Exploring Mechanisms of Alpha-Synuclein Spread in Parkinson's and Atypical Parkinson's Diseases
Author Valdinocci, Dario
Published 2020-04-15
Thesis Type Thesis (PhD Doctorate)
School School of Medical Science
DOI https://doi.org/10.25904/1912/1951
Copyright Statement The author owns the copyright in this thesis, unless stated otherwise.
Downloaded from http://hdl.handle.net/10072/393604
Griffith Research Online https://research-repository.griffith.edu.au
PhD Thesis
Exploring Mechanisms of Alpha-Synuclein Spread
in Parkinson‟s and Atypical Parkinson‟s Diseases
Student Name: Dario Valdinocci, BBiomedSc(Hons) s2843976 Principal Supervisor: Dr. Dean L. Pountney Associate Supervisor: A/Prof Gary Grant
School of Medical Science Griffith University Gold Coast
Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy
December 2019
Abstract
The development of neurodegenerative diseases is an ever increasing risk faced by modern society as aging populations rise globally. For complex diseases such as Alzheimer‟s Disease (AD) or Parkinson‟s Disease (PD) the prospect of finding a cure within the immediate future is unlikely. These complexities arise primarily from the interactions of the protein of interest each of the diseases, which in the case of typical and atypical PD is α-synuclein (α-syn). Thought to be involved in neuronal vesicle recycling in its monomeric α-helical state, change to a β-sheet conformation allows α-syn to misfold, creating oligomers and eventually aggregates which form the initial bedrock for inclusion bodies. One of the more fascinating features of pathological α- syn is the difference in inclusion body composition and cell type affected in typical and atypical PD variants. For instance within Multiple System Atrophy (MSA), an atypical PD variant, α-syn aggregates form Glial Cytoplasmic Inclusions (GCIs) the composition of which differs from the Lewy Bodies (LB) formed in PD, and are largely found in oligodendrocytes. Unlike neurons, oligodendrocytes normally express only minimal amounts of α-syn which does not alter even after GCIs are formed, indicating acquisition of α-syn from an external source. How oligodendrocytes acquire pathological α-syn and the mechanisms by which the protein is able to spread within the central nervous system (CNS) are topics currently with more questions than answers. Thus the primary focus of the thesis is to investigate potential models of α-syn spread in order to develop future novel targets to slow disease progression.
The investigations within Chapters 2 and 3 focus on exploring microglial cells as a potential vehicle for α-syn migration within the CNS. Along with the presence of α-syn inclusion bodies, part of the degeneration taking place within MSA is due to the chronic inflammatory activation and actions of the CNS resident immune cell, the microglia. Whilst microglial inflammatory responses play a role in PD, the severity of inflammation is greater in MSA. Microglia possess many traits which contribute to their ability to become a potential transporter of α-syn, including the lack of ability to degrade internalised pathological α-syn variants such as oligomers and aggregates. Post-mortem human MSA brain tissues were examined for evidence of microglial interactions with GCI-affected oligodendrocytes. Not only were interactions between these cells noted in a variety of different tissue regions and patients, but qualitative evidence of microglial migration with internalised α-syn distal from GCI-affected cells was obtained. Whilst these findings suggest that microglia could play a potential role in α-syn spread within the CNS and α-syn occurrence within oligodendrocytes, post-mortem examination is limited to detailing what may occur following the observed interactions.
An in vitro assay was developed to investigate α-syn uptake and mobilisation by cells from a point source of immobilized protein. In this assay, monomeric and aggregated α-syn uptake and migration were assessed when exposured to THP-1 monocytes and microglial-like differentiated
i
THP-1 cells. Microglial-like differentiated THP-1 cells were found to show uptake and migration with internalised aggregate α-syn to distal regions, not observed with undifferentiated THP-1 monocytes or monomeric protein. This result indicates that microglia/microglial-like cells are more likely to internalise pathological α-syn such as aggregates than the normal, monomeric form. The greater numbers of differentiated THP-1 with internalised aggregated α- syn indicates that once the protein has been taken up by microglia, then it becomes a transported to distal brain regions. The inability of microglia to degrade aggregated α-syn, similar to microglia within the CNS, likely contributes microglial-mediated α-syn spread. Further testing using highly aggressive proliferating immortalised (HAPI) cells, a rat microglial cell line, was conducted which corroborated the previous findings, indicating that microglia take up the pathological variant of α-syn, mobilizing the protein rather than degrading it. Utilising the same α-syn mobilization assay, inhibition of microtubule instability through the use of the microtubule stabilising agent Epothilone D (EpoD) proved successful in lowering both uptake and mobilization of aggregated α-syn, showing promise as a novel therapy against α-syn spread. Inhibition of the microglial inflammatory response through TAK-242 treatment, a Toll-like Receptor 4 (TLR4) inhibitor, was also trialled to investigate if TLR4 plays a significant role with regards to α-syn uptake and mobilisation. However, unlike EpoD, TAK-242 proved ineffective.
For Chapter 4 experimentation focussed on investigating the role of intercellular mitochondrial transfer through tunnelling nanotubes (TnTs) as a potential means of α-syn spread. Pathological α-syn species have been described to bind to intercellularly migrating organelles such as lysosomes for spread to adjacent sites. The role of mitochondria in typical and atypical PD has predominantly focussed on their role in degeneration and as such gaps exist as to mitochondrial function in relation to α-syn propagation. Utilising Stimulated Emission Depletion (STED) super resolution microscopy, SH-SY5Y, 1321N1 and differentiated THP-1 monocultures, as models of neurons, astrocytes and microglia, were each inoculated with aggregated α-syn to examine transfer to adjacent cells via TnTs whilst bound to mitochondria. Under each cell culture condition, the formation of TnT-like structures was observed between adjacent cells, each containing migrating mitochondria with α-syn aggregates bound to the outer mitochondrial membrane. Further exploration of this result through 1321N1 / differentiated THP-1 co-culture showed that this mechanism of α-syn spread is not limited to monoculture conditions and may be a potential mechanism relevant to typical and atypical PD. Knockdown of Miro1, a critical protein bridging mitochondria to the motor adaptor complex for intercellular migration, was performed in order to ascertain if its role is significant in the α-syn spread process and a potential therapeutic target. Miro1 silencing however proved qualitatively ineffective in the prevention of α-syn spread mediated by mitochondria, indicating other factors in addition to Miro1 may be involved in bridging mitochondria to intercellular motor complexes. Taken
ii together these results illustrate the great complexity of mechanisms of α-syn spread and the inherent difficulties associated with attempting to combat them.
