar study using

Sprague-Dawley rats showed that supplementation with stabilised docosahexaenoic acid

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 44 rich oil, but not eicosapentaenoic acid enriched oil, increased NOS activity in brain homogenates [157]. Importantly, WT α-syn has been shown to induce mitochondrial

NO when it is associated with mitochondria [158]. This indicates that not only will normal increases in oxidative stress cause aggregation but that aggregation of α-syn also induces more oxidative stress within the cell forming a positive feedback loop.

However, this is in contrary to other research which shows that α-syn protects cells from oxidative stress by inactivating the c-jun N-terminal kinase (JNK) pathway, although this data was from cells challenged with exogenous H2O2 [159]. The combination of oxidative stress and α-syn expression has been used to generate a model of MSA in mice, whereby the over-expression of α-syn in glial cells is combined with 3- nitropropionic acid. Treatment to induce mitochondrial oxidative stress was sufficient to induce MSA like pathology. Results obtained by Radford et al. (2015) also support the notion that a combination of oxidative stress and raised Ca2+ could also influence glial cells. It promotes the glial α-syn pathology observed in MSA and offers the potential for an anti- Ca2+/antioxidant combination therapy in this disease.

There is further evidence that oxidative stress is increased in the normal aged brain, however the level of oxidative stress is greatly increased in patients with neurodegenerative diseases [137]. Moreover, Kume et al. (2012) found urinary 8-Oxo-

2'-deoxyguanosine levels were significantly higher in DLB cases compared to controls, indicating systemically increased oxidative stress. The major contribution to oxidative stress in ageing primates originates from mitochondrial complexes I and III of the electron transport chain leading to greater mitochondrial DNA damage compared to nuclear DNA damage [136]. Indeed, widespread mitochondrial DNA damage occurs at early stages of DLB [161]. Oxidative stress characterized by α-syn lipoxidation precedes the formation of α-syn aggregates and the development of neocortical LB

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 45 pathology in DLB [162]. Furthermore, Quilty et al. (2006) showed that when mouse primary neocortical cells were incubated in the absence of antioxidants, mild oxidative stress caused α-syn accumulation in a subset of neurons. Indeed, recent studies have found the oxidized form of the endogenous oxidative stress sensor, DJ-1, progressively increased in the later stages of PD and more highly oxidized forms were likely present in DLB [163]. Furthermore, it has been demonstrated that Ca2+ influx can interact with

α-syn to mediate increased oxidative stress [164] and a synergistic effect of combined oxidative stress and raised intracellular free Ca2+ on α-syn aggregation [110].

Recent studies have examined the role of oxidative stress in the formation of potentially toxic α-syn oligomeric species in conjunction with Ca2+ binding. It was found that although there was no significant difference in the number of α-syn aggregates between cells, however, when treated with Ca2+ ionophore or thapsigargin and H2O2 in combination, there was a dramatic increase in the number of α-syn aggregates per cell. From these experiments using H2O2 to induce ROS in 1321N1 cells, in combination with raised intracellular free Ca2+, significantly increase intracellular α- syn aggregation resulted. This was also reflected in in vitro experiments which showed that the combination of Ca2+ treatment and oxidation of recombinant α-syn monomers caused the formation of stable, oligomeric α-syn aggregates, indicating a cooperative interaction between Ca2+ binding to α-syn and α-syn side-chain oxidation [110].

1.17 α-Synuclein oligomerization induces raised Ca2+ and oxidative stress

Hettiarachchi et al. (2009) demonstrated than elevated levels of intracellular α- syn have been shown to elevate levels of intracellular Ca2+. Secreted α-syn induces increase in capacitive Ca2+ entry in differentiated SH-SY5Y [166]. A study investigated the Ca2+ dynamics in transgenic mice expressing human WT α-syn showed that α-Syn

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 46 transgenic mice exhibited augmented, long-lasting Ca2+ transients characterized by considerable deviation from the exponential decay. Furthermore control and α-syn knock out groups demonstrated low percentages of neurons with Ca2+ abnormalities whereas the α-syn transgenic group showed Ca2+ response alteration, suggesting these alterations are related to α-syn expression [167].

Other studies have shown that α-syn overexpression augments mitochondrial

Ca2+ transients by enhancing ER-mitochondria interactions. α-Syn with acidic C- terminal domain overexpression increases mitochondrial Ca2+ in SH-SY5Y and HeLa cells. Moreover, treatment with naturally secreted α-syn increases Ca2+ entry in primary rat cortical neurons and induces mitochondrial Ca2+ uptake. Significantly higher levels of mitochondrial Ca2+ content in α-syn treated cells were observed compared to control cells [168].

Dryanovski et al. (2013) found that in dopaminergic neurons bearing inclusions, mitochondrial oxidant stress levels were higher in the soma and proximal dendrites than in neurons without inclusions. Treatment with isradipine significantly diminished the oxidative stress levels in CB-negative (CB−) dopaminergic neurons when compared to

CB-positive (CB+) dopaminergic neurons. This suggests that the formation of α-syn inclusions stimulates ROS production in the cytosol. Buttner et al. (2013) established that α-syn provokes an elevation of cytosolic Ca2+ in yeast that coincides with an increase in oxidative stress. This suggests that α-syn aggregation leads to an increase in mitochondrial Ca2+transient and then to oxidative stress. WT α-syn has also been shown to induce mitochondrial NO when it is associated with mitochondria [158]. This demonstrates that normal increase in oxidative stress can cause aggregation and that aggregation of α-syn also can induce further oxidative stress within the cell forming a positive feedback loop. Koch et al (2016) study indicates that α-syn fibrils increase

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 47

SOD1 aggregation in vitro and in vivo. A more recent study has shown that the antioxidant capacity of SH-SY5Y cells was decreased by alterations in SOD1 and

SOD2 enzyme activity and a decrease in gluthathione levels following the overexpression of α-syn [171] (Perfeito et al., 2016).). Though, this is contrary to other research which shows that α-syn protects cells from oxidative stress by inactivating the

JNK pathway [159]. Furthermore, Navarria et al (2015) described how overexpression of α-syn reduces NMDAR-mediated Ca(II) influx.

Figure 6: Mechanism of α-syn aggregation induced by raised Ca2+ and oxidative stress. (Store-operated channels (SOC), Receptor operated channels (ROC), Na+-Ca2+ exchanger (NCX))

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 48

1.18 Synergistic effect of Ca2+ and oxidative stress

Recent findings have indicated that increased intracellular free Ca2+ and oxidative stress work synergistically to induce α-syn aggregation. It has been shown that a dramatic increase in the number α-syn+ protein aggregates occurs in glial cells when oxidative stress and raised Ca2+ occur simultaneously. Moreover, recombinant protein experiments demonstrated that Ca2+ binding alone was able to induce non-stable α-syn

2+ aggregates, which could be stabilised via H2O2 treatment. Co-treatments with Ca and oxidant resulted in the formation of larger, stable aggregates. This may be extremely important in the pathogenic mechanisms behind α-syn aggregation and α- synucleinopthathy disease progression. Wood et al. (1999) showed that the fibrillation of α-syn is highly dependent on nucleation centres, showing that the addition of 1% pre- aggregated α-syn dramatically increased the rate of α-syn aggregation. Furthermore, kinetic studies of α-syn aggregation by Nath et al (2010) were consistent with an auto- catalytic mechanism. Thus, Ca2+/oxidation stabilized α-syn aggregates which serve to increase nucleation centres in disease. This is supported by Krishnan et al. (2003) that show that di-tyrosine cross linked α-syn dimers were the rate limiting step in the fibrillation process in forming nucleation centres. Importantly, this indicates that Ca2+ influx into neurons can induce oxidative stress in the mitochondria of mouse dopaminergic neurons [174]. This group also showed that oxidative stress induced by

Ca2+ influx was exacerbated in DJ-1 mutant mice.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 49

Figure 7: Synergistic effect of α-syn aggregation, Ca2+ regulation and oxidative stress (positive feedback loop).

1.19 Voltage-Gated Ca2+ Channels in the Brain

Ca2+ regulation is vital to the health and functioning of cells. Although N-type

Ca2+ channels are the primary regulators of Ca2+ influx and corresponding neurotransmitter release in neurons [175] [176], evidence highlights the broader range

2+ of Ca channels found throughout the CNS. There are three types of Cavs [177].

Firstly, there are four subtypes of Cav1 channels, each are L-type configuration.

Cav1 channels can be found in several areas of the brain as well other cells outside of the brain, such as cardiac myocytes, retinal rod cells, and smooth muscle myocytes

[177].

Secondly, there are three types of Cav2 channels and which are distributed throughout the brain. The Cav2 channel subtypes are P/Q, N, and R respectively. Cav2.1

2+ and Cav2.2 are responsible for the Ca involved in neurotransmitters release. Inhibition

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 50 of Cav1, 2.1, and 2.2 channels, separately, have each been shown to decrease neuronal damage upon injury [178-180]. This implication is particularly important to note given that traumatic brain injury is related to an increase in the expression of α-syn, α-syn aggregation, and Parkinson’s-like pathology [181].

Thirdly, the family of low-voltage activated T-type channels (Cav3) is comprised of three distinct members: Cav3.1, Cav3.2 and Cav3.3 [177]. The physiological importance of T-type Ca2+ channels is they are predominantly found in neurons [182,

183], where they contribute to the pathogenesis of epilepsy [184], but have also been reported in glial cells and the pacemaker cells of the myocardium [185]. Moreover, neurological pathologies associated with T-type Ca2+ channels, such as epilepsy, require selective pharmacological blockade to regulate and maintain Ca2+ homeostasis. Thus,

Wildburger et al (2009) investigated the neuroprotective properties of the T-type Ca2+ channel blocker, TMO, in cultured hippocampal and cortical neurons from C57BL/6 and α1H -/- mice. The results indicated a significant decrease in the levels of lactate dehydrogenase representing cell death following TMO treatment [186]. Follett et al.

(2013) showed that SH-SY5Y human neuroblastoma cells undergo membrane depolarization induced by KCl, resulting in Ca2+ influx and in turn stimulate the formation of alpha-synuclein aggregates. They also found that such α-syn positive cytoplasmic aggregates could be blocked by TMO pre-treatment.

1.20 Ca2+ buffering proteins in the brain

Since the regulation of Ca2+ is vital for cell survival and function, it is not surprising that the process is tightly regulated. There are a number of proteins that are capable of Ca2+ buffering in neurons known as CBP. CB, calretinin (CR) and parvalbumin (PV) are three cytosolic CBP that are capable of Ca2+ buffering in neurons.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 51 In a study conducted by Bu et al. (2003) with human brain tissue, they found a decrease in both CR and CB in aged compared to young cortical neurons, but no difference in PV positive neurons. CBP are intracellular Ca2+ acceptors that belong to two different families: the EF-hand proteins and the annexins. The annexin family is characterized by proteins that bind Ca2+ in the presence of phospholipids-containing membranes. The former family consists of proteins showing a conserved structure in the Ca2+-binding domain called the EF-hand, which is a stretch of amino acids forming a typical helix- loop-helix structure [188]. The EF-hand family of CBP contains about forty known

Ca2+-regulated proteins, of which several are found in the CNS. The EF-hand proteins may function either as triggers, starting a cascade of reactions or as Ca2+ buffers, decreasing the free cytoplasmic concentration of this ion [189]. The prototype of a trigger protein is the ubiquitous calmodulin that activates at least twenty different enzymes. The buffer proteins, such as PV, CB, and CR, represent a more passive system responsible for decreasing the amplitude of Ca2+ signals. The importance of these proteins has been implicated by CR which has an important role as a modulator of neuronal excitability including the induction of long term potentiation. Loss of expression of CR in hippocampal interneurons has been suggested to be relevant in temporal lobe epilepsy.

The importance of these proteins has been implicated by German et al. (1992) who studied the brains of human patients with PD, MPTP monkey or in C57BL/6 mice.

They found that in both idiopathic PD and in the above models, neurons that contained

CB were spared while neurons in CB- regions were lost. Tsuboi et al. (2000) and Kim et al. (2000) found that CR expression in dopaminergic neurons of the SnPc exhibited higher protection against 6-hydroxydopamine [191, 192]. Lysosomes, aside from their role in the recycling of proteins, also act as intracellular Ca2+ stores. Using glycyl-L-

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 52 phenylalanine-beta-naphthylamide (GPN) to permeabilise lysosomes Haller et al.

(1996) showed they act as Ca2+ stores capable of a 60nM to 2mM increase in intracellular Ca2+, the homeostasis of which also can be affected by LRRK2, a gene involved in familial PD.

1.21 Calbindin D28K

Recently interest has been shown in neuronal cells in regards to their Ca2+ buffering, Ca2+ chelators and Ca2+-dependent proteases, especially due the established relationship between Ca2+ and PD aetiology [194].

Figure 8: NMR solution superimposition of the ten lowest-energy structures of Ca2+-loaded Calbindin-D28K, (CB) showing representative pair-pair interactions between EF1 (Red)-EF2 (Orange) and EF3 (Yellow)-EF4 (green) [195]

The functional significance of CBP has only started to be recently understood.

This is due to their complex interplay with other Ca2+ controlling mechanisms and the inherent technical difficulties in studying biophysical properties of individual proteins

[196]. Buffers are characterized by more or less specific binding/chelating of Ca2+

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 53 without further Ca2+-dependent target interactions. Their function is in the control of the spatio-temporal extent of Ca2+ signalling domains [197].

CB is a CBP and functions as a buffer for cytosolic Ca2+ which may stimulate a membrane Ca2+-ATPase and a 3',5'-cyclic nucleotide phosphodiesterase. CB has six EF- hands, of which two are non- metal binding [195, 198, 199]. EF-2 and EF-6 are considered non-metal binding domains [198, 200], while EF-1, 3, 4, 5 are mixed metal binding sites. However, these sites have a much higher affinity for Ca2+ [201] than for

Mg2+ [199] and especially under normal physiological conditions, CB functions predominantly as a Ca2+ buffer. CB makes a major contribution to the total buffer capacitance in neurons [202]. Lack of CB can result in alteration of motor coordination which is consistent with the strong CB expression in cerebellar cortex and its impact on synaptically mediated Ca2+ transients [203, 204].

The importance of Ca2+ buffering in neurons, especially of that localised to the

SnPc has been demonstrated. CB+ dopaminergic neurons are relatively spared in the PD brain compared to CB- neurons [205], suggesting that Ca2+ buffer channels may be protective against neurodegeneration. Yamada et al (1990) also demonstrated the importance of Ca2+ buffering in neurons localized to the SnPc. Dopaminergic neurons contained in the mediodorsal region of the SN were found to harbour the Ca2+ buffering protein, CB, whereas dopaminergic neurons isolated from the ventrolateral SN were found to lack CB. This evidence led to the hypothesis that CB+ dopaminergic neurons are spared, as opposed to CB- dopaminergic neurons being destroyed in PD cases [194].

However, no current evidence exists investigating a decrease, if any, in CB levels in aged patients.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 54 Guo et al. (1998) showed that amyloid-β-induced (Aβ) elevations of Ca2+ and

ROS are blocked by CB overexpression. Overexpression of CB prevents apoptosis in cultured neural cells expressing mutant PS-1, a gene encoding for presenilin (L286V and M146V mutations). CB-overexpressing cells showed to supress the elevated levels of intracellular Ca2+ concentration and decrease the generation of ROS induced by Aβ.

CB was shown to be upregulated by the hormonal form of vitamin D,

1,25OH2D3 Calcitriol, in cytotrophoblasts [208]. The potential of CB to be induced by vitamin D could be used to prevent or stop PD or DLB.

1.22 Vitamin D and the brain in Parkinson’s disease

Major metabolites of vitamin D were found to be present in human cerebrospinal fluid which include calcitriol, 25OHD3 (Calcifediol) and 24,25OH2D3 (24,25-

Dihydroxycholecalciferol) [209]. As other neurosteroids, vitamin D metabolites have been shown to be able to cross the blood brain barrier [210]. Two enzymes involved with the conversion of 25OHD3 to 1,25OH2D3 and 1,25OH2D3 to 24,25OH2D3

(CYP27B1 and CYP24A1, respectively) are present in the brain; therefore blood brain barrier (BBB) permeability may not be essential.

Figure 9: Vitamin D3 metabolizing pathway

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 55 The major limitation surrounding the delivery of therapeutics to the brain is due to the impermeability of the BBB. The enzyme, CYP27B1, has been identified in embryonic neural and glial cells throughout the adult human brain [211-213]. Eyles et al. (2005) found CYP27B1 to be present in both neurons and glial cells. It was most strongly expressed in the SN and the supraoptic and paraventricular nuclei of the hypothalamus. These results point towards the potential of the brain to synthesize the active metabolite 1,25OH2D3. Furthermore, Naveilhan et al. (1993) found the expression of CYP24A1 mRNA was increased in rat primary glial cell culture, in a dose-dependent manner upon addition of 1,25OH2D3, indicating that levels of active

1,25OH2D3 can also be reduced in the brain.

Further research has found that administration of 1,25OH2D3 protects against damage from a neurotoxin that specifically lesions dopaminergic and noradrenergic cells, 6-hydroxydopamine (6OHDA), [216, 217]. Furthermore, the administration of

1,25OH2D3 has found to be protective against neurotoxic doses of methamphetamine by preserving dopamine and serotonin levels [218]. Such neuroprotection of dopaminergic neurons may be relevant to the loss of dopaminergic neurons in the SN in PD. In addition, some investigations have linked vitamin D insufficiency with an increased risk of PD [219, 220]. Moreover, higher levels of 25OHD3 were associated with a reduced risk of developing PD in later life [221]. Indeed, several investigations have linked vitamin D insufficiency with an increased risk of PD [219, 220]. Moreover, in a large patient cohort study, higher levels of 25OHD3 were associated with a reduced risk of developing PD in later life [221]. Abnormalities in the vitamin D receptor have also been linked with a risk of developing PD [222]. On the other hand a recent study by

Shrestha et al. (2016) have found an association between serum 25-hydroxyvitamin D

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 56 concentration in mid-adulthood and PD risk, which suggested that vitamin D might not help reduced the risk of PD.

1.23 Challenges for future therapeutics

A major area of concern for PD treatment is early detection, as when PD symptoms first manifest, 50% of dopaminergic signalling is lost [224]. At this stage, the

α-syn protein is readily secreted from the neurons through exosomes where it is then taken up by other cells within the CNS. This ultimately leads to its spread to neighbouring neurons, exacerbating cell death [129]. A number of non-invasive detection methods have recently been used in early diagnosis, such as a smell test to detect hyposmia, or speech analysis. Biomarkers have also been investigated in which

α-syn has been found in the gut of PD patients. Transcranial sonography and 18-Fluoro- dopa positron emission tomography have also been used to probe for potential biomarkers [225].

Figure 10: PET scan from control and patient subject showing high uptake of 18-fluoro-dopa in the striatum [226].

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 57

Due to the spread of α-syn between neurons [130] it might have access to the extracellular space; α-syn has been seen in detectable amounts in the blood, cerebrospinal fluid [227] and saliva [228]. Some tests also differentiate between monomeric and oligomeric species, and the Phospho-129 post translational modification has been associated with LBs. Other biomarkers also tested for PD association are neurofilaments, interleukins, osteopontin and hypocretin, plasma ApoA1, cerebrospinal fluid Aβ, plasma urate (antioxidant).

Targeting free Ca2+ in neurons could be another potential target for future treatments of PD or DLB. However Ca2+ lowering medications such as anti-epileptic drugs have significant side effects, as they act directly on Ca2+ channels. Selectively targeting raised intracellular free Ca2+ in the brain could be used as a powerful neuroprotective strategy which will depend on the development of reliable inducement of CBP through drugs or gene therapy.

1.24 Aims

Ca2+ regulation has been shown to influence the progression of neurodegeneration in PD. Findings show the over-representation of cells positive for the

CBP, CB, amongst surviving neurons in PD and hence is a key protein to study in order to understand the pathophysiology of PD. Upon examination of human diseased tissue from DLB which shares some common pathological features to PD can provide additional information. Direct unilateral injection of the pesticide, rotenone, a mitochondrial complex 1 inhibitor that causes oxidative stress, provides a facile mouse model of α-synucleinopathies which permits a direct comparison between the two brain hemispheres in each lesioned animal studied as the untreated hemisphere serves as a

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 58 control. Thus, the influence of CB on neuronal survival in response to an oxidative stress lesion can be examined pair-wise between hemispheres of individual α- synucleinopathy mouse animals, thereby reducing variability and permitting a relatively small sample studied. Complementary to animal model studies, cell culture studies using a dopaminergic cell line, the SH-SY5Y human neuroblastoma cell line, have previous demonstrated the ability to provide quantitative data on the interactive between

Ca2+, CB and α-syn aggregation [110, 152, 153].

The overall objective for this study was to determine the relationship between

CB, α-syn aggregation and neurodegeneration. Experiments investigating human DLB disease tissue, a rotenone lesion mouse model and neuroblastoma cell culture model were combined to provide complementary data regarding DLB and PD pathogenesis. It was expected that neurons with efficient Ca2+ buffering would show a greater rate of survival than those that poorly regulate Ca2+. As such, the specific aims of this project were:

1.24.1 Aim one: To investigate the proportion of CB and α-syn aggregates positive neurons in DLB and healthy, non-DLB tissue. It is therefore proposed to determine whether there is a correlation between CB expression, α-syn aggregation and neuronal survival in this disease. In human tissue, this was investigated through the use of immunofluorescence and subsequent visualisation through fluorescence microscopy.

Primary antibodies for CB and α-syn we used in conjunction with fluorescently-labelled secondary antibodies in order to observe co-localisation. Digital fluorescent images were then analysed by cell counting to quantify the incidence of α-syn+ and CB+ neurons among cell populations in DLB and normal tissue in specific brain regions as well as any correlation with neurodegeneration.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 59 1.24.2 Aim two: To investigate the proportion of CB and neurons positive for α- syn aggregates in a mouse model of α-synucleoinpathy. Immunofluorescence experiments were performed on tissue sections obtained from the unilateral rotenone- treated aged and young mice cohorts. Primary antibodies for CB and α-syn were used in conjunction with fluorescently labelled secondary antibodies in order to observe co- localisation. Digital fluorescent images were then analysed and cell counting were performed to quantify the incidence of α-syn+ and CB+ neurons among cell populations as well as any correlation with neurodegeneration.

To investigate the total level of expression of CB in PD mouse model, the CBP,

CB, expression of which results in relative sparing of CB+ neurons in PD, was investigated by Western blot (WB) analyses in brain tissue homogenates obtained from the unilateral rotenone-treated mice in aged and young adult cohorts. PAGE was used to separate the proteins in the brain homogenates which were then analysed by WB.

1.24.3 Aim three: To investigate the correlation between the overexpression of

CB and α-syn aggregation in SH-SY5Y cells. Previous studies showed that cells that undergo membrane depolarization induced by KCl, result in Ca2+ influx and that α-syn cytoplasmic aggregates form 36 hours post-depolarization. We hypothesized that this

Ca2+ dependent aggregation could be blocked by the overexpression of the CBP, CB.

We increased CB expression upon treating the cells with Calcipotriol (Cp) (vitamin D

Analogue) in order to conduct quantitative cell counting experiments. CB expression was measured using WB analyses and immunofluorescence. Digital fluorescent images were then analysed to quantify the incidence of α-syn aggregates as Cp concentration increased.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 60 1.25 Hypotheses

Experiments were conducted to test the following specific hypotheses:

1.25.1 Aim one: Investigation of the proportion of CB and α-syn aggregates positive neurons in DLB and healthy, non-DLB tissue

First hypothesis

- Is there a difference in frequency of CB+ in DLB compared to normal case groups?

H0: There is no difference in the proportion of CB+ neurons between DLB and normal groups.

H1: There is a difference in the proportion of CB+ neurons between DLB and normal groups.

Second hypothesis

- Is there a difference in frequency of α-syn aggregates in DLB compared to normal case groups?

H0: There is no difference in the proportion of α-syn aggregates positive cells between DLB and normal groups.

H1: There is a difference in the proportion of cell positive for α-syn aggregates between DLB and normal groups.

Third hypothesis

- Is the frequency of α-syn aggregates similar in CB+ and CB- neurons (DLB/control tissue)?

H0: There is no difference in the amount/levels of α-syn aggregation between the CB+ and CB- neurons.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 61

H1: There is difference in α-syn levels between the CB+ and CB- neurons.

1.25.2 Aim two: Investigation of the proportion of CB and neurons positive for α-syn aggregates in a mouse model of α-synucleinopathy

First hypothesis

- Is there a difference in frequency of CB+ in lesioned compared to non-lesioned and

Sham case groups?

H0: There is no difference in CB levels between the two hemispheres.

H1: There is a difference in CB levels between the treated and non-treated hemispheres

Second hypothesis

- Is there a difference in frequency of α-syn aggregates in lesioned compared to non- lesioned and Sham case groups?

H0: There is no difference in the proportion of α-syn aggregates positive cells between lesioned compared to non-lesioned and Sham case groups.

H1: There is a difference in the proportion of cell positive for α-syn aggregates between lesioned compared to non-lesioned and Sham case groups.

Third hypothesis

- Is the frequency of α-syn aggregate similar in CB+ and CB- neurons?

H0: There is no difference in α-syn aggregation between the CB+ and CB- neurons.

H1: There is a difference in α-syn levels between the CB+ and CB- neurons.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 62 1.25.3 Aim three: Investigation of the correlation between the overexpression of CB and α-syn aggregation in SH-SY5Y cells

First hypothesis

- Does Cp treatment of SH-SY5Y human neuroblastoma cells increases levels of

CB?

H0: There is no difference in CB levels after Cp treatment.

H1: There is a difference in CB levels after Cp treatment.

Second hypothesis

- Is α-syn aggregation modified after Cp treatment in SH-SY5Y neuroblastoma cells?

H0: There is no difference in the proportion of α-syn aggregates positive cells after Cp treatment

H1: There is a difference in the proportion of α-syn aggregates positive cells after Cp treatment.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 63 1.26 Significance

In Australia, 245,000 people are diagnosed with neurodegenerative diseases, costing 5.4 billion dollars per annum. This number is predicted to rise to 1.13 million individuals by 2050 [229], many of which will be suffering from PD as it affects 2% of the population over 65 years old of age. The relentless and devastating nature of PD is apparent as it has no treatment and cannot be prevented, reversed nor slowed [230]. The grand magnitude of PD has inevitably made it a burden on our society affecting healthcare systems, families and patients. Prevention and subsequent treatment of this disease would save billions of dollars in healthcare and insurance, and prevent premature loss of family members and friends.

In Australia, 353,800 Australians live with dementia and sadly, this number is expected to increase to 400,000 in less than five years and expected to be over 900,000 in 2050. DLB accounts for 15 to 30% of those dementias. By the 2060s, spending on dementia is set to exceed that of any other health condition. The financial burden on our economy and healthcare systems is predicted to be $83 billion, and will represent around 11% of health and residential aged care sector spending.

Even though there are many speculations on what causes PD and DLB, most of the aetiologies are unknown, and it is widely accepted that it can be either genetically inherited or sporadic [2].Further research is imperative to fully understand PD and DLB aetiologies.

Currently, PD and DLB aetiology is not fully known, as is the molecular progression of neurodegeneration. The role of Ca2+ regulation has not definitively been linked to PD and DLB, however the role of Ca2+ buffering proteins such as CB and their

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 64 presence in neurons selectively spared in diseased states has stimulated this research.[194]. A better understanding of this protein will allow a much needed leap forward in the understanding of PD and its link to Ca2+ regulation.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 65 Chapter 2: Interactions between Calcium and Alpha-Synuclein in

Neurodegeneration

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 66 STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED PAPER

This chapter includes a co-authored paper. The bibliographic details published for publication of the co-authored paper, including all authors, are:

Alexandre Rcom-H’cheo-Gauthier

Dr Jacob Goodwin

Dr Dean Pountney

My contribution to the paper involved:

These authors contributed equally to this work

Rcom-H'cheo-Gauthier, A., Goodwin, J., Pountney, D.L. Interactions between Calcium and Alpha-Synuclein in Neurodegeneration. Biomolecules 2014, 4,795-811, doi:10.3390/biom4030795.

(Signed) ______(Date)______

Alexandre Rcom-H’cheo-Gauthier

(Countersigned)______(Date)__16/08/2016_

Corresponding author of paper: Dr Dean Pountney

(Countersigned) _ __ (Date)_16/08/2016___

Supervisor: Dr Dean Pountney

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 67 Biomolecules 2014, 4, 795-811; doi:10.3390/biom4030795 OPEN ACCESS biomolecules ISSN 2218-273X Review www.mdpi.com/journal/biomolecules/ Interactions between Calcium and Alpha-Synuclein in Neurodegeneration

Alex Rcom-H’cheo-Gauthier †, Jacob Goodwin † and Dean L. Pountney * Griffith Health Institute, School of Medical Science, Griffith University, Gold Coast, Queensland 4222, Australia; E-Mails: [email protected] (A.R.-H.-G.); [email protected] (J.G.)

† These authors contributed equally to this work.

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +61-7-5552-7273; Fax: +61-7-5552-8908.

Received: 26 March 2014; in revised form: 25 June 2014 / Accepted: 14 July 2014 / Published: 14 August 2014

Abstract: In Parkinson’s disease and some atypical Parkinson’s syndromes, aggregation of the α-synuclein protein (α-syn) has been linked to neurodegeneration. Many triggers for pathological α-syn aggregation have been identified, including port-translational modifications, oxidative stress and raised metal ions, such as Ca2+. Recently, it has been found using cell culture models that transient increases of intracellular Ca2+ induce cytoplasmic α-syn aggregates. Ca2+-dependent α-syn aggregation could be blocked by the Ca2+ buffering agent, BAPTA-AM, or by the Ca2+ channel blocker, Trimethadione. Furthermore, a greater proportion of cells positive for aggregates occurred when both raised Ca2+ and oxidative stress were combined, indicating that Ca2+ and oxidative stress cooperatively promote α-syn aggregation. Current on-going work using a unilateral mouse lesion model of Parkinson’s disease shows a greater proportion of calbindin-positive neurons survive the lesion, with intracellular α-syn aggregates almost exclusively occurring in calbindin-negative neurons. These and other recent findings are reviewed in the context of neurodegenerative pathologies and suggest an association between raised Ca2+, α-syn aggregation and neurotoxicity.

Keywords: alpha-synuclein; Parkinson’s disease; calcium; multiple system atrophy; neurodegeneration; oxidative stress

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 68

Biomolecules 2014, 4 796

1. Introduction

1.1. Neurodegeneration and α-Synuclein

Under normal cellular conditions, proteins have a stable fold that is appropriate to perform their biological function. In general, misfolded proteins that cannot be refolded are degraded by the ubiquitin proteasome system (UPS), or via lysosomal degradation. In disease, however, the misfolded protein may be resistant to degradation and undergo aggregate formation. Abnormal aggregation of the protein α-synuclein (α-syn) has been implicated in a number of neurological diseases, including Parkinson’s disease (PD), characterised by cytoplasmic aggregates in multiple cell types throughout the central nervous system. Collectively, these diseases are termed α-synucleinopathies and include PD and the atypical Parkinson’s syndrome, multiple system atrophy (MSA). In this report, key features of PD and MSA will be reviewed, two diseases that characterize the range of cell types affected by α-syn aggregates. Moreover, mechanisms of and factors influencing α-syn aggregation in the CNS will be discussed, with a special emphasis on the role of Ca2+ interactions [1–3].

1.2. Parkinson’s Disease and α-Synuclein

PD has progressive clinical symptoms, including slight weakness, tremors, forward posture, sleep disturbance, constipation, the inability to walk unaided, speech impairment, difficulty swallowing, tremor, loss of urinary and gastrointestinal control and extreme exhaustion. The three main symptoms of PD are bradykinesia, or a slowing of voluntary controlled movement, rigidity, and tremor. Pathological examination of brain tissues from PD sufferers shows that there is a loss of dopaminergic neurons in the Substantia nigra (SN), a region of the brain that, through neural connections with the striatum, is responsible for controlled muscle movements. PD can be classified into two main groups: idiopathic and familial. Idiopathic PD, the larger of the two groups, accounts for roughly 85%–90% of all PD cases. Familial PD accounts for the remaining 10%–15% of cases and is due to mutations in a number of genes, including SNCA (PARK1/4), the gene responsible for the expression of α-syn. To date, five PD-linked point mutations in SNCA, have been identified [1], comprising the A30P [4], A53T [5], E46K [6], G51D [7] and H50Q [8] amino acid substitutions that disrupt the neurotransmitter vesicle binding domain (see Figure 1). The A53T and A30P mutations also affect the response to oxidative stress with expression of these mutant isoforms significantly increasing cytotoxicity induced by hydrogen peroxide and 1-methyl-4-phenylpyridinium (MPP+) in comparison to cells expressing wild-type α-syn and control cells [9]. Moreover, the A30P, A53T and H50Q mutations result in increased oligomerization and fibril formation compared to wild-type [10,11]. Furthermore, gene duplication [12] and triplication of α-syn [13] have also been found in familial PD, implicating gene dosage effects in pathogenesis.

1.3. Parkinson’s Disease and Environmental Factors

Environmental factors, such as exposure to pesticides and insecticides, may play a role in the pathogenesis of idiopathic PD. Rotenone, 1-methyl-4-phenylpyridinium (MPP+) and Paraquat have all been linked to PD [14,15]. These chemicals generate oxidative stress through the inhibition of complex I of the mitochondrial electron transport chain (rotenone, MPP+) or by acting as a general inducer of The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 69

Biomolecules 2014, 4 797 reactive oxygen species (ROS) (paraquat). Interestingly, two environmental factors have the opposite affect and are protective for PD. Cigarette smoking and caffeine have both been shown to be protective [16], and while the mechanism of caffeine protection is unclear the caloric restriction associated with smoking with a subsequent decrease in metabolic oxidative stress may be a factor.

Figure 1. α-Synuclein protein (α-syn) domain structure. α-Syn contains three putative domains. KTK repeats in the N-terminus are involved in lipid interaction, the hydrophobic NAC domain is important for aggregation and the C-terminal Ca2+ binding site can increase the rate of oligomerization [1,2]. The Parkinson’s disease (PD)-linked point mutations are indicated within the neurotransmitter vesicle binding domain.

1.4. Pathology of Parkinson’s Disease and Multiple System Atrophy

A characteristic pathological feature of PD is a severe loss of the neuromelanin-positive dopaminergic neurons of the SN, located in the midbrain. Signalling between the SN and the striatum is involved in controlling muscle movements; therefore the resultant loss of nigro-striatal pathway signalling can explain the classical symptoms of PD. Of the neurons that remain, large protein aggregates called Lewy bodies (LB) are often observed. While LB’s are made up of numerous proteins, including proteasome components, lysosome components and chaperone proteins, α-syn immunoreactivity defined α-syn as a major component of the LB in PD and in dementia with Lewy bodies [1,17]. Multiple system atrophy (MSA) is also late onset, neurodegenerative, idiopathic and progressive [3]. However unlike PD, where aggregates form in neuronal cells, α-syn positive protein inclusions are formed within the cytoplasm of oligodendrocytes and are therefore termed glial cytoplasmic inclusions (GCI). MSA also differs from PD in the distribution of these cytoplasmic aggregates throughout the central nervous system where in addition to the SN, the locus coeruleus, putamen, inferior olives, pontine nuclei, Purkinje cells and the intermediolateral columns of the spinal cord are also affected. Classified into two subgroups, MSA-P (Parkinsonian) is characterized by GCIs within the SN, which results in the Parkinsonian symptoms, whereas, MSCA-C (cerebellar ataxia) affects the cerebellum and results in gait and limb ataxia and oculomotor disturbances. In each sub-type, the severity and type of symptoms is dependent on the distribution and density of α-syn inclusions. MSA has no strong genetic link, although a number of single nucleotide polymorphisms, including the SNCA gene, have been

Biomolecules 2014, 4 798 identified with an increased risk of MSA [18]. A number of studies have looked at environmental and other risk factors for MSA, although with no consensus findings [19,20].

1.5. Properties of α-Synuclein

α-Syn is a 14 kDa protein encoded by the SNCA gene that is highly conserved in vertebrate species. Although the exact role of α-syn remains unclear, the protein is primarily expressed in the olfactory bulb, frontal cortex, striatum and the hippocampus with lower expression levels also observed in the hypothalamus, thalamus, midbrain, cerebellum and pons [21] where it localises to presynaptic terminals of dopaminergic neurons [22,23]. It is believed to be involved in neurotransmitter vesicle recycling and dopamine neurotransmission through interaction with soluble NSF attachment protein receptor (SNARE) [24]. Three putative domains of the α-syn protein have been identified (see Figure 1). The N-terminal domain comprises seven 11 amino acid imperfect repeat sequences, predicted to form aliphatic helices (KTK repeats) allowing α-syn to associate with lipid membranes. The C-terminal domain contains a number of acidic residues identified as a Ca2+ binding site [25]. The third domain (NAC domain) contains hydrophobic amino acids and is important for aggregation [26]. The conformation of α-syn is highly dependent on environmental conditions. In the aqueous cellular environment, α-syn adopts a random coiled structure, but adopts a helical conformation upon binding to acidic phospholipid vesicles [27–29]. α-Syn is also prone to nucleation dependent aggregation [30] through the N-terminus [31] and this aggregation, inhibited upon membrane binding, transforms α-syn from the random coiled conformation to beta-pleated sheets [32,33]. Deletion of the hydrophobic 12 aa central region of α-syn results in the loss of α-syn aggregation and the hydrophobic 12 aa region alone is sufficient to form aggregates. The α-syn protein has been shown to interact with membranes [34] and both the N- and C- terminus of the protein can be bound to membranes. This membrane interaction, and the function as a SNARE associated protein is mediated by Rab3a [35]. Gene triplication and duplication of SNCA in autosomal dominant forms of PD suggests the importance of α-syn gene dosage and protein concentration in aggregation. Interestingly, mouse studies have shown that there is an age-dependent decline in both α-syn protein and mRNA levels [36]. Whereas, analysis of single dopaminergic neurons from tissue of PD affected individuals and controls have revealed that there is a significant increase in α-syn mRNA levels [37].

1.6. α-Synuclein Oligomerization and Cytotoxicity

Abnormal oligomeric α-syn species have been implicated in the pathogenesis of α-synucleinopathies [38–43]. Danzer and co-workers found that small annular α-syn species but not monomeric protein were able to increase cytosolic Ca2+ levels in SH-SY5Y cells [43]. This increase of 2+ cytosolic Ca2+ was rapid, reaching a plateau around 200 s, and dependent on extracellular Ca , indicating a pore forming ability of α-syn oligomeric species. Treatment with α-syn oligomers resulted in an increased level of cleaved (active) caspase 3 indicating α-syn oligomers induced apoptosis. Outeiro et al. [38] used protein fragment complementation assay (PCA) and bimolecular fluorescence complementation (BiFC) assay to monitor α-syn monomer interaction in vitro and determined that the most favourable α-syn interaction was anti-parallel, resulting in cytotoxicity that could be suppressed by HSP70. 71

Biomolecules 2014, 4 799

One possible mechanism for increased rate of α-syn oligomerization is an increase in mitochondrial mediated ROS production. Using PD cybrids, which have relatively high levels of oxidative stress and an enhancement in oligomer formation, treatment with CoQ10 and GSH antioxidants resulted in a decrease in oligomer formation [39]. Moreover, α-syn oligomer association with mitochondria may be linked to mitochondrial dysfunction [42]. Recently, using both non-denaturing gel electrophoresis and MALDI-TOF mass spectroscopy, α-syn has been shown to exist as α-syn tetramers in M17D, HEK293, HeLa and COS-7 cells [44,45], with predominantly helical structure. These normally occurring tetramers are proposed to be non-toxic, with the abnormal, toxic oligomeric species produced via interconversion first to the monomer.

1.7. α-Synuclein Post-Translational Modifications

Phosphorylation of α-syn has been shown to be important in increasing the rate of aggregation [46] and has also been shown to aid metal ion association [47,48]. Truncation and proteolytic processing of α-syn have also been implicated in aggregate pathology [49]. Oxidation leads to other common post- translational modifications, including nitrosylation, and it has been shown that oxidative stress can stabilise oligomeric α-syn species via the formation of di-tyrosine cross links [50,51]. However, oxidation of recombinant human α-syn results primarily in oxidation of methionine residues [52] and at normal physiological pH this oxidation abolishes α-syn aggregation [53]. One of the most striking characteristics of PD is the selective loss of dopaminergic neurons in the SN which has been mimicked in a drosophila model system [54]. However, α-syn has been shown to play a cyto-protective role in dopaminergic cells. The N27 dopaminergic cell line transfected with human α-syn is protected against MPP+ induced apoptosis via inhibition of PKCδ cleavage and inhibition of BAD, and the concentration of ROS is reduced, in comparison to non-transfected cells [55]. Dopamine has also been shown to inhibit the formation of α-syn fibrils via oxidative modification of the protein and enhances formation of protofibrillar α-syn species [56].

1.8. Exosomes and the Cell to Cell Spread of α-Synuclein

One factor contributing to the toxicity of oligomeric α-syn species is cell to cell spread of α-syn via exosomes [57]. Exosomes are membranous vesicles released from mammalian cells and have been shown to contain mRNA, microRNA and proteins. Alvarez-Erviti et al. [58] demonstrated that α-syn positive exosomes isolated from a SHSY-5Y neuroblastoma α-syn overexpressing cell line were capable of transferring the protein to other SHSY-5Y cells. They also concluded that inhibition of the lysosomes that are involved in α-syn degradation resulted in an increase in α-syn exosome-mediated release into the culture media. Danzer et al. [59] demonstrated in H4 cells and primary cortical neurons that α-syn in exosomes was oligomeric. They also deduced that α-syn was predominantly either outside or associated with the outer membrane of the exosome, but not totally excluded from the lumen. Furthermore, processing of disease-associated α-syn in the human brain is consistent with prion-like cell-to-cell spread [60]. Microglial cells have also been shown to secrete α-syn positive exosomes [61]. Moreover, it was found in foetal tissue graft recipients that return of symptoms of a long term graft survivor correlated with classic PD markers such as α-syn and ubiquitin aggregation within the grafted region, replicated in patients who received foetal mesencephalic dopaminergic neurons [62,63]. Biomolecules 2014, 4 800

1.9. Oxidative Stress

There is evidence that oxidative stress is increased in normal aged brain however the level of oxidative stress is greatly increased in patients with neurodegenerative diseases [64]. The major contribution to oxidative stress in ageing primates originates from mitochondrial complexes I and III of the electron transport chain leading to greater mitochondrial DNA damage compared to nuclear DNA damage [65]. Quilty et al. [66] showed that when mouse primary neocortical cells were incubated in the absence of antioxidants a subset of neurons exhibited a higher α-syn expression and decreased apoptosis. Whereas, Selkoe and co-workers found that prefibrillar α-syn promoted complex I-dependent mitochondrial dysfunction [43]. Moreover, in human SHSY-5Y cells, there was no significant difference in the viability of cells with WT α-syn overexpression; however the A53T and A30P mutations were more susceptible to oxidative insult [9].

2. Increased Intracellular Ca2+ Induces α-Synuclein Oligomers

2.1. The Role of Ca2+ in the Neuron and Age Related Changes

Ca2+ plays many important roles in normal cellular processes, such as apoptosis, metabolism, signal transduction, gene expression and cell death, and intracellular Ca2+ homeostasis is tightly regulated between the cytoplasm, intracellular Ca2+ stores, such as the endoplamic reticulum (ER), and between the intracellular environment and the extracellular environment. Resting intracellular Ca2+ is found not to be increased with age of the neuron, with studies indicating no difference between intracellular Ca2+ levels of young and aged neurons. However, the return time to resting levels after a stimulus is greatly reduced in aged neurons. Thus, the possible link between Ca2+ and α-syn aggregation in neurodegenerative diseases is a current focus of research [67,68]. Indeed, α-syn oligomers have been shown to promote Ca2+ influx [42]. Furthermore Ca2+ and Co2+ binding have been shown to accelerate the formation of α-syn annular oligomeric species [69]. Although the precise Ca2+ binding site has not yet been defined, truncation at residue 125 was found to abolish Ca2+ binding and Ca2+-dependent aggregation. The major sources of intracellular Ca2+ include Ca2+ influx through ligand-gated glutamate receptors, 2+ such as N-methyl-D-aspartate receptor (NMDAR) or various voltage-dependent Ca channels (VDCCs), as well as the release of Ca2+ from intracellular stores. Clearance of Ca2+ after stimulation is achieved either by intracellular Ca2+ binding, uptake into the ER and mitochondria or pumping into the extracellular space via plasma membrane Ca2+ ATPases, which have been shown to be impaired in aged neurons [70–72]. Aged neurons also exhibit a decreased capacity to recover from Ca2+ stimulus 2+ 2+ 2+ through uptake into the intracellular Ca stores with a decline in sarcoplasmic ER Ca ATPase Ca function [73]. The mitochondrion is a second intracellular Ca2+ store and like the ER, the ability of this organelle to act as a reservoir for Ca2+ is also decreased with age [74]. Calbindin (CB), calretinin and parvalbumin are three cytosolic calcium binding proteins that are capable of Ca2+ buffering in neurons. Bu et al. [75] found a decrease in both calretinin and CB in aged compared to young cortical neurons; put no difference in parvalbumin positive neurons. German et al. [76] found that in both idiopathic PD and in MPTP monkey or mouse models that CB+ neurons were spared. Moreover, calretinin expression in dopaminergic neurons of the SN protected The Protective effect of Calbindin D28K on α-synuclein aggregation 73 Biomolecules 2014, 4 801 against 6-hydroxydopamine [77,78]. Furthermore, Yamada et al. [79] found relative sparing of SN neurons positive for CB in PD cases.

2.2. Increased Intracellular Ca2+ Induces α-Synuclein Oligomers

Recent studies have shown that a transient increase in the intracellular free Ca2+ concentration induced in cultured 1321N1 glioma cells by thapsigargin or Ca2+ ionophore (CI) chemical treatments caused a significant increase in the proportion of cells bearing microscopically-visible α-syn aggregates (Figure 2A). It was also demonstrated that chelating free Ca2+ with BAPTA, resulted in no significant difference in the number of inclusions between control and CI/BAPTA cells, indicating that raised intracellular free Ca2+ directly induces α-syn aggregates. Moreover, supporting studies with recombinant protein indicated that direct binding of Ca2+ ion to α-syn promoted rapid oligomer formation in vitro [80], which was not observed with the C-terminally truncated protein (1–125) that lacks the glutamate-rich putative Ca2+ binding domain [69] (see Figure 1). Further studies are needed to map the Ca2+ binding site by mutating each of the putative glutamate residues in this region thought to represent potential metal ligands (see Figure 1). More recently, Follett et al. [81], demonstrated that potassium depolarization of the plasma membrane in HEK293T and SH-SY5Y human cell lines resulted in raised intracellular free Ca2+ and α-syn aggregate formation under more physiologically relevant cellular conditions ([81]; Figure 2B). Both raised free Ca2+ and α-syn aggregation were blocked by BAPTA chelation treatment (Figure 2B, centre). Potassium depolarization was observed especially to trigger formation of frequent large, Lewy body-like perinuclear α-syn inclusion bodies (Figure 2B, right). It is clear that raised Ca2+ is an important factor influencing α-syn aggregation, and potentially in the formation of the cytotoxic oligomeric species seen in disease. Thus, addition of Ca2+ to α-syn monomer in vitro could promote α-syn oligomerization and resulted in the rapid formation of potentially toxic annular α-syn oligomeric structures [80]. This provides a potential therapeutic target, by using drugs that modulate the amount of free Ca2+ in the cell. Ca2+ channel blockers, such as those from the dihydropyridine family, may be used to lessen the increase in intracellular Ca2+ seen in aged neurons [82]. A step towards replicating the complex architecture of the CNS and assessing Ca2+ blockade was performed by Chan et al. [83] who used brain slices prepared from a MPTP mouse model for PD. 2+ They found that by using blocking L-type Cav1.3 Ca channels with Isradipine, a common drug used to treat high blood pressure, they could recover dopaminergic neural activity. This supports the data of Yamada et al. [79] that dopaminergic neurons of the SN, rich in the Ca2+ binding protein CB, were preferentially spared in control brain sections compared with PD patients and the PD mouse model data showing neurons expressing CB were spared from pathological loss [84]. Consistent with this, Trimethadione (TMO), a Ca2+ channel blocker with broad selectivity commonly used as an anti-epileptic + drug, blocked K -depolarization induced Ca2+ influx into SH-SY5Y cells resulting in loss of α-syn positive aggregate formation post-depolarization [81]. Furthermore, recent studies using a unilateral rotenone (oxidative stress) lesion mouse model of PD (described in [85]), also showed improved survival of CB+ neurons and almost exclusive partitioning of α-syn aggregates in the CB− cell population (Figure 3; [86]). In this model, injection of rotenone into the medial forebrain bundle of one brain hemisphere only, allows for comparison of α-syn inclusion body positive and CB+ neurons between treated and untreated hemispheres. 74 Biomolecules 2014, 4 802

Figure 2. Raised intracellular Ca2+ promotes α-syn aggregation. (A) 1321N1 human glioma cells treated with either thapsigargin or Ca2+ ionophore caused raised intracellular free Ca2+ and induced α-syn aggregates (arrows) after 12–24 h [80]; (B) Potassium depolarization of SH-SY5Y human neuroblastoma and HEK293T cells resulted in transiently raised intracellular free Ca2+ and Lewy body-like large α-syn aggregates (arrows) that could be blocked by the BAPTA-AM Ca2+ chelator [81]. (C) Co-treatment of 1321N1 cells with thapsigargin (TG) or Ca2+ ionophore (CI) and hydrogen peroxide resulted in increased α-syn aggregates (arrows; graph, right); consistent with a cooperative interaction between raised free Ca2+ and oxidative stress [87]. Scale bars, 10 μm. Biomolecules 2014, 4 803

Figure 3. Unilateral rotenone lesion mouse (oxidative stress) model of PD shows α-syn aggregates primarily in calbindin-negative neurons. (A) CB+ neurons (arrowheads) showed relative protection in the unilateral rotenone lesion (oxidative stress) model of PD (as detailed in [85]), with more CB+ neurons surviving in the treated than in the untreated hemisphere and partitioning of α-syn aggregates (arrow) in the CB− neurons [86]. Scale bar, 50 μm. (B) Graph of cell counting data shows a significantly greater number of α-syn aggregates occur in CB− neurons than in CB+ neurons.

2.3. α-Synuclein Oligomerization Induces Raised Ca2+ and Oxidative Stress

Elevated levels of intracellular α-syn have been shown to elevate levels of intracellular Ca2+ [88]. Secreted α-syn induces increase in capacitive Ca2+ entry in differentiated SH-SY5Y [89]. Reznichenko et al. [90] investigated Ca2+ dynamics in transgenic (tg) mice expressing human WT α-syn. α-Syn-tg mice exhibited augmented, long-lasting Ca2+ transients characterized by considerable deviation from the exponential decay. Furthermore control and α-syn KO groups demonstrated low percentages of neurons with Ca2+ abnormalities whereas the α-syn tg group showed Ca2+ response alteration, suggesting these alterations are related to α-syn expression. Other studies have shown that α-syn overexpression augments mitochondrial Ca2+ transients by enhancing ER-mitochondria interactions. Cali et al. [91] demonstrated that α-syn with acidic C-terminal domain overexpression increases mitochondrial Ca2+ in SH-SY5Y and HeLa cells. Additionally, treatment with naturally secreted α-syn increases Ca2+ entry in primary rat cortical neurons and induces mitochondrial Ca2+ uptake. Significantly higher levels of mitochondrial Ca2+ content in α-syn treated cells were observed compared to control cells. Dryanovsky et al. [92] found that in CB− and CB+ dopaminergic neurons having inclusions, mitochondrial oxidant stress levels were higher in the soma and proximal dendrites than in neurons without inclusions. Treatment with isradipine significantly diminished the oxidative stress levels in CB− dopaminergic neurons. This suggests that the formation of α-syn inclusions stimulates ROS production in the cytosol. α-Syn provokes an elevation of cytosolic Ca2+ in yeast that coincides with an increase in oxidative stress [93], suggesting that α-syn aggregation leads to an increase in mitochondrial Ca2+ transient and then to oxidative stress. WT α-syn has also been shown to induce mitochondrial NO when it is associated with mitochondria [94]. This indicates that not only will normal increases in oxidative stress cause aggregation but that aggregation of α-syn also induces more oxidative stress

Biomolecules 2014, 4 804 within the cell forming a positive feedback loop. However, this is contrary to other research which shows that α-syn protects cells from oxidative stress by inactivating the c-Jun N-terminal kinase (JNK) pathway [95].

2.4. Synergistic Effect of Ca2+ and Oxidative Stress

Oxidative stress is strongly implicated in α-synucleinopathy and may combine synergistically with other factors, such as α-syn expression and raised Ca2+, to promote α-syn aggregation and neurodegeneration. The combination of oxidative stress and α-syn expression has been used to generate a model of MSA in mice, whereby the overexpression of α-syn in glial cells is combined with 3- nitropropionic acid [96]. Moreover, recent studies have examined the role of oxidative stress in the formation of potentially toxic α-syn oligomeric species in conjunction with Ca2+ binding. It was found 2+ that, when treated with Ca ionophore (CI) or thapsigargin (TG) and H2O2 in combination, there was a dramatic increase in the number of protein aggregates per cell in 1321N1 cells (Figure 2). This was also reflected in in vitro experiments that showed that the combination of Ca2+ treatment and oxidation of recombinant α-syn monomer caused the formation of stable, oligomeric α-syn aggregates, indicating a cooperative interaction between Ca2+ binding to α-syn and α-syn oxidation [87]. Thus, these recent findings have indicated that increased intracellular free Ca2+ and oxidative stress work synergistically to induce α-syn aggregation. This may be extremely important in the pathogenic mechanisms behind α- syn aggregation and α-synopthathy disease progression as fibrillization of α-syn is highly dependent on nucleation centres, with pre-aggregated α-syn dramatically increasing the rate of α-syn aggregation. Furthermore, kinetic studies of α-syn aggregation by Nath et al. [97] were consistent with an auto-catalytic mechanism. Thus, Ca2+/oxidation stabilized α-syn aggregates may serve to increase nucleation centres in disease. Indeed, the formation of di-tyrosine cross linked α-syn dimers have been found previously to be a rate-limiting step in the fibrillation process and the formation of nucleation centres [98]. Moreover, Ca2+ influx into neurons can induce oxidative stress in mitochondria of mouse dopaminergic neurons [99], as oxidative stress induced by Ca2+ influx was exacerbated in DJ-1 mutant mice.

3. Conclusions: Targeting Calcium with Future Therapeutics

The link between PD and some atypical Parkinson’s syndromes and aggregation of α-syn makes this process a major target for the development of future neurodegenerative therapies. Many triggers for pathological α-syn aggregation have been identified, including raised Ca2+ and oxidative stress. Recent studies have found that transient increases of intracellular Ca2+ induce cytoplasmic α-syn aggregates, that can be blocked by Ca2+ buffering or Ca2+ channel blocking agents. Furthermore, it has been shown that Ca2+ and oxidative stress cooperatively promote α-syn aggregation. These recent findings suggest an association between raised intracellular Ca2+, α-syn aggregation and neurotoxicity paving the way for the development of therapeutics that target raised Ca2+ [100]. Since Ca2+ lowering medications, such as anti-epileptic drugs, often have significant side-effects, successfully targeting raised intracellular free Ca2+ in the brain as a neuroprotective strategy will depend on the development of reliable genetic, 2+ imaging or biochemical tests. It is clear that in order to differentiate disease sub-types with strong Ca involvement, multiple marker evaluation will be necessary.

Biomolecules 2014, 4 805

Acknowledgments

We are grateful to the Australian Research Council, Griffith Health Institute and the Clem Jones Foundation for financial support.

Author Contributions

Alex Rcom-H’cheo-Gauthier, Jacob Goodwin and Dean L. Pountney each contributed equally to the writing of this review article. Alex Rcom-H’cheo-Gauthier and Dean L. Pountney contributed unpublished data as indicated.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Goedert, M.; Spillantini, M.G.; del Tredici, K.; Braak, H. 100 years of Lewy pathology. Nat. Rev. Neurol. 2013, 9, 13–24. 2. Eschbach, J.; Danzer, K.M. α-Synuclein in Parkinson’s disease: Pathogenic function and translation into animal models. Neurodegener. Dis. 2014, 14, 1–17. 3. Radford, R.; Wong, M.B.; Pountney, D.L. Neurodegenerative aspects of multiple system atrophy. In Handbook of Neurotoxicity; Kostrzewa, R.M., Ed.; Springer: New York, NY, USA, 2014; pp. 2157–2180. 4. Krüger, R.; Kuhn, W.; Müller, T.; Woitalla, D.; Graeber, M.; Kösel, S.; Przuntek, H.; Epplen, J.T.; Schöls, L.; Riess, O. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat. Genet. 1998, 18, 106–108. 5. Polymeropoulos, M.H.; Lavedan, C.; Leroy, E.; Ide, S.E.; Dehejia, A.; Dutra, A.; Pike, B.; Root, H.; Rubenstein, J.; Boyer, R.; et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 1997, 276, 2045–2047. 6. Zarranz, J.J.; Alegre, J.; Gómez-Esteban, J.C.; Lezcano, E.; Ros, R.; Ampuero, I.; Vidal, L.; Hoenicka, J.; Rodriguez, O.; Atarés, B.; et al. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 2004, 55, 164–173. 7. Lesage, S.; Anheim, M.; Letournel, F.; Bousset, L.; Honore, A.; Rozas, N.; Pieri, L.; Madiona, K.; Durr, A.; Melki, R.; et al. G51D α-Synuclein mutation causes a novel parkinsonian-pyramidal syndrome. Ann. Neurol. 2013, 73, 459–471. 8. Proukakis, C.; Dudzik, C.G.; Brier, T.; MacKay, D.S.; Cooper, J.M.; Millhauser, G.L.; Houlden, H.; Schapira, A.H. A novel α-synuclein missense mutation in Parkinson disease. Neurology 2013, 80, 1062–1064. 9. Kanda, S.; Bishop, J.F.; Eglitis, M.A.; Yang, Y.; Mouradian, M.M. Enhanced viability to oxidative stress by alpha-synuclein mutations and C-terminal truncation. Neuroscience 2000, 97, 279–284. 10. Narhi, L.; Wood, S.J.; Steavenson, S.; Jiang, Y.; Wu, G.M.; Anafi, D.; Kaufman, S.A.; Martin, F.; Sitney, K.; Denis, P.; et al. Both familial Parkinson’s disease mutations accelerate alpha-synuclein aggregation. J. Biol. Chem. 1999, 274 , 9843 –9846. 78

Biomolecules 2014, 4 806

11. Khalaf, O.; Fauvet, B.; Oueslati, A.; Dikiy, I.; Mahul-Mellier, A.L.; Ruggeri, F.S.; Mbefo, M.; Vercruysse, F.; Dietler, G.; Lee, S.J.; et al. The H50Q mutation enhances α-synuclein aggregation, secretion and toxicity. J. Biol. Chem. 2014, doi:10.1074/jbc.M114.553297. 12. Ibáñez, P.; Bonnet, A.M.; Débarges, B.; Lohmann, E.; Tison, F.; Pollak, P.; Agid, Y.; Dürr, A.; Brice, A. Causal relation between alpha-synuclein gene duplication and familial Parkinson’s disease. Lancet 2004, 364, 1169–1171. 13. Singleton, A.B.; Farrer, M.; Johnson, J.; Singleton, A.; Hague, S.; Kachergus, J.; Hulihan, M.; Peuralinna, T.; Dutra, A.; Nussbaum, R.; et al. alpha-Synuclein locus triplication causes Parkinson’s disease. Science 2003, doi:10.1126/science.1090278. 14. Tanner, C.M.; Kamel, F.; Ross, W.; Hoppin, J.A.; Goldman, S.M.; Korell, M.; Marras, C.; Bhudhikanok, G.S.; Kasten, M.; Chade, A.R.; et al. Rotenone, paraquat, and Parkinson’s disease. Environ. Health Perspect. 2011, 119, 866–872. 15. Wang, X.; Su, B.; Liu, W.; He, X.; Gao, Y.; Castellani, R.J.; Perry, G.; Smith, M.A.; Zhu, X. DLP1-dependent mitochondrial fragmentation mediates 1-methyl-4-phenylpyridinium toxicity in neurons: Implications for Parkinson’s disease. Ageing Cell. 2011, 10, 807–823. 16. Hernán, M.A.; Takkouche, B.; Caamaño-Isorna, F.; Gestal-Otero, J.J. A meta-analysis of coffee drinking, cigarette smoking, and the risk of Parkinson’s disease. Ann. Neurol. 2002, 52, 276–284. 17. Spillantini, M.G.; Schmidt, M.L.; Lee, V.M.; Trojanowski, J.Q.; Jakes, R.; Goedert, M. Alpha- synuclein in Lewy bodies. Nature 1997, 388, 839–840. 18. Al-Chalabi, A.; Dürr, A.; Wood, N.W.; Parkinson, M.H.; Camuzat, A.; Hulot, J.S.; Morrison, K.E.; Renton, A.; Sussmuth, S.D.; Landwehrmeyer, B.G.; et al. Genetic variants of the alpha-synuclein gene SNCA are associated with multiple system atrophy. PLoS One 2009, 4, e7114. 19. Hanna, P.A.; Jankovic, J.; Kirkpatrick, J.B. Multiple system atrophy: The putative causative role of environmental toxins. Arch. Neurol. 1999, 56, 90–94. 20. Vidal, J.S.; Vidailhet, M.; Elbaz, A.; Derkinderen, P.; Tzourio, C.; Alpérovitch, A. Risk factors of multiple system atrophy: A case-control study in French patients. Mov. Disord. 2008, 23, 797– 803. 21. Iwai, A.; Masliah, E.; Yoshimoto, M.; Ge, N.; Fianagan, L.; Rohan de Silva, H.A.; Kittei, A.; Saitoh, T. The precursor protein of non-aβ component of Alzheimer’s disease amyloid is a presynaptic protein of the central nervous system. Neuron 1995, 14, 467–475. 22. Maroteaux, L.; Campanelli, J.T.; Scheller, R.H. Synuclein: A neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J. Neurosci. 1998, 8, 2804–2815. 23. Clayton, D.F.; George, J.M. Synucleins in synaptic plasticity and neurodegenerative disorders. J. Neurosci. Res. 1999, 58, 120–129. Burré, J.; Sharma, M.; Tsetsenis, T.; Buchman, V.; Etherton, M.R.; Südhof, T.C. Alpha-synuclein 24. promotes SNARE-complex assembly in vivo and in vitro. Science 2010, 329, 1663–1667.

25. Nielsen, M.S.; Vorum, H.; Lindersson, E.; Jensen, P.H. Ca2+ binding to alpha-synuclein regulates ligand binding and oligomerization. J. Biol. Chem. 2001, 276, 22680–22684. 26. Giasson, B.I.; Murray, I.V.; Trojanowski, J.Q.; Lee, V.M. A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J. Biol. Chem. 2001, 276, 2380–2386. 79

Biomolecules 2014, 4 807

27. Eliezer, D.; Kutluay, E.; Bussell, R., Jr.; Browne, G. Conformational properties of alpha-synuclein in its free and lipid-associated states. J. Mol. Biol. 2001, 307, 1061–1073. 28. Davidson, W.S.; Jonas, A.; Clayton, D.F.; George, J.M. Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 1998, 273, 9443–9449. 29. Wietek, J.; Haralampiev, I.; Amoussouvi, A.; Herrmann, A.; Stöckl, M. Membrane bound α- synuclein is fully embedded in the lipid bilayer while segments with higher flexibility remain. FEBS Lett. 2013, 587, 2572–2577. 30. Wood, S.J.; Wypych, J.; Steavenson, S.; Lousis, J-C.; Citron, M.; Biere, A.L. α-Synuclein fibrillogenesis is nucleation dependent: Implications for the pathogenesis of Parkinson’s disease. J. Biol. Chem. 1999, 274, 19509–19512. 31. Zibaee, S.; Jakes, R.; Fraser, G.; Serpell, L.C.; Crowther, R.A.; Goedert, M. Sequence determinants for amyloid fibrillogenesis of human alpha-synuclein. J. Mol. Biol. 2007, 374, 454–464. 32. Narayanan, V.; Scarlata, S. Membrane binding and self-association of alpha-synucleins. Biochemistry 2001, 40, 9927–9934. 33. Lokappa, S.B.; Suk, J.E.; Balasubramanian, A.; Samanta, S.; Situ, A.J.; Ulmer, T.S. Sequence and membrane determinants of the random coil-helix transition of α-synuclein. J. Mol. Biol. 2014, 426, 2130–2144. 34. McLean, P.J.; Kawamata, H.; Ribich, S.; Hyman, T. Membrane association and protein conformation of α-synuclein in intact neurons. J. Biol. Chem. 2000, 275, 8812–8816. 35. Chen, R.H.; Wislet-Gendebien, S.; Samuel, F.; Visanji, N.P.; Zhang, G.; Marsilio, D.; Langman, T.; Fraser, P.E.; Tandon, A. α-Synuclein membrane association is regulated by the Rab3a recycling machinery and presynaptic activity. J. Biol. Chem. 2013, 288, 7438–7449. 36. Mak, S.R.; McCormack, A.L.; Langston, J.W.; Kordower, J.H.; di Monte, D.A. Decreased α- synuclein expression in the ageing mouse substantia nigra. Exp. Neurobiol. 2009, 220, 359–365. 37. Grundemann, J.; Schlaudraff, F.; Haeckel, O.; Liss, B. Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in Idiopathic Parkinson’s disease. Nucleic Acids Res. 2008, 36, e38. 38. Outeiro, T.F.; Putcha, P.; Tetzlaff, J.E.; Spoelgen, R.; Koker, M.; Carvalho, F.; Hyman, B.T.; McLean, P.J. Formation of toxic oligomeric alpha-synuclein species in living cells. PLoS One 2008, 3, e1867. 39. Esteves, A.R.; Arduíno, D.M.; Silva, D.F.; Oliveira, C.R.; Cardoso, S.M. Mitochondrial dysfunction: The Road to alpha-synuclein oligomerization in PD. Parkinsons Dis. 2011, doi:10.4061/2011/693761. 40. Kalia, L.V.; Kalia, S.K.; McLean, P.J.; Lozano, A.M.; Lang, A.E. α-Synuclein oligomers and clinical implications for Parkinson disease. Ann. Neurol. 2013, 73, 155–169. 41. Yasuda, T.; Nakata, Y.; Mochizuki, H. α-Synuclein and neuronal cell death. Mol. Neurobiol. 2013, 47, 466–483. 42. Luth, E.S.; Stavrovskaya, I.G.; Bartels, T.; Kristal, B.S.; Selkoe, D.J. Soluble, prefibrillar α- synuclein oligomers promote complex I-dependent, Ca2+-induced mitochondrial dysfunction. J. Biol. Chem. 2014, doi:10.1074/jbc.M113.545749.