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Statement of Originality
I, Dario Valdinocci, declare that this thesis entitled “Exploring Mechanisms of Alpha-Synuclein Spread in Parkinson’s and Atypical Parkinson’s Diseases” is of my own works and 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.
Dario Valdinocci
December 2019
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Contents List
Abstract pg.i
Statement of Originality pg.iv
List of Figures pg.xi
Publications During Candidacy pg.xiv
Abbreviations pg.xix
Acknowledgements pg.xxii
Chapter 1: Introduction pg.1
1.1 Literature Review pg.2
1.1.1 Multiple System Atrophy pg.2
1.1.2 Alpha-Synuclein pg.4
1.1.2.1 Alpha-Synuclein in the Brain pg.4
1.1.2.2 Alpha-Synuclien in Disease pg.4
1.1.2.3 MSA as a Prion Disease pg.7
1.1.3 Microglia pg.8
1.1.3.1 Origin and Normal Role of Microglia pg.8
1.1.3.2 Iba-1 pg.11
1.1.3.3 Microglia in MSA pg.12
1.1.3.4 Toll-like Receptor 4 pg.15
1.1.3.5 Toll-like Receptor 4 and Its Role in Alpha Synucleinopathy pg.16
1.1.4 Transport pg.18
1.1.4.1 Extracellular Transport of Alpha Synuclein pg.18
1.1.4.2 Exosomes and Endocytosis pg.19
1.1.4.3 Tunnelling Nanotubes pg.21
1.1.4.4 Microglia as a Possible Transporter pg.23
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1.1.4.5 Microtubules pg.25
1.1.4.6 Epothilones pg.26
1.1.5 Mitochondria pg.27
1.1.5.1 Mitochondria Normal Role pg.27
1.1.5.2 Role of Mitchondria in Neurons, Microglia and Astrocytes pg.31
1.1.5.3 Mitochondrial Transport pg.33
1.1.5.4 Role of Mitochondria in Synucleinopathies pg.35
1.2 Project Aims Overview pg.41
1.3 Specific Aims and Hypotheses pg.42
1.3.1 Chapter 2 - Epothilone D Inhibits Microglial-Mediated Spread of Alpha Synuclein Aggregates pg.42
1.3.1.1 Post-Mortem MSA Tissue Analysis pg.42
1.3.1.2 Alpha-Synuclein Mobilization Assay pg.42
1.3.1.3 Epothilone D Treatment pg.43
1.3.2 Chapter 3 - TAK-242 Has No Effect on Microglial Mediated Transport of Alpha Synuclein Species pg.44
1.3.2.1 TAK-242 Treatment pg.44
1.3.3 Chapter 4 - Mitochondria mediate cell-to-cell transfer of alpha-synuclein aggregates via Tunnelling Nanotubes pg.44
1.3.3.1 Confocal Immunofluorescence Examination of α-syn Transfer via TnT pg.44
1.3.3.2 Stimulated Emission Depletion Microscopy Examinations of TnT-based α-syn transfer in SH-SY5Y Monocultures pg.45
1.3.3.3 Stimulated Emission Depletion Microscopy Examinations of TnT-based α-syn transfer in 1321N1 and Differentiated THP-1 Mono and Co-Cultures pg.46
1.3.3.4 Miro1 siRNA Treatment pg.46
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Chapter 2: Epothilone D Inhibits Microglial-Mediated Spread of Alpha Synuclein Aggregates (Publication) pg.47
2.0 Statement of Contribution to Co-Authored Published Paper pg.48
Abstract pg.49
Introduction pg.49
Materials and Methods pg.51
Cell Culture pg.51
Alpha Synuclein Mobilisation Assay pg.51
EpoD treatment pg.51
Human tissue pg.51
Confocal microscopy pg.51
Quantitative Analysis pg.51
Cell counting pg.51
Data Analysis pg.51
Results pg.52
Microglial cytosplasmic α-syn in MSA pg.52
Differentiated THP-1 cells take up immobilized α-syn and migrate away pg.52
Microglial-like differentiated THP-1 cells do not degrade α-syn aggregate, but transport it from the site of deposition pg.53
THP-1 cells take up immobilized control BSA proteins, but do not transport it pg.53
Microglial HAPI cells display similar uptake and transport of α-syn aggregates to differentiated THP-1 cells pg.57
Astrocyte-like 1321N1 take up immobilized α-syn to a lesser extent than microglial-like cells and do not transport α-syn aggregates pg.57
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Distal cells containing α-syn are decreased following EpoD treatment pg.57
Discussion pg.59
Acknowledgements pg.62
References pg.62
Chapter 3: TAK-242 Has No Effect on Microglial Mediated Transport of Alpha Synuclein Species pg.64
3.0 Abstract pg.65
3.1 Introduction pg.66
3.2 Materials and Methods pg.67
3.2.1 Cell Culturing – THP-1 and HAPI pg.67
3.2.2 Alpha-Synuclein Mobilization Assay pg.68
3.2.3 Confocal Microscope pg.68
3.2.4 Data Acquisition – Fluorescent Intensity and Cell Counting pg.69
3.2.5 Statistical analysis methods – Student t-test and ANOVA utilised pg.69
3.3 Results pg.69
3.3.1 No significant reduction in protein uptake following TAK-242 treatment pg.69
3.3.2 No significant difference in proportion of α-syn positive bearing cells on coverslip edge from TAK-242 treatment pg.72
3.4 Discussion pg.74
3.5 Conclusion pg.78
Chapter 4: Mitochondria mediate cell-to-cell transfer of alpha synuclein aggregates via Tunnelling Nanotubes pg.79
4.0 Abstract pg.80
4.1 Introduction pg.81
4.2 Materials and Methods pg.83 viii
4.2.1 Cell Lines Utulised and Culture Conditions pg.83
4.2.2 SDS-PAGE and Western Blotting Protocol pg.84
4.2.3 Miro1 and Control siRNA Transfection pg.85
4.2.4 Sample Preparation for Fixation and Fluorescent Staining pg.86
4.2.5 Fixation and Fluorescent Staining Protocol pg.86
4.2.6 Microscope Analysis and Fiji Software pg.87
4.3 Results pg.88
4.3.1 Confocal Analysis Revelaed Formation og TnTs in KCl Treated SH-SY5Y Mono-Cultures and Potential α-syn Exchange in 1321N1and Differentiated THP-1 Co-Cultures pg.88
4.3.2 STED Analysis Shows Formation of TnT-like Structures Between SH-SY5Y Cells in Addition to α-syn Migrating Within These Structures Whilst Bound to Mitochondria pg.91
4.3.3 1321N1 and THP-1 Mono-Culture Experiments Displayed Formation of TnT-like Structures and Confirmed α-syn- Mitochondria-mediated Migration As a Mechanism of Spread Within Other Cell Types pg.94