80

Biomolecules 2014, 4 808

43. Danzer, K.M.; Haasen, D.; Karow, A.R.; Moussaud, S.; habeck, M.; Giese, A.; Kretzschmar, H.; Hengerer, B.; Kostka, M. Different species of alpha-synuclein oligomers induce calcium influx and seeding. J. Neurosci. 2007, 27, 9220–9232. 44. Bartels, T.; Choi, J.C.; Selkoe, D.J. α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature 2011, 477, 107–111. 45. Wang, W.; Perovic, W.; Chittuluru, J.; Kaganovich, A.; Nguyen, L.T.T.; Liao, J.; Auclair, J.R.; Johnson, D.; Landeru, D.; Simorellis, A.K.; et al. A soluble α-synuclein construct forms a dynamic tetramer. Proc. Natl. Acad. Sci. USA 2011, 108, 17797–17802. 46. Smith, W.W.; Margolis, R.L.; Li, X.; Troncoso, J.C.; Lee, M.K.; Dawson, V.L.; Dawson, T.M.; Iwatsubo, T.; Ross, C.A. Alpha-synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells. J. Neurosci. 2005, 25, 5544–5552. 47. Lu, Y.; Prudent, M.; Fauvet, B.; Lashuel, H.A.; Girault, H.H. Phosphorylation of α-Synuclein at Y125 and S129 alters its metal binding properties: Implications for understanding the role of α- synuclein in the pathogenesis of Parkinson’s disease and related disorders. ACS Chem. Neurosci. 2011, 2, 667–675. 48. Liu, L.L.; Franz, K.J. Phosphorylation of an alpha-synuclein peptide fragment enhances metal binding. J. Am. Chem. Soc. 2005, 127, 9662–9663. 49. Anderson, J.P.; Walker, D.E.; Goldstein, J.M.; de Laat, R.; Banducci, K.; Caccavello, R.J.; Barbour, R.; Huang, J.; Kling, K.; Lee, M.; et al. Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 2006, 281, 29739–29752. 50. Uversky, V.N.; Yamin, G.; Munishkina, L.A.; Karymov, M.A.; Millett, I.S.; Doniach, S.; Lyubchenko, Y.L.; Fink, A.L. Effects of nitration on the structure and aggregation of alpha- synuclein. Mol. Brain Res. 2005, 134, 84–102. 51. Chavarría, C.; Souza, J.M. Oxidation and nitration of α-synuclein and their implications in neurodegenerative diseases. Arch. Biochem. Biophys. 2013, 533, 25–32. 52. Glaser, C.B.; Yamin, G.; Uversky, V.N.; Fink, A.L. Methionine oxidation, alpha-synuclein and Parkinson’s disease. Biochim. Biophys. Acta 2005, 1703, 157–169. 53. Uversky, V.N.; Yamin, G.; Souillac, P.O.; Goers, J.; Glaser, C.B.; Fink, A.L. Methionine oxidation inhibits fibrillation of human alpha-synuclein in vitro. FEBS Lett. 2002, 517, 239–244. 54. Feany, M.B.; Bender, W.W. A Drosophila model of Parkinson’s disease. Nature 2000, 404, 394– 398. 55. Kaul, S.; Anantharam, V.; Kanthasamy, A.; Kanthasamy, A.G. Wild-type alpha-synuclein interacts with pro-apoptotic proteins PKCdelta and BAD to protect dopaminergic neuronal cells against MPP+-induced apoptotic cell death. Brain Res. Mol. Brain Res. 2005, 139, 137–152. 56. Pham, C.L.; Cappai, R. The interplay between lipids and dopamine on α-synuclein oligomerization and membrane binding. Biosci. Rep. 2013, 33, e00074. 57. Lee, H.J.; Bae, E.J.; Lee, S.J. Extracellular α-synuclein—A novel and crucial factor in Lewy body diseases. Nat. Rev. Neurol. 2014, 10, 92–98. 58. Alvarez-Erviti, L.; Seow, Y.; Schapira, A.H.; Gardiner, C.; Sargent, I.L.; Wood, M.J.; Cooper, J.M. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol. Dis. 2011, 42, 360–367. 81

Biomolecules 2014, 4 809

59. Danzer, K.M.; Kranich, L.R.; Ruf, W.P.; Cagsal-Getkin, O.; Winslow, A.R.; Zhu, L.; Vanderburg, C.R.; McLean, P.J. Exosomal cell-to-cell transmission of alpha synuclein oligomers. Mol. Neurodegener. 2012, doi:10.1186/1750-1326-7-42. 60. Kovacs, G.G.; Breydo, L.; Green, R.; Kis, V.; Puska, G.; Lőrincz, P.; Perju-Dumbrava, L.; Giera, R.; Pirker, W.; Lutz, M.; et al. Intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread. Neurobiol. Dis. 2014, doi:10.1016/j.nbd.2014.05.020. 61. Chang, C.; Lang, H.; Geng, N.; Wang, J.; Li, N.; Wang, X. Exosomes of BV-2 cells induced by alpha-synuclein: Important mediator of neurodegeneration in PD. Neurosci. Lett. 2013, 26, 190–195. 62. Kordower, J.H.; Chu, Y.; Hauser, R.A.; Freeman, T.B.; Olanow, C.W. Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson’s disease. Nat. Med. 2008, 14, 504–506. 63. Li, J.Y.; Englund, E.; Holton, J.L.; Soulet, D.; Hagell, P.; Lees, A.J.; Lashley, T.; Quinn, N.P.; Rehncrona, S.; Bjorklund, A.; et al. Lewy bodies in grafted neurons in subjects with Parkinson’s disease suggest host-to-graft disease propagation. Nat. Med. 2008, 14, 501–503. 64. Sims-Robertson, C.; Hur, J.; Hayes, J.M.; Dauch, J.R.; Keller, P.J.; Brooks, S.V.; Feldman, E.L. The role of oxidative stress in nervous system aging. PLoS One 2013, 8, e68011. 65. Castro, R.; Suarez, E.; Kraiselburd, E.; Isidro, A.; Paz, J.; Ferder, L.; Ayala-Torres, S. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys. Exp. Gerentol. 2012, 47, 29–37. 66. Quilty, M.C.; King, A.E.; Gai, W.P.; Pountney, D.L.; West, A.K.; Vickers, J.C.; Dickson, T.C. Alpha-synuclein is upregulated in neurons in response to chronic oxidative stress and is associated with neuroprotection. Exp. Neurol. 2006, 199, 249–256. 67. Surmeier, D.J.; Schumacker, P.T. Calcium, bioenergetics, and neuronal vulnerability in Parkinson’s disease. J. Biol. Chem. 2013, 288, 10736–10741. 68. Fairless, R.; Williams, S.K.; Diem, R. Dysfunction of neuronal calcium signalling in neuroinflammation and neurodegeneration. Cell Tissue Res. 2013, doi:10.1007/s00441-013-1758-8. 69. Lowe, R.; Pountney, D.L.; Jensen, P.H.; Gai, W.P.; Voelcker, N.H. Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with the C-terminal domain. Protein Sci. 2004, 13, 3245–3252. 70. Brini, M.; Calì, T.; Ottolini, D.; Carafoli, E. Neuronal calcium signaling: Function and dysfunction. Cell. Mol. Life Sci. 2014, doi:10.1007/s00018-013-1550-7. 71. Michaelis, M.L.; Bigelow, D.J.; Schöneich, C.; Williams, T.D.; Ramonda, L.; Yin, D.; Hühmer, A.F.; Yao, Y.; Gao, J.; Squier, T.C. Decreased plasma membrane calcium transport activity in aging brain. Life Sci. 1996, 59, 405–412. 72. Duckles, S.P.; Tsai, H.; Buchholz, J.N. Evidence for decline in intracellular calcium buffering in adrenergic nerves of aged rats. Life Sci. 1996, 58, 2029–2035. 73. Schwaller, B. Calretinin: From a “simple” Ca2+ buffer to a multifunctional protein implicated in many biological processes. Front. Neuroanat. 2014, doi:10.3389/fnana.2014.00003. 74. Perier, C.; Vila, M. Mitochondrial biology and Parkinson’s disease. Cold Spring Harb. Prospect. Med. 2012, doi:10.1101/cshperspect.a009332.

The Protective effect of Calbindin D28K on α-synuclein aggregation 82

Biomolecules 2014, 4 810

75. Bu, J.; Sathyendra, V.; Nagykery, N.; Geula, C. Age-related changes in calbindin-D28k, calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex. Exp. Neurol. 2003, 182, 220–231. 76. German, D.C.; Manaye, K.F.; Sonsalla, P.K.; Brooks, B.A. Midbrain dopaminergic cell loss in Parkinson’s disease and MPTP-induced Parkinsonism: Sparing of calbindin-D 28K containing cells. Ann. NY Acad. Sci. 1992, 648, 42–62. 77. Tsuboi, K.; Kimber, T.A.; Shults, C.W. Calretinin-containing axon and neurons are resistant to intrastriatal 6-hydroxydopamine. Brain Res. 2000, 866, 55–64. 78. Kim, B.G.; Shin, D.H.; Jeon, G.S.; Seo, J.H.; Kim, Y.W.; Jeon, B.S.; Cho, S.S. Relative sparing of calretinin containin neurons in the substantia nigra of 6-OHDA treated rat Parkinsonian model. Brain Res. 2000, 7, 162–165. 79. Yamada, T.; McGeer, P.L.; Bainbridge, K.G.; McGeer, E.C. Relative sparing in Parkinson’s disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain Res. 1990, 526, 303–307. 80. Nath, S.; Goodwin, J.; Engelborghs, Y.; Pountney, D.L. Raised calcium promotes α-synuclein aggregate formation. Mol. Cell. Neurosci. 2011, 46, 516–526. 81. Follett, J.; Darlow, B.; Wong, M.B.; Goodwin, J.; Pountney, D.L. Potassium depolarization and raised calcium induces α-synuclein aggregates. Neurotox. Res. 2013, 23, 378–392. 82. Kopecky, B.J.; Liang, R.; Bao, J. T-type calcium channel blockers as neuroprotective agents. Eur. J. Physiol. 2014, 466, 757–765. 83. Chan, S.C.; Guzman, J.N.; Ilijic, E.; Mecer, J.N.; Rick, C.; Tkatch, T.; Meredith, G.E.; Surmeier, D.J. “Rejuvenation” protects neurons in mouse models of Parkinson’s disease. Nature 2007, 447, 1081–1089. 84. Gasper, P.; Ben Jelloun, N.; Febvret, A. Sparing of the dopaminergic neurons containing calbindin-D28k and the loss of dopaminergic mesocortical projections in weaver mice. Neuroscience 1994, 61, 293–305. 85. Weetman, J.; Wong, M.B.; Sharry, S.; Rcom-H’cheo-Gauthier, A.; Gai, W.P.; Meedeniya, A.; Pountney, D.L. Increased SUMO-1 expression in the unilateral rotenone-lesioned mouse model of Parkinson’s disease. Neurosci. Lett. 2013, 544, 119–124. 86. Rcom-H’cheo-Gauthier, A.; Meedeniya, A.; Pountney, D.L. School of Medical Science, Griffith University, Gold Coast, Australia. Unpublished work, 2014. 87. Goodwin, J.; Nath, S.; Engelborghs, Y.; Pountney, D.L. Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation. Neurochem. Int. 2013, 62, 703–711. 88. Hettiarachchi, N.T.; Parker, A.; Dallas, M.L.; Pennington, K.; Hung, C.C.; Pearson, H.A.; Boyle, J.P.; Robinson, P.; Peers, C. α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells. J. Neurochem. 2009, 111, 1192–1201. 89. Melachroinou, K.; Xilouri, M.; Emmanouilidou, E.; Masgrau, R.; Papazafiri, P.; Stefanis, L.; Vekrellis, K. Deregulation of calcium homeostasis mediates secreted alpha-synuclein-induced neurotoxicity. Neurobiol. Aging 2013, 34, 2853–2865. 90. Reznichenko, L.; Cheng, Q.; Nizar, K.; Gratiy, S.L.; Saisan, P.A.; Rockenstein, E.M.; González, T.; Patrick, C.; Spencer, B.; Desplats, P.; et al. In vivo alterations in calcium buffering capacity in transgenic mouse model of synucleinopathy. J. Neurosci. 2012, 32, 9992–9998. 83 Biomolecules 2014, 4 811

91. Cali, T.; Ottolini, D.; Negro, A.; Brini, M. alpha-Synuclein controls mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions. J. Biol. Chem. 2012, 287, 17914–17929. 92. Dryanovski, D.I.; Guzman, J.N.; Xie, Z.; Galteri, D.J.; Volpicelli-Daley, L.A.; Lee, V.M.; Miller, R.J.; Scumacker, P.T.; Surmeier, D.J. Calcium entry and alpha-synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons. J. Neurosci. 2013, 33, 10154–10164. 93. Buttner, S.; Faes, L.; Reichelt, W.N.; Broeskamp, F.; Habernig, L.; Benke, S.; Kourtis, N.; Ruli, D.; Carmona-Gutierrez, D.; Eisenberg, T.; et al. The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca2+ levels to alpha-synuclein toxicity in Parkinson’s disease models. Cell Death Differ. 2013, 20, 465–477. 94. Parihar, M.S.; Parihar, A.; Fujita, M.; Hashimoto, M.; Ghafourifar, P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell. Mol. Life Sci. 2008, 65, 1272–1284. 95. Hashimoto, M.; Hsu, L.J.; Rockenstein, E.; Takenouchi, T.; Mallory, M.; Masliah, E. α- Synuclein protects against oxidative stress via inactivation of the c-Jun N-terminal kinase stress- signalling pathway in neuronal cells. J. Biol. Chem. 2002, 277, 11465–11472. 96. Stefanova, N.; Reindl, M.; Neumann, M.; Haass, C.; Poewe, W.; Kahle, P.J.; Wenning, G.K. Oxidative stress in transgenic mice with oligodendroglial α-synuclein overexpression replicates the characteristic neuropathology of multiple system atrophy. Am. J. Pathol. 2005, 166, 869–876. 97. Nath, S.; Meuvis, J.; Hendrix, J.; Carl, S.A.; Engelborghs, Y. Early aggregation steps in alpha- synuclein as measured by FCS and FRET: Evidence for a contagious conformational change. Biophys. J. 2010, 98, 1302–1311. 98. Krishnan, S.; Chi, E.Y.; Wood, S.J.; Kendrick, B.S.; Li, C.; Garzon-Rodrigues, W.; Wypych, J.; Randolph, T.W.; Narhi, L.O.; Biere, A.L.; et al. Oxidative dimer formation is the critical rate- limiting step for Parkinson’s disease α-synuclein fibrillogenesis. Biochemistry 2003, 42, 829– 837. 99. Goldberg, J.A.; Guzman, J.N.; Estep, C.M.; Ilijic, E.; Kondapalli, J.; Sanchez-Padilla, J.; Surmerier, D.J. Calcium entry induces mitochondrial oxidant stress in vagal neurons at risk in Parkinson’s disease. Nat. Neurosci. 2012, 15, 1414–1421. 100. Schapira, A.H.; Olanow, C.W.; Greenamyre, J.T.; Bezard, E. Slowing of neurodegeneration in Parkinson’s disease and Huntington’s disease: Future therapeutic perspectives. Lancet 2014, doi:10.1016/S0140-6736(14)61010-2.

2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 84

Chapter 3: Methods

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 85

3.1 Research Plan

Aim 1: Investigation of the proportion of CB and α-syn aggregates positive neurons in

DLB and healthy, non-DLB tissue

DLB tissue staining

Fluorescence microscopy: Determine the expression of CB and α-syn in neurons and levels of expressions in those neurons.

Statistical analysis of results: to establish the percentage of CB+ neurons to other neurons, in lesioned and control side, as well as the correlation between CB neurons and α-syn aggregation.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 86

Aim 2: Investigation of the proportion of CB and neurons positive for α-syn aggregates in a mouse model of α-synucleinopathy

Surgery: Unilateral lessiong of the MFB with rotenone

Brain harvest , Fixation and Brain harvest and sectionning homogenised

Fluoresence microscopy: Determine the expression of Western Blot analysis: CB and α-syn in neurons and Investigation of overall levels of expressions in those protein expression of CB. neurons.

Statistical analysis of results: to establish the percentage of CB+ Statistical analysis of results: neurons to other neurons, in to establish the difference in lesioned and control side. As CB expression between well as the correlation between lesioned and control side. CB neurons and α-syn aggregation.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 87

Aim 3: Investigation of the correlation between the overexpression of CB and α-syn aggregation in SH-SY5Y cells

Cell culture: SH-SY5Y. Treatement with Cp

KCl, KCL/H2O2 and Cell lysate, protein rotenone extraction

Fluoresence microscopy: Western Blot analysis: Determine the Investigation of overall expression of CB and α- protein expression of syn aggregation CB.

Statistical analysis of results: to establish the Statistical analysis of expression of CB,. As well results: to establish the as the correlation between difference in CB CB expression and α-syn expression. aggregation.

Figure 11: Overview of methods used to accomplish aims

In the first part of this study, CB expression and α-syn aggregation in DLB was studied in the first part of the study (Aim 1). DLB tissue from human patients were used for

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 88 analyses and compared to normal human brain tissue. The correlation between CB+ neurons and α-syn aggregates was studied using immunohistochemistry. Statistical analyses were performed to find a correlation between CB+ neurons and α-syn aggregation. DLB tissues were also stained with doublecortin (DCX) to confirm neuronal expression of inclusion bodies.

Secondly (Aim 2), the experimental design comprised of two cohorts of mice, each subjected to unilateral rotenone injection, with one cohort examined for CB expression by

Western analysis and the second cohort subjected to immunohistochemistry analysis. Figure

11 provides an overview of the experimental plan.

Unilateral rotenone injections were given to each animal in the medial forebrain bundle (MFB) and were left 14 days to recover and to develop appropriate neurodegeneration.

Mice were then sacrificed and the brains harvested following perfusion and fixation for the immunofluorescence studies. Tissue homogenates from each hemisphere were prepared in a previous study.

The frequency of CB+ neurons in treated hemispheres compared to those untreated was determined by the use of immunofluorescence of the brain tissue section, as well as the correlation between CB+ neurons and α-syn aggregates. Mice tissues were also stained with

NEUronal Nuclei (NeuN) to confirm neuronal expression of CB. In parallel, the overall expression of CB was determined using tissue homogenates prepared from the treated and untreated brain tissue by WB analysis. For immunohistochemistry, statistical analysis was performed between sham and control mice, between aged and young mice, between treated and control hemisphere and also between different cortices within the same hemisphere. For

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 89

WB, statistical analysis was performed between aged and young mice and between treated and control hemisphere.

α-Syn aggregation in SH-SY5Y cells were studied as the third part of the study (Aim

3). SH-SY5Y cells were treated with Cp and α-syn aggregation was compared between different Cp concentrations and different stress factors such as KCl depolarisation, KCl/H2O2 combination and rotenone treatment to induce oxidative stress, using immunohistochemistry and WB analysis. Statistical analyses were performed to find any correlation between different Cp concentrations and α-syn aggregation.

3.2 Resources & health and safety

DLB and normal human brain tissues were received from the South Australian Brain

Bank and were stained for analyses (five cases each, six brain regions each case). These were analysed through immunohistochemistry for CB and α-syn expression.

Homogenates from five aged and five young mice WT have already been prepared in previous studies and were analysed through WB for CB expression.

Tissues from five young WT mice had already been prepared and were analysed through immunohistochemistry for CB and α-syn expression. Ten aged mice and three young mice were required to complete this study. Data for TH and Neuronal Nuclei (NeuN) were obtained in a previous study using to two young, two young adult and two aged mice.

SH-SY5Y cells, Cp, a vitamin D analogue and CB siRNA were required to complete this study.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 90

3.3 Overview of ethics requirements

Ethics approval was required for the rotenone lesioning. The protocol used was from similar procedure previously approved for rats (BBS/08/04/AEC, SCE/01/06/AEC, and

ESK/05/08/AEC), and mice (BPS/01/07/AEC). Human ethics approval: MSC/16/11/HREC.

Animal ethics to perform the rotenone lesioning was approved on the 10th of June 2014

(MSC/06/14/AEC).

3.4 Methods and Materials

3.4.1 Human Model

3.4.1.1 Human Tissue

Human brain tissue was acquired from the South Australian Brain Bank. Diagnosis of disease was conducted at autopsy by neuropathologist Professor Peter Blumbergs according to disease specific criteria. Autopsy was conducted 7-30 hours post-mortem where the brains were bisected into the hemispheres and one formalin fixed, whilst the other was kept in frozen storage. The formalin fixed tissue were then embedded in blocks of paraffin before being cut into 5μm thick slices and mounted on gelatine-coated glass slides. Six DLB (age at death: 70

± 7, Post-mortem interference [PMI] 8 ± 3 hours), and six age matched normal cases (age at death 73 ± 9; PMI 14 ± 8 hours) were obtained. Six brain regions (motor cortex, visual cortex, frontal lobe, temporal lobe, hippocampus and cingulate gyrus) in each case were analysed.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 91

CASE # Sex Age PMI (hr) Neuropathological Diagnosis Frozen

SA0031 F 61 8 NORMAL - CONTROL NONE SA0038 F 86 25 NO NEUROPATHOLOGY REPORT NONE

SA0036 F 84 15 NO NEUROPATHOLOGY REPORT NONE

SA0050 M 47 8 NO NEUROPATHOLOGY REPORT NONE

SA0063 F 81 7 Diffuse Lewy Body Disease (DLBD), Right INFARCTS SA0094 M 74 24 Diffuse Lewy Body Disease (DLBD) Left

SA0113 F 81 12 Diffuse Lewy Body Disease (DLBD) Right

SA0234 M 79 24 Diffuse Lewy Body Disease (DLBD) Right

SA0249 M 72 12 Diffuse Lewy Body Disease (DLBD) + Left AD SA0162 M 72 30 NORMAL - CONTROL (BRAIN + Left SPINAL CORD) SA0225 M 86 23 Diffuse Lewy Body Disease (DLBD) + None - OTHER Perfusion SA0230 M 86 22 NORMAL - CONTROL Right

Table 2: Dementia with Lewy Body and control Post-Mortem Tissue

3.4.1.2 Fluorescence microscopy

Formalin fixed DLB and normal human brain tissues received from the South

Australian Brain Bank and were stained for analyses (6 cases each, 6 brain regions each case).

These were then analysed through immunohistochemistry for CB and α-syn expression.

Firstly, tissue slices of ≈0.5cm thick were dehydrated using an Shandon™ Excelsior™ ES

Tissue Processor in 70% ethanol (1 hour) then in 80%, 90% and 100% ethanol consecutively

(1 hour each). Then the sections were placed in paraffin wax (56-58ºC) (3 changes, 45mins, 1 hour and 1.15 hour each) and then embed into paraffin blocks. Sections were cut (5µm

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 92 sections), mounted on gelatine-coated glass slides and dried 24 hours in an oven at 40ºC.

Sections were stored in a fridge at 4ºC until use.

Tissue sections were deparaffinised by xylene (2 washes for 10 minutes), then rehydrated in 100%, 95%, and 70% ethanol (5 minutes per wash). Heat induced antigen retrieval was performed in 1mM EDTA pH8 solution at 100ºC for 10mins in microwave, to try and recover any antigens which may be masked during tissue fixation [231]. After allowing the slides to cool (<50ºC), the sections were flushed and incubated for 5 minutes in

Tris-buffered saline (TBS)( NaCl 125mM,tris 25mM, pH 7.5). Excess moisture was removed by drying the area around the tissue before non-specific binding sites were blocked by immersing the tissue in 20% normal horse serum (NHS) in TBS for 1 hour. The area around the tissues were dried and then incubated with primary antibodies (α-syn 1:200, DCX 1:50 or

CB 1:200 all cases, for DLB and normal) in 1% NHS-TBS overnight at 4ºC. The tissues were then washed for 3x5 minutes in TBS then again the area around the tissues was dried.

Secondary antibodies (1:200 for both Alexafluor (AF) 488 anti-mouse and AF-568 anti- rabbit) in 1% NHS-TBS were applied to the tissues and left to incubate for 90 minutes at room temperature. The tissues were washed again with TBS (3x5 minutes), before drying the slide around the tissue and applying Prolong GOLD mounting medium with 4',6-diamidino-2- phenylindole (DAPI) (Life Technologies) and a cover slip. Finally nail varnish was applied to the edges of the coverslip to limit its movement.

3.4.1.3 Cell counting

Immunostained cells were counted using image J software and classified as either

CB+ or CB- or α-syn aggregate positive or α-syn aggregate negative. To be classified as a

CB+ cell, bright immunostaining needed to be visualized throughout the cytoplasm for CB

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 93

(mean pixel intensity 3-fold over background intensity). α-Syn aggregate bearing cells were counted as bearing one or more bright cytoplasmic puncta clearly visualised by its immunofluorescence for α-syn over the entire aggregate (mean pixel intensity 4-fold over background cytoplasmic intensity). Total cell count per area imaged was determined based on

DAPI nucleus stain. A standardized pattern of three random areas containing on average 40 cells (210µm x 210µm) each in the motor cortex, visual cortex, frontal lobe, temporal lobe, cingulate gyrus and the hippocampus was imaged for each of the six cases and six controls.

3.4.1.4 Statistical analysis

CB+ and CB- cells were counted on each image along with α-syn+ and α-syn-negative

(α-syn-) cells. Then, a percentage of CB+ cells, a percentage of α-syn+ cells and a percentage of CB/α-syn+ neurons were established for each image. Data was analysed using an ANOVA test analysis between CB/α-syn and α-syn+ neurons, DLB/Normal tissues and brain areas.

3.4.1.5 Antibodies

Two different primary antibodies were used in this experiment. One type of anti-CB antibody (Swant) was used to stain human tissue while a mouse monoclonal anti-α-syn antibody (Invitrogen) was used to detectα-syn aggregates.

In order to visualise the primary antibody binding, AF conjugated secondary antibodies were used. AF-488 anti-mouse and AF-568 anti-rabbit fluorescent secondary antibodies were used to visualise primary antibodies in human DLB and normal tissue. AF conjugated secondary antibodies were purchased from Molecular Probes, Invitrogen.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 94 Antibody Use Antigen Host Reactivity Source Human, CB38 Primary CB Rabbit Swant rodent Human, LB509 Primary α-syn Mouse Invitrogen rodent Double- Human, DCX Primary Rabbit Abcam cortin rodent Mouse Fc Molecular AF-488 Secondary Donkey Mouse receptor Probes Rabbit Fc Molecular AF-568 Secondary Goat Rabbit receptor Probes

Table 3: Antibodies used for immunofluorescence labelling

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 95

3.4.2 Animal Model

3.4.2.1 Animals

This study comprised of five aged (1.75-2 years) and five young adult (8-12 months) male C57BL/6 mice for the WB analysis and two young ten young adult and twelve aged WT

C57BL/6 mice for immunohistochemistry. Mice were located at Griffith University, Gold

Coast Campus, and the animals were housed in standard cages, on a 12-hour light cycle with ad libitum access to food and water.

Label Age Group Gender/ Breed Tissue source 1Y Young (12months) Male – C57 Previous study 2Y Young (12months) Male – C57 Previous study 3Y Young (12months) Male – C57 Previous study 4Y Young (12months) Male – C57 Current study 5Y Young (12months) Male – C57 Current study 1A Aged (1.75–2 years) Male – C57 Previous study 2A Aged (1.75–2 years) Male – C57 Previous study 3A Aged (1.75–2 years) Male – C57 Previous study 4A Aged (1.75–2 years) Male – C57 Previous study 5A Aged (1.75–2 years) Male – C57 Previous study

Table 4: Animal use for Western analysis

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 96

Label Age Group Gender/Breed Lesion Tissue Source #2 8–12 months Female – C57 WT Rotenone Previous Study #3 8–12 months Female – C57 WT Rotenone Previous Study #66 8-12 months Male – C57 WT Rotenone Previous Study #67 8-12 months Male – C57 WT Rotenone Previous Study #86 8–12 months Female – C57 WT Rotenone Previous study #11 1.75–2 years Male – C57 WT Rotenone Current Study #102 1.75–2 years Male – C57 WT Rotenone Current Study #103 1.75–2 years Male – C57 WT Rotenone Current Study #109 1.75–2 years Female – C57 WT Rotenone Current Study #111 1.75–2 years Female – C57 WT Rotenone Current Study #0 1.75–2 years Male – C57 WT Sham Current Study #100 1.75–2 years Male – C57 WT Sham Current Study #105 1.75–2 years Male – C57 WT Sham Current Study #106 1.75–2 years Female – C57 WT Sham Current Study #107 1.75–2 years Female – C57 WT Sham Current Study #13 8–12 months Female – C57 WT Sham Current Study #14 8–12 months Male – C57 WT Sham Current Study #15 8-12 months Male – C57 WT Sham Current Study

Table 5: Animal use for immunohistochemistry

3.4.2.2 Surgery

Surgery involved stereotaxic injection of a rotenone solution into the MFB (figure 8) of 15 aged and 13 young mice, which allows for the infusion of rotenone into the SnPc.

Pre-operatively, the mice were injected subcutaneously with temgesic 20 minutes prior to the completion of the surgical procedure using a 1ml syringes size attached to a 27G needle.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 97 MFB

Figure 12: Target for stereotaxic surgery ((MFB) medial forbrain bundle).

Two techniques were used to anaesthetise the mice. The first with isoflurane (level 3)

which was fitted into a nosecone apparatus to sustain delivery of isoflurane (level 1.5–2.5)

and oxygen throughout the surgery. The other technique consists of a chemical anaesthesia

using a solution of 350µl of ketamine and xylazine (Table 5). The mice were placed within a

stereotaxic frame, on a heat pad. Ear bars and teeth holder were utilised to ensure placement

stability throughout the surgery. Due to the inability of the sedated mouse to close its eyes,

viscotears was applied to prevent irritation from various debris and surgical lamps. Reflex

tests were performed to verify the success of the anaesthesia which, coupled with a

determination of the animals’ respiratory function, were repeated throughout the procedure to

ensure the isoflurane was kept at optimal levels. In preparation of the surgery, the surface of

the head which would allow access to the brain was shaved and treated with the topical

antiseptic solution, betadine.

A medial-sagittal incision was made, so the location of the physiological markers of

lambda and bregma to be in sight (figure 9). These sutures were used to calibrate and “zero”

MFB The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 98 the stereotaxic apparatus for positioning at the injection point as given by an x (1.25mm) and y (- 0.94mm) axis. A single burr hole was drilled with stereotaxic drill, gaining access to the right hemisphere and allowing for placement of a 5µL Hamilton syringe attached to a dental

27G needle direct superior to the target point. The needle was then slowly advanced along the z axis 5.35mm then retracted to 5.25mm creating a cavity into which the treatment could be injected.

Figure 13: Location of the sutures bregma and lambda in mouse

The toxin solution (0.25mg/ml of rotenone in 1:1 DMSO and PEG) was injected over a period of four minutes into the brain at rate of 0.5µL/sec which gave a final volume injected of 2µL. The needle was then slowly retracted over a period of four minutes to prevent rotenone from being drawn out of the treatment cavity. The burr hole was filled with spongostan gelatine matrix to promote regrowth of the tissues. The incision was closed using

2-3 square knot sutures with a needle holder and the animal was given a 0.4mL antibiotic injection of Enrofloxacin subcutaneously before being taken off anaesthetic and its removal from the stereotaxic apparatus.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 99

Post operatively, mice were placed on a heating mat to maintain body heat whilst they recover from the isoflurane or the solution of xylazine and ketamine. Once awake, the mouse were placed in a new, clean and separate cage subcutaneous injection of analgesic (temgesic) is administrated for pain management every 6-12 hours over a three day periods. Enrofloxacin was injected subcutaneously every 12 hours over five days.

Dilutions of Injectable Anaesthetic Mixtures for Use in Mouse Dosage Ketamine 120mg/kg Xylazine 16.0mg/kg Dose 0.1ml/10gm body Rate weight Volumes Ketamine 0.60ml Xylazine at 20mg/ml 0.40ml Water for Injection 4.00ml Total volume 5.0ml ≈ 50 doses

Table 6: Chemical anaesthetic (For euthanasia Ketamine/xylazine combinations was utilized at 4 times the anaesthetic dose by the normal route)

Dilutions of Injectable Analgesic for Use in Mouse Dosage Temgesic 0.1mg/kg Dose Rate 0.1ml/15gm body weight Volumes Temgesic at 0.3mg/ml 0.50ml Water for Injection 9.50ml Total volume 10.0ml ≈ 50 doses

Table 7: Analgesic Temgesic

Dilution of Injectable Antibiotic for use in Mouse Dosage Enrofloxacin 5.00mg/kg Dose Rate 0.02ml/10g Body weight Volumes Enrofloxicin at 22.7mg/ml 0.50 ml Water for Injection 4.50 ml Total 5.00 ml ≈ 50 doses

Table 8: Antibiotic Enrofloxacin

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 100

3.4.2.3 Movement analysis

Previous studies have indicated that two weeks post-surgery was sufficient for the development of motor dysfunction and disease pathology, such as protein aggregation [232].

As such, movement repertoire was observed after two weeks to investigate the phenotypic expression of clinical Parkinson’s, namely motor dysfunction. Previous movement studies employed by our lab utilised the grasp test to analyse potential unilateral motor deficits. This entails encouraging the mice to walk along a suspended bar whilst noting the number of left paw placements in comparison to total paw placements. The resultant number of left paw placements was then calculated as a ratio of overall paw placements and statistically analysed. The group of animals to undergo surgery included enough subjects for meaningful quantitation of movement dysfunction. As such, motor skills were observed qualitatively, specifically, examining the favouring of one direction of movement, indicating unilateral hemisphere motor control dysfunction, or the demonstration of other documented

Parkinsonian symptoms.

3.4.2.4 Tissue Harvest and fixation

Animals were euthanized after 2 weeks via injection of ketamine (320μL) and xylazine (80μL). Mice then received an upper abdominal incision and lateral incision through the rib cage to expose the heart. Transcardial perfusion with sodium nitrite was performed with a 27G needle and an incision was made in the right ventricle allowing drainage of blood and perfusate. Sodium nitrite is a vasodilator and facilitates removal of red blood cells perfusion. Modified Zamboni’s fixative was then perfused into the animals to stabilize the tissues. Formaldehyde fixative such as modified Zamboni’s fixative has a minor effect on

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 101 antigens resulting in a higher quality of immunofluorescence. The brains were then removed and placed into modified Zamboni’s fixative for 24 hours at 4˚C.

Chemical For 1L of fixative dH20 477.6mL Paraformaldehyde 20g 28.39gNa2HPO4/L 400mL 31.2gNaH2OPO4.2H2O/L 120mL Picric Acid 2mL NaOH 10M 0.4mL

Table 9: Modified Zamboni’s fixative

3.4.2.5 Tissue harvest and homogenisation

Animals were sacrificed via injection with ketamine (320μL) and xylazine (80μL).

Transcardial perfusion with sodium nitrite eliminated all blood and extraneous materials from the brain. The brain were then isolated and dissected sagittally to separate the two hemispheres. Tissues were diluted 1 in 5 (g/mL) in buffer (tricene 20mM, sucrose 0.25mM,

EDTA 1mM, ph7.6) and manually homogenised with a handheld homogenizer, using 24 strokes for each sample. Prior to use, the sections were stored at -80˚C.

3.4.2.6 Optical clearing

Following fixation, the fixative was removed from tissues by extended washed in

Phosphate buffered saline (PBS) 0.1% Triton X-100, followed by washing in 100% Dimethyl sulfoxide (DMSO) for 2 hours on bench top rockers and under vacuum as thick sections were cut later. Triton X-100 dissolved lipids and facilitated the removal of optically opaque materials while DMSO interacted with the lipid membrane in which it formed water pores and displaced the water. This facilitated the removal of water and the intake of mounting medium

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 102 by the tissues. The use of vacuum lowered the surface tension of the lipids and the use of bench top rockers prevented the formation of diffusion gradients.

3.4.2.7 Western blot

WB analysis was performed to determine whether there was a change in the protein expression profile in response to the rotenone injection (PD inducement). WB was carried out to investigate CB expression. As WB required integration of band intensity control, actin was used as a loading control in those WB.

Chemical 12% separating gel (1 4% stacking gel (1 gel) gel) Acrylamide Solution 1.5mL 125µL Tris HCL (1.5MpH 8.8) 1.25mL (0.5MpH 6.8) 315µL Water 2.175mL 700µl 10% SDS 50µL 12.5µL Tetramethylethylenediamine 2.5µL 1.25µL 10% Ammonium persulfate 25µL 6.25µL

Table 10: 12% SDS-PAGE (quantity for 1 gel)

The crude mouse brain homogenates was diluted 1:2 in loading buffer (Table 11). The samples were boiled for 5 minutes to reduce and denature the proteins. Then 12µL of each sample was loaded into a 0.75mm, 12% SDS-PAGE with 4% loading gel (Table 10) with a

10µL Benchmark Ore-stained protein ladder. The gels were run in running buffer (Table 11) at a voltage of 100V for 1 hour and 40 minutes. After cleaning the gel, it was placed in the transfer cassette with a nitrocellulose membrane and run at a constant voltage of 100V for 45 minutes in cold transfer buffer (Table 11).

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 103 0.3g BSA 0.05g Skim Milk Blocking Solution 20µL Tween 20 make up to 10mL of tris buffered saline, 0.1%tween 20 (TBS-T) Loading 5% 2-mercaptoethanol Buffer 95% Laemmli sample Buffer 25mM Tris Running 192mM Glycine Buffer 0.1% w/v SDS pH8.3 100mL 10x Stock Transfer 695mL Water Buffer 200mL Methanol 5mL 10% SDS 10x Transfer 30.2g/L Tris stock 144.13 g/L

Table 11: Solutions used in Western blot analysis

The nitrocellulose was blocked using blocking solution (Table 11) for 45 minutes with shaking. Primary antibodies (Rb-ab CB) were diluted 1:1000 in blocking solution. The nitrocellulose was incubated with the primary antibody overnight at 4˚C then washed in TBS-

T for five minutes, 3 times and incubated with secondary antibody (anti-rabbit IgG) at a dilution of 1:1000 in blocking solution for an hour at room temperature, with shaking.

Following this, the membrane was then washed as previously done, to remove excess secondary antibody. Then WB chemiluminescence substrate was prepared (1:1 enhancer solution: peroxide solution) and the membrane was incubated for 5 minutes (0.125ml for each cm²). An image was then taken using a Biorad camera.

WB images were analysed using the Alpha Innotech software, after having been cropped and rotated in order to orientate the membranes correctly. Bands were found from a designated area and then calculated by the software, which provides a un-biased analysis. The

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 104 software measures the intensity of each band in relation to the background and a line graph of band intensity was calculated. To quantify these results, the software calculated the area under the curve. To determine the relative intensity of each band, actin loading controls were performed and analysed.

3.4.2.8 Sectioning

In order to facilitate sectioning of biological materials, the support of a matrix was required. The freezing of a liquid such as Optimal Cutting Temperature compound (OCT) formed a hardened block which provides rigidity to the tissue. The tissues were placed in 30% sucrose PBS azide (0.02% w/v) and stored at 4˚C overnight. Tissues were then placed in a series of OCT solutions of increasing concentration (20%, 50% and 70% in 30% sucrose in

PBS azide) for one hour each. The brains were then placed in a 100% OCT solution for 1 hour to remove any sucrose from the tissues. The brains were then mounted in 100% OCT and stored at -80˚C until sectioning. The brains were sectioned at 30µm using a microtome- cryostat. Sections were kept as adherent sections on coated slides at -80˚C until processed. All washes and incubations were done on a bench top rocker and in vacuo at room temperature.

3.4.2.9 Fluorescence microscopy

Immunofluorescence was used to determine the expression of CB and its level of expression in neurons, to compare the lesioned and control hemispheres of the brain and to observe the presence of α-syn aggregates within neurons.

The brain sections were first brought to room temperature for 20 minutes, washed with

TBS for 10 minutes and then incubated for 30 minutes in 20% NHS in TBS. In a parallel set of slides, tissues were first incubated for 15 mins at 37°C with proteinase K (20µg/mL), prior

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 105 to blocking. The sections were then incubated overnight with rabbit anti-CB antibody (Rb-ab

CB) and mouse anti-α-syn or (Ms-ab α-syn) or mouse anti-NeuN (Ms-ab NeuN) diluted 1:200 in 1% NHS-TBS. On the following day, the sections were washed 3 times in TBS and then incubated with the secondary anti-body (donkey anti-rabbit IgG with fluorescent dye AF-568 and donkey anti-mouse IgG with fluorescent dye AF-488) for 45 minutes diluted 1:200 in 1%

NHS-TBS at room temperature in the dark. Then the sections were dried, Prolong GOLD mounting medium containing DAPI was applied, covered with a coverslip, then stored at 4˚C before being viewed on a confocal fluorescence microscope, the Olympus FV1000 (Filter based system). It was configured for the acquisition of digital multi-channel fluorescence images using a laser excitation and filter based detection system. Pictures from a random spot were taken in the motor and somatosensory cortices and the striatum from the treated and control hemispheres.

3.4.2.10 Cell counting

Figure 14: Area of the brain analysed showing motor cortex (blue), somatosensory cortex

(green) and striatum (red).

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 106 Immunostained cells were counted and classified as either CB+ or CB- or α-syn+ or α-syn-

. To be classified as CB+ cell or α-syn+ cell, and CB+ cell or NeuN+ cells, the prescence of clear and bright immunostaining needed to be visualized. Total cell count per area imaged was determined based on the DAPI nuclear stain. Random areas in the motor cortex, somatosensory cortex and the striatum were analysed. The percentage of CB+ cells was calculated in comparison to CB- cells. Experiments were then repeated on the other hemisphere utilizing identical cell counting procedures. For mouse tissue, a standardized pattern of three random areas containing on average 50 cells (210µm x 210µm) each in the motor cortex, somatosensory cortex, and striatum was imaged for each of the 8 young adult and 10 aged hemispheres. Image files were then de-identified for counting. The proportion of

CB+ cells was calculated along with α-syn aggregate positive cells.

3.4.2.11 Statistical analysis

Comparison of CB expression in the treated and untreated hemisphere using the protein band intensities obtained from WB were analysed for statistical significance via two- tailed, paired t-tests. A paired t-test was chosen due to the equal number of subjects in each of the groups tested. The data was assumed to be parametric as treated and control conditions were obtained from the same animal. CB expression between aged and young mice was determined using this method.

CB+ and CB- cells were counted on each image along with α-syn+ and α-syn- cells.

Then, a percentage of CB+ cells, a percentage of α-syn+ cells and a percentage of CB/α-syn+ neurons were established for each image. Data was analysed using an ANOVA test between

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 107 CB/α-syn and α-syn+ neurons, age (young adults and aged mice), different hemispheres

(Sham, control and lesioned) and brain areas.

3.4.2.12 Antibodies

Three different primary antibodies were used in the present study. One type of anti-CB antibody (Swant) was used to stain in the rotenone lesioned tissue while a mouse monoclonal anti-α-syn antibody from Invitrogen was used to detect α-syn aggregate. NeuN (clone A60) antibodies were used to recognize the DNA-binding, neuron-specific protein NeuN, which is present in most CNS and peripheral nervous system (PNS) neuronal cell types of all vertebrates tested. In order to visualise the primary antibody binding, AF conjugated secondary antibodies were used. AF-488 anti-mouse and AF-568 anti-rabbit fluorescent secondary antibodies were used to visualise primary antibodies in the immunofluorescence experiment. Horseradish peroxidase conjugate antibodies were used to visualise primary antibodies in the WB analyses.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 108 Antibody Use Antigen Host Reactivity Use Source Human, CB38 Primary CB Rabbit WB/IF Swant rodent Human, LB509 Primary α-syn Mouse IF Invitrogen rodent BD Human, 42/α-syn Primary α-syn Mouse IF Transduction mouse Laboratories Human, Chemicon NeuN Primary NeuN Mouse IF rodent (Millipore) Human, Sigma- AC74 Primary β-Actin Mouse WB rodent Aldrich Mouse Fc Molecular AF-488 Secondary Donkey Mouse IF receptor Probes Rabbit Fc Molecular AF-568 Secondary Goat Rabbit IF receptor Probes HRP Rabbit Secondary Goat Rabbit WB Biorad Conjugate IgG (H+L) HRP Mouse Secondary Goat Mouse WB Biorad Conjugate IgG (H+L)

Table 12: Antibodies used for immunofluorescence labelling

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 109 3.4.3 Cell culture

3.4.3.1 Cell culture

SH-SY5Y cells were seeded at 20,000 cells/24 well plates on 10mm glass coverslips.

Cells were incubated for 24 hours at 37°C with 5% CO2 in 1mL of 15% FBS, 82.96% Hams

DMEM:F12 (Invitrogen), 0.04% Amphotericin B and 1% Strep/Penicillin prior to drug treatment.

For siRNA transfection cells were treated and seeded simultaneously at 20,000cells/24 well plates on 10mm glass cover slips. Cells were placed in 5µL of transfection reagent and

5µL of CB siRNA (Santa Cruz Biotechnology, Inc.) in 500µL of transfection reagent for 6 hours. Then the medium was changed to a transfection reagent and siRNA free medium and cells were left incubating for 48 hours.

+ 3.4.3.2 Calcipotriol, K , H2O2 and rotenone treatments

SH-SY5Y cells were incubated in the presence of increasing concentrations of Cp (0,

01, 0.5, 1, 5 or 10nM) in serum free DMEM for 5 hours at 37C with 5% CO2 then washed twice with PBS. The medium was then replaced with medium supplemented with 50mM KCl for 1 hour or 50mM KCl and 10µM H2O2 for 1 hour. For control cells, un-supplemented medium was used. Following treatment, cells were incubated with fresh medium for 36 at

2 37C with 5% CO before fixing. For rotenone, after Cp treatment cells were placed in 500nM of rotenone and left incubating for 24 hours at 37ºC with 5% CO2 before fixing.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 110 3.4.3.3 Fluorescence microscopy and analysis

Cells were rinsed in PBS prior to fixation. Cells were fixed and permeabilised using

1:3 methanol:acetone and blocked with or 20% NHS. Monoclonal mouse anti-α-syn antibody

(LB509, Invitrogen) and monoclonal rabbit anti-CB antibody were used 1:100 for immunostaining. AF conjugated secondary antibodies (Molecular Probes, Invitrogen) were used for immunofluorescence and mounted using Prolong Gold with DAPI mounting medium

(Invitrogen). Microscope slides were stored in the dark at 4°C.

Immunostained coverslips were imaged using the Nikon A1R+ confocal laser microscope. Negative control slides were imaged to set microscope settings (laser voltage, electronic gain and offset) to appropriate levels. The 60x oil immersion lens were used for fixed cell imaging. DAPI, AF-488 and 568 were imaged together, on separate channels.

3.4.3.4 Cell counting

Immunostained cells were counted (n= 30) and classified as either α-syn inclusion body-positive or body-negative. Random 60x fields of view (n= 5) were imaged and counted using Columbus PerkinElmer image analysis software. To be classified as α-syn inclusion body-positive cell, clear and bright focal immunostaining needed to be observed in one or more confocal slice. The percentage of α-syn inclusion body positive cells was calculated along with inclusion body-negative cells. Experiments were then repeated with n= 5 for SH-

SY5Y/CB cells utilizing identical cell counting procedures. CB intensity was measured using

Columbus PerkinElmer analysis software.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 111 3.4.3.5 Western Blot Analysis

Treated cells were rinsed in cold PBS prior to be treated with cold RIPA buffer.

Adherent cells were scraped off the dish and maintained at constant agitation for 30mins at

4°C. Cells were centrifuge at 4°C for 5mins at 20,000rpm; the supernatant was kept and stored at -20ºC.

20μL of each sample was run in replicate on 8-16% Mini-PROTEAN® TGX Stain-Free™

Gels (200V, 30mins), transferred to nitrocellulose using Trans-Blot® Turbo™ Transfer

System (25V, 30 mins), assessed staining of proteins, blocked (1 hour, 5% skim milk powder), incubated with primary antibody (1:1000 overnight) rabbit anti-CB (Swant); mouse anti-α-syn (Invitrogen), washed (3x, TBS–T), incubated with HRP-conjugated secondary antibody (Biorad, 1:2000; 1 hour) washed (3x, TBS–T) and developed with West Pico

SuperSignal Chemiluminescent Substrate (Thermo Scientific). Membranes were imaged by

ChemiDoc™ MP System. For re-probing, blots were stripped with 0.2N NaOH for 30 min, then re-blocked and probed for actin. Bands were quantitated by area under the curve relative to actin loading controls (Image Lab™ software).

3.4.3.6 Statistical analysis

Data were analysed using SPSS Version 22.0. α-Syn aggregation in SH-SY5Y/CB and

SH-SY5Y cells were analysed for statistical significance using an ANOVA test.

3.4.3.7 Antibodies

Two different primary antibodies were used for the present study. One type of anti-CB antibodies from Swant were used to stain in the SH-SY5Y neuroblastoma cells and anti-α-syn antibodies from Invitrogen.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 112 In order to visualise the primary antibody binding, AF conjugated secondary antibodies were used. AF-488 anti-mouse and AF-568 anti-rabbit Fluorescent secondary antibodies were used to visualise primary antibodies in human MSA, normal and the rotenone mouse model. AF conjugated secondary antibodies were purchased from Molecular Probes,

Invitrogen.

Antibody Use Antigen Host Reactivity Use Source Human, CB38 Primary CB Rabbit WB/IF Swant rodent

Human, Primary α-syn Mouse LB509 rodent IF Invitrogen

Human, Sigma- AC74 Primary β-Actin Mouse WB rodent Aldrich

Mouse Fc Molecular AF-488 Secondary Donkey Mouse IF receptor Probes

Rabbit Fc Molecular AF-568 Secondary Goat Rabbit IF receptor Probes

HRP Rabbit IgG Secondary Goat Rabbit WB Biorad Conjugate (H+L)

HRP Mouse IgG Secondary Goat Mouse WB Biorad Conjugate (H+L)

Table 13: Antibodies used for immunofluorescence labelling

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 113 Chapter 4: Alpha-Synuclein aggregates are excluded from Calbindin-D28k-

positive neurons in dementia with Lewy bodies and a unilateral rotenone

mouse model

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 114 STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED PAPER

This chapter includes a co-authored paper. The bibliographic details published of the co-authored paper, including all authors, are:

Alexandre Rcom-H’cheo-Gauthier

Amelia Davis

Dr Adrian Meedenyia

Dr Dean Pountney

My contribution to the paper involved:

Tissue processing, sectioning, staining and statistical analysis for immunofluorescence study on human tissue.

Surgery, tissue collection, sectioning, staining and statistical analyses for immunofluorescence study on mice for α-syn and CB (all).

Surgery, tissue collection for 2 young mice in Western blot analyses.

Running, and statistical analyses for Western blot analyses for CB expression.

Rcom-H’Cheo-Gauthier, A., Davis, A., Meedeniya, A., Pountney, D.L. Alpha-Synuclein aggregates are excluded from Calbindin-D28k-positive neurons in dementia with Lewy bodies and a unilateral rotenone mouse model. Molecular and Cellular Neuroscience, submitted June 17, 2015, accepted Fri, Sep 11, 2015

(Signed) ______(Date)______

Alexandre Rcom-H’cheo-Gauthier

(Countersigned)______(Date)__16/08/2016__

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 115 Corresponding author of paper: Dr Dean Pountney

(Countersigned)______(Date)__16/08/2016__ Supervisor: Dr Dean Pountney

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 116 4.1 Introduction

DLB like PD are known as α-synucleinopathies which are characteristic of their abnormal aggregation of the pre-synaptic protein α-syn within neurons. In DLB, unlike PD,

LB pathology occurs throughout the brain. This aggregation may be associated with disturbed

Ca2+ homeostasis and oxidative stress within neurons. The α-syn gene have been identified with amino acid substitutions E46K having both PD and DLB variants [34]. Recent studies have shown that a transient increase in the intracellular free Ca2+ concentration and oxidative stress induced by chemical treatments caused a significant increase in the proportion of cells bearing microscopically-visible α-syn aggregates [110]. Multiple CBP, such as CB, PV and

CR, have been found to be over represented in dopaminergic neurons in the SN of PDs post mortem brain tissue [194, 233]. This indicates that CBP may protect some neurons against degeneration in PD. CB might also be overexpressed in DLB which indicates that it could also be protective in this disease.

In the current study, we investigated the proportion of CB and α-syn positive neurons in DLB and normal tissue. It was proposed therefore to investigate whether there was a correlation between CB expression, α-syn aggregation and neuronal survival in this disease.

In addition, we investigated the proportion of CB and α-syn positive neurons in an animal model of α-synucleinopathy. It was proposed therefore to determine if there was a correlation between CB expression, α-syn aggregation and neuronal survival in this model.

Finding exclusion of α-syn aggregates from CB+ neurons could indicate the Ca2+ buffering effect of CB on α-syn aggregation. It could confirm that free intracellular Ca2+ is a major factor influencing α-syn aggregation, and potentially the formation of the cytotoxic oligomeric species seen in disease. Successfully targeting raised intracellular free Ca2+ in the

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 117 brain by promoting the expression of CB at the transcriptional level might be effective at reducing Ca2+-dependent neuronal α-syn aggregation and cytotoxicity.

4.2 PDF of paper

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 118

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 119

MCN

Alpha-Synuclein aggregates are excluded from Calbindin-D28k-positive neurons in dementia with Lewy bodies and a unilateral rotenone mouse model

Alexandre N. Rcom-H’cheo-Gauthier, Amelia Davis, Adrian C. B. Meedeniya

and Dean L. Pountney*

Menzies Health Institute Queensland, School of Medical Science, Griffith University, Gold Coast, Queensland 4222, Australia

*Corresponding author: Dean L. Pountney Griffith Health Institute, School of Medical Science, Griffith University, Gold Coast, Queensland 4222, Australia

Tel: +61 7 5552 7273 Fax: +61 7 5552 8908 Email: [email protected]

7136 words, 7 figures

Running title: alpha-synuclein and calbindin

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 120

Keywords: alpha-synuclein; Parkinson’s disease; Dementia with Lewy Bodies; calcium; calbindin-D28k; neurodegeneration; oxidative stress

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 121

Abstract:

α-Synuclein (α-syn) aggregates (Lewy bodies) in Dementia with Lewy Bodies (DLB) may be associated with disturbed calcium homeostasis and oxidative stress. We investigated the interplay between α-syn aggregation, expression of the calbindin-D28k (CB) calcium- buffering protein and oxidative stress, combining immunofluorescence double labelling and

Western analysis, and examining DLB and normal human cases and a unilateral oxidative stress lesion model of α-syn disease (rotenone mouse). DLB cases showed a greater proportion of CB+ neurons in affected brain regions compared to normal cases with Lewy bodies largely present in CB- neurons and virtually undetected in CB+ neurons. The unilateral rotenone-lesioned mouse model showed a greater proportion of CB+ cells and α-syn aggregates within the lesioned hemisphere than the control hemisphere, especially proximal to the lesion site, and α-syn inclusions occurred primarily in CB- cells and were almost completely absent in CB+ cells. Consistent with the immunofluorescence data, Western analysis showed the total CB level was 20% higher in lesioned compared to control hemisphere in aged animals that are more sensitive to lesion and 25 % higher in aged compared to young mice in lesioned hemisphere, but not significantly different between young and aged in the control hemisphere. Taken together, the findings show α-syn aggregation is excluded from CB+ neurons, although the increased sensitivity of aged animals to lesion was not related to differential CB expression.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 122

Introduction

Dementia with Lewy Bodies (DLB) is the most common non-Alzheimer’s dementia, showing acetylcholine and dopamine depletion and associated disruption of perception, thinking and behavior. Degeneration of multiple brain systems leads also to atypical

Parkinsonism characterized by rigidity of movement combined with cognitive/memory disorder and neuropsychiatric symptoms, including hallucinations and behavioural problems.

Factors including oxidative stress, gene mutations and elevated calcium ion concentrations are known to impact on the formation of the intraneuronal protein lesions (Lewy bodies) characteristic of the disease, in which α-synuclein (α-syn) (14 kDa) is a key protein component, leading to its classification as an α-synucleinopathy (Breydo et al, 2012;

Cookson, 2009; Jellinger 2009a; Marques & Outeiro, 2012; Rcom-H’cheo-Gauthier et al,

2014). Unlike in Parkinson’s disease (PD), where pathology occurs primarily in the substantia nigra (sn), Lewy body pathology in DLB is widespread throughout the brain, including cortical areas (Jellinger, 2009b; Jenner et al, 2013). Although α-synucleinopathy is predominantly idiopathic, to date, six mutations in PARK1/4, the α-syn gene, have been identified with amino acid substitutions, A30P (Kruger et al, 1998), A53T (Polymeropoulos et al, 1997), E46K (Zarranz et al, 2004), H50Q Proukaksi et al, 2013), G51D (Lesage et al,

2013) and A53E (Pasanen et al, 2014), most linked to PD, but E46K having both PD and

DLB variants.

Recent studies have shown that a transient increase in the intracellular free Ca2+ concentration induced by chemical treatments caused a significant increase in the proportion of cells bearing microscopically-visible α-syn aggregates (Nath et al, 2011). It was also demonstrated that chelation of intracellular free Ca2+ could block α-syn aggregate induction,

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 123 implicating the direct involvement of raised intracellular free Ca2+ . Moreover, in vitro protein studies showed that direct binding of Ca2+ ion to α-syn promoted rapid oligomer formation.

Furthermore, Follett et al demonstrated that potassium depolarization of the plasma membrane in HEK293T and SH-SY5Y human cell lines resulted in raised intracellular free

Ca2+ and caused α-syn aggregate formation, with both raised free Ca2+ and α-syn aggregation blocked by chelation treatment (Follett et al, 2013].

Ca2+ regulation is vital for cell survival and function and there are specific proteins responsible for Ca2+ buffering in neurons, including calbindin-D28K (CB), calretinin and parvalbumin. Yamada et al. found that dopaminergic neurons of the sn that were high in CB were preferentially spared in PD patients, indicating that increased free Ca2+ may be a major factor in the pathogenesis of α-synucleinopathies (Yamada et al, 1990). Bu et al found decreases in both calretinin and CB in aged compared to young cortical neurons, put no difference in parvalbumin positive neurons (Bu et al, 2003). Moreover, German et al found in both MPTP monkey or C57BL mouse models that CB+ neurons were spared whilst neurons in CB- regions were lost (German et al, 1992). In other studies, calretinin expressing dopaminergic neurons of the sn were more protected against 6-hydroxydopamine (Kim et al,

2000; Tsuboi et al, 2000). Ca2+ blockade was performed by Chan et al using brain slices

2+ prepared from a MPTP mouse model, who found that by blocking L-type Cav1.3 Ca channels with Isradipine, they could recover dopaminergic neural activity (Chan et al, 2007).

There is evidence that oxidative stress is increased in normal aged brain however the level of oxidative stress is greatly increased in patients with neurodegenerative diseases (Sims-

Robinson et al, 2013). Moreover, Kume et al found urinary 8-OHdG levels were significantly higher in DLB cases compared to controls, indicating systemically increased oxidative stress

(Kume et at, 2012). The major contribution to oxidative stress in ageing primates originates

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 124 from mitochondrial complexes I and III of the electron transport chain leading to greater mitochondrial DNA damage compared to nuclear DNA damage (Castro et al, 2012). Indeed, widespread mtDNA damage occurs at early stages of Lewy body disease (Lin et al, 2012).

Oxidative stress characterized by α-syn lipoxidation precedes the formation of -syn aggregates and the development of neocortical Lewy body pathology in DLB (Dalfo & Ferrer,

2008). Furthermore, Quilty et al showed that when mouse primary neocortical cells were incubated in the absence of antioxidants, mild oxidative stress caused raised α-syn accumulation in a subset of neurons (Quilty et al, 2006). Indeed, recent studies have found the oxidized form of the endogenous oxidative stress sensor, DJ-1, progressively increased in the later stages of PD and more highly oxidized forms were likely present in DLB (Saito et al,

2014). Furthermore, Surmeier et al have shown that calcium influx can interact with α-syn to mediate increased oxidative stress (Dryanovski et al, 2013) and Goodwin et al demonstrated a synergistic effect of combined oxidative stress and raised intracellular free calcium on a-syn aggregation (Goodwin et al, 2013). In animal models, the complex I inhibitor, rotenone, results in oxidative stress and replicates behavioural, anatomical and biochemical characteristics of α-synucleinopathy (Betarbet et al, 2000; Vila et al, 2001; Sanders &

Greenmyre, 2013). Unilateral stereotactic injection of rotenone into the MFB at low dose resulted in widespread -syn inclusion bodies, primarily in neurons, and permits pair-wise- comparison between brain hemispheres of protein expression between treated and untreated hemispheres, with a more severe phenotype apparent in aged over young adult animals

(Weetman et al, 2013; Radford et al, 2015). Whereas acute, targeted rotenone injection, rather than systemic exposure allowed for diffusion of rotenone whilst avoiding gross physical trauma resulting from the surgery and produced a gradient of α-syn and neuro-inflammation pathology (Radford et al, 2015).

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 125

In the current study, we investigated the interplay between α-syn aggregation, expression of CB and oxidative stress by examining CB-positive neurons and α-syn inclusion bodies in

DLB and normal cases and in the unilateral rotenone mouse model. DLB cases showed an increased proportion of CB+ neurons compared to normal cases, whilst Lewy bodies, that were widespread in DLB tissue, were not detected in CB+ neurons. In the mouse model, CB+ neurons were over-represented in the rotenone lesioned compared to control hemisphere, especially proximal to the lesion site, whilst α-syn aggregates were increased and occurred almost exclusively in CB- cells. Comparing aged (1.75 years-plus) animals with young (6-12 months) animals, both the number of CB+ cells and total CB level was greater in the lesioned tissue, especially of the aged animals, but not significantly different in unlesioned tissue between aged and young animals, indicating that decreased CB expression does not underlie the increased susceptibility to lesion observed for aged animals.

Materials and Methods

Human tissue

Human brain tissue was acquired from the South Australian Brain Bank. Diagnosis of disease was conducted at autopsy by neuropathologist Professor Peter Blumbergs according to disease specific criteria. Autopsies were conducted 4-17 hours post-mortem where the brains were bisected into the hemispheres with one hemisphere formalin fixed, whilst the other was stored at -80 °C. The formalin fixed tissue was then embedded in paraffin, sectioned (5μm) and mounted on gelatine-coated glass slides. Six DLB (age at death: 70 ± 7, post-mortem

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 126

interval, PMI 8 ± 3 hours), and six age matched normal cases (age at death 73 ± 9; PMI 14 ± 8

h) were analysed, six brain regions in each case.

Animals

Experiments conducted under the approval of the Griffith University Ethics Committee

comprised of 17 aged (1.75 year-plus), 15 young adults (6-12 months) and two young (8

weeks) wild-type (WT) C57BL/6 mice. Mice were housed in standard cages, on a 12-hour MFB light cycle with ad libitum access to food and water. Surgery involved stereotaxic injection of

a rotenone solution into the MFB which allows for the diffusion of rotenone into the SnPc, as

described (Weetman, et al, 2013). A medial-sagittal incision was made, to locate the

physiological markers of lambda and bregma, and these sutures were used to calibrate and

zero the stereotaxic apparatus for positioning at the injection point as given by an x (1.25mm)

and y (- 0.94mm) axis. A single burr hole was drilled gaining access to the right hemisphere

and allowing for placement of a 5µL Hamilton syringe connected to a dental 27G needle

directly above the target point. The needle was then slowly advanced along the z axis 5.35mm

then retracted to 5.25mm creating a cavity into which, the treatment could be injected. The

toxin solution (0.25mg/ml of rotenone in 1:1 DMSO:PEG) or vehicle only was injected over a

period of four minutes into the brain at a rate of 0.5µL/sec which gives a final volume injected

of 2µL. The needle was slowly retracted over a period of four minutes to prevent rotenone

from being drawn out of the treatment cavity.

Animals were sacrificed after two weeks via injection with Ketamine (320μL) and

Xylazine (80μL), then mice were perfused transcardially with 0.5% sodium nitrite in 0.1M

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 127 phosphate buffered saline to eliminate blood and extraneous material followed by Zamboni's fixative.

Immunofluorescence

Human tissue sections were deparaffinized by xylene (2 washes for 10 minutes), then rehydrated in 100%, 95%, and 70% ethanol. Heat induced antigen retrieval was performed in

1mM EDTA pH8 solution at 100ºC [231]. After allowing the slides to cool (<50ºC), non- specific binding sites were blocked by immersing the tissue in 20% normal horse serum

(NHS) in TBS for 1 hour then incubated with rabbit anti-calbindin-D28K (1:200; Swant) and mouse anti-α-synuclein (1:200; LB 509 invitrogen) primary antibodies and Alexafluor 488 and 568 (1:200; Life Technologies) secondary antibodies and imaged using an Olympus

FV1000 confocal laser scanning microscope. Negative control slides incubated only with secondary antibodies were imaged to set microscope settings (laser voltage, electronic gain and offset) to appropriate levels. The 60x oil immersion lens was used for image acquisition.

Neuronal CB and α-syn staining was confirmed by use of the doublecortin (DCX) antibody marker (1:50; Abcam).

For mouse tissue, following fixation, the fixative was removed from tissues by extended washing in PBS, 0.1% Triton X-100, then tissues were incubated in DMSO for 2 hours on bench top rockers and in vacuum. The tissues were placed in 30% sucrose PBS-azide at 4˚C overnight, then a series of OCT solutions of increasing concentration (20%, 50% and 70% in

30% sucrose in PBS azide) for one hour each, then 100% OCT solution for 1 hour. Tissue was sectioned at 30µm using a microtome-cryostat and kept as adherent sections on coated slides at -80˚C until processed for immunofluorescence. Non-specific binding sites were blocked by

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 128 immersing the tissue in 20% NHS in TBS. In a parallel set of slides, tissues were first incubated for 15 mins at 37°C with proteinase K, prior to blocking. Tissues were then stained using rabbit anti-calbindin-D28K (1:200; Swant) and mouse anti-α-synuclein (1:400; BD

Transduction Laboratories) primary antibodies and Alexafluor 488 and 568 (1:200; Life

Technologies) secondary antibodies and imaged using an Olympus FV1000 confocal laser scanning microscope. Negative control slides were imaged to set microscope settings (laser voltage, electronic gain and offset) to appropriate levels. The 60x oil immersion lens was used for image acquisition. Neuronal CB staining was confirmed by use of the NeuN antibody marker (1:400; Chemicon).