4.3.4 Evidence of TnT-like Structure Formation and α-syn- Mitochondria mediated transfer between 1321N1 and Differentiated THP-1 pg.98
4.3.5 Miro1 knockdown does not affect a-syn transfer via TnTs pg.100
4.4 Discussion pg.102
Chapter 5: Discussion pg.108
Chapter 6: Conclusion pg.118
Appendix pg.121
Item I – Potential Modes of Intercellular α-synuclein Transmission pg.122
Item II – Extracellular Interactions of Alpha-Synuclein in Multiple System Atrophy pg.139
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Item III - Intracellular and Intercellular Mitochondrial Dynamics in Parkinson‟s Disease pg.159
Item IV - Gel Electrophoresis of recombinant α-syn revealed consistent molecular weight across most samples (Figure 22) pg.167
Item V – Dilution control tests revealing monomeric α-syn is significantly diluted in cellular media whilst aggregated α-syn is not (Figure 23) pg.168
Item VI - Whole blots of bands displayed in Figure 20 (Figure 24) pg.169
Reference List pg.171
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List of Figures
Chapter 1: Introduction
Figure 1 – Predicted Models of Neurodegeneration of MSA-P and MSA-C pg.3
Figure 2 – The role of Oligodendrocytes in Neuronal signalling and how the presence of pathological α-syn affects this pg.6
Figure 3 – The Differing Morphological States of Microglia pg.8
Figure 4 – Summarized Differences in Microglial Activation State pg.11
Figure 5 – Interactions of Iba-1 with the Cytoskeleton pg.12
Figure 6 – Pathways of TLR4 activation pg.18
Figure 7 – Visual Illustration of Both Forms of Exosome Synthesis and how α-syn can take advantage of this system for spread pg.20
Figure 8 – Visual representation of materials that utilise TnTs for interceullar spread pg.22
Figure 9 – Summary of transportation mechanisms pg.24
Figure 10 – Microtubules and MTOC pg.26
Figure 11 – Summary of Mitochondrial Dynamics within Neurons in Synucleinopathies pg.40
Chapter 2: Epothilone D Inhibits Microglial-Mediated Spread of Alpha Synuclein Aggregates (Publication)
Figure 1 – Confocal immunofluorescence of MSA Frontal Cortex pg.52
Figure 2 - Potential time series that may occur in MSA based on three nearby regions of the same cerebellum pg. 53
Figure 3 – Differentiated THP-1 cells take up immobilized α-syn aggregate and migrate away pg.54
Figure 4 - Cell counting analysis reveals differentiated THP-1 do not degrade α-syn aggregate, but transport away from deposition site pg.55
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Figure 5 - THP-1 cells take up immobilized control BSA protein, but do not transport it pg.56
Figure 6 - HAPI Microglial-like cells display similar α-syn uptake and transport features to tha of differentiated THP-1 cells pg.58
Figure 7 – EpoD inhibits microglial-mediated α-syn mobilization pg.60
Figure 8 – Distal cells containing α-syn are decreased following EpoD treatment pg.61
Chapter 3: TAK-242 Has No Effect on Microglial Mediated Transport of Alpha Synuclein Species
Figure 12 - TAK-242 treatment failed to lead to increases of immobilised protein in the central spot region pg.71
Figure 13 - TAK-242 treatment does not significantly affect microglia- mediated microglia-mediated alpha-synuclein migration pg.73
Figure 14 - Immobilized central spots and coverslip regions of both differentiated THP-1 and HAPI cell controls and TAK-242 treated groups reveal TAK-242 to be ineffective in preventing α-syn engulfment and mobilisation pg.75
Chapter 4: Mitochondria mediate cell-to-cell transfer of alpha synuclein aggregates via Tunnelling Nanotubes
Figure 15 – Confocal Immunofluorescence shows α-syn in TnT-like Structures pg.90
Figure 16 – STED immunofluorescence Imaging of SH-SY5Y Cells Revealed The Formation of TnT-like Structures and Migration of α-syn Bound to Mitochondria pg.92
Figure 17 - Imaging of 1321N1 Cells monocultures via STED Revealed The Formation of TnT-like Structures and Migration of α-syn Bound to Mitochondria in Treated Samples pg.95
Figure 18 - Imaging of Differerentiated THP-1 Cells monocultures via STED Revealed The Formation of TnT-like Structures and Migration of α-syn Bound to Mitochondria in Treated Samples pg.18
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Figure 19 – TnT-like Structure Formation with Migrating α-syn between 1321N1 and Differentiated THP-1 cells pg.99
Figure 20 –Miro1 siRNA knockdown does not affect Mitochondria- mediated α-Syn migration in TnT-like Structures pg.101
Chapter 5: Discussion
Figure 21 - Mitochondrial Intercellular Transfer of α-syn via TnTs pg.115
Appendix
Item IV
Figure 22 - Gel Electrophoresis of recombinant α-syn revealed consistent molecular weight across most samples pg.167
Item V
Figure 23 - Dilution control tests revealing monomeric α-syn is significantly diluted in cellular media whilst aggregated α-syn is not pg.168
Item VI
Figure 24 - Whole blots of bands displayed in Figure 20 pg.169
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Publications During Candidacy
Included in this thesis are both review and research articles, all of which were published during my PhD candidature and each of which I am first author for. Chapter 2 is a copy of a published research article. Contributions of myself and other co-authors are outlined as follows:
Chapter 2
Valdinocci, D., Grant, G., Dickson, T. and Pountney, D. (2018). Epothilone D inhibits microglia-mediated spread of alpha-synuclein aggregates. Molecular and Cellular Neuroscience, 89, pp.80-94.