Cell counting

Immunostained cells were counted using image J software and classified as either CB+ or

CB- or α-syn aggregate positive or α-syn aggregate negative. To be classified as a CB+ cell, bright immunostaining needed to be visualized throughout the cytoplasm for CB (mean pixel intensity 3-fold over background intensity). α-Syn aggregate bearing cells were counted as bearing one or more bright cytoplasmic puncta clearly immunofluorescent for α-syn over the entire aggregate (mean pixel intensity 4-fold over background cytoplasmic intensity). Total cell count per area imaged was determined based on DAPI nuclear stain. For human tissue, a standardized pattern of three random areas containing on average 40 cells (210µm x 210µm) each in the motor cortex, visual cortex, frontal lobe, temporal lobe, cingulate gyrus and the hippocampus was imaged for each of the six cases and six controls. For mouse tissue a standardized pattern of three random areas containing on average 50 cells (210µm x 210µm) each in the motor cortex, somatosensory cortex, and striatum was imaged for each of the 10

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 129 young adult and 10 aged hemispheres. Image files were then de-identified for counting. The proportion of CB+ cells was calculated along with α-syn aggregate positive cells.

Western Blotting

Unfixed mouse brains were dissected sagitally to separate the two hemispheres and homogenized by handheld homogenizer, as described (Weetman et al, 2013), at 1:5 g/mL

50mM Tris–HCl pH 7.4/0.32M sucrose/5mM EDTA/Sigma protease inhibitor cocktail then stored at −80 ◦C. Crude mouse brain homogenates were diluted 1:3 in loading buffer and boiled then run in triplicate on 12% SDS-PAGE (100 V, 2 h), transferred to nitrocellulose (80

V, 40 min), assessed via Ponceau Red staining of proteins, blocked (1h, 5% skim milk powder), incubated with primary antibody overnight (1:1000; rabbit anti-Calbindin-D28K); washed (3×, TBST), incubated with HRP-conjugated secondary antibody (1:1000; 1 h) washed 4× and developed with West Pico SuperSignal Chemiluminescent Substrate (Thermo

Scientific). Membranes were imaged by Alpha Innotech Fluorochem FC2. For re-probing, blots were stripped with 0.2N NaOH for 30 min, then re-blocked and probed with mouse anti- actin primary antibodies (1:1000; Sigma). Bands were quantitated by area under curve relative to actin loading control (Alpha Innotech software).

Statistical Methods

All data were analyzed using SPSS Version 22.0. Standard error of the mean for graphs

휎 was calculated using( ). Two-tailed t and ANOVA tests were conducted to test for statistical √푛 significance.

Results

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 130

2+ In the current study, we were interested in probing the interaction between Ca , α-syn aggregates and oxidative stress. We analyzed the juxtaposition of neurons positive for the calcium buffering protein, CB, and -syn inclusion bodies in Dementia with Lewy Bodies tissue and in the unilateral rotenone mouse model of α-syn disease. The unilateral rotenone mouse model reproduces features of α-syn disease common to DLB, MSA and PD, including motor dysfunction and α-syn deposits, primarily in neurons (Weetman et al, 2013; Radford et al, 2015).

Calbindin-D28k-positive neurons are increased in DLB and exclude -synuclein inclusions

In order to examine CB and α-syn distribution in brain tissue sections from DLB cases and normal controls, immunofluorescence confocal microscopy was performed on formalin-fixed sections from cortical and non-cortical brain regions (visual and motor cortex, temporal and frontal lobe grey matter tracts, cingulate gyrus and cerebellum). Representative images are shown in Fig. 1B-H from sections (5 m) from DLB (Fig. 1B, C, E-H) and control cases (Fig.

1D). Fig. 1B represents a section of DLB frontal lobe stained for DCX, a neuronal marker, and α-syn (enlargement in Fig. 1E). Over the DLB cases examined, α-syn aggregates occurred predominantly in neuronal cells (> 90%), in DCX-positive neurons (79.1% ±6.7) and other cells with neuronal morphology. Consistent with previous studies (Baba et al., 1998; Beyer et al, 2007), few α-syn aggregates were detected in glial cells (Fig 1 H). CB was expressed within the cytoplasm of a subset of neurons, whilst the majority of cells were observed not to contain cytoplasmic CB, especially within the control cases. Fig. 1C shows the characteristic more intense uniform CB expression within CB+ cells in DLB cases than normal cases.

Neuronal cytoplasmic α-syn deposits (arrowheads; Fig. 1C and 1D) were observed frequently in DLB cases, but were rare in normal cases. Most inclusion bodies were observed in CB-

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 131 neurons, but occasional cells weakly positive for CB were observed with cytoplasmic -syn inclusions Fig. 1F shows an enlarged example of an α-syn aggregate inside a weakly CB+ neuron. Additional example data from different brain regions is presented in Supplementary

Figure 1.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 132

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 133

Figure 1: Calbindin-D28k (CB) expression and α-synuclein (α-syn) aggregation in dementia with Lewy body (DLB) and control cases. (A) Negative control slides with secondary antibodies only. (B) Immunofluorescence of the frontal lobe showing DCX+ cells (red) and α-syn aggregates (green). (C-D) Immunofluorescence (DLB and normal cases of the visual cortex) showing CB+ cells (red; arrow) are over-represented in DLB cases (B) compared to control cases (C) and α-syn aggregate positive cells (green; arrowhead) are over- represented in DLB cases (B) compared to control cases (C). Thick arrows (yellow dots in merged) represent auto-fluorescence. Scale bar, 50µm. (E) Representative image of a DCX/α-syn+ neuron (F) Representative image of a CB+/α-syn+ neuron. (G) Representative image of a CB-/α-syn+ neuron.. (H) Representative image of a α-syn+ glial cell. Scale bar 5µm.

To obtain a more quantitative view, cell counting was conducted across the six DLB cases and six control cases over six brain regions. The cell counting data is summarized in Fig. 2 A-

D. The mean proportion of α-syn aggregate bearing cells was significantly higher in DLB cases than the control cases (frontal lobe, cingulate gyrus (p, 0.0001); temporal lobe, (p, 0.01), hippocampus, visual cortex, motor cortex (p, 0.05)); α-syn background staining was less intense compared to focal intracellular accumulations. The mean proportion of CB+ cells was also higher in DLB cases than the control cases, reaching significance in the visual cortex (p,

0.01), motor cortex and temporal lobe (p, 0.05). Other brain regions showed the same trend of an increased proportion of CB+ cells in DLB compared to normal, but did not reach statistical significance. The overall mean proportion of cells containing α-syn aggregates was significantly higher in DLB cases than normal cases (p, 0.0001; Fig. 2 C) with α-syn aggregate bearing cells 7-fold more frequent in DLB patients compared to normal cases. The overall mean proportion of CB+ cells was 45% greater in DLB patients compared to normal cases, and reached statistical significance (p, 0.05). We then compared the proportion of α-syn

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 134 inclusion bodies in CB+ compared with CB- neurons. Inclusion bodies were categorized as neuronal based on the distinctive nuclear morphology and represented greater than 90% of total inclusions (Fig. 1B, E - G). Fig. 2D shows that there is a significantly higher proportion of α-syn aggregate bearing CB- than α-syn aggregate bearing CB+ neurons across the six brain regions examined in both the DLB and normal groups (normal, p, 0.05; DLB, p, 0.001).

Thus, α-syn inclusions were almost completely absent in CB+ cells.

Figure 2: Cell counting of CB-positive and -syn aggregate bearing cells in DLB and normal cases. (A-B) Mean proportion of CB+ and total α-syn aggregate bearing cells of different brain regions. (C) Overall mean proportion of CB+ and total α-syn aggregate bearing cells. (D) Mean proportion of of α-syn aggregate bearing CB+ and CB- neurons in different brain regions of DLB and control cases. Overall, the graph indicates predominant α-syn aggregate

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 135

localization within CB- cells especially in DLB cases. *p < 0.05 (or 95%); **p < 0.01 (or 99%); ***p < 0.001 (or 99.9%); Error bars indicate the SEM.

Calbindin-D28k-positive cells are increased in the unilateral rotenone mouse model, especially in aged mice, and exclude -synuclein aggregates

In order to investigate the influence of CB expression on cell loss and α-syn aggregation in the unilateral rotenone mouse (oxidative stress) model, male C57 black mice (6-12 months) were lesioned by unilateral injection of rotenone in the left MFB. We have previously established this unilateral lesion model (Weetman et al, 2013; Radford et al, 2015) and have shown that animals exhibited side bias by grasp test and rotation after two weeks post-lesion.

When graded for severity of movement dysfunction, aged animals had exhibited a more severe phenotype in our previous study and were unable to securely grasp the suspended wire track (Weetman et al, 2013). Animals were sacrificed 2 weeks post-lesion and brain tissue was prepared for immunofluorescence. Brains were fixed by perfusion in situ, after which free-floating sections (30µm) were incubated with primary antibodies and imaged by confocal microscopy. Figure 3 shows the loss of TH-positive neurons in rotenone lesioned, but not vehicle-lesioned hemispheres, in animals 8 weeks (N=2), 7-10 months (N=2) and 1.75 years of age (N=2).

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 136

Figure 3. TH-staining of lesioned and control hemisphere shows neuronal loss. A-F). Coronal striatal PEG sections of 3 age groups one week post lesion stained with NeuN (red) and TH (green) –50x magnification.(A) A young (8 weeks) lesioned striatal section, TH absent with the Striatum ipsilateral of the lesion site. (B) A young (8 weeks) vehicle striatal section, TH expression is abundant on both sides in the Striatum. (C) A young adult (7-10months) lesioned striatal section, TH absent within the striatum ipsilateral to the lesion site. (D) A young adult (7-10 months) vehicle striatal section, TH expression is abundant on both sides in the striatum. (E) An aged (1.75years) lesioned striatal section, TH

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 137

absent within the striatum ipsilateral to the lesion site. (F) An aged (1.75years old) vehicle striatal section, TH expression is abundant on both sides in the Striatum. Scale Bar 100µm.

Representative images are shown in Fig. 4B-F of fixed frozen sections from the control and rotenone-lesioned hemispheres of the young adult lesioned mice (N=5) and aged mice

(N=5). Proteinase K pre-treatment made little difference to the α-syn imaging, however less background was observed after Proteinase K pre-treatment (Supplementary Fig. 2). Data presented are with proteinase K pre-treatment, unless otherwise noted. Not represented in the figures are five aged mice and 3 aged young adults mice were also treated with sham injection

(vehicle only). Immunofluorescence revealed the presence of clusters of CB+ cells. In parallel, co-staining with the neuronal marker, NeuN, confirmed CB expression was exclusively in neuronal cell types. Imaging revealed localization of CB within the cytoplasm of a subset of neurons. A large proportion of cells were observed not to contain cytoplasmic

CB, especially within the control hemisphere. Fig. 4B-F shows the characteristic more intense uniform CB expression within CB+ cells within the lesioned hemisphere than the control hemisphere. Furthermore, the proportion of CB+ cells was apparently greater within the lesioned hemisphere than the control hemisphere. α-Syn deposits (arrowhead; Fig. 4B-F) were observed frequently in the lesioned hemisphere, but were rare in the non-lesioned hemisphere.

Immunohistochemistry also revealed the presence of CB and α-syn clusters. Imaging revealed colocalisation of CB and α-syn aggregates within the cytoplasm of a small subset of cells,

(CB+/α-syn+). However, a far greater proportion of cells did not contain CB within the cytoplasm, especially within the control hemisphere. Cell counting was conducted on the mouse tissue from both lesioned and control hemispheres (40 hemispheres) in three brain

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 138 regions (somatosensory cortex (SC), motor cortex (MC) and striatum (ST)).

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 139

Figure 4: CB expression and α-syn aggregation in the rotenone-lesioned and control hemispheres of wild-type young and aged mice. (A1-3) negative control slides with secondary antibodies only. (B) CB expression and NeuN expression in the control hemisphere of a young mouse (C–G) Immunofluorescence (rotenone-lesioned, Sham and non-lesioned hemisphere of the somatosensory cortex of an aged and young adult mouse) showing CB+ cells (red; arrow) are over-represented in lesioned hemisphere (D,F) compared to non- lesioned and sham hemisphere (C,E,G). Scale bar, 50µm.

The cell counting data on the unilateral rotenone-lesioned and sham-lesioned mouse tissues is summarized in Figure 5. The overall mean proportion of CB+ cells was significantly greater in lesioned compared to control and sham-lesioned hemispheres (p, 0.001) (Fig. 5 B). In our previous work on the unilateral rotenone mouse model, we had found that aged animals

(>1.75 yr) showed a far more severe movement dysfunction phenotype when compared to the young mice (6-12 months) animals subjected to the same treatment (Weetman et al, 2013).

Fig 5 B also shows the overall mean proportion of CB+ cells was significantly higher in the lesioned hemisphere of aged mice compared to the young mice. The overall mean proportion of cells bearing α-syn aggregates was also significantly greater in the lesioned hemisphere than the control hemisphere (p<0.001; Fig. 5 A). Thus, α-syn aggregates were 4-fold more frequent in the lesioned hemisphere compared to the non-lesioned hemisphere. Fig. 5C-D shows the mean proportion of the CB+ and α-syn aggregate bearing cells present for each of the different brain regions examined. For somatosensory cortex, striatum and motor cortex (p,

0.05) the proportion of CB+ cells was significantly higher in the lesioned hemisphere than the control hemisphere in aged and young adult animals. The mean proportion of cells bearing α- syn aggregates was also significantly different in these brain regions (p, 0001) when comparing lesion and control hemispheres.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 140

Figure 5: Cell counting of CB-positive and α-syn aggregate bearing cells in unilateral rotenone-lesioned and sham-lesioned mouse. (A) Mean proportion of α-syn aggregate bearing cells of lesioned and sham animals. (B) Mean proportion of CB+ cells of young and aged animals. (C) Mean proportion of CB+, total α-syn aggregate bearing cells of different brain regions. Somatosensory cortex (SC), mortor cortex (MC) and striatum (ST). *p < 0.05 (or 95%); **p < 0.01 (or 99%); ***p < 0.001 (or 99.9%); Error bars indicate the standard error of the mean (SEM).

In order to investigate further the relationship between CB expression and α-syn aggregates in the unilateral rotenone mouse model, we conducted immunocolocalization studies. Fig. 6A shows representative images from fixed frozen sections from the rotenone- lesioned hemisphere. Fig. 6A shows CB+ neurons (arrow) and typical perinuclear cytoplasmic

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 141

α-syn accumulations (arrowhead) in the lesioned hemisphere. Cytoplasmic CB was rarely associated with α-syn deposits (arrowhead; Fig. 6A) in the lesioned hemisphere. Aggregates of -syn detected in CB+ cells were typically small, multipunctate (Fig. 6 B), compared to the large concentric -syn aggregates characteristic of CB- cells (Fig. 6 C). Typically, large α-syn aggregates were observed only in CB- neurons not in CB+ neurons. Thus, α-syn aggregates appeared to be excluded from CB+ neurons. In order to test this hypothesis, we counted the relative proportion of α-syn aggregate bearing CB+ cells compared to α-syn aggregate bearing

CB- cells. The counting data is summarized graphically in Fig. 6D and shows a significantly higher proportion of α-syn aggregate positive / CB- than α-syn aggregate positive / CB+ cells across the four brain regions examined. There were no significant differences in the cell counting data without proteinase K digestion. Overall, α-syn aggregates were significantly more frequent in CB- cells (unlesioned hemisphere, 0.05; lesioned hemisphere, p, 0.01) with large α-syn aggregates being almost exclusively found in CB- cells (p, 0.01). Thus, large α- syn inclusions were almost completely absent in CB+ cells.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 142

Figure 6: α-syn aggregates are excluded from CB+ cells. (A) Representative CB expression and α-syn aggregation in the lesioned hemisphere of young mouse (striatum). α-syn perinuclear deposits (green, arrow); CB cytoplasmic expression (red). Scale bar, 50µm. (B) Representative image of a CB+/α-syn+ neuron. (C) Representative image of a CB-/α-syn+ neuron. Scale bar, 5µm. (D) Mean of α-syn aggregate frequency in CB+ and CB- cells in different brain regions of control and rotenone-lesioned or sham-lesioned hemispheres. Overall, the graph indicates far greater frequency of α-syn aggregates within CB- cells. Overall, the graph indicates an almost total exclusion of α-syn aggregates from CB+ cells. *p < 0.05 (or 95%); **p < 0.01 (or 99%); ***p < 0.001 (or 99.9%). Error bars indicate the SEM.

Calbindin-D28k level higher in lesioned hemisphere, especially in aged mice, but not significantly different between young and aged in control hemisphere

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 143

In order to investigate CB expression profiles, Western blot analysis was performed for CB in parallel with the tissue immunofluorescence analyses for the unilateral rotenone mouse model. Previous results comparing the mean of the total α-syn lane integral between the aged and young mice group had demonstrated that, as well as a statistically significant increase in total α-syn accumulation between the treated and control hemispheres, α-syn expression showed a statistically significant increase in the mean in aged over young animals (Weetman et al, 2013). We hypothesized that the greater sensitivity of the aged animals to lesion could be due to reduced CB+ expression. In total, three repeat experiments per hemisphere were conducted for each of the ten animals examined. Fig. 7A shows representative CB Western blots and actin loading controls for lesioned and control hemispheres from aged (N=5) and young animals (N=5). Examination of the Western blot data indicates the consistent presence of a major band immunopositive for CB at 28KDa. In general, the treated band was proportionately more intense than its control counterpart, especially in aged animals. Western blots were subjected to integration of band intensity and were normalized to the band intensities of actin loading controls. Fig. 7B shows a graph of mean integrated intensity of the

CB bands present for the aged and young animal group at 28KDa as a ratio of the actin loading control. There is an overall positive trend observed for CB levels between lesioned and control hemisphere, consistent with the immunofluorescence data. The lesioned hemisphere had a significantly higher CB expression level than the control hemisphere in aged animals (p, 0.01). Fig. 7B also shows that in the lesioned hemisphere, aged mice had a significantly higher level of total CB than the young lesioned hemisphere (p, 0.05) which follows the same trend as the cell counting data. In summary, Western analysis showed total

CB expression was 25% higher (p, 0.01) in lesioned compared to control hemisphere in the aged animals and 20 % higher in aged compared to young mice in the lesioned hemisphere (p,

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 144

0.05), but not significantly different between young and aged in the unlesioned hemisphere.

Figure 7: Western analysis of CB in crude brain tissue homogenates of the rotenone-lesioned and control hemispheres of young and aged mice. (A) Representative CB and actin Western bands for the lesioned and control hemispheres. (B) Mean band integrals as a ratio to the actin loading controls of aged and young mice. The mean CB level was higher in the lesioned hemispheres compared to the control hemispheres, reaching statistical significance in the aged animals. The CB level in the lesioned hemisphere was significantly greater in the aged animals compared to the young animals. *p < 0.05 (or 95%); **p < 0.01 (or 99%). Error bars indicate the SEM.

Discussion

We investigated the relationship between the expression of the endogenous neuronal calcium buffering protein, CB, and aggregation of the pre-synaptic protein, α-syn, linked to

DLB in DLB and normal cases and in a mouse model. We examined changes in the proportion of CB-positive cells and α-syn protein aggregates between DLB and normal cases

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 145 and between lesioned, sham-lesioned and unlesioned brain hemispheres in a unilateral rotenone-lesioned mouse model that involved stereotactic rotenone injection into the MFB in young adult (6–12 months) mice. Immunofluorescence of DLB cases showed an increased proportion of CB+ neurons compared to normal cases, reaching statistical significance in visual and motor cortex, temporal lobe and cingulate gyrus. Neuronal Lewy bodies were widespread in DLB tissue, but were rarely detected in CB+ neurons.

Oxidative stress is implicated as a causative factor in neurodegenerative disease development and α-syn aggregation (Breydo et al, 2012; Cookson, 2009; Jellinger 2009a;

Marques & Outeiro, 2012). Unilateral lesion of the rodent MFB with the mitochondrial complex I inhibitor, rotenone, results in evidence of oxidative stress in the lesioned hemisphere, intracellular α-syn aggregates and movement dysfunction (Weetman et al, 2013;

Norazit et al, 2010; Sanders & Greenamyre, 2013). There was a greater proportion of CB+ cells in the treated hemispheres compared to the unlesioned and sham-lesioned hemispheres.

The greater proportion of CB+ cells between the treated and control hemisphere may indicate a greater survival in response to cellular stress of CB+ over CB- neurons. As CB is a Ca2+ buffering protein, the greater proportion of surviving CB+ neurons could reflect the link between Ca2+ regulation and neuronal survival after rotenone lesioning. This supports the earlier data of Yamada et al. (Yamada et al, 1990) who found that dopaminergic neurons of the Sn that were high in CB were preferentially spared in control brain sections compared with PD patients, indicating that increased free Ca2+ (not buffered by CB) may be a major factor in the pathogenesis of α-synucleinopathies. Also, in a mouse model with Parkinsonian- like pathological features and loss of dopaminergic neurons, it was found that neurons expressing CB were spared from this pathological loss (Gaspar et al, 1994).

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 146

Since Yamada et al. demonstrated the importance of Ca2+ buffering in neurons localized to the Sn, increasing evidence has linked Ca2+ with PD aetiology (Yamada et al, 1990). Thus, mediodorsal dopaminergic Sn neurons were found to express the Ca2+ buffering protein, CB, whereas dopaminergic neurons of the ventrolateral Sn were found to lack CB. CB+ dopaminergic neurons were relatively spared compared to the CB- dopaminergic neurons lost in PD cases. Ca2+ is an essential intracellular metal ion involved in post-synaptic neuronal activation and neurotransmitter release with a highly regulated intracellular concentration.

Soluble α-syn oligomer formation represents a crucial process in the pathogenesis of

Parkinson’s disease and other α-synucleinopathy diseases (Danzer et al, 2009; Putcha et al,

2010). Moreover, Nielsen et al. reported the selective binding of Ca2+ to recombinant α-syn

(Nielsen et al, 2001). By using atomic force microscopy to visualize aggregates in vitro, Lowe et al showed that Ca2+ binding to the C-terminus of α-syn could induce annular oligomers

(Lowe et al, 2004). More recently, it was demonstrated that raised Ca2+ promotes α-syn aggregation both in vivo and in vitro (Nath et al, 2011; Follett et al, 2013) and that Ca2+ and oxidative stress can cooperatively induce α-syn aggregates in a cell culture model system

(Goodwin et al, 2013) . The link between DLB and aggregation of α-syn makes this process a major target for the development of future neurodegenerative therapies. Though the role of α- syn is not completely understood, there is a well-documented increase in α-syn high molecular weight species and previous results have indicated analogous changes in the protein expression profile of α-syn, between the treated and control hemispheres of the rotenone mouse model (Weetman et al, 2013). Many triggers for pathological α-syn aggregation have been identified, including oxidative stress and raised free Ca2+. CB+ cells with α-syn intracytoplasmic aggregates were rare and α-syn aggregates were observed to occur almost exclusively in CB- cells. The results show almost exclusive partitioning of α-syn aggregates

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 147 in the CB- cell population in both human and mouse brain tissue. This finding is consistent with previous cell culture model studies that showed that raised intracellular free Ca2+ induced

α-syn aggregate formation that was could be blocked by the low molecular weight Ca2+ buffer, BAPTA (Follett et al, 2013). The findings show α-syn aggregation is excluded from

CB+ neurons, indicating that binding of Ca2+ to CB protects against Ca-dependent α-syn aggregation.

In the current study, the results showed a consistently greater CB level in DLB compared to normal cases. In the mouse model, the lesioned hemisphere showed a greater level of CB protein compared to the control hemisphere; especially in the aged animals, but no significant difference in CB level between young and aged animals in the control hemisphere, in spite of the greater evidence of cell loss in aged animals in response to lesion found previously

(Weetman et al, 2013). The findings suggest that the increased sensitivity of aged animals to lesion is not related to differential CB expression, but may instead relate to factors, such as reduced trophic support.

It is clear that free intracellular Ca2+ is a major factor influencing α-syn aggregation, and potentially the formation of the cytotoxic oligomeric species seen in disease. Future studies will be required to map the molecular species of aggregates and oligomers in DLB and rotenone mouse tissue. However, recent findings suggest an association between raised intracellular Ca2+, α-syn aggregation and neurotoxicity paving the way for the development of therapeutics that target raised Ca2+ (Schapira et al, 2014). This provides a potential therapeutic target, by using drugs that modulate the amount of free Ca2+ in the cell. Ca2+ channel blockers, such as those from the dihydropyridines family, may be used to lessen the increase in intracellular Ca2+ seen in aged neurons, however, the current study suggests that induction of endogenous CB might also be effective at reducing Ca2+-dependent neuronal α-syn

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 148 aggregation and cytotoxicity. Since Ca2+ lowering medications, such as anti-epileptic drugs, often have significant side-effects, successfully targeting raised intracellular free Ca2+ in the brain by promoting the expression of CB at the transcriptional level by using inducers, such as calcitriol (Halhali et al, 2010), could therefore represent an alternative neuroprotective strategy.

Acknowledgments

This study was supported by the Australian Research Council, Menzies Health Institute,

Clem Jones Foundation, Parkinson’s Queensland and NHMRC. The authors gratefully acknowledge Jenna Weetman and Stuart Sharry for providing the mouse brain tissue homogenates.

References

Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, Trojanowski JQ, Iwatsubo T. 1998. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. The American Journal of Pathology 152(4): 879–84 Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. 2000. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nature neuroscience 3: 1301-6

Breydo L, Wu JW, Uversky VN. 2012. Alpha-synuclein misfolding and Parkinson's disease. Biochimica et biophysica acta 1822: 261-85

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 149 Bu J, Sathyendra V, Nagykery N, Geula C. 2003. Age-related changes in calbindin-D28k, calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex. Exp Neurol 182: 220-31 Castro Mdel R, Suarez E, Kraiselburd E, Isidro A, Paz J, et al. 2012. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys. Experimental gerontology 47: 29-37

Chan CS, Guzman JN, Ilijic E, Mercer JN, Rick C, et al. 2007. 'Rejuvenation' protects neurons in mouse models of Parkinson's disease. Nature 447: 1081-6 Cookson MR. 2009. alpha-Synuclein and neuronal cell death. Molecular neurodegeneration 4: 9

Dalfó E, Ferrer I. 2008 Early alpha-synuclein lipoxidation in neocortex in Lewy body diseases. Neurobiol Aging. 29(3):408-17.

Danzer KM, Krebs SK, Wolff M, Birk G, Hengerer B. 2009. Seeding induced by alpha-synuclein oligomers provides evidence for spreading of alpha- synuclein pathology. Journal of neurochemistry 111: 192-203 Dryanovski DI, Guzman JN, Xie Z, Galteri DJ, Volpicelli-Daley LA, Lee VM, Miller RJ, Schumacker PT, Surmeier DJ. 2013 Calcium entry and α- synuclein inclusions elevate dendritic mitochondrial oxidant stress in dopaminergic neurons. J Neurosci. 33(24):10154-64. Follett J, Darlow B, Wong MB, Goodwin J, Pountney DL. 2013. Potassium depolarization and raised calcium induces alpha-synuclein aggregates. Neurotoxicity research 23: 378-92 Gaspar P, Ben Jelloun N, Febvret A. 1994. Sparing of the dopaminergic neurons containing calbindin-D28k and of the dopaminergic mesocortical projections in weaver mutant mice. Neuroscience 61: 293-305

German DC, Manaye KF, Sonsalla PK, Brooks BA. 1992. Midbrain dopaminergic cell loss in Parkinson's disease and MPTP-induced parkinsonism: sparing of calbindin-D28k-containing cells. Ann N Y Acad Sci 648: 42-62

Goodwin J, Nath S, Engelborghs Y, Pountney DL. 2013. Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation. Neurochem Int. 62:703-11

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 150

Halhali A, Figueras AG, Diaz L, Avila E, Barrera D, et al. 2010. Effects of calcitriol on calbindins gene expression and lipid peroxidation in human placenta. The Journal of steroid biochemistry and molecular biology 121: 448-51

Heuckeroth, R.O. Enomoto, H. Grider, J.R. Golden, J.P. Hanke, J.A. Jackman, A. Molliver, D.C. Bardgett, M.E. Snider, W.D. Johnson, E.M.J. Milbrandt, J. 1999. Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons. Neuron 22:253–263 Jellinger KA. 2009a. Formation and development of Lewy pathology: a critical update. J Neurol. 256 Suppl 3:270-9.

Jellinger KA. 2009b. A critical evaluation of current staging of alpha-synuclein pathology in Lewy body disorders. Biochimica et biophysica acta 1792: 730-40 Jenner P, Morris HR, Robbins TW, Goedert M, Hardy J, Ben-Shlomo Y, Bolam P, Burn D, Hindle JV, Brooks D. (2013) Parkinson's disease--the debate on the clinical phenomenology, aetiology, pathology and pathogenesis. J Parkinsons Dis.;3(1):1-11. doi: 10.3233/JPD-130175.

Kim BG, Shin DH, Jeon GS, Seo JH, Kim YW, et al. 2000. Relative sparing of calretinin containing neurons in the substantia nigra of 6-OHDA treated rat parkinsonian model. Brain Res 855: 162-5 Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, et al. 1998. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet 18: 106-8

Lesage S, Anheim M, Letournel F, Bousset L, Honore A, et al. 2013. G51D alpha-synuclein mutation causes a novel Parkinsonian-pyramidal syndrome. Ann Neurol 73: 459-71 Lin MT, Cantuti-Castelvetri I, Zheng K, Jackson KE, Tan YB, Arzberger T, Lees AJ, Betensky RA, Beal MF, Simon DK. 2012 Somatic mitochondrial DNA mutations in early Parkinson and incidental Lewy body disease. Ann Neurol. 71(6):850-4.

Lowe R, Pountney DL, Jensen PH, Gai WP, Voelcker NH. 2004. Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 151

the C-terminal domain. Protein science : a publication of the Protein Society 13: 3245-52 Marques O, Outeiro TF. 2012. Alpha-synuclein: from secretion to dysfunction and death. Cell death & disease 3: e350

Nath S, Goodwin J, Engelborghs Y, Pountney DL. 2011. Raised calcium promotes alpha-synuclein aggregate formation. Molecular and cellular neurosciences 46: 516-26 Nielsen MS, Vorum H, Lindersson E, Jensen PH. 2001. Ca2+ binding to alpha- synuclein regulates ligand binding and oligomerization. The Journal of biological chemistry 276: 22680-4 Norazit A, Meedeniya AC, Nguyen MN, Mackay-Sim A. 2010. Progressive loss of dopaminergic neurons induced by unilateral rotenone infusion into the medial forebrain bundle. Brain research 1360: 119-29

Pasanen P, Myllykangas L, Siitonen M, Raunio A, Kaakkola S, Lyytinen J, Tienari PJ, Pöyhönen M, Paetau A. 2014. A novel α-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson's disease-type pathology. Neurobiol Aging. 35(9):2180.e1-5.

Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, et al. 1997. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 276: 2045-7 Proukakis C, Dudzik CG, Brier T, MacKay DS, Cooper JM, Millhauser GL, Houlden H, Schapira AH. 2013. A novel α-synuclein missense mutation in Parkinson disease. Neurology. 80(11):1062-4. Putcha P, Danzer KM, Kranich LR, Scott A, Silinski M, et al. 2010. Brain- permeable small-molecule inhibitors of Hsp90 prevent alpha-synuclein oligomer formation and rescue alpha-synuclein-induced toxicity. The Journal of pharmacology and experimental therapeutics 332: 849-57 Quilty MC, King AE, Gai WP, Pountney DL, West AK, et al. 2006. Alpha- synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection. Experimental neurology 199: 249-56

Radford R, Rcom-H'cheo-Gauthier A, Wong MB, Eaton ED, Quilty M, Blizzard C, Norazit A, Meedeniya A, Vickers JC, Gai WP, Guillemin GJ, West

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 152

AK, Dickson TC, Chung R, Pountney DL. 2015 The degree of astrocyte activation in multiple system atrophy is inversely proportional to the distance to α-synuclein inclusions.Mol Cell Neurosci. 65:68-81. Rcom-H’cheo-Gauthier A, Goodwin J, Pountney DL (2014) Interactions between calcium and alpha-synuclein in Neurodegeneration, Biomolecules, 4, doi:10.3390/biom40x000x.

Saito Y, Miyasaka T, Hatsuta H, Takahashi-Niki K, Hayashi K, Mita Y, Kusano-Arai O, Iwanari H, Ariga H, Hamakubo T, Yoshida Y, Niki E, Murayama S, Ihara Y, Noguchi N. 2014 Immunostaining of oxidized DJ- 1 in human and mouse brains. J Neuropathol Exp Neurol. 73(7):714-28. Schapira AH, Olanow CW, Greenamyre JT, Bezard E. 2014. Slowing of neurodegeneration in Parkinson's disease and Huntington's disease: future therapeutic perspectives. Lancet

Sanders LH, Greenamyre JT. 2013 Oxidative damage to macromolecules in human Parkinson disease and the rotenone model. Free Radic Biol Med. 62:111-20. Sims-Robinson C, Hur J, Hayes JM, Dauch JR, Keller PJ, et al. 2013. The role of oxidative stress in nervous system aging. PloS one 8: e68011

Tsuboi K, Kimber TA, Shults CW. 2000. Calretinin-containing axons and neurons are resistant to an intrastriatal 6-hydroxydopamine lesion. Brain Res 866: 55-64 Vila M, Jackson-Lewis V, Guegan C, Wu DC, Teismann P, et al. 2001. The role of glial cells in Parkinson's disease. Current opinion in neurology 14: 483-9

Weetman J, Wong MB, Sharry S, Rcom-H'cheo-Gauthier A, Gai WP, et al. 2013. Increased SUMO-1 expression in the unilateral rotenone-lesioned mouse model of Parkinson's disease. Neuroscience letters 544: 119-24 Yamada T, McGeer PL, Baimbridge KG, McGeer EG. 1990. Relative sparing in Parkinson's disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain research 526: 303-7

Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E, Ros R, et al. 2004. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann Neurol 55: 164-73

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 153

Supplementary Figures

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 154

Supplementary figure 1: CB expression and α-syn aggregation in DLB cases. (A - E) Immunofluorescence of DLB cases (motor cortex, temporal lobe, frontal lobe, hippocampus and cigulate gyrus) showing CB+ cells (red) and α-syn aggregate positive cells (green). Scale bar, 50µm.

Supplementary figure 2: CB and α-syn staining in the rotenone-lesioned hemispheres of wild-type young mice, comparing pre-treatment with and without proteinase K. Immunofluorescence (motor cortex) showing CB+ cells (red) are α- syn aggregate positive cells (green). Scale bar, 50µm.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 155

STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED PAPER

This chapter includes a co-authored paper. The bibliographic details published for publication of the co-authored paper, including all authors, are:

Alexandre Rcom-H’cheo-Gauthier

Dr Adrian Meedenyia

Dr Dean Pountney

My contribution to the paper involved:

All the experimental procedures and data analyses

Rcom-H’Cheo-Gauthier, A., Meedeniya, A., Pountney, D.L. Cacipotriol inhibits α-synuclein aggregation in SH-SY5Y neuroblastoma cells by a Calbindin-D28K- dependent mechanism. Journal of Neurochemistry, Submitted August 12, 2016.

(Signed) ______(Date)______

Alexandre Rcom-H’cheo-Gauthier

(Countersigned)______(Date)__16/08/2016_

Corresponding author of paper: Dr Dean Pountney

(Countersigned) _ __ (Date)_16/08/2016___

Supervisor: Dr Dean Pountney

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 156

Chapter 5: Calcipotriol inhibits α-synuclein aggregation in SH-SY5Y

neuroblastoma cells by a Calbindin-D28K-dependent mechanism

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 157

5.1 Introduction

PD and DLB are the two most frequent α-synucleinopathies, which are characterized by the presence of LB mostly in neurons of the SN affected in PD and throughout the brain in

DLB. LBs are predominantly composed of α-syn aggregates. α-Syn has been shown to aggregates with raised levels of Ca2+ in neurons [152] as well as due to oxidative stress. It also been demonstrated that the combination of Ca2+ increase and oxidative stress further promotes α-syn aggregation [110]. Treatment directly targeting Ca2+ voltage gated channels was shown to prevent α-syn aggregation in cells [153], but as a therapeutic treatment, it would involve multiple side effects as those ions gated channels are active in multiple cellular mechanisms. In the previous study, we showed that CB was found to be overexpressed in the post mortem brain of DLB patients and Yamada et al. (1990) found that CB was overexpressed in the SN on PD patients. Furthermore, we also observed the almost complete exclusion of α-syn aggregates from CB+ neurons. Therefore, triggering the overproduction of

CB could be achieved to prevent α-syn aggregation and provide neuroprotection in PD and

DLB. CB was shown to be upregulated in cells when they were treated with the hormonal form of vitamin D, Calcitriol [234].

To study the correlation between the overexpression of CB and α-syn aggregation in a cell model of PD, CB over-production was triggered by Cp, a vitamin D analogue which is a highly stable structure in comparison to Calcitriol. Previous studies also showed that cells that undergo membrane depolarization induced by KCl, result in Ca2+ influx and that α-syn cytoplasmic aggregates form 36 hours post-depolarization. In this study, we observed whether such Ca2+ dependent aggregation could be blocked by the overexpression of CB.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 158

5.2 PDF of paper

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 159

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 160

Journal of Neurochemistry Calcipotriol induction of calbindin-D28k inhibits calcium- dependent α-synuclein aggregation in SH-SY5Y neuroblastoma cells

Alexandre N. Rcom-H’cheo-Gauthier, Adrian C. B. Meedeniya and Dean L. Pountney*

Menzies Health Institute Queensland, School of Medical Science, Griffith University, Gold Coast, Queensland 4222, Australia

*Corresponding author: Dean L. Pountney Menzies Health Institute Queensland, School of Medical Science, Griffith University, Gold Coast, Queensland 4222, Australia Tel: +61 7 5552 7273 Fax: +61 7 5552 8908 Email: [email protected]

Keywords: alpha-synuclein; Parkinson’s disease; calcium; calbindin-D28k;

Calcipotriol; oxidative stress

Abbreviations: α-syn Alpha-synuclein CB Calbindin-D28k Cp Calcipotriol PD Parkinson’s disease Sn Substantia nigra TMO Trimethadione

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 161

Abstract

Many neurodegenerative diseases are characterized by the formation of microscopically visible intracellular protein aggregates. α-Synuclein is the key aggregating protein in

Parkinson’s disease which is characterized by neuronal cytoplasmic inclusion bodies, termed Lewy bodies. Previous post-mortem studies have shown relative sparing of neurons in Parkinson’s disease that are positive for the neuronal calcium buffering protein, Calbindin-D28k, the expression of which can be induced by the hormonal form of vitamin D, calcitriol. Recent cell culture studies have shown that α-synuclein aggregation can be induced by raised intracellular free Ca(II) and demonstrated that raised intracellular calcium and oxidative stress can act synergistically to promote α- synuclein aggregation. We hypothesized that calcipotriol, a potent vitamin D analogue used pharmaceutically, may be able to suppress calcium-dependent α-synuclein aggregation by inducing Calbindin-D28k expression. Immunofluorescence and Western blot analysis showed that calcipotriol induces Calbindin-D28k in a dose dependent manner in SH-SY5Y neuroblastoma cells. Calcipotriol significantly decreased the frequency of α-synuclein aggregate positive cells subjected to treatments that cause raised intracellular free Ca(II) (potassium depolarization, KCl/H2O2 combined treatment and rotenone) in a dose dependent manner. Suppression of Calbindin-D28k expression in calcipotriol-treated cells using Calbindin-D28k-specific siRNA showed significantly higher α-synuclein aggregation levels, indicating that calcipotriol -mediated blocking of calcium-dependent α-synuclein aggregation was largely due to induction of Calbindin-

D28k expression. Targeting raised intraneuronal free Ca(II) in the brain by promoting the expression of Calbindin-D28k could be a novel approach to prevent α-synuclein aggregate formation.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 162

Introduction

Parkinson’s disease (PD) has progressive clinical symptoms, including tremors, the inability to walk unaided, speech impairment and loss of urinary and gastrointestinal control.

Brain tissue from PD sufferers shows a loss of dopaminergic neurons in the Substantia nigra

(Sn), a region of the brain that, through neural connections with the striatum, is responsible for controlled muscle movements. Factors including oxidative stress, gene mutations and elevated calcium ion concentrations are known to impact on the formation of the intraneuronal protein lesions (Lewy bodies) characteristic of the disease, in which α-synuclein

(α-syn) (14 kDa) is a key protein component (Breydo et al. 2012, Cookson 2009, Jellinger

2009, Marques & Outeiro 2012, Rcom-H'cheo-Gauthier et al. 2014). Although approximately

95% of PD cases are not attributed to genetic abnormality, several specific gene mutations have been implicated in familial PD. To date, six mutations in PARK1/4, the α-syn gene, have been identified with amino acid substitutions, A30P (Kruger et al. 1998), A53T

(Polymeropoulos et al. 1997), E46K (Zarranz et al. 2004), G51D (Lesage et al. 2013), H50Q

(Proukakis et al. 2013) and A53E (Pasanen et al. 2014).

Recent studies have shown that a transient increase in the intracellular free Ca(II) concentration induced in cultured 1321N1 glioma cells by thapsigargin or Ca(II) ionophore chemical treatments caused a significant increase in the proportion of cells bearing microscopically-visible α-syn aggregates (Nath et al. 2011). It was also demonstrated that chelating free Ca(II) with BAPTA, resulted in no significant difference in the number of inclusions between control and CI/BAPTA cells, indicating that raised intracellular free Ca(II) directly induces α-syn aggregates (Goodwin et al. 2013). Moreover, supporting studies with

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 163 recombinant protein indicated that direct binding of Ca(II) ion to α-syn promoted rapid oligomer formation in vitro. More recently, Follett et al (2012), demonstrated that potassium depolarization of the plasma membrane in HEK293T and SH-SY5Y human cell lines resulted in raised intracellular free Ca(II) by triggering the opening of the voltage-gated calcium channels and α-syn aggregate formation. Both raised free Ca(II) and α-syn aggregation could be blocked by BAPTA chelation treatment. Potassium depolarization was observed especially to promote formation of frequent large, Lewy body-like peri-nuclear α-syn inclusion bodies

(Follett et al. 2013). Consistent with this, Trimethadione (TMO), a calcium channel blocking drug inhibited potassium depolarization induced Ca(II) influx resulting in loss of α-syn positive aggregate formation post-depolarization (Follett et al. 2013). The mitochondrial complex I inhibitor, rotenone, causes oxidative stress, autophagy dysfunction and also promotes α-syn aggregation in a dose- and time-dependent manner. Calcium has also been implicated recently in the mechanism of rotenone-mediated α-syn aggregation. Thus, BAPTA has been found to attenuate rotenone-induced impairments of autophagy by calcium chelation and rotenone-induced α-syn aggregation was found to be mediated by the calcium/GSK3b signaling pathway (Yuan et al. 2015).

Early evidence for a role of calcium in PD pathogenesis was provided by Yamada et al.

(1990), who found that dopaminergic neurons of the SnPc that were high in the calcium buffering protein Calbindin-D28k (CB) were preferentially spared in control brain sections compared with PD patients, indicating that increased Ca(II) may be a major factor in the pathogenesis of α-synucleinopathies. Later, Guo et al. (1998) showed that Aβ-induced elevation of Ca(II) and reactive oxygen species were blocked by CB over-expression. Also, in a mouse model with Parkinsonian-like pathological features, including the loss of

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 164 dopaminergic neurons, it was found that neurons expressing CB were spared from this pathological loss (Rcom-H'cheo-Gauthier et al. 2014).

Since the regulation of Ca(II) is vital for cell survival and function, it is not surprising that the process is tightly regulated and that there are a number of proteins that are capable of

Ca(II) buffering in neurons, such as CB, calretinin and parvalbumin (Bu et al. 2003). The importance of these proteins has been further implicated by German et al. (1998) who studied the brains of patients with PD or in MPTP monkey or C57BL mouse models. They found that in both idiopathic PD and in the animal models that neurons that were CB-positive were spared while neurons in CB-negative regions were lost (German et al. 1992). CB was also shown to be upregulated by the hormonal form of vitamin D, Calcitriol, in cytotrophoblasts

(Halhali et al. 2010).

In this study, we show that SH-SY5Y neuroblastoma cells subjected to the vitamin D analogue, Calcipotriol (Cp), show raised CB expression that is dose-dependent. We show that

Cp suppresses cytoplasmic α-syn aggregates promoted by potassium depolarization,

KCl/hydrogen peroxide and rotenone treatments that result in raised Ca(II) by a CB-dependent mechanism. We propose that by promoting the expression of CB at the transcriptional level,

Cp may also be able to target raised intracellular free Ca(II) in the brain.

Materials and Methods

Cell culture

SH-SY5Y cells were seeded at 20,000 cells/well in 24-well plates on 10mm glass cover slips. Cells were incubated for a period of 24h at 37°C with 5% CO2 in 1mL of 15% FBS,

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 165

82.96% Hams DMEM:F12 (Invitrogen), 0.04% Amphotericin B and 1% Strep/Penicillin prior to drug treatment. siRNA Transfection

For siRNA transfection, cells were treated and seeded simultaneously at 20,000 cells/well on 10mm glass cover slips. Cells were placed in 5µL of transfection reagent (Santa Cruz, sc-

29528) and 5µL of CB siRNA (sc-29879) in 500µL of transfection medium (sc-36868) for 7 hours. Then, the medium was replaced and cells were left incubated for 48h. Transfection efficiency was measured using control siRNA-A (sc-37007) and control siRNA (FITC

Conjugate)-A (sc-36869).

+ Calcipotriol, K , H2O2 and rotenone treatments

SH-SY5Y cells were incubated in the presence of increasing concentrations of Cp (0, 01, 0.5,

1, 5 or 10nM) in serum free DMEM for 5h at 37C with 5% CO2 then washed twice with PBS.

The medium was then replaced with medium supplemented with 50µM KCl for 1h or 50mM

KCl and 10µM H202 for 1h. For control cells, un-supplemented medium was used. Following treatment, cells were incubated with fresh medium for 36h at 37 ºC with 5% CO2 before fixing. For rotenone treatments, following Cp treatment, cells were placed in 500nM of rotenone and incubated for 24h at 37 ºC with 5% CO2 before fixing. Cell viability post- treatment was determined by MTT assay.

Immunohistochemistry

Cells were rinsed in PBS prior to fixation. Cells were fixed and permeabilized using 1:3 methanol: acetone and blocked with 20% normal horse serum. Monoclonal mouse anti-α-syn

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 166 antibody (LB509, Invitrogen) and monoclonal rabbit anti-CB antibody (CB38, swant) were used 1:100 for immunostaining. Alexa fluor-conjugated secondary antibodies (Molecular

Probes, Invitrogen) were used for immunofluorescence and mounted using Prolong Gold with

DAPI mounting medium (Invitrogen). Microscope slides were stored in the dark at 4°C.

Minimal non-specific staining was observed without primary antibody.

Confocal Microscopy and Image Analysis

Immunostained coverslips were imaged using a Nikon A1R+ confocal laser microscope.

Negative control slides were imaged to set microscope settings (laser voltage, electronic gain and offset) to appropriate levels. The 60x oil immersion lens was used for fixed cell imaging.

DAPI, AF-488 and 568 were imaged together, on separate channels.

Immunostained cells were counted (n= 30) and classified as either α-syn inclusion body- positive or body-negative. A standardized pattern 60x fields of view (n= 5) were imaged and counted using Columbus PerkinElmer image analysis software. To be classified as α-syn inclusion body-positive cell, clear and bright focal immunofluorescence needed to be observed in one or more confocal slice. The percentage of α-syn inclusion body positive cells was calculated along with inclusion body-negative cells. Experiments were then repeated with n= 5 for CB siRNA treated cells. CB intensity was measured using Columbus PerkinElmer analysis software. Low CB (siRNA transfected) or high CB (siRNA negative) cells in the siRNA transfection experiments were categorized using Columbus PerkinElmer image analysis software using the CB immunofluorescence intensity levels in the cytoplasm.

Western Blot Analysis

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 167

Cells were rinsed in cold PBS prior to be lysis in cold RIPA buffer. Adherent cells were scraped off the dish and maintained at constant agitation for 30mins at 4°C. Cells were centrifuged at 4°C for 5mins at 20,000rpm; the supernatant was then stored at -20ºC.

Each sample (20μL) was run in triplicate on 8-16% Mini-PROTEAN® TGX Stain-Free™

Gels (200V, 30mins), transferred to nitrocellulose using Trans-Blot® Turbo™ Transfer

System (25V, 30mins), assessed staining of proteins, blocked (1h, 5% skim milk powder), incubated with primary antibody (1:1000, overnight) rabbit anti-CB (Swant) washed (3x,

TBS-T), incubated with HRP-conjugated secondary antibody (BioRad, 1:2000; 1H) washed

(3x, TBS-T) and developed with West Pico SuperSignal Chemiluminescent Substrate

(ThermoFisher Scientific). Membranes were imaged by BioRad ChemiDoc™ MP System.

For re-probing, blots were stripped with 0.2N NaOH for 30mins, then re-blocked and probed for actin (mouse anti-actin; Sigma). Bands were quantitated by area under the curve relative to actin loading controls (Image Lab™ software).

Statistical analysis

All data were analysed using SPSS Version 22.0. Standard error of the mean for graphs

휎 was calculated using ( ). ANOVA tests were conducted to test for statistical significance. √푛

Results

In the current study, we were interested in probing the interaction between the calcium buffering protein, CB, Ca(II), α-syn aggregates and oxidative stress. We investigated whether

Cp treatment induces the expression of CB in SH-SY5Y neuroblastoma cells. We analyzed the expression of CB, and α-syn aggregation after potassium depolarisation, KCl/H2O2 and

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 168 rotenone treatments, with and without Cp pre-treatment. We also treated the cells with CB- specific siRNA to knock down CB expression to observe the effect of Cp on α-syn aggregation in the absence of CB induction.

Calcipotriol potently induces Calbindin-D28k expression in SH-SY5Y cells

SH-SY5Y cells were treated with Cp at concentrations of 0, 0.1, 0.5, 1, 5 and 10nM. After incubation for 5h, cells appeared to retain normal morphology and MTT assay showed no significant difference in cell viability post-treatment at concentrations below 50nM Cp (p =

0.001). At 50nM Cp and above, bright field microscopy revealed some detachment of cells from cover slips. Representative confocal immunofluorescence images are shown in Fig. 1A-

C from cells treated with Cp 0-10nM, showing that CB was expressed within the cytoplasm of

SH-SY5Y cells. Fig. 1A-C shows the characteristic more intense uniform CB expression within cells treated with higher concentrations of Cp.

To obtain a more quantitative view, immunofluorescence intensity of CB was measured for

6 concentrations of Cp. The data summarized in Fig. 1E shows significant differences in CB immunofluorescence intensity between the different concentrations of CP used. We observed an increase of 2-fold in the intensity of CB when treated with 1nM Cp (p<0.0001) compared to control. We also observed an increase of 2-fold between the intensity of CB when treated with 10 nM Cp compared to 1nM Cp (p<0.0001).

To investigate the protein expression profile, Western blot analysis was performed for CB.

Western blots were subjected to integration of bands and were normalized to the band intensities of actin loading controls. Fig. 1D shows representative CB Western blots for different concentrations of Cp treatment. Examination of the Western blot data indicates the

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 169 consistent presence of a major 28kDa molecular weight band immunopositive for CB. The data summarized in Fig. 1E shows significant differences in CB integral between the different concentrations of CP used. We observed an increase of 2-fold between the amount of CB when treated with 1nM Cp compared to control with a significance value of p<0.0001. We also observed an increase of 2-fold between the CB integral when treated with 10 nM Cp compared with 1nM with a significance value of p<0.0001. These findings correlate well with the measurements of CB expression using immunofluorescence. We did not observed statistically significant differences between the two methods of measurement after normalizing the results.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 170

Figure 1: Calbindin-D28K (CB) protein expression profiles after Calcipotriol (Cp) treatment. (A-C) Representative images of SH-SY5Y cells pre-treated with 0-10nM Cp for 5h showing CB (red) and DAPI (blue). (Scale bar 30µm). (D) Representative CB and Actin Western bands after Cp treatment. (E) Graph of the normalized CB band intensity and CB fluorescent intensity for different

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 171

concentrations of Cp. Error bars indicates the SEM. *p<0.05 (or 95%); **p<0.01 (or 99%); ***p<0.001 (or 99.9%); ****p<0001 (or 99.99%).

Calcipotriol pre-treatment suppresses the formation of -synuclein cytoplasmic aggregates

SH-SY5Y cells were pre-treated with Cp at concentrations of 0, 0.1, 0.5, 1, 5 and 10nM to induce CB expression. Depolarization with 50mM KCl, 50mM KCl/10µM H2O2 combined treatment or 500nM rotenone was employed to induce α-syn cytoplasmic aggregates. Cells retained normal morphology, showing no signs of clumping or rounding by differential interference microscopy. In order to examine CB expression and α-syn aggregation after treatments, immunofluorescence confocal microscopy was performed on fixed cells. α-Syn aggregates were counted using Columbus software and cells were classified as α-syn aggregate positive or negative, then the proportion of the α-syn aggregate positive cells was calculated. ANOVA statistical analysis was used to compare the different proportion of α-syn aggregate positive cells under each treatment condition. After incubation for 36h following

KCl, KCl/H2O2 or rotenone treatment, cells pre-treated with 0-0.5nM Cp for 5h retained normal morphology but showed large cytoplasmic α-syn aggregates. No large α-syn focal aggregates were observed by immunofluorescence analysis in cells pre-treated with 1-10nM

Cp when α-syn aggregation was triggered by either KCl or KCl/H2O2 as illustrated in Fig. 2C and 2F for 10nM Cp. We then treated cells with the mitochondrial complex I inhibitor, rotenone, which triggers both oxidative stress and increased intracellular free Ca(II) (Yuan et al. 2015). Few α-syn focal aggregates were observed by immunofluorescence analysis in the

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 172 cells pre-treated with 1-10nM Cp compared to cells that did not receive pre-treatment when α- syn aggregation was induced by rotenone as illustrated in Fig. 2 I for 10nM Cp.

To obtain a more quantitative view, α-syn aggregates were counted using Columbus

PerkinElmer image analysis software for 6 concentrations of Cp. Cells containing cytoplasmic

α-syn aggregates were counted as α-syn aggregate-positive and the proportion of total cells was then calculated. The data summarized in Fig. 2 B shows significant differences in α-syn aggregation between the different concentrations of CP used.

Figure 2: α-Synuclein (α-Syn) aggregation following potassium

depolarization, KCl/H2O2 combined treatment and rotenone treatment with and without Cp pre-treatment and in SH-SY5Y cells. (A-L) Representative images of pre-treated with 0-10nM Cp for 5h, KCl treated (A-C), sham-treated

(D-F), KCl and H2O2 treated (G-I) and rotenone (J-L) cells immunostained for α- syn (Green) and DAPI (Blue). Scale bar 30µm.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 173

Firstly, we observed a significant difference (p<0.001) between the proportion of α-syn aggregate bearing cells when cells were treated with only KCl compared to KCl/H2O2 combined treatment. There were proportionately 30% fewer α-syn aggregate positive cells with KCl treatment compared to treatment with a combination of KCl and H2O2. In addition, cells treated with KCl only showed fewer, larger aggregates in the cytoplasm, while cells treated with KCl/H2O2 showed a greater multiplicity of aggregates of smaller size. These results were consistent with previous studies which examined the cooperative effect on α-syn aggregation of Ca(II) and oxidative stress (Goodwin et al. 2013). When cells were treated with Cp, we observed a decrease in the frequency of α-syn aggregate positive cells. Cells that were treated with the combination of KCl/H2O2 showed a significant (p<0.0001) decrease in the proportion of α-syn aggregate positive cells of 40% when pre-treated with a concentration

0.1nM Cp. When treated with a Cp concentration of 0.5nM, KCl-treated cells showed a significant (p<0.001) decrease in the proportion of α-syn aggregate positive cells by 25%.

There was no significant difference in the proportion α-syn aggregate positive cells at 0.1nM

Cp with KCl treatment compared to control. Cp pre-treatment decreased significantly the proportion of α-syn aggregate positive cells with either KCl or KCl/H2O2 treatments when the

Cp concentration was increased to 5nM.

After cells were pre-treated with Cp, we did not observe any significant difference in the proportion of α-syn aggregate positive cells between KCl or KCl/H2O2 experiments. Cells treated with KCl/H2O2 showed more α-syn aggregation than cells treated with KCl only, but when cells were pre-treated with Cp, we observed similar aggregation in both groups.

When treated with rotenone only, we observed 100% cells containing α-syn aggregates.

The α-syn aggregates observed were not often multiple per cell but large (2 µM), similar that that observed in the KCl treated cells. When treated with concentrations of Cp from 0-10nM,

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 174 we observed a significant decrease in the proportion of α-syn aggregate positive cells. There was a significant (p<0.001) decrease of 50% in α-syn aggregate positive cells when cells were treated with 1nM Cp.

Figure 3: (A) Summay of cell counting data of treated and control cells showing the proportion of α-Syn inclusion body positive SH-SY5Y cells. (B) Graph of cell counting data of treated and control cells showing the proportion of inclusion body positive SH-SY5Y cells. A statistically significant (p = 0.001) difference

was found when comparing treated (50mM KCl, 50mM KCl/10µM H2O2 or 500nM rotenone) to sham-treated cells 36 h after incubation for 1 h. Proportion of

total cells bearing aggregates in KCl, KCl/ H2O2, rotenone treated and sham- treated cells with 5h pre-treatment of 0–10nM Cp as indicated. Error bars indicates the SEM.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 175

Cabindin-D28k siRNA blocks the inhibition of -synuclein aggregation by calcipotriol

In order to determine if Cp-mediated suppression of calcium-dependent α-syn aggregation is due to increased CB expression, we employed CB-specific siRNA to inhibit CB expression in Cp treated cells. To examine CB expression in SH-SY5Y cells after siRNA and Cp treatments, immunofluorescence confocal microscopy was performed on fixed cells.

Representative images are shown in Fig. 4 A-C from cells treated with 0-10nM Cp and siRNA. CB was expressed within the cytoplasm of SH-SY5Y cells. Fig. 4A-C shows the characteristic more intense uniform CB expression within cells treated with higher concentrations of Cp and lower CB expression in cells transfected with siRNA.

To investigate the protein expression profile after siRNA and Cp treatments, Western blot analysis was performed for CB. Western blots were subjected to integration of bands and were normalized to the band intensities of actin loading controls. Fig. 4D shows representative CB Western blots for different concentrations of Cp treatment. The data summarized in Fig. 4A shows significant differences in CB intensity between the different concentrations of CP used. We observed a significant (p<0.001) decrease of 30% between the cells treated with siRNA and untreated. This confirms the down regulation of CB by the CB siRNA as seen in the immunofluorescence data.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 176

Figure 4: CB protein expression profiles after Cp and CB SiRNA treatments.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 177

(A-C) Representative images of SH-SY5Y cells treated with siRNA for 6 h and 0- 10nM Cp for 5h showing CB (red) and DAPI (blue) (Scale bar 30µm). (D) Representative CB and actin Western bands after siRNA and Cp treatment. (E) Graph of the corrected relative CB band intensity for different concentrations of CP comparing siRNA treated and non-treated cells. Error bars indicates ± SEM. (F) Summary of CB immunofluorescence intensity and CB Western blot band integral in SH-SY5Y cells treated with Cp and and in cells co-treated with Cp/ and CB-specific siRNA.

To delineate the effect of CB induction by Cp on α-syn aggregation, cells were treated with

CB-specific siRNA before Cp and KCl treatments. Fig. 5A-I shows the characteristic more intense uniform CB expression within cells treated with higher concentrations of Cp and lower CB expression in cells transfected with siRNA. Fig. 5J-K represents the output from the

Columbus software determining a cell as siRNA+ or siRNA- based on mean immunofluorescence intensity of cells with red being cells that are below the CB expression threshold and green for cells above the threshold. The apparent rate of siRNA transfection determined by immunofluorescence analysis was 70%, which was consistent with the transfection efficiency determined with the siRNA control construct. In the siRNA transfected cells, we observed higher α-syn aggregation than in cells that were not transfected, especially at 0.5nM Cp. Furthermore, when α-syn aggregates were present in cells overexpressing CB, their sizes were small and they were few in number.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 178

Figure 5: CB and α-syn immunofluorescence in SH-SY5Y cells following, CB- specific siRNA, Cp and KCl treatments. (A-I) Representative images of cells treated with CB-specific siRNA for 6 h and 0-10nM Cp for 5h then KCl for 1H and 36h post-incubation showing CB (red, arrowhead), α-syn (green, arrow) and DAPI (blue). (J- L) Representative images of cells selected by Columbus software analysis and false coloured to indicate high CB immunofluorescence (green) consistent with uptake of CB-siRNA or low CB immunofluorescence indicating no siRNA uptake (scale bar 30µm).

The data summarized in Fig. 6A shows significant differences in α-syn aggregation between cells that were pre-treated with CB-siRNA compare to cells that were not subjected to siRNA treatment. We observed a significant decrease in α-syn aggregation p<0.05 at 0, 0.1 and

0.5nM Cp, p<0.01 at 5 and 10nM Cp and p<0.001 at 1nM Cp. The data summarized in Fig.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 179

6B shows significant differences in α-syn aggregation between siRNA+ and siRNA- cells at all the concentrations of CP used. We observed at all concentrations, a significant (p<0.0001) increase of α-syn aggregation in cells that were judged to have taken up the CB-specific siRNA compared to cells that had not at each of the 6 different Cp concentrations. We observed a 30% decrease (p<0.0001) of α-syn aggregate positive cells between 1 and 5nM Cp in siRNA-treated cells, indicating that high Cp concentration can compensate for the effect of the siRNA knock down.

Figure 6: Cell counting data of CB-specific siRNA pre-treated SH-SY5Y cells showing the proportion of cells that are α-syn inclusion body positive. (A) Graph of proportion of cells bearing aggregates in potassium depolarized cells following 5h Cp treatment (0-10nM) comparing total cells pre-treated with 6h pre- treatment with CB-siRNA and unpre-treated cells. (B) Graph of cell counting data for cells bearing α-syn aggregates (Large, small, total) in potassium depolarized cells with 6h pre-treatment with CB-siRNA and 5h of 0–10nM Cp, shown as the proportion of cells with either high CB expression (siRNA-) or low CB expression (siRNA+). Cells scored as either siRNA- or siRNA+ by Columbus software analysis on CB immunofluorescence as described in Methods. Error bars indicates the SEM.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 180

Discussion

Yamada et al. (1990) first demonstrated the over-representation of neurons positive for the endogenous, vitamin D dependent neuronal calcium buffering protein, CB, in the Sn of PD patients. Subsequent studies using animal models have provided additional data to support a neuroprotective role for CB. Moreover, cell culture models have established a causative link between raised intracellular free calcium and α-syn aggregation. In this study, we sought to investigate a potential inhibitory effect of CB against α-syn aggregation in cell culture models of PD. We hypothesized that the vitamin D analogue, calcipotriol, may efficiently induce CB expression in a neuron-like cell line. We further hypothesized that increased CB expression could suppress the formation of cytoplasmic -synuclein aggregates promoted by raised intracellular free Ca(II) and oxidative stress.

SH-SY5Y neuroblastoma cells were subjected to multiple treatments that trigger α-syn aggregation. Vitamin D and Vitamin D analogues have shown to induce CB expression

(Halhali et al. 2010), which we hypothesised, could reduce α-syn aggregation induced by potassium depolarization, combined KCl/H2O2 treatment and rotenone treatment. We have shown that in the SH-SY5Y human neuroblastoma cell line, endogenous CB expression could be increased potently by low, non-toxic concentrations of Cp (1 nM range). We examined changes in CB levels and α-syn protein aggregates between different Cp concentration treatments. Immunofluorescence and Western blotting showed that Cp induced increased CB expression in a dose dependent manner and this led to a reduction in α-syn aggregate bearing cells under each of the treatments used.

We hypothesized that the decrease in α-syn aggregation observed is directly linked to the increased CB expression. To test this hypothesis, we have pre-treated cells with CB-specific

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 181 siRNA to prevent CB overexpression upon Cp treatment. The results of the siRNA transfection experiments showed that cells that had taken up the CB-specific siRNA and consequently showed significantly lower levels of CB after Cp treatment also showed significantly more α-syn aggregates than cells that showed the level of CB induction observed in the absence of siRNA. These results indicate that the reduced aggregation of α-syn observed upon Cp treatment is due largely to the increase in CB expression and is likely a consequence of increased calcium buffering.

It is clear that free intracellular Ca(II) is a major factor influencing α-syn aggregation, and potentially the formation of the cytotoxic oligomeric species seen in PD. Lowe et al showed that Ca(II) binding to the C-terminus of α-syn could induce annular oligomers (Lowe et al.

2004). It was demonstrated that raised Ca(II) promotes α-syn aggregation both in vivo and in vitro (Nath et al. 2011, Follett et al. 2013) and that Ca(II) and oxidative stress can cooperatively induce α-syn aggregates in a cell culture model system (Goodwin et al. 2013).

More recently it was found that α-syn oligomers disrupt calcium regulation in cells and lead to calcium dependent cell death (Angelova et al. 2016). Moreover, recent findings suggest an association between raised intracellular Ca(II), α-syn aggregation and neurotoxicity paving the way for the development of therapeutics that target raised Ca(II) (Schapira et al. 2014).

Targeting Ca(II) dysregulation, by using drugs that modulate the amount of intracellular free

Ca(II) provides a therapeutic target to treat PD. Ca(II) channel blockers, such as those from the dihydropyridines family, that target the calcium channels in neurons may be used to lessen the increase in intracellular Ca(II). However, the current study suggests that induction of endogenous CB might also be effective at reducing Ca(II)-dependent neuronal α-syn aggregation and cytotoxicity.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 182

Major metabolites of vitamin D have been found to be present in human cerebrospinal fluid, including 25OHD3 (Calcifediol), 1,25OH2D3 (Calcitriol) and 24,25OH2D3 (24,25-

Dihydroxycholecalciferol) (Balabanova et al. 1984). Furthermore, vitamin D metabolites have been shown to cross the blood brain barrier (Pardridge et al. 1985). CYP27B1 is present in both neurons and glia and is most strongly expressed in the Sn and the supraoptic and paraventricular nuclei of the hypothalamus in the adult brain (Eyles et al. 2005). This potentially indicates that the brain may be able to synthesize the active metabolite

1,25OH2D3. Since Ca(II) lowering medications, such as anti-epileptic drugs, often have significant side-effects, efficiently buffering raised intracellular free Ca(II) in the brain by promoting the expression of CB at the transcriptional level by using Cp may offer an effective alternative.