My Contribution:
- Experiment conductor, data analysis, manuscript writing, preparation and revision
Gary Grant Contribution:
- Manuscript writing, preparation and revision
Tracey Dickson Contribution:
- Provider of EpoD, manuscript writing, preparation and revision
Dean Pountney‟s Contribution:
- Manuscript writing, preparation and revision
Signed: Date: December 2019 Dario Valdinocci
Signed: Date: December 2019 Dean Pountney (Co-Author)
Signed: Date: December 2019 Dean Pountney (Principal Supervisor)
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Review Articles within Appendix
Item I
Valdinocci, D., Radford, R., Siow, S., Chung, R. and Pountney, D. (2017). Potential Modes of Intercellular α-Synuclein Transmission. International Journal of Molecular Sciences, 18(2), p.469.
My Contribution:
- Manuscript writing, preparation and revision
Rowan Radford Contribution:
- Manuscript writing, preparation and revision
Sue Maye Siow Contribution:
- Manuscript writing, preparation and revision
Roger Chung Contribution
- Manuscript writing, preparation and revision
Dean Pountney‟s Contribution:
- Manuscript writing, preparation and revision
Signed: Date: December 2019 Dario Valdinocci
Signed: Date: December 2019 Dean Pountney (Co-Author)
Signed: Date: December 2019 Dean Pountney (Principal Supervisor)
xv
Item II
Valdinocci, D., Radford, R., Goulding, M., Hayashi, J., Chung, R. and Pountney, D. (2018). Extracellular Interactions of Alpha-Synuclein in Multiple System Atrophy. International Journal of Molecular Sciences, 19(12), p.4129.
My Contribution:
- Manuscript writing, preparation and revision
Rowan Radford Contribution:
- Manuscript writing, preparation and revision
Michael Goulding Contribution:
- Manuscript writing, preparation and revision
Junna Hayashi Contribution:
- Manuscript writing, preparation and revision
Roger Chung Contribution:
- Manuscript writing, preparation and revision
Dean Pountney‟s Contribution:
- Manuscript writing, preparation and revision
Signed: Date: December 2019 Dario Valdinocci
Signed: Date: December 2019 Dean Pountney (Co-Author)
Signed: Date: December 2019 Dean Pountney (Principal Supervisor)
xvi
Item III
Valdinocci, D., Simões, R., Kovarova, J., Cunha-Oliveira, T., Neuzil, J. and Pountney, D. (2019). Intracellular and Intercellular Mitochondrial Dynamics in Parkinson‟s Disease. Frontiers in Neuroscience, 13, p.930.
My Contribution:
- Manuscript writing, preparation and revision
Rui Simões Contribution:
- Manuscript writing, preparation and revision
Jaromira Kovarova Contribution:
- Manuscript writing, preparation and revision
Teresa Cunha-Oliveira Contribution:
- Manuscript writing, preparation and revision
Jiri Neuzil Contribution:
- Manuscript writing, preparation and revision
Dean Pountney‟s Contribution:
- Manuscript writing, preparation and revision
Signed: Date: December 2019 Dario Valdinocci
Signed: Date: December 2019 Dean Pountney (Co-Author)
Signed: Date: December 2019 Dean Pountney (Principal Supervisor)
xvii
Abbreviations
AD = Alzheimer‟s Disease
α-syn = α-synuclein
ATP = Adenosine Tri-Phosphate
BSA = Bovine Serum Albumin
CNS = Central Nervous System
DA = Dopamine
DAMPs = Damage Associated Molecular Patterns
dH2O = Deionised H2O
DLB = Dementia with Lewy Bodies
Drp1 = Dynamin-related Protein 1
EpoD = Epothilone D
ER = Endoplasmic Reticulum
FACS = Fluorescence Activated Cell Sorting
GCI = Glial Cytoplasmic Inclusion
HAPI = Highly Aggressive Proliferating Immortalised
Hsp = Heat-Shock Proteins
IL = Interleukin
IFN-γ = Interferon-γ
IMM = Inner Mitochondrial Membrane
LB = Lewy Body
LPS = Lipopolysaccharides
Mfn = Mitofusin
MGV = Mean Gray Value
MMSCs = Mesenchymal Multipotent Stromal Cells
xviii
MPP = Mitochondrial Processing Peptidase
MPP+ = 1-Methyl-4-Phenylpyridinium
MSA = Multiple System Atrophy
MTOC = Microtubule Organising Centre
NGS = Normal Goat Serum
NHS = Normal Horse Serum
OMM = Outer Mitochondrial Membrane
OPA1 = Optic Atrophy Protein 1
PAMPs = Pathogen-Associated Molecular Patterns
PARL = Presenilin-Associated Rhomboid-Like Protein
PBS = Phosphate Buffered Saline
PD = Parkinson‟s Disease
PINK1 = PTEN-Induced Kinase 1
PMA = Phorbol-12-Myristate-13-Acetate
PrPC = Prion Protein (Normal)
PrPSC = Prion Protein (Converted)
RIPA = Radioimmunoprecipitation Assay
ROS = Reactive Oxygen Species
RT = Room Temperature
SDS = Sodium Dodecyl-Sulfate siRNA = Small interfering RNA
SNARE = SNAP Receptor
STED = Stimulated Emission Depletion
TGF-β = Transforming Growth Factor β
TH = T-Helper
xix
TIM = Translocase of Inner Membrane Receptor
TIR = Toll/Interleukin-1 Receptor
TLR = Toll-Like Receptor
TNF-α = Tumour Necrosis Factor-α
TnTs =Tunnelling Nanotubes
TOM = Translocase of Outer Membrane Receptor
VDAC = Voltage-Dependent Anion Channel
WB = Western Blot
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Acknowledgements
There are a multitude of people I wish to thank, each of whom have been instrumental in being able to complete my Thesis since commencing in 2016.
I would like to first thank my principal supervisor Dr Dean Pountney. Dean‟s support over the last few years has been invaluable not only for completing my thesis but for improving my abilities as both a researcher and scientist. From graciously making time and helping me if trouble ever arose to helping find solutions when experiments were not functioning as intended, I am truly thankful to have been under his supervision for this project. Additionally I would also like to thank my secondary supervisor A/Prof Gary Grant. Gary has been incredibly generous in advice and support over the years, providing perspectives I would not have considered or thought to explore had I not consulted with him. I am incredibly thankful to have been under his wing for this project.