Previous research has found that administration of 1,25OH2D3 protects against damage from the neurotoxin, 6-hydroxydopamine (6OHDA), that specifically lesions dopaminergic and noradrenergic cells (Wang et al. 2001, Smith et al. 2006). Furthermore, administration of

1,25OH2D3 has been shown to protect against neurotoxic doses of methamphetamine by preserving dopamine and serotonin levels (Cass et al. 2006). Such neuroprotection of dopaminergic neurons may be relevant to the loss of dopaminergic neurons in the Sn in PD. In addition, some studies have linked vitamin D insufficiency with increased risk of PD (Evatt et al. 2008, Newmark & Newmark 2007). Moreover, higher levels of 25OHD3 were associated with a reduced risk of developing PD in later life (Knekt et al. 2010). Abnormalities in the vitamin D receptor have also been linked with risk of developing PD (Kim et al. 2005). On the other hand a recent study by Shrestha et al. (2016) has investigated the possible association between serum 25-hydroxyvitamin D concentration in mid-adulthood and PD risk, and found no association between vitamin D and the risk of PD. Our experimental results

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 183 indicate that Cp could be neuroprotective by reducing α-syn aggregate formation, however further animal model studies would be required to determine if Cp can induce increased CB expression in differentiated neurons in the brain tissue context. This study demonstrates that

CB can be induced in neuron-like cells using the vitamin D analogue, Cp, and that increased

CB expression induced by Cp treatment can directly suppress intracellular α-syn aggregation caused by high levels of free Ca(II) and oxidative stress.

Acknowledgments

This study was supported by the Australian Research Council, Menzies Health Institute

Queensland, Parkinson’s Queensland.

References

Angelova, P. R., Ludtmann, M. H. R., Horrocks, M. H. et al. (2016) Calcium is a key factor in α-synuclein induced neurotoxicity. Journal of cell science.

Balabanova, S., Richter, H. P., Antoniadis, G., Homoki, J., Kremmer, N., Hanle, J. and Teller, W. M. (1984) 25-hydroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid. Klinische Wochenschrift, 62, 1086-1090.

Breydo, L., Wu, J. W. and Uversky, V. N. (2012) Alpha-synuclein misfolding and Parkinson's disease. Biochimica et biophysica acta, 1822, 261-285.

Bu, J., Sathyendra, V., Nagykery, N. and Geula, C. (2003) Age-related changes in calbindin-D28k, calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex. Experimental neurology, 182, 220-231.

Cass, W. A., Smith, M. P. and Peters, L. E. (2006) Calcitriol protects against the dopamine- and serotonin-depleting effects of neurotoxic doses of

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 184 methamphetamine. In: Annals of the New York Academy of Sciences, Vol. 1074, pp. 261-271.

Cookson, M. R. (2009) alpha-Synuclein and neuronal cell death. Molecular neurodegeneration, 4, 9.

Evatt, M. L., DeLong, M. R., Khazai, N., Rosen, A., Triche, S. and Tangpricha, V. (2008) Prevalence of vitamin D insufficiency in patients with Parkinson disease and Alzheimer disease. Archives of Neurology, 65, 1348-1352.

Eyles, D. W., Smith, S., Kinobe, R., Hewison, M. and McGrath, J. J. (2005) Distribution of the Vitamin D receptor and 1α-hydroxylase in human brain. Journal of Chemical Neuroanatomy, 29, 21-30.

Follett, J., Darlow, B., Wong, M. B., Goodwin, J. and Pountney, D. L. (2013) Potassium depolarization and raised calcium induces alpha-synuclein aggregates. Neurotoxicity research, 23, 378-392.

German, D. C., Manaye, K. F., Sonsalla, P. K. and Brooks, B. A. (1992) Midbrain Dopaminergic Cell Loss in Parkinson's Disease and MPTP-Induced Parkinsonism: Sparing of Calbindin-D25k—Containing Cellsa. Annals of the New York Academy of Sciences, 648, 42-62.

Goodwin, J., Nath, S., Engelborghs, Y. and Pountney, D. L. (2013) Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation. Neurochemistry international, 62, 703-711.

Guo, Q., Christakos, S., Robinson, N. and Mattson, M. P. (1998) Calbindin D28k blocks the proapoptotic actions of mutant presenilin 1: Reduced oxidative stress and preserved mitochondrial function. Proceedings of the National Academy of Sciences, 95, 3227-3232.

Halhali, A., Figueras, A. G., Díaz, L., Avila, E., Barrera, D., Hernández, G. and Larrea, F. (2010) Effects of calcitriol on calbindins gene expression and lipid peroxidation in human placenta. The Journal of steroid biochemistry and molecular biology, 121, 448-451.

Jellinger, K. A. (2009) A critical evaluation of current staging of alpha- synuclein pathology in Lewy body disorders. Biochimica et biophysica acta, 1792, 730-740.

Kim, J. S., Kim, Y. I., Song, C., Yoon, I., Park, J. W., Choi, Y. B., Kim, H. T. and Lee, K. S. (2005) Association of vitamin D receptor gene polymorphism

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 185 and Parkinson's disease in Koreans. Journal of Korean Medical Science, 20, 495-498.

Knekt, P., Kilkkinen, A., Rissanen, H., Marniemi, J., Sääksjärvi, K. and Heliövaara, M. (2010) Serum vitamin D and the risk of Parkinson disease. Archives of Neurology, 67, 808-811.

Kruger, R., Kuhn, W., Muller, T. et al. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet, 18, 106-108.

Lesage, S., Anheim, M., Letournel, F. et al. (2013) G51D alpha-synuclein mutation causes a novel parkinsonian-pyramidal syndrome. Annals of neurology, 73, 459-471.

Lowe, R., Pountney, D. L., Jensen, P. H., Gai, W. P. and Voelcker, N. H. (2004) Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with the C-terminal domain. Protein science : a publication of the Protein Society, 13, 3245-3252.

Marques, O. and Outeiro, T. F. (2012) Alpha-synuclein: from secretion to dysfunction and death. Cell death & disease, 3, e350.

Nath, S., Goodwin, J., Engelborghs, Y. and Pountney, D. L. (2011) Raised calcium promotes alpha-synuclein aggregate formation. Molecular and cellular neurosciences, 46, 516-526.

Newmark, H. L. and Newmark, J. (2007) Vitamin D and Parkinson's disease - A hypothesis. Movement Disorders, 22, 461-468.

Pardridge, W. M., Sakiyama, R. and Coty, W. A. (1985) Restricted transport of Vitamin D and A derivatives through the rat blood-brain barrier. Journal of neurochemistry, 44, 1138-1141.

Pasanen, P., Myllykangas, L., Siitonen, M., Raunio, A., Kaakkola, S., Lyytinen, J., Tienari, P. J., Pöyhönen, M. and Paetau, A. (2014) A novel α-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson's disease-type pathology. Neurobiology of aging, 35, 2180.e2181-2180.e2185.

Polymeropoulos, M. H., Lavedan, C., Leroy, E. et al. (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science, 276, 2045-2047.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 186

Proukakis, C., Dudzik, C. G., Brier, T., MacKay, D. S., Cooper, J. M., Millhauser, G. L., Houlden, H. and Schapira, A. H. (2013) A novel α-synuclein missense mutation in Parkinson disease. Neurology, 80, 1062-1064.

Rcom-H'cheo-Gauthier, A., Goodwin, J. and Pountney, D. L. (2014) Interactions between calcium and alpha-synuclein in neurodegeneration. Biomolecules, 4, 795-811.

Schapira, A. H., Olanow, C. W., Greenamyre, J. T. and Bezard, E. (2014) Slowing of neurodegeneration in Parkinson's disease and Huntington's disease: future therapeutic perspectives. Lancet, 384, 545-555.

Shrestha, S., Lutsey, P. L., Alonso, A., Huang, X., Mosley, T. H., Jr. and Chen, H. (2016) Serum 25-hydroxyvitamin D concentrations in Mid-adulthood and Parkinson's disease risk. Movement disorders : official journal of the Movement Disorder Society.

Smith, M. P., Fletcher-Turner, A., Yurek, D. M. and Cass, W. A. (2006) Calcitriol protection against dopamine loss induced by intracerebroventricular administration of 6-hydroxydopamine. Neurochemical Research, 31, 533-539.

Wang, J. Y., Wu, J. N., Cherng, T. L., Hoffer, B. J., Chen, H. H., Borlongan, C. V. and Wang, Y. (2001) Vitamin D3 attenuates 6-hydroxydopamine-induced neurotoxicity in rats. Brain research, 904, 67-75.

Yamada, T., McGeer, P. L., Baimbridge, K. G. and McGeer, E. G. (1990) Relative sparing in Parkinson's disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain research, 526, 303-307.

Yuan, Y.-h., Yan, W.-f., Sun, J.-d., Huang, J.-y., Mu, Z. and Chen, N.-H. (2015) The molecular mechanism of rotenone-induced α-synuclein aggregation: Emphasizing the role of the calcium/GSK3β pathway. Toxicology Letters, 233, 163-171.

Zarranz, J. J., Alegre, J., Gomez-Esteban, J. C. et al. (2004) The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Annals of neurology, 55, 164-173.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 187

Chapter 6: General Discussion

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 188

Ca2+ is an essential intracellular metal ion involved in post-synaptic neuronal activation and neurotransmitter release with a tightly regulated intracellular concentration. It has been established that the buffering capacity of neurons decreases in ageing neurons [148,

149]. Studies have also shown that after neuronal stimulation, the concentration of Ca2+ decreases; however the time taken to recover to resting levels is dramatically increased.

Overtime, this lag in Ca2+ regulation could be a major factor triggering the α-syn aggregation process.

Moreover, Nielsen et al. (2001) reported the selective binding of Ca2+ to recombinant

α-syn. By using atomic force microscopy to visualize aggregates in vitro, Lowe et al showed that Ca2+ binding to the C-terminus of α-syn could induce annular oligomers [235]. More recently, it was demonstrated that raised Ca2+ promotes α-syn aggregation both in vivo and in vitro [110, 152, 153] and that Ca2+ and oxidative stress can cooperatively induce α-syn aggregates in a cell culture model system [110]. Soluble α-syn oligomer formation represents a crucial process in the pathogenesis of PD and DLB and other α-synucleinopathy diseases

[236, 237]. The link between DLB, PD and aggregation of α-syn makes this process a major target for the development of future neurodegenerative therapies. Though the role of α-syn is not completely understood, there is a well-documented increase in the high molecular weight species of α-syn and previous results have indicated analogous changes in the protein expression profile of α-syn, between the treated and control hemispheres of the rotenone mouse model [238]. Many triggers for pathological α-syn aggregation have been identified, including oxidative stress and raised free Ca2+ levels.

Yamada et al. (1990) have demonstrated the overexpression of the CBP in the SN of

PD patients. We predicted the importance of CB in neuroprotection in α-synucleinopathies

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 189 due to this protein’s ability to buffer the intracellular levels of Ca2+ and could potentially prevent the aggregation of α-syn.

We investigated the relationship between the expression of the endogenous neuronal protein, CB, and aggregation of the endogenous pre-synaptic protein, α-syn, in DLB and non-

DLB, healthy cases. We examined the changes in the proportion of CB+ cells and α-syn protein aggregates between DLB and normal cases. Immunofluorescence of DLB cases showed an increased proportion of CB+ neurons compared to normal cases, reaching statistical significance in the visual and motor cortex, temporal lobe and cingulate gyrus.

Neuronal LBs were widespread in DLB tissue, but were rarely detected in CB+ neurons. As

CB is a CBP, the greater proportion of surviving CB+ neurons could reflect the link between

Ca2+ regulation and neuronal survival. This survival rate of CB+ neurons could be due to the action of the CBP in downregulating Ca2+ mediated cell death or by preventing the aggregation of α-syn. This supports the earlier data of Yamada et al. (1990) who found that dopaminergic neurons of the SN that were high in CB and were preferentially spared in the control brain sections compared to PD patients, indicating that increased free Ca2+ (not buffered by CB, CR and PV) may be a major factor in the pathogenesis of α- synucleinopathies.

We investigated the same mechanism in lesioned, sham-lesioned and unlesioned brain hemispheres in a unilateral rotenone-lesioned mouse model which involved a stereotactic rotenone injection into the MFB in young adult (6–12 months old) and aged (1.5-1.75 years old) mice. Oxidative stress is implicated as a causative factor in neurodegenerative disease development and α-syn aggregation [239-242]. The unilateral lesion of the rodent MFB with the mitochondrial complex I inhibitor, rotenone, resulted in oxidative stress in the lesioned

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 190 hemisphere, the formation of intracellular α-syn aggregates and movement dysfunction [232,

238, 243]. There was a greater proportion of CB+ cells in the treated hemispheres compared to the un-lesioned and sham-lesioned hemispheres. The greater proportion of CB+ cells between the treated and control hemisphere may indicate a greater survival in response to cellular stress of CB+ over CB- neurons. Rotenone was also shown to increase α-syn aggregates in a dose-dependent manner and time-dependent manner in cells [244]. Rotenone induced α-syn aggregation was found to be mediated by Ca2+ [244]. BAPTA, a Ca2+ chelating agent, attenuated rotenone-induced impairments of autophagy [244]. Oxidative stress can cause Ca2+ release by the mitochondria and cause elevated intracellular Ca2+ in cells.

However, Guo et al. (1998) showed that Aβ-Induced elevations of ROS are blocked by CB overexpression. It is possible that CB acts as both CBP or anti-oxidant preventing Ca2+ mediated cell death, α-syn aggregation, or oxidative stress by preventing the elevations of the intracellular Ca2+concentration and generation of ROS [206] induced by α-syn aggregation

[245].

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 191

Figure 15: Protective effect of CB in the mechanism of α-syn aggregation. Overexpressed CB binds to free intracellular Ca2+ which does not allow it to bind to α-syn monomers and to trigger α-syn aggregation. CB also buffers the influx of Ca2+ in the cytoplasm caused by α-syn oligomers forming transmembrane pores or from intracellular Ca2+ stores (Mitochondria or ER).

High levels of intracellular Ca2+ may cause α-syn aggregation and in time, the formation of LBs. This dysregulation could be due to elevated ROS levels, α-syn aggregation or dysregulation of VDCC, PMCA or Na+-Ca2+ exchanger (NCX). The return duration to resting levels after a stimulus is greatly increased in aged neurons. This lag in Ca2+ uptake/removal over a long period of time could be the cause of α-syn aggregation. This could also explain why PD and DLB are mostly a disease of the elderlies.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 192

We have demonstrated in this project that neurons which express CB seem to bear significantly less inclusion bodies in comparison to other neurons. The proposed mechanism of this neuronal protection is that CB sequesters intracellular free Ca2+ which cannot bind to

α-syn monomers and facilitate aggregation through its metal binding domain. Furthermore,

Peng et al. (2010) have demonstrated that oxidative stress can be caused by mitochondrial

Ca2+ overload, which could explain how CB decreases oxidative stress in cells, and also decrease α-syn aggregation. Inversely, Ca2+ dysregulation and increase can be triggered by increased intracellular ROS [244]. Therefore increasing CB production in neurons using vitamin D could be a potent target to prevent α-syn aggregation.

It is clear that free intracellular Ca2+ is a major factor influencing α-syn aggregation, and potentially the formation of the cytotoxic oligomeric species seen in disease. Future studies will be required to map the molecular species of aggregates and oligomers in DLB and rotenone mouse tissue. However, recent findings suggest an association between raised intracellular Ca2+, α-syn aggregation and neurotoxicity highlighting the importance for the development of therapeutics that target raised Ca2+ [247]. This provides a potential therapeutic target, by using drugs that chelate/decrease the amount of free Ca2+ in the cell.

Ca2+ channel blockers, such as those from the dihydropyridines family, may be used to mitigate the increase in intracellular Ca2+ levels as seen in aged neurons.

Treatment directly targeting Ca2+ voltage gated channels was shown to prevent α-syn aggregation in cells [153], but development into a therapeutic treatment would involve multiple side effects as those ions gated channels are activated in multiple cellular mechanisms. In the previous study, we showed that CB was found to be over represented in the post mortem brain of DLB patients and Yamada et al. (1990) found that CB was over- represented in the SN on PD patients. Furthermore, we also observed the almost complete

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 193 exclusion of α-syn aggregates from CB+ neurons. Therefore, triggering overproduction of CB could be achieved to prevent α-syn aggregation and provide neuroprotection in PD and DLB.

CB was shown to be upregulated in cells when treated with the hormonal form of vitamin D,

Calcitriol [234].

We investigated the overexpression of CB and α-syn aggregation in a cell model of

PD, CB over-production was triggered by Cp treatment, which is a vitamin D analogue and has a more stable structure than Calcitriol. After treatment with Cp, we found that CB was induced in a dose dependent manner. We examined changes in CB levels and α-syn protein aggregates between different Cp concentration treatments. We observed a reduction of α-syn aggregates in cells when the concentration was increased. We believe this decrease is due to

α-syn aggregation being directly linked to the increase in the levels of CB in those cells. We believe that over production of CB which is triggered by vitamin D could be used to prevent or stop the progression of diseases such as DLB and PD.

Major metabolites of vitamin D were found to be present in human cerebrospinal fluid which include 25OHD3, 1,25OH2D3 and 24,25OH2D3 [209]. Furthermore, vitamin D metabolites have been found to.be able to cross the blood brain barrier [210]. CYP27B1 was present in both neurons and glia and was most strongly expressed in the SN and the supraoptic and paraventricular nuclei of the hypothalamus in the adult brain [214]. This potentially indicates that the brain may be able to synthesize the active metabolite 1,25OH2D3.

Further research has found that administration of 1,25OH2D3 is protective against damage from a neurotoxin that specifically lesions dopaminergic and noradrenergic cells

6OHDA [216, 217]. Furthermore, the administration of 1,25OH2D3 has found to be protective against neurotoxic doses of methamphetamine by preserving dopamine and serotonin levels

[218]. Such neuroprotection of dopaminergic neurons may be relevant in the loss of

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 194 dopaminergic neurons in the SN in PD. In addition, some investigations have linked vitamin

D insufficiency with an increased risk of PD [219, 220]. Moreover, higher levels of 25OHD3 were associated with a reduced risk of developing PD in later life [221]. Abnormalities in the vitamin D receptor have also been linked with risk of developing PD [222]. On the other hand, a recent study by Shretha et al. (2016) has found an association between serum 25- hydroxyvitamin D concentration in mid-adulthood and PD risk, which suggested that vitamin

D may not help reduce the risk of PD. The experimental results obtained could explain this effect of neuroprotection of vitamin D on the brain, however as previously stated this could also be due to other effects of vitamin D on cell regulation that were not measured in this set of experiments.

Following the cell experiments, further studies involving Cp and primary neural cell culture could be conducted to see if Cp also increases CB expression in this cell line. Future studies on an animal model could also be done by pre-treating mice with a vitamin D analogue to induce CB production prior to the unilateral lesion of rotenone. Different modes of vitamin D delivery could be used, such a cutaneous (Cp as a cream to treat psoriasis), as an injection at the same time as the rotenone lesion, or orally using vitamin D tablets. To verify the effectiveness of the treatment, the total level of CB expression could be measured through

WB and through immunofluorescence. Levels of TH neurons and NeuN could also be measured to study the direct influence of vitamin D on neurodegeneration.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 195

References

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 196

[1] Parkinson J. An essay on the shaking palsy. 1817. J Neuropsychiatry Clin Neurosci 2002;14:223-36; discussion 2.

[2] Lesage S, Brice A. Parkinson's disease: from monogenic forms to genetic susceptibility factors. Hum Mol Gen 2009;18:R48-59.

[3] Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat. Neurosci 2000;3:1301-6.

[4] Gelb DJ, Oliver E, Gilman S. Diagnostic criteria for Parkinson disease. JAMA Neurol 1999;56:33-9.

[5] Marella M, Seo BB, Yagi T, Matsuno-Yagi A. Parkinson's disease and mitochondrial complex I: a perspective on the Ndi1 therapy. J Bioenerg Biomembr 2009;41:493-7.

[6] Fasano A, Daniele A, Albanese A. Treatment of motor and non-motor features of Parkinson's disease with deep brain stimulation. Lancet neurol 2012;11:429-42.

[7] Jenner P. Pharmacology of dopamine agonists in the treatment of Parkinson's disease. Neurology 2002;58:S1-8.

[8] Barbeau A, Murphy GF, Sourkes TL. Excretion of dopamine in diseases of basal ganglia. Science 1961;133:1706-7.

[9] Smith Y, Wichmann T, Factor SA, DeLong MR. Parkinson's Disease Therapeutics: New Developments and Challenges Since the Introduction of Levodopa. Neuropsychopharmacology 2012;37:213-46.

[10] Tanner CM, Kamel F, Ross GW, Hoppin JA, Goldman SM, Korell M, et al. Rotenone, paraquat, and Parkinson's disease. Environ Health Perspect 2011;119:866-72.

[11] Dick FD, De Palma G, Ahmadi A, Scott NW, Prescott GJ, Bennett J, et al. Environmental risk factors for Parkinson's disease and parkinsonism: the Geoparkinson study. Occup Environ Med 2007;64:666-72.

[12] Wang X, Su B, Liu W, He X, Gao Y, Castellani RJ, et al. DLP1-dependent mitochondrial fragmentation mediates 1-methyl-4-phenylpyridinium toxicity in neurons: implications for Parkinson's disease. Aging Cell 2011;10:807-23.

[13] Hernan MA, Takkouche B, Caamano-Isorna F, Gestal-Otero JJ. A meta-analysis of coffee drinking, cigarette smoking, and the risk of Parkinson's disease. Ann Neurol 2002;52:276-84.

[14] Hornykiewicz O. Brain monoamines and Parkinsonism. Psychopharmacol Bull 1975;11:34-5.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 197 [15] Jellinger KA. Pathology of Parkinson's disease. Changes other than the nigrostriatal pathway. Mol Chem Neuropathol 1991;14:153-97.

[16] Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 2003;24:197-211.

[17] Bloch A, Probst A, Bissig H, Adams H, Tolnay M. Alpha-synuclein pathology of the spinal and peripheral autonomic nervous system in neurologically unimpaired elderly subjects. Neuropathol Appl Neurobiol 2006;32:284-95.

[18] Braak H, de Vos RA, Bohl J, Del Tredici K. Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease- related brain pathology. Neurosci Lett 2006;396:67-72.

[19] Klos KJ, Ahlskog JE, Josephs KA, Apaydin H, Parisi JE, Boeve BF, et al. Alpha- synuclein pathology in the spinal cords of neurologically asymptomatic aged individuals. Neurology 2006;66:1100-2.

[20] Braak H, Sastre M, Bohl JR, de Vos RA, Del Tredici K. Parkinson's disease: lesions in dorsal horn layer I, involvement of parasympathetic and sympathetic pre- and postganglionic neurons. Acta Neuropathol 2007;113:421-9.

[21] Barbeau A, Mars H, Botez MI, Joubert M. Levodopa combined with peripheral decarboxylase inhibition in Parkinson's disease. Can Med Assoc J 1972;106:1169-74.

[22] Rinne UK, Marttila R, Sonninen V. Brain dopamine turnover and the relief of parkinsonism. Arch Neurol 1977;34:626-9.

[23] Schrag A. Psychiatric aspects of Parkinson's disease--an update. J Neurol 2004;251:795- 804.

[24] Hauser RA, Zesiewicz TA. Advances in the pharmacologic management of early Parkinson disease. Neurologist 2007;13:126-32.

[25] Blandini F, Bazzini E, Marino F, Saporiti F, Armentero MT, Pacchetti C, et al. Calcium homeostasis is dysregulated in parkinsonian patients with L-DOPA-induced dyskinesias. Clin Neuropharmacol 2009;32:133-9.

[26] Lees AJ. The Parkinson chimera. Neurology 2009;72:S2-11.

[27] Hirsch E, Graybiel AM, Agid YA. Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson's disease. Nature 1988;334:345-8.

[28] Good CH, Hoffman AF, Hoffer BJ, Chefer VI, Shippenberg TS, Backman CM, et al. Impaired nigrostriatal function precedes behavioral deficits in a genetic mitochondrial model of Parkinson's disease. FASEB J 2011;25:1333-44.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 198

[29] Ekstrand MI, Terzioglu M, Galter D, Zhu S, Hofstetter C, Lindqvist E, et al. Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons. Proc Natl Acad Sci U S A 2007;104:1325-30.

[30] Galter D, Pernold K, Yoshitake T, Lindqvist E, Hoffer B, Kehr J, et al. MitoPark mice mirror the slow progression of key symptoms and L-DOPA response in Parkinson's disease. Genes Brain Behav 2010;9:173-81.

[31] Jellinger KA. Neuropathological spectrum of synucleinopathies. Mov Disorders 2003;18:2-12.

[32] McKeith IG, Galasko D, Kosaka K, Perry EK, Dickson DW, Hansen LA, et al. Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop. Neurology 1996;47:1113- 24.

[33] Neef D, Walling AD. Dementia with Lewy bodies: an emerging disease. Am fam physician 2006;73:1223-9.

[34] Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E, Ros R, Ampuero I, et al. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann of neurol 2004;55:164-73.

[35] Marx FP, Holzmann C, Strauss KM, Li L, Eberhardt O, Gerhardt E, et al. Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson's disease. Hum mol gen 2003;12:1223-31.

[36] Ohtake H, Limprasert P, Fan Y, Onodera O, Kakita A, Takahashi H, et al. Beta-synuclein gene alterations in dementia with Lewy bodies. Neurology 2004;63:805-11.

[37] McKeith I, Mintzer J, Aarsland D, Burn D, Chiu H, Cohen-Mansfield J, et al. Dementia with Lewy bodies. The Lancet Neurol 2004;3:19-28.

[38] Merdes AR, Hansen LA, Jeste DV, Galasko D, Hofstetter CR, Ho GJ, et al. Influence of Alzheimer pathology on clinical diagnostic accuracy in dementia with Lewy bodies. Neurology 2003;60:1586-90.

[39] Lopez OL, Becker JT, Kaufer DI, Hamilton RL, Sweet RA, Klunk W, et al. Research evaluation and prospective diagnosis of dementia with Lewy bodies. Arch Neurol 2002;59:43-6.

[40] Del Ser T, Hachinski V, Merskey H, Munoz DG. Clinical and pathologic features of two groups of patients with dementia with Lewy bodies: effect of coexisting Alzheimer-type lesion load. Alzheimer disease and associated disorders 2001;15:31-44.

[41] Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathologica 1991;82:239-59.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 199

[42] Braak H, Tredici KD, Rüb U, de Vos RAI, Jansen Steur ENH, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol of Aging;24:197-211.

[43] McKeith IG, Dickson DW, Lowe J, Emre M, O'Brien JT, Feldman H, et al. Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium. Neurology 2005;65:1863-72.

[44] Weisman D, Cho M, Taylor C, Adame A, Thal LJ, Hansen LA. In dementia with Lewy bodies, Braak stage determines phenotype, not Lewy body distribution. Neurology 2007;69:356-9.

[45] Hohl U, Tiraboschi P, Hansen LA, Thal LJ, Corey-Bloom J. Diagnostic accuracy of dementia with Lewy bodies. Arch Neurol 2000;57:347-51.

[46] Walker MP, Ayre GA, Cummings JL, Wesnes K, McKeith IG, O'Brien JT, et al. The Clinician Assessment of Fluctuation and the One Day Fluctuation Assessment Scale. Two methods to assess fluctuating confusion in dementia. Br J Psychiatry 2000;177:252-6.

[47] Aarsland D, Andersen K, Larsen JP, Lolk A, Kragh-Sorensen P. Prevalence and characteristics of dementia in Parkinson disease: an 8-year prospective study. Arch Neurol 2003;60:387-92.

[48] McKeith IG. Spectrum of Parkinson's disease, Parkinson's dementia, and Lewy body dementia. Neurol clin 2000;18:865-902.

[49] Barber R, Panikkar A, McKeith IG. Dementia with Lewy bodies: diagnosis and management. Int J of Geriatr Psychatry 2001;16 Suppl 1:S12-8.

[50] McKeith I, Mintzer J, Aarsland D, Burn D, Chiu H, Cohen-Mansfield J, et al. Dementia with Lewy bodies. The Lancet Neurol;3:19-28.

[51] Duda JE, Giasson BI, Mabon ME, Lee VMY, Trojanowski JQ. Novel antibodies to synuclein show abundant striatal pathology in Lewy body diseases. Ann of neurol 2002;52:205-10.

[52] Samuel W, Caligiuri M, Galasko D, Lacro J, Marini M, McClure FS, et al. Better cognitive and psychopathologic response to donepezil in patients prospectively diagnosed as dementia with Lewy bodies: a preliminary study. Int J of Geriatr Psychiatry 2000;15:794-802.

[53] Mosimann UP, McKeith IG. Dementia with lewy bodies--diagnosis and treatment. Swiss Med Wkly 2003;133:131-42.

[54] McLaren AT, Allen J, Murray A, Ballard CG, Kenny RA. Cardiovascular effects of donepezil in patients with dementia. Dem geriatr cogn dis 2003;15:183-8.

[55] Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A 1998;95:6469-73.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 200

[56] Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M. Alpha- synuclein in Lewy bodies. Nature 1997;388:839-40.

[57] Ross CA, Poirier MA. Opinion: What is the role of protein aggregation in neurodegeneration? Nat Rev Mol cel biol 2005;6:891-8.

[58] Tanaka M, Kim YM, Lee G, Junn E, Iwatsubo T, Mouradian MM. Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective. J Biol Chem 2004;279:4625-31.

[59] Tompkins MM, Hill WD. Contribution of somal Lewy bodies to neuronal death. Brain Res 1997;775:24-9.

[60] Olanow CW, Perl DP, DeMartino GN, McNaught KS. Lewy-body formation is an aggresome-related process: a hypothesis. Lancet Neurol 2004;3:496-503.

[61] Shults CW. Lewy bodies. Proc Natl Acad Sci USA 2006;103:1661-8.

[62] Recasens A, Dehay B, Bové J, Carballo-Carbajal I, Dovero S, Pérez-Villalba A, et al. Lewy body extracts from Parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. Ann Neurol 2014;75:351-62.

[63] Campbell BC, McLean CA, Culvenor JG, Gai WP, Blumbergs PC, Jakala P, et al. The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease. J Neurochem 2001;76:87-96.

[64] Gomez-Tortosa E, Newell K, Irizarry MC, Sanders JL, Hyman BT. alpha-Synuclein immunoreactivity in dementia with Lewy bodies: morphological staging and comparison with ubiquitin immunostaining. Acta neuropathol 2000;99:352-7.

[65] Sakamoto M, Uchihara T, Hayashi M, Nakamura A, Kikuchi E, Mizutani T, et al. Heterogeneity of nigral and cortical Lewy bodies differentiated by amplified triple-labeling for alpha-synuclein, ubiquitin, and thiazin red. Exp neurol 2002;177:88-94.

[66] Wakabayashi K, Tanji K, Mori F, Takahashi H. The Lewy body in Parkinson's disease: molecules implicated in the formation and degradation of alpha-synuclein aggregates. Neuropathology 2007;27:494-506.

[67] Beyer K, Ariza A. Protein aggregation mechanisms in synucleinopathies: commonalities and differences. J neuropathol exp neurol2007;66:965-74.

[68] Iwai A, Masliah E, Yoshimoto M, Ge N, Flanagan L, de Silva HA, et al. The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system. Neuron 1995;14:467-75.

[69] Maroteaux L, Campanelli JT, Scheller RH. Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J Neurosci 1988;8:2804-15.

[70] Clayton DF, George JM. Synucleins in synaptic plasticity and neurodegenerative disorders. J Neurosci Res 1999;58:120-9.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 201

[71] Burre J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, Sudhof TC. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 2010;329:1663-7.

[72] Nielsen MS, Vorum H, Lindersson E, Jensen PH. Ca2+ binding to alpha-synuclein regulates ligand binding and oligomerization. J Biol Chem 2001;276:22680-4.

[73] Giasson BI, Murray IV, Trojanowski JQ, Lee VM. A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem 2001;276:2380-6.

[74] Eliezer D, Kutluay E, Bussell R, Jr., Browne G. Conformational properties of alpha- synuclein in its free and lipid-associated states. J Mol Biol 2001;307:1061-73.

[75] Davidson WS, Jonas A, Clayton DF, George JM. Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J Biol Chem 1998;273:9443-9.

[76] Han H, Weinreb PH, Lansbury PT. The core Alzheimer's peptide NAC forms amyloid fibrils which seed and are seeded by β-amyloid: is NAC a common trigger or target in neurodegenerative disease? Chem Biol 1995;2:163-9.

[77] Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M, Biere AL. alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson's disease. J Biol Chem 1999;274:19509-12.

[78] Crowther RA, Jakes R, Spillantini MG, Goedert M. Synthetic filaments assembled from C-terminally truncated alpha-synuclein. FEBS Lett 1998;436:309-12.

[79] Narayanan V, Scarlata S. Membrane binding and self-association of alpha-synucleins. Biochemistry 2001;40:9927-34.

[80] Hu HY, Li Q, Cheng HC, Du HN. beta-sheet structure formation of proteins in solid state as revealed by circular dichroism spectroscopy. Biopolymers 2001;62:15-21.

[81] George S, Mok SS, Nurjono M, Ayton S, Finkelstein DI, Masters CL, et al. alpha- Synuclein transgenic mice reveal compensatory increases in Parkinson's disease-associated proteins DJ-1 and parkin and have enhanced alpha-synuclein and PINK1 levels after rotenone treatment. J Mol Neurosci 2010;42:243-54.

[82] McLean PJ, Kawamata H, Ribich S, Hyman BT. Membrane association and protein conformation of alpha-synuclein in intact neurons. Effect of Parkinson's disease-linked mutations. J Biol Chem 2000;275:8812-6.

[83] Chen RH, Wislet-Gendebien S, Samuel F, Visanji NP, Zhang G, Marsilio D, et al. alpha- Synuclein membrane association is regulated by the Rab3a recycling machinery and presynaptic activity. J Biol Chem 2013;288:7438-49.

[84] Eschbach J, Danzer KM. alpha-Synuclein in Parkinson's disease: pathogenic function and translation into animal models. Neurodegener Dis 2014;14:1-17.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 202

[85] Goedert M, Spillantini MG, Del Tredici K, Braak H. 100 years of Lewy pathology. Nat Rev Neurol 2013;9:13-24.

[86] Radford R, Rcom-H'cheo-Gauthier A, Wong MB, Eaton ED, Quilty M, Blizzard C, et al. The degree of astrocyte activation in multiple system atrophy is inversely proportional to the distance to alpha-synuclein inclusions. Mol Cell neurosci 2015;65:68-81.

[87] Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, et al. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat Genet 1998;18:106-8.

[88] Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997;276:2045-7.

[89] Proukakis C, Dudzik CG, Brier T, MacKay DS, Cooper JM, Millhauser GL, et al. A novel α-synuclein missense mutation in Parkinson disease. Neurology 2013;80:1062-4.

[90] Pasanen P, Myllykangas L, Siitonen M, Raunio A, Kaakkola S, Lyytinen J, et al. A novel α-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson's disease-type pathology. Neurobio aging 2014;35:2180.e1-.e5.

[91] Lesage S, Anheim M, Letournel F, Bousset L, Honore A, Rozas N, et al. G51D alpha- synuclein mutation causes a novel parkinsonian-pyramidal syndrome. Ann Neurol 2013;73:459-71.

[92] Kanda S, Bishop JF, Eglitis MA, Yang Y, Mouradian MM. Enhanced vulnerability to oxidative stress by alpha-synuclein mutations and C-terminal truncation. Neuroscience 2000;97:279-84.

[93] Ibanez P, Bonnet AM, Debarges B, Lohmann E, Tison F, Pollak P, et al. Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease. Lancet 2004;364:1169-71.

[94] Chartier-Harlin MC, Kachergus J, Roumier C, Mouroux V, Douay X, Lincoln S, et al. Alpha-synuclein locus duplication as a cause of familial Parkinson's disease. Lancet 2004;364:1167-9.

[95] Conway KA, Harper JD, Lansbury PT. Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat Med 1998;4:1318-20.

[96] Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury PT, Jr. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha- synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy. Proc Natl Acad Sci U S A 2000;97:571-6.

[97] Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM, Anafi D, et al. Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation. J Biol Chem 1999;274:9843-6.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 203

[98] Oliveira LMA, Falomir-Lockhart LJ, Botelho MG, Lin KH, Wales P, Koch JC, et al. Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells. Cell death dis 2015;6:e1994.

[99] Mak SK, McCormack AL, Langston JW, Kordower JH, Di Monte DA. Decreased alpha- synuclein expression in the aging mouse substantia nigra. Exp Neurol 2009;220:359-65.

[100] Wirdefeldt K, Bogdanovic N, Westerberg L, Payami H, Schalling M, Murdoch G. Expression of alpha-synuclein in the human brain: relation to Lewy body disease. Brain Res Mol Brain Res 2001;92:58-65.

[101] Grundemann J, Schlaudraff F, Haeckel O, Liss B. Elevated alpha-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease. Nucleic Acids Res 2008;36:e38.

[102] Tan EK, Chandran VR, Fook-Chong S, Shen H, Yew K, Teoh ML, et al. Alpha- synuclein mRNA expression in sporadic Parkinson's disease. Mov Disord 2005;20:620-3.

[103] Recasens A, Dehay B, Bove J, Carballo-Carbajal I, Dovero S, Perez-Villalba A, et al. Lewy body extracts from Parkinson disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys. Ann Neurol 2014;75:351-62.

[104] Danzer KM, Haasen D, Karow AR, Moussaud S, Habeck M, Giese A, et al. Different species of alpha-synuclein oligomers induce calcium influx and seeding. J Neurosci 2007;27:9220-32.

[105] Outeiro TF, Putcha P, Tetzlaff JE, Spoelgen R, Koker M, Carvalho F, et al. Formation of toxic oligomeric alpha-synuclein species in living cells. PLoS ONE 2008;3:e1867.

[106] Esteves AR, Arduino DM, Swerdlow RH, Oliveira CR, Cardoso SM. Oxidative Stress involvement in alpha-synuclein oligomerization in Parkinsons disease cybrids. Antioxid Redox Signal 2008.

[107] Bartels T, Choi JG, Selkoe DJ. alpha-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Nature 2011;477:107-10.

[108] Coelho-Cerqueira E, Carmo-Gonçalves P, Sá Pinheiro A, Cortines J, Follmer C. α- Synuclein as an intrinsically disordered monomer – fact or artefact? FEBS Journal 2013;280:4915-27.

[109] Coelho-Cerqueira E, Carmo-Goncalves P, Pinheiro AS, Cortines J, Follmer C. alpha- Synuclein as an intrinsically disordered monomer--fact or artefact? FEBS J 2013;280:4915- 27.

[110] Goodwin J, Nath S, Engelborghs Y, Pountney DL. Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation. Neurochem Intl 2013;62:703-11.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 204

[111] Uversky VN, Yamin G, Munishkina LA, Karymov MA, Millett IS, Doniach S, et al. Effects of nitration on the structure and aggregation of alpha-synuclein. Brain Res Mol Brain Res 2005;134:84-102.

[112] Chavarría C, Souza JM. Oxidation and nitration of α-synuclein and their implications in neurodegenerative diseases. Arch Biochem Biophys 2013;533:25-32.

[113] Glaser CB, Yamin G, Uversky VN, Fink AL. Methionine oxidation, α-synuclein and Parkinson's disease. Biochim Biophys 2005;1703:157-69.

[114] Uversky VN, Yamin G, Souillac PO, Goers J, Glaser CB, Fink AL. Methionine oxidation inhibits fibrillation of human alpha-synuclein in vitro. FEBS letters 2002;517:239- 44.

[115] Feany MB, Bender WW. A Drosophila model of Parkinson's disease. Nature 2000;404:394-8.

[116] Kaul S, Anantharam V, Kanthasamy A, Kanthasamy AG. Wild-type alpha-synuclein interacts with pro-apoptotic proteins PKCdelta and BAD to protect dopaminergic neuronal cells against MPP+-induced apoptotic cell death. Brain Res Mol Brain Res 2005;139:137-52.

[117] Pham CL, Cappai R. The interplay between lipids and dopamine on alpha-synuclein oligomerization and membrane binding. Biosci Rep 2013;33.

[118] Smith WW, Margolis RL, Li X, Troncoso JC, Lee MK, Dawson VL, et al. Alpha- synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH- SY5Y cells. J neurosci 2005;25:5544-52.

[119] Lu Y, Prudent M, Fauvet B, Lashuel HA, Girault HH. Phosphorylation of alpha- Synuclein at Y125 and S129 alters its metal binding properties: implications for understanding the role of alpha-Synuclein in the pathogenesis of Parkinson's Disease and related disorders. ACS Chem Neurosci 2011;2:667-75.

[120] Liu LL, Franz KJ. Phosphorylation of an alpha-synuclein peptide fragment enhances metal binding. J Am Chem Soc2005;127:9662-3.

[121] Anderson JP, Walker DE, Goldstein JM, de Laat R, Banducci K, Caccavello RJ, et al. Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J Biol Chem 2006;281:29739-52.

[122] Kleinknecht A, Popova B, Lazaro DF, Pinho R, Valerius O, Outeiro TF, et al. C- Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of alpha-Synuclein in a Yeast Model of Parkinson's Disease. PLoS genetics 2016;12:e1006098.

[123] Lee HJ, Bae EJ, Lee SJ. Extracellular alpha--synuclein-a novel and crucial factor in Lewy body diseases. Nat Rev Neurol 2014;10:92-8.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 205 [124] Alvarez-Erviti L, Seow Y, Schapira AH, Gardiner C, Sargent IL, Wood MJ, et al. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol Dis 2011;42:360-7.

[125] Danzer KM, Kranich LR, Ruf WP, Cagsal-Getkin O, Winslow AR, Zhu L, et al. Exosomal cell-to-cell transmission of alpha synuclein oligomers. Mol Neurodegener 2012;7:42.

[126] Chang C, Lang H, Geng N, Wang J, Li N, Wang X. Exosomes of BV-2 cells induced by alpha-synuclein: important mediator of neurodegeneration in PD. Neurosci Lett 2013;548:190-5.

[127] Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW. Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease. Nat Med 2008;14:504-6.

[128] Li JY, Englund E, Holton JL, Soulet D, Hagell P, Lees AJ, et al. Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation. Nat med 2008;14:501-3.

[129] Jones DR, Delenclos M, Baine AT, DeTure M, Murray ME, Dickson DW, et al. Transmission of Soluble and Insoluble α-Synuclein to Mice. Journal of Neuropathology & Experimental Neurology 2015;74:1158-69.

[130] Prusiner SB, Woerman AL, Mordes DA, Watts JC, Rampersaud R, Berry DB, et al. Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism. Proc. Natl. Acad. Sci. USA 2015;112:E5308-E17.

[131] Reyes JF, Olsson TT, Lamberts JT, Devine MJ, Kunath T, Brundin P. A cell culture model for monitoring α-synuclein cell-to-cell transfer. Neurobiol Dis2015;77:266-75.

[132] Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol 1956;11:298-300.

[133] Ku HH, Brunk UT, Sohal RS. Relationship between mitochondrial superoxide and hydrogen peroxide production and longevity of mammalian species. Free Radic Biol Med 1993;15:621-7.

[134] Abdul HM, Sultana R, St Clair DK, Markesbery WR, Butterfield DA. Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age. Free Radic Biol Med 2008;45:1420-5.

[135] Turrens JF. Superoxide production by the mitochondrial respiratory chain. Biosci Rep 1997;17:3-8.

[136] Castro Mdel R, Suarez E, Kraiselburd E, Isidro A, Paz J, Ferder L, et al. Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys. Exp gerontol 2012;47:29-37.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 206

[137] Sims-Robinson C, Hur J, Hayes JM, Dauch JR, Keller PJ, Brooks SV, et al. The role of oxidative stress in nervous system aging. PloS one 2013;8:e68011.

[138] Quilty MC, King AE, Gai WP, Pountney DL, West AK, Vickers JC, et al. Alpha- synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection. Exp Neurol 2006;199:249-56.

[139] Mandal PK, Tripathi M, Sugunan S. Brain oxidative stress: detection and mapping of anti-oxidant marker 'Glutathione' in different brain regions of healthy male/female, MCI and Alzheimer patients using non-invasive magnetic resonance spectroscopy. Biochem Biophys Res Commun 2012;417:43-8.

[140] Sulzer D, Surmeier DJ. Neuronal vulnerability, pathogenesis, and Parkinson's disease. Mov Dis 2013;28:41-50.

[141] Fairless R, Williams SK, Diem R. Dysfunction of neuronal calcium signalling in neuroinflammation and neurodegeneration. Cell Tissue Res 2014;357:455-62.

[142] Goswami P, Gupta S, Biswas J, Joshi N, Swarnkar S, Nath C, et al. Endoplasmic Reticulum Stress Plays a Key Role in Rotenone-Induced Apoptotic Death of Neurons. Mol Neurobiol 2016;53:285-98.

[143] Farajnia S, Meijer JH, Michel S. Age-related changes in large-conductance calcium- activated potassium channels in mammalian circadian clock neurons. Neurobiol aging 2015;36:2176-83.

[144] Ghosh A, Ginty DD, Bading H, Greenberg ME. Calcium regulation of gene expression in neuronal cells. J Neurobiol 1994;25:294-303.

[145] Geiger JR, Melcher T, Koh DS, Sakmann B, Seeburg PH, Jonas P, et al. Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron 1995;15:193-204.

[146] Berridge MJ. Neuronal calcium signaling. Neuron 1998;21:13-26.

[147] Michaelis ML, Bigelow DJ, Schoneich C, Williams TD, Ramonda L, Yin D, et al. Decreased plasma membrane calcium transport activity in aging brain. Life Sci 1996;59:405- 12.

[148] Villa P, Miehe M, Sensenbrenner M, Pettmann B. Synthesis of specific proteins in trophic factor-deprived neurons undergoing apoptosis. J Neurochem 1994;62:1468-75.

[149] Duckles SP, Tsai H, Buchholz JN. Evidence for decline in intracellular calcium buffering in adrenergic nerves of aged rats. Life Sci 1996;58:2029-35.

[150] Pottorf WJ, Duckles SP, Buchholz JN. SERCA function declines with age in adrenergic nerves from the superior cervical ganglion. J Auton Pharmacol 2000;20:281-90.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 207

[151] Perier C, Vila M. Mitochondrial biology and Parkinson's disease. Cold Spring Harb Perspect Med 2012;2:a009332.

[152] Nath S, Goodwin J, Engelborghs Y, Pountney DL. Raised calcium promotes alpha- synuclein aggregate formation. Mol Cell neurosci 2011;46:516-26.

[153] Follett J, Darlow B, Wong MB, Goodwin J, Pountney DL. Potassium depolarization and raised calcium induces alpha-synuclein aggregates. Neurotox Res 2013;23:378-92.

[154] Chan CS, Guzman JN, Ilijic E, Mercer JN, Rick C, Tkatch T, et al. 'Rejuvenation' protects neurons in mouse models of Parkinson's disease. Nature 2007;447:1081-6.

[155] Gaspar P, Ben Jelloun N, Febvret A. Sparing of the dopaminergic neurons containing calbindin-D28k and of the dopaminergic mesocortical projections in weaver mutant mice. Neurosci 1994;61:293-305.

[156] Avramovic N, Dragutinovic V, Krstic D, Colovic M, Trbovic A, de Luka S, et al. The effects of omega 3 fatty acid supplementation on brain tissue oxidative status in aged wistar rats. Hippokratia 2012;16:241-5.

[157] Engstrom K, Saldeen AS, Yang B, Mehta JL, Saldeen T. Effect of fish oils containing different amounts of EPA, DHA, and antioxidants on plasma and brain fatty acids and brain nitric oxide synthase activity in rats. Ups J Med Sci 2009;114:206-13.

[158] Parihar MS, Parihar A, Fujita M, Hashimoto M, Ghafourifar P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci 2008;65:1272-84.

[159] Hashimoto M, Hsu LJ, Rockenstein E, Takenouchi T, Mallory M, Masliah E. alpha- Synuclein protects against oxidative stress via inactivation of the c-Jun N-terminal kinase stress-signaling pathway in neuronal cells. J Biol Chem 2002;277:11465-72.

[160] Kume K, Kikukawa M, Hanyu H, Takata Y, Umahara T, Sakurai H, et al. Telomere length shortening in patients with dementia with Lewy bodies. Eur J Neurol 2012;19:905-10.

[161] Lin MT, Cantuti-Castelvetri I, Zheng K, Jackson KE, Tan YB, Arzberger T, et al. Somatic mitochondrial DNA mutations in early Parkinson and incidental Lewy body disease. Ann Neurol 2012;71:850-4.

[162] Dalfo E, Ferrer I. Early alpha-synuclein lipoxidation in neocortex in Lewy body diseases. Neurobiol Aging 2008;29:408-17.

[163] Saito Y, Miyasaka T, Hatsuta H, Takahashi-Niki K, Hayashi K, Mita Y, et al. Immunostaining of oxidized DJ-1 in human and mouse brains. J Neuropathol Exp Neurol 2014;73:714-28.

[164] Dryanovski DI, Guzman JN, Xie Z, Galteri DJ, Volpicelli-Daley LA, Lee VM, et al. Calcium entry and alpha-synuclein inclusions elevate dendritic mitochondrial oxidant stress in

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 208 dopaminergic neurons. The Journal of neuroscience : the official journal of the Society for Neurosci 2013;33:10154-64.

[165] Hettiarachchi NT, Parker A, Dallas ML, Pennington K, Hung CC, Pearson HA, et al. alpha-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells. J Neurochem 2009;111:1192-201.

[166] Melachroinou K, Xilouri M, Emmanouilidou E, Masgrau R, Papazafiri P, Stefanis L, et al. Deregulation of calcium homeostasis mediates secreted alpha-synuclein-induced neurotoxicity. Neurobiol Aging 2013;34:2853-65.

[167] Reznichenko L, Cheng Q, Nizar K, Gratiy SL, Saisan PA, Rockenstein EM, et al. In vivo alterations in calcium buffering capacity in transgenic mouse model of synucleinopathy. J Neurosci 2012;32:9992-8.

[168] Cali T, Ottolini D, Negro A, Brini M. alpha-Synuclein controls mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions. J Biol Chem 2012;287:17914-29.

[169] Buttner S, Faes L, Reichelt WN, Broeskamp F, Habernig L, Benke S, et al. The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca(2+) levels to alpha-synuclein toxicity in Parkinson's disease models. Cell death Dif 2013;20:465-77.

[170] Koch Y, Helferich AM, Steinacker P, Oeckl P, Walther P, Weishaupt JH, et al. Aggregated α-Synuclein Increases SOD1 Oligomerization in a Mouse Model of Amyotrophic Lateral Sclerosis. Am J Pathol 2016;186:2152-61.

[171] Perfeito R, Ribeiro M, Rego AC. Alpha-synuclein-induced oxidative stress correlates with altered superoxide dismutase and glutathione synthesis in human neuroblastoma SH- SY5Y cells. Arch Toxico 2016:1-15.

[172] Navarria L, Zaltieri M, Longhena F, Spillantini MG, Missale C, Spano P, et al. Alpha- synuclein modulates NR2B-containing NMDA receptors and decreases their levels after rotenone exposure. Neurochem Intl 2015;85–86:14-23.

[173] Krishnan S, Chi EY, Wood SJ, Kendrick BS, Li C, Garzon-Rodriguez W, et al. Oxidative dimer formation is the critical rate-limiting step for Parkinson's disease alpha- synuclein fibrillogenesis. Biochem 2003;42:829-37.

[174] Goldberg JA, Guzman JN, Estep CM, Ilijic E, Kondapalli J, Sanchez-Padilla J, et al. Calcium entry induces mitochondrial oxidant stress in vagal neurons at risk in Parkinson's disease. Nat Neurosci 2012;15:1414-21.

[175] Simms Brett A, Zamponi Gerald W. Neuronal Voltage-Gated Calcium Channels: Structure, Function, and Dysfunction. Neuron 2014;82:24-45.

[176] Kurihara T, Tanabe T. N-type Ca2+ channel. Nihon yakurigaku zasshi Folia pharmacologica Japonica 2003;121:211-22.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 209

[177] Catterall WA, Perez-Reyes E, Snutch TP, Striessnig J. International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev 2005;57:411-25.

[178] Babu CS, Ramanathan M. Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics. Int J Dev Neurosci 2011;29:93-105.

[179] Kim TY, Yoshimoto T, Aoyama Y, Niimi K, Takahashi E. Analysis of the protective effects of a neuronal Cav2.1 calcium channel in brain injury. Neuroscience 2016;313:110-21.

[180] Perez-Pinzon MA, Yenari MA, Sun GH, Kunis DM, Steinberg GK. SNX-111, a novel, presynaptic N-type calcium channel antagonist, is neuroprotective against focal cerebral ischemia in rabbits. J Neurol Sci 1997;153:25-31.

[181] Acosta SA, Tajiri N, de la Pena I, Bastawrous M, Sanberg PR, Kaneko Y, et al. Alpha- synuclein as a pathological link between chronic traumatic brain injury and Parkinson's disease. J Cell Physiol 2015;230:1024-32.

[182] Talley EM, Cribbs LL, Lee JH, Daud A, Perez-Reyes E, Bayliss DA. Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19:1895-911.

[183] Heady TN, Gomora JC, Macdonald TL, Perez-Reyes E. Molecular pharmacology of T- type Ca2+ channels. Jpn J Pharmacol 2001;85:339-50.

[184] Chemin J, Monteil A, Perez-Reyes E, Nargeot J, Lory P. Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide. The EMBO journal 2001;20:7033-40.

[185] Yunker AM, Sharp AH, Sundarraj S, Ranganathan V, Copeland TD, McEnery MW. Immunological characterization of T-type voltage-dependent calcium channel CaV3.1 (alpha 1G) and CaV3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 2003;117:321-35.

[186] Wildburger NC, Lin-Ye A, Baird MA, Lei D, Bao J. Neuroprotective effects of blockers for T-type calcium channels. Molecular neurodegeneration 2009;4:44.

[187] Bu J, Sathyendra V, Nagykery N, Geula C. Age-related changes in calbindin-D28k, calretinin, and parvalbumin-immunoreactive neurons in the human cerebral cortex. Exp Neurol 2003;182:220-31.

[188] Andressen C, Blümcke I, Celio MR. Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 1993;271:181-208.

[189] Dalgarno D, Klevit RE, Levine BA, Williams RJP. The calcium receptor and trigger. Trends Pharmacol Sci 1984;5:266-71.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 210

[190] German DC, Manaye KF, Sonsalla PK, Brooks BA. Midbrain dopaminergic cell loss in Parkinson's disease and MPTP-induced parkinsonism: sparing of calbindin-D28k-containing cells. Ann N Y Acad Sci 1992;648:42-62.

[191] Tsuboi K, Kimber TA, Shults CW. Calretinin-containing axons and neurons are resistant to an intrastriatal 6-hydroxydopamine lesion. Brain Res 2000;866:55-64.

[192] Kim BG, Shin DH, Jeon GS, Seo JH, Kim YW, Jeon BS, et al. Relative sparing of calretinin containing neurons in the substantia nigra of 6-OHDA treated rat parkinsonian model. Brain Res 2000;855:162-5.

[193] Haller T, Dietl P, Deetjen P, Volkl H. The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release. Cell Calcium 1996;19:157-65.

[194] Yamada T, McGeer PL, Baimbridge KG, McGeer EG. Relative sparing in Parkinson's disease of substantia nigra dopamine neurons containing calbindin-D28K. Brain Res 1990;526:303-7.

[195] Kojetin DJ, Venters RA, Kordys DR, Thompson RJ, Kumar R, Cavanagh J. Structure, binding interface and hydrophobic transitions of Ca2+-loaded calbindin-D(28K). Nat Struct Mol Biol2006;13:641-7.

[196] Neher E. Calcium buffers in flash-light. Biophysical journal 2000;79:2783-4.

[197] Augustine GJ, Santamaria F, Tanaka K. Local calcium signaling in neurons. Neuron 2003;40:331-46.

[198] Akerfeldt KS, Coyne AN, Wilk RR, Thulin E, Linse S. Ca2+-binding stoichiometry of calbindin D28k as assessed by spectroscopic analyses of synthetic peptide fragments. Biochem 1996;35:3662-9.

[199] Berggard T, Miron S, Onnerfjord P, Thulin E, Akerfeldt KS, Enghild JJ, et al. Calbindin D28k exhibits properties characteristic of a Ca2+ sensor. J Biol Chem2002;277:16662-72.

[200] Cedervall T, Andre I, Selah C, Robblee JP, Krecioch PC, Fairman R, et al. Calbindin D28k EF-hand ligand binding and oligomerization: four high-affinity sites--three modes of action. Biochem 2005;44:13522-32.

[201] Faas GC, Raghavachari S, Lisman JE, Mody I. Calmodulin as a direct detector of Ca2+ signals. Nat Neuro 2011;14:301-4.

[202] Fierro L, Llano I. High endogenous calcium buffering in Purkinje cells from rat cerebellar slices. Journal Physio1996;496 ( Pt 3):617-25.

[203] Airaksinen MS, Eilers J, Garaschuk O, Thoenen H, Konnerth A, Meyer M. Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene. Proc Natl Acad Sci USA 1997;94:1488-93.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 211 [204] Barski JJ, Hartmann J, Rose CR, Hoebeek F, Morl K, Noll-Hussong M, et al. Calbindin in cerebellar Purkinje cells is a critical determinant of the precision of motor coordination. J Neuro 2003;23:3469-77.

[205] Mizuta I, Tsunoda T, Satake W, Nakabayashi Y, Watanabe M, Takeda A, et al. Calbindin 1, fibroblast growth factor 20, and alpha-synuclein in sporadic Parkinson's disease. Human genetics 2008;124:89-94.

[206] Guo Q, Christakos S, Robinson N, Mattson MP. Calbindin D28k blocks the proapoptotic actions of mutant presenilin 1: Reduced oxidative stress and preserved mitochondrial function. Proc Natl Acad Sci USA 1998;95:3227-32.

[207] Guo Q, Christakos S, Robinson N, Mattson MP. Calbindin D28k blocks the proapoptotic actions of mutant presenilin 1: Reduced oxidative stress and preserved mitochondrial function. Proc Natl Acad Sci USA 1998;95:3227-32.

[208] Halhali A, Figueras AG, Díaz L, Avila E, Barrera D, Hernández G, et al. Effects of calcitriol on calbindins gene expression and lipid peroxidation in human placenta. J Steroid Biochem Mol Biol 2010;121:448-51.

[209] Balabanova S, Richter HP, Antoniadis G, Homoki J, Kremmer N, Hanle J, et al. 25- hydroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid. Klinische Wochenschrift 1984;62:1086-90.

[210] Pardridge WM, Sakiyama R, Coty WA. Restricted transport of Vitamin D and A derivatives through the rat blood-brain barrier. J Neurochem 1985;44:1138-41.

[211] Fu GK, Lin D, Zhang MYH, Bikle DD, Shackleton CHL, Miller WL, et al. Cloning of human 25-hydroxyvitamin D-1α-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol 1997;11:1961-70.

[212] Neveu I, Naveilhan P, Menaa C, Wion D, Brachet P, Garabedian M. Synthesis of 1,25- dihydroxyvitamin D3 by rat brain macrophages in vitro. J Neurosci Res 1994;38:214-20.

[213] Zehnder D, Bland R, Williams MC, McNinch RW, Howie AJ, Stewart PM, et al. Extrarenal expression of 25-hydroxyvitamin D3-1α-hydroxylase. J Clin Endocrinol Metab 2001;86:888-94.

[214] Eyles DW, Smith S, Kinobe R, Hewison M, McGrath JJ. Distribution of the Vitamin D receptor and 1α-hydroxylase in human brain. J Cheml Neuroanat 2005;29:21-30.

[215] Naveilhan P, Neveu I, Baude C, Ohyama KY, Brachet P, Wion D. Expression of 25(OH) vitamin d3 24-hydroxylase gene in glial cells. NeuroReport 1993;5:255-7.

[216] Wang JY, Wu JN, Cherng TL, Hoffer BJ, Chen HH, Borlongan CV, et al. Vitamin D3 attenuates 6-hydroxydopamine-induced neurotoxicity in rats. Brain Res 2001;904:67-75.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 212

[217] Smith MP, Fletcher-Turner A, Yurek DM, Cass WA. Calcitriol protection against dopamine loss induced by intracerebroventricular administration of 6-hydroxydopamine. Neurochem Res 2006;31:533-9.

[218] Cass WA, Smith MP, Peters LE. Calcitriol protects against the dopamine- and serotonin-depleting effects of neurotoxic doses of methamphetamine. Ann N Y Acad Sci 2006. p. 261-71.

[219] Evatt ML, DeLong MR, Khazai N, Rosen A, Triche S, Tangpricha V. Prevalence of vitamin D insufficiency in patients with Parkinson disease and Alzheimer disease. Archives of Neurology 2008;65:1348-52.

[220] Newmark HL, Newmark J. Vitamin D and Parkinson's disease - A hypothesis. Mov Dis 2007;22:461-8.

[221] Knekt P, Kilkkinen A, Rissanen H, Marniemi J, Sääksjärvi K, Heliövaara M. Serum vitamin D and the risk of Parkinson disease. Arch Neurol 2010;67:808-11.

[222] Kim JS, Kim YI, Song C, Yoon I, Park JW, Choi YB, et al. Association of vitamin D receptor gene polymorphism and Parkinson's disease in Koreans. J Korean Med Sci 2005;20:495-8.

[223] Shrestha S, Lutsey PL, Alonso A, Huang X, Mosley TH, Jr., Chen H. Serum 25- hydroxyvitamin D concentrations in Mid-adulthood and Parkinson's disease risk. Mov Dis 2016; 31:972-8.

[224] Guttman M, Burkholder J, Kish SJ, Hussey D, Wilson A, DaSilva J, et al. [11C]RTI-32 PET studies of the dopamine transporter in early dopa-naive Parkinson's disease: implications for the symptomatic threshold. Neurology 1997;48:1578-83.

[225] Sharma S, Moon CS, Khogali A, Haidous A, Chabenne A, Ojo C, et al. Biomarkers in Parkinson’s disease (recent update). Neurochem Intl 2013;63:201-29.

[226] Redgrave P, Rodriguez M, Smith Y, Rodriguez-Oroz MC, Lehericy S, Bergman H, et al. Goal-directed and habitual control in the basal ganglia: implications for Parkinson's disease. Nat Rev Neurosci 2010;11:760-72.

[227] Parnetti L, Castrioto A, Chiasserini D, Persichetti E, Tambasco N, El-Agnaf O, et al. Cerebrospinal fluid biomarkers in Parkinson disease. Nat Rev Neurol 2013;9:131-40.

[228] Vivacqua G, Latorre A, Suppa A, Nardi M, Pietracupa S, Mancinelli R, et al. Abnormal Salivary Total and Oligomeric Alpha-Synuclein in Parkinson’s Disease. PloS one 2016;11:e0151156.

[229] Wirdefeldt K, Adami HO, Cole P, Trichopoulos D, Mandel J. Epidemiology and etiology of Parkinson's disease: a review of the evidence. Eur J Epidemiol 2011;26 Suppl 1:S1-58.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 213 [230] Lim KL, Lim TM. Molecular mechanisms of neurodegeneration in Parkinson's disease: clues from Mendelian syndromes. IUBMB life 2003;55:315-22.

[231] Cattoretti G, Becker MH, Key G, Duchrow M, Schluter C, Galle J, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992;168:357-63.

[232] Norazit A, Meedeniya AC, Nguyen MN, Mackay-Sim A. Progressive loss of dopaminergic neurons induced by unilateral rotenone infusion into the medial forebrain bundle. Brain Res 2010;1360:119-29.

[233] Mouatt-Prigent A, Agid Y, Hirsch EC. Does the calcium binding protein calretinin protect dopaminergic neurons against degeneration in Parkinson's disease? Brain Res 1994;668:62-70.

[234] Halhali A, Figueras AG, Diaz L, Avila E, Barrera D, Hernandez G, et al. Effects of calcitriol on calbindins gene expression and lipid peroxidation in human placenta. J Steroid Biochem Mol Biol 2010;121:448-51.

[235] Lowe R, Pountney DL, Jensen PH, Gai WP, Voelcker NH. Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with the C-terminal domain. Protein science : a publication of the Protein Society 2004;13:3245-52.

[236] Danzer KM, Krebs SK, Wolff M, Birk G, Hengerer B. Seeding induced by alpha- synuclein oligomers provides evidence for spreading of alpha-synuclein pathology. J Neurochem 2009;111:192-203.

[237] Putcha P, Danzer KM, Kranich LR, Scott A, Silinski M, Mabbett S, et al. Brain- permeable small-molecule inhibitors of Hsp90 prevent alpha-synuclein oligomer formation and rescue alpha-synuclein-induced toxicity. J Pharmacol Exp Ther 2010;332:849-57.

[238] Weetman J, Wong MB, Sharry S, Rcom-H'cheo-Gauthier A, Gai WP, Meedeniya A, et al. Increased SUMO-1 expression in the unilateral rotenone-lesioned mouse model of Parkinson's disease. Neurosci Lett 2013;544:119-24.

[239] Breydo L, Wu JW, Uversky VN. Alpha-synuclein misfolding and Parkinson's disease. Biochim Biophys acta 2012;1822:261-85.

[240] Cookson MR. alpha-Synuclein and neuronal cell death. Mol Neurodegener 2009;4:9.

[241] Jellinger KA. Absence of alpha-synuclein pathology in postencephalitic parkinsonism. Acta neuropathol 2009;118:371-9.

[242] Marques O, Outeiro TF. Alpha-synuclein: from secretion to dysfunction and death. Cell Death Dis 2012;3:e350.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 214

[243] Sanders LH, Greenamyre JT. Oxidative damage to macromolecules in human Parkinson disease and the rotenone model. Free Radic Biol Med 2013;62:111-20.

[244] Yuan Y-h, Yan W-f, Sun J-d, Huang J-y, Mu Z, Chen N-H. The molecular mechanism of rotenone-induced α-synuclein aggregation: Emphasizing the role of the calcium/GSK3β pathway. Toxicol Lett 2015;233:163-71.

[245] Calì T, Ottolini D, Negro A, Brini M. α-Synuclein Controls Mitochondrial Calcium Homeostasis by Enhancing Endoplasmic Reticulum-Mitochondria Interactions. J Biol Chem 2012;287:17914-29.

[246] Peng TI, Jou MJ. Oxidative stress caused by mitochondrial calcium overload. Ann N Y Acad Sci 2010;1201:183-8.

[247] Schapira AH, Olanow CW, Greenamyre JT, Bezard E. Slowing of neurodegeneration in Parkinson's disease and Huntington's disease: future therapeutic perspectives. Lancet 2014;384:545-55.

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 215

Appendices

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 216

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 217

The protective effect of Calbindin D28K on α-synuclein aggregation in α-synucleinopathies 218