A special acknowledgement for both Prof Tracey Dickson and Prof Jiri Neuzil who have been incredible collaborators to work with over the last few years. Thank you to Tracey Dickson for providing Epothilone D, which was instrumental in not only forming my first chapter but first published research paper as first author. Thank you to Jiri Neuzil for not only allowing me to work as part of the Molecular Therapy group at BIOCEV in Vestec, Czech Republic but for also showing me the wonders of scientific collaboration.
I would also like to thank everyone who has helped within the DLP Neurodegeneration lab over the years since I first joined back in 2015 which include Alex Rcom-H‟cheo-Gauthier, Bruno Vieira, Shamini Vijayakumaran, Fleur McLeary, Junna Hayashi, Samantha Osborne, Tracie Cheng, Myra De Smet and Yuho Okita. Each and every one of these wonderful individuals not only provided assistance when I needed it, but have made working in the DLP lab the last few years a joy.
I also want to give a very special thank you to all the members of the Molecular Therapy Group at BIOCEV at Vestec, Czech Republic which include Jiri Neuzil, Jaromíra Kovářová, Renata Zobalová, Jakub Rohlena, Ladislav Anděra, Štěpána Boukalová, Soňa Hubáčková, Kateřina Váňova, Zuzana Ezrová, Linda Krobová, Eliška Davidová, Zuzana Nahácka, Mária Dubišová, Sílvia Novais, Kateřina Jarošová, Rui Simões, Michal Kraus and Mirko Milošević. Thank you all for being extremely accommodating during my stay at the Czech Republic as well as putting up with my very regular questions on procedures and protocols. Both the training provided and friendships made during my stay were able to elevate my work to the next level and I am truly thankful for that.
I would like to additionally acknowledge Tia Griffith of the School of Medical Science and Shailendra Dukie of the School of Pharmacology at Griffith University Gold Coast Campus for xxi graciously supplying the THP1 cells used within the experiments. Additionally I would like to acknowledge and thank Griffith University‟s School of Medical Science for approving the grant allowing for the collaboration to take place between the DLP group of Griffith University and the Molecular Therapy group at BIOCEV.
I would also like to thank Rowan Radford of Macquarie University for his assistance in understanding and using the Imaris software. Additionally a special acknowledgement and thank you for Marie Olšinová of BIOCEV‟s Imaging Core Facility for providing both training of the STED microscope and assistance in the acquisition of images using the STED microscope. Both of these individuals were instrumental in expanding my skillset and taking the potential of what could be imaged to the next level.
Lastly I‟d like to thank my personal support network which includes my family, friends, my dog Whiskey and my wonderful partner Rebecca Williams for their generous support over the last few years as I‟ve been hard at work trying to complete this Thesis. You have all been incredibly understanding of the amount of stress and free time I don‟t have, and I‟m not sure if I would still be sane at the end of this entire process without you all. Your support has meant more to me than I can put into words and from the very bottom of my heart, I thank you all so much.
In memory of Frances Doohan (1938-2018).
- Dario Valdinocci, BBiomedSc(Hons)
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Chapter 1
Introduction
1
1.1 Literature Review
1.1.1 Multiple System Atrophy
Multiple System Atrophy (MSA) is a sporadic neurodegenerative disease characterised by the presence of Parkinsonian-like symptoms in the affected patient. These symptoms include bradykinesia, motor rigidity, gait and ataxia to name a few (Longo, Fanciulli and Wenning, 2015; Ubhi, Low and Masliah, 2011). As such MSA is regularly misdiagnosed as Parkinson‟s Disease (PD) due to these similarities presented by patients, making an accurate diagnosis impossible without post-mortem brain examination (Longo, Fanciulli and Wenning, 2015; Ubhi, Low and Masliah, 2011). Additional signs which can help determine whether an individual may be suffering from MSA include presence of autonomic disorders such as urinary incontinence, or rapid loss of motor functions. MSA patients have a more widespread and rapid degeneration profile in the brain compared to PD patients, with an individual succumbing to death on average around 8-10 years with MSA after diagnosis compared to PD >10 years (Marttila and Rhine, 1991; Wenning and Stefanova, 2009). Prevalence of MSA is estimated to be around 2-7 per 100,000 people per year, a considerably smaller number than for PD at 8-18 per 100,000/year or ~1% of the total adult population (de Lau and Breteler, 2006, de Rijk et al., 1995). It is worth noting that the prevalence number for MSA may in fact be higher due to the inability to correctly diagnose a patient with the disease whilst they are alive.
There are 2 subcategories of MSA based on pattern of spread and symptoms exhibited by patients, MSA-P and MSA-C (Figure 1). In MSA-P (or MSA-SND), the more Parkinsonian-like of the 2, patients display symptoms such as tremors and bradykinesia as well as a similar spread pattern that is exhibited by PD (Longo, Fanciulli and Wenning, 2015; Ubhi, Low and Masliah, 2011; Kiely et al., 2018). This degeneration is thought to occur in the substantia nigra and putamen where it then later spreads towards the rest of the midbrain, infecting the globus pallidus, caudate and the brain stem (Paviour et al., 2007). Degeneration in these latter regions is initially mild but can rapidly accelerate to severe levels. Degeneration of regions such as the putamen and substantia nigra is still ongoing whilst spread of the disease to neighbouring regions is underway (Halliday et al., 2011). The last sites seen to be heavily affected by MSA degeneration are those involved in motor functions, the frontal cortex and the cerebellum (Paviour et al., 2007; Halliday et al., 2011). Environmental and genetic factors may be implicated in the sub-type of MSA. A study based in Europe revealed that 68.2% of all MSA cases in analysed European countries were MSA-P, a similar statistic to which was also observed in a U.S. population study (60%) (Stefanova et al., 2009; May et al., 2007).
By contrast, MSA-C (or MSA-OPCA) appears to be the predominant type in Japanese populations, with a Japanese study revealing MSA-C was the leading type of MSA at 83.8% (Yabe et al., 2006). This is almost in direct contrast to MSA-P, which is predicted to make up 2 for ~80% of all Caucasian cases of MSA-P (Wenning et al., 2004). MSA-C displays a four stage mechanism of spread that affects different regions to different degrees than to MSA-P. Stage 1 is thought to begin in cerebellular subcortical white matter, various regions of the medulla such as the olivo-cerebellar fibres, inferior cerebellar peduncle and pontocerebellar fibres (Brettschneider et al., 2016). It is thought to begin here as MSA-C with the lowest burden of pathology displayed both degeneration and formation of MSA inclusion bodies (Section 1.2.2 goes into greater detail) within these brain regions. In stage 2 the degeneration begins to occur in deeper regions of the medulla and white matter of the cerebellum as well as beginning to spread to the spinal cord (Brettschneider et al., 2016). In stage 3 degeneration and presence of inclusion bodies occurs in the cortex in regions such as the frontal and parietal cortices all whilst degeneration in previously affected areas gets progressively worse. Lastly in stage 4 the amygdala and hippocampus begin to undergo degeneration (Brettschneider et al., 2016). MSA- C patients typically display symptoms of ataxia and gait. After some time however both MSA types do overlap in regions affected and begin to show additional symptoms of autonomic dysfunctions such as urinary incontinence and/or orthostatic hypotension (Ubhi, Low and Masliah, 2011). Figure 1 below provides a visual description of the spread as well as the severity of neurodegeneration that occurs as the disease progresses. MSA is classified as a synucleinopathy, a disease in which degeneration is brought about by the presence of a misfolded form of a protein termed α-synuclein (α-syn) (Fernagut et al., 2014).
Figure 1- Predicted models of neurodegeneration of MSA-P and MSA-C
The brains depict the differences in the areas of degeneration and rates between MSA-P and MSA-C.
From left to right, degeneration gets progressively worse, with darker colours signifying severity of loss of cells and GCI abundance. Adapted from Halliday et al., 2011.
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1.1.2 Alpha-Synuclein
1.1.2.1 Alpha-Synuclein in the Brain
α-syn is a soluble 140 amino acid protein found predominantly to exist within the Central Nervous System (CNS) but is also known to be found in the peripheral nervous system as well as circulating erythrocytes (Barbour et al., 2008; Theillet et al., 2016; Weinreb et al., 1996). There is much contention as to the correct conformation of the protein. Early discoveries and synthesis via recombinant methods using prokaryotic cells described α-syn as an unfolded monomer (14kDa) (Jakes, Spillantini and Goedert, 1994). Eukaryote extractions however yields multimeric α-syn, such as tetramers, with α-helical content (58kDa) (Bartels, Choi and Selkoe, 2011; Luth et al., 2015). Recently there have been studies to suggest that α-syn can exist in both states and that confirmation is based on whether α-syn is bound to a lipid membrane, adopting a multimeric confirmation from the monomeric one (Burré et al., 2013; Theillet et al., 2016). As of yet the current normal role for α-syn is unknown however there has been many reports and speculations as to its function. α-Syn has been revealed to exist in high concentrations around the presynaptic terminals of neurons, implicating it in roles of synaptic transmission (Cheng, Vivacqua and Yu, 2011). Other roles include dopamine (DA) synthesis and regulation of vesicle trafficking and SNAP Receptor (SNARE) protein activity (Burre et al. 2010; Perez et al., 2002; Larsen et al., 2006; Yavich et al., 2004; Yavich, Jäkälä and Tanila, 2006).
1.1.2.2 Alpha-Synuclein in Disease
As previously alluded to, in synucleinopathies α-syn becomes misfolded and thus changes from a normal healthy protein to a pathological one. There are a multitude of triggers that can cause this, such as oxidative stress, increased concentration of metal ions such as Ca2+ and post- translational modification (Hashimoto et al., 1999; Rcom-H'cheo-Gauthier et al., 2016; Fujiwara et al., 2002; Reynolds et al., 2007). In particular phosphorylation of Serine 129 has been implicated as a major step in the formation of the oligomeric β-sheet α-syn found in MSA (Cookson, 2009; Anderson et al., 2006). Oligomeric α-syn is the toxic form of α-syn, causing cellular death by damaging cellular organelles through loss of normal cellular function or excessive production of ROS (Winner et al., 2011). An additional trait oligomeric α-syn possesses is the ability to aggregate into fibrils which then, in combination with other proteins, form an inclusion body. Each synucleinopathy has a differing inclusion body based on its composition. In MSA the inclusion body is termed a Glial Cytoplasmic Inclusion (GCI) and is composed of ubiquitin, αB-crystallin and tau in addition to α-syn (Miki et al., 2010; Tu et al., 1998; Pountney et al., 2005). These inclusion bodies typically take on a triangular or sickle cell shape inside affected cells. Rare missense mutations in the α-syn gene SNCA have also produced cases of individuals developing synucleinopathies, some with resemblance to MSA implicating another method by which fibrils and in turn inclusion bodies can form (Lesage et 4 al., 2013; Polymeropoulos, 1997). Isoform levels of α-syn (114 and 120), Parkin and Synphillin- 1 are also altered in MSA brains and displayed distinct alterations compared to both normal and PD brains (Brudek et al., 2015).
One of the distinguishing factors of MSA in comparison to other synucleinopathies such as PD or Dementia with Lewy Bodies (DLB), is that α-syn and GCIs are shown to exist and persist within different cells. In PD α-syn and Lewy Bodies (LB), the inclusion body found in PD, have been shown to selectively form within and degrade neurons. In MSA however not only are GCIs found within oligodendrocytes but unlike LB do not actively degrade or kill the cell they are within (Ahmed et al., 2012). GCI formation and presence does still affect the function of oligodendritic cells however (Brück et al., 2016). Previously it was thought that this inclusion body in separate cells indicated selective over expression in those particular cell types however this has been disproven. Recent work by Sekia et al., revealed oligomeric α-syn within both neurons and oligodendrocytes within MSA brains (Sekiya et al., 2019). Whilst inclusion bodies were still formed exclusively within oligodendrocytes, such a finding denies α-syn over- expression is exclusive to cells displaying formation of inclusion bodies. This is especially true considering that oligodendrocytes only express small quantities of α-syn, with no significant change in this expression in MSA (Asi et al., 2014). Oligodendrocytes are glial cells involved in the insulation of neuronal axons in the CNS with a protein-phospholipid mixture termed myelin. This insulation allows for the effective firing of signals down the axon towards the synapse at the axonal terminals, signalling for neurotransmitter release to neighbouring neurons (Bradl and Lassmann, 2009). Myelin increases the electrical resistance in the axon, allowing for rapid firing of signals (Bradl and Lassmann, 2009). The axons have small nodes where myelination does not take place, termed the Nodes of Ranvier, and these nodes contain the only ion-gated channels within the axon. This lack of ion-gated channels along the axon prevents leakage of Na+ into the extracellular environment and keeps a high Na+ concentration allowing for rapid signalling to take place via depolarisation (Hartline, 2008; Poliak and Peles, 2003). Depolarisation is less effective in unmyelinated axons, producing slower signalling times (Figure 2).
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Figure 2 – The role of oligodendrocytes in neuronal signalling and how the presence of pathological α-syn affects this
The neuronal axons (blue) are mostly covered in myelin (green) synthesized by surrounding oligodendrocytes (purple). Myelin insulates the axon allowing for more effective action potential
signalling toward the nerve terminal for neurotransmitter release. Nodes of Ranvier, the gaps between the sheaths contain Na+, Ca2+ and K+ channels which further aid in action potential
propagation. Presence of α-syn aggregates and GCIs negatively affect oligodendrocytes ability to synthesise myelin leading to its degradation around the axon over time, thus leading to less efficient
signalling at the nerve terminals (Poliak and Peles, 2003).
In PD motor control is impaired due to the loss of dopaminergic neurons, the main neurons associated with motor control due to the release of the neurotransmitter DA (Naoi and Maruyama, 1999). This neurotransmitter acts on nearby neurons, causing them to activate and signal for musculoskeletal movement. This loss of neurotransmitter signalling is also associated with the development of tremors, the most well-known clinical phenotype of PD. In MSA the loss of motor function is a result of both poorly insulated motor neurons and loss of neurons. Loss of neurons is partially a result of oxidative stress placed upon them by chronically activated immune cells such as microglia. This is further discussed later in the microglial section (Stefanova et al., 2009). GCI formation within oligodendrocytes affects the synthesis of myelin and thus the cells ability to insulate surrounding motor neurons, significantly reducing their efficiency in releasing and signalling other neighbouring neurons with DA (Wong, Halliday and Kim, 2014; Don et al., 2014). In PD the decreased levels of DA due to loss of dopaminergic neurons is treatable by L-DOPA, an amino acid and a precursor to DA, whereby it helps increase the concentration of DA in the brain (Dorszewska et al., 2014). This increase in concentration helps alleviate some of the symptoms presented by PD whilst the disease is still in
6 its early stages. In MSA however L-DOPA is rendered ineffective as unlike PD, the development of the movement disorders are not due to low DA concentrations but in part due to the inability of motor neurons to effectively signal due to lack of myelin insulation.
1.1.2.3 MSA as a Prion Disease
It is currently unknown why α-syn and GCIs in MSA selectively target oligodendrocytes, however it could be related to MSA‟s classification as a prion disease. The current model of prions within prion disease proposes that proteins exist as one of two types, normal (PrPC) and prion (PrPSC). If a prion protein makes contact/interacts with a non-prion protein, it converts it from PrPC into PrPSC, leading to continuous infection if allowed to spread (Cushman et al., 2010; Angot et al., 2010). Prior to 2015, the only other prion disease in humans was Creutzfeldt-Jacobs Disease and as such much of what encompasses MSA‟s role as a prion disease is still unknown (Gousset et al., 2009). Work by Prusiner et al. (2015), went on to speculate that the strain of α-syn found in MSA may be different than that of other synucleinopathies, potentially explaining the difference observed in rate of spread and as to why oligodendrocytes are predominantly affected (Prusiner et al., 2015). Spread of pathological α- syn and its inclusion bodies are present in other synucleinopathies as well, hinting that other prion disease status may not just be limited to MSA. For example, Recasens et al. (2014) devised an animal model whereby human LB were stereo-tactically inoculated into the substantia nigra and striatum of both mice and macaque monkeys (Recasens et al., 2014). It was found that after 14 months both mice and monkeys had increased expression of α-syn in inoculated regions as well as increased loss of dopaminergic neurons as well (Recasens et al., 2014). Both developed loss of muscular function however this was not severe enough to be classified as PD. Somewhat similar results were seen in humans as well. Reported by Li et al. (2008), LB‟s were seen infecting brain sites where neurons were grafted due to PD degeneration over a decade after the procedure (Li et., 2008). Similarly this was observed in another case by Kordower et al. (2008) the same year, reporting a similar time of onset at 14 years (Kordower et al., 2008). It may be possible that these cases are just examples of the aggressiveness by which α-syn and its inclusion bodies spread in disease rather than evidence of potential actions of a prion disease. Currently there are no similar reported graft examples for MSA and as such is difficult to comment if α-syn and GCI spread occurs within grafted sites and what their rate of spread is in comparison to PD. Given MSA‟s somewhat recent classification from 2015, it is expected more research will be conducted to answer such long-standing questions.
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1.1.3 Microglia
1.1.3.1 Origin and Normal Role of Microglia
Much like the rest of the human body, the CNS also possesses an immune system to protect the brain from potentially damaging molecules or species. Microglia are one of the specialised immune cells found within the CNS, accounting for ~10% of total glial cell population and acting as a macrophage-like cell. Microglia possess many qualities of a macrophage such as engulfment of pathogens and molecules via phagocytosis and the recruitment of additional immune cells via inflammatory factor release (Fu et al., 2014; Kettenmann et al., 2011). They also possess a high degree of plasticity, allowing for microglia to successfully change from a surveillant/resting state to a phagocytic/reactive one as well as assisting the cell with transport around the brain (Amadio et al., 2013; Figure 3). Microglia are highly mobile and constantly survey the brain for Pathogen Associated Molecular Patterns (PAMPs) such as bacterial species or Damage Associated Molecular Patterns (DAMPs) such as misfolded α-syn (Su et al., 2008). Whilst in its surveillant state microglia are ramified, with long thin pseudopodia used for the detection of PAMPs and DAMPs (Neumann, Kotter and Franklin, 2008). If activated, microglia change into an amoebic state whereby degradation of engulfed material can successfully take place as well as releasing factors such as interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) for additional microglia recruitment. This activation of microglia is typically carried out by Toll-Like Receptor‟s (TLRs) such as TLR2 and TLR4 as well as scavenger receptors like CD36 (Stefanova et al., 2011; Kim et al., 2013; Fellner et al., 2013; Tang and Le, 2015). In addition to pro-inflammatory factor release, anti-inflammatory factors such as IL-1 and transforming growth factor-β (TGF-β) are released to control the inflammatory process to prevent it from becoming chronic (Neumann, Kotter and Franklin, 2008; Kettenmann et al., 2011).
Figure 3 – The differing morphological states of microglia
The top microglial cell is in a ramified state. This is
the inactivated surveillant state that microglial cells
predominantly exist in. When activated the protrusions from the ramified state begin to shrink, leading to a bigger, rounder microglial cell whereby
inflammatory factors are released for recruitment of
additional cells. In this amoebic, phagocytic state the cell can engulf and degrade pathological and dangerous materials within the CNS environment
(Amadio et al., 2013). 8
It was previously thought that active microglia were strictly classified as 1 of 2 sub-categories; M1 microglia which are predominantly associated with pro-inflammatoryfactor synthesis and release and M2 microglia which are required for neuro-protection due to synthesis and release of anti-inflammatory factors (Figure 4). In addition to anti-inflammatory factor release, M2 microglia are thought to work in debris clearance from inflammatory events preventing potential damage to neighbouring cells (Tang and Le, 2015). Stimulation by lipopolysaccharide (LPS) or Interferon-γ (IFN-γ) is required to signal for activation of M1 microglia, a process termed „classical activation‟. When activated, M1 microglia signal for synthesis and release of pro- inflammatory factors which include TNF-α, IL-1β and IL-6 as well as ROS generation (Le et al., 2001; Tang and Le, 2015). IFN-γ is typically released by T-Helper (TH) 1 cells and astrocytes, however it can also be secreted by microglia in an autocrine manner during acute inflammation (Kawanokuchi et al., 2006; Suzuki et al., 2005; Cherry, Olschowka and O‟Banion, 2014). In addition to pro-inflammatory cytokine release, up-regulation of surface markers CD16/32, CD40 and CD86 are all involved as stimulatory factors of immune response activation (Kalkman and Feuerbach, 2016; Zhou et al., 2017). Stimulation of M2 microglia can occur via one of two fashions, termed „Alternative Activation‟ and „Acquired Deactivation‟. Alternative activation requires IL-4 and/or IL-13 from TH 2 cells which stimulates for an autocrine IL-4 and IL-13 response regulating acute inflammation (Gordon and Martinez, 2010). Equally „Acquired Deactivation‟ of M2 microglia also work to alleviate inflammation, however they have the added role of apoptotic cell phagocytosis. This is an innate response that induces IL-10 and TGF-β release (Tang and Le, 2015). Both M2 activation pathways additionally release factors like Arg1, FIZZ1 and Chi313 as well as promote CD206 surface receptor expression, which all further aid in immunosuppression and neuro-protection through various functions such as tissue repair and or debris clearance (Tang and Le, 2015). In this model both microglial types act to regulate the other preventing inflammation from becoming chronic or increasing the presence of pro-inflammatory factors to combat PAMPS/ DAMPS. This concept has shifted the last few years however as M1 microglia still present with factors that are mainly associated with M2 microglia and vice versa. Thus the idea of a spectrum was proposed, in that active microglia can shift from a pro-inflammatory profile to an anti-inflammatory profile depending on the needs of the system/situation due to microglial cells plasticity profile (Mosser and Edwards, 2008).
Microglia can originate from one of two sources, the mesenchymal yolk sac in the brain or from monocytes derived from hematopoietic stem cells in the bone marrow (Gomez Perdiguero et al., 2014; Ritter et al., 2006; Ifergan et al., 2008). Bone marrow derived microglia originate as monocytes which later differentiate into microglia as they are crossing the blood-brain barrier, differentiating due to exposure of factors such as TGF-β and granulocyte macrophage colony stimulating factor (M-CSF) (Ifergan et al., 2008). Microglia originating from the brain are also
9 independent of the transcription factor Myb whilst monocyte derived microglia require Myb for differentiation and continual maintenance (Gomez Perdiguero, Schulz and Geissmann, 2012). It‟s been proposed that depending on site of origin the role of microglia will differ, with monocyte derived microglia involved more in maintenance such as disposal of damaged myelin whilst brain-derived microglia are involved in debris clearance through phagocytosis (Yamasaki et al., 2014). Cells such as oligodendrocytes have also been shown to interact with microglia for the disposal of toxic/waste materials, releasing these via exocytosis for microglia to engulf and degrade through macropinocytosis (Fitzner et al., 2011). Microglia internalise PAMPS and DAMPS once they have made contact with the immune cell by forming a phagocytic cup before placing them in an endosome. This endosome, termed a „phagosome‟, will begin degradation through the use of enzymes to make the environment toxic to the engulfed material before fusing with the lysosome to complete phagocytosis (Wake and Fields, 2011). Lysosomes are the main organelles involved in the degradation of macromolecules in the cell and thus are a required component for phagocytosis and unwanted material disposal. An alternative method for engulfment is macropinocytosis whereby membrane ruffling, due to actin-dependent manipulation, engulfs large materials or solutes (Holmes et al., 2013). These materials are placed in a macropinosome and undergo a similar process as endosomes where they begin degradation of contents before fusing with lysosomes to finish the process. Additionally monocytes can infifltrate the brain, crossing the blood-brain barrier to assist in inflammatory activity and clearance. Worth noting however is that their activation can take up to 72 hrs compared to 24 hrs for residual microglia, thus acting as more of a supplemental factor to inflammation and clearance as opposed to a major one (Xu et al., 2018b).
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Figure 4 – Summarized differences in microglial activation state
Displayed on the left is a summary of the differences and factors associated with M1 and M2 microglial cells. M1 microglia will undergo classical activation, and this from DAMP/PAMP related factors which result in an increase in pro-inflammatory microglial cell presence. This presence is naturally accompanied by increased pro-inflammatory factor presence, ROS and Nitric Oxide species generation. M2 microglia require alternative activation to occur, which requires the presence of pro-inflammatory factors such as IL-4. This activation is accompanied with anti-inflammatory factor release used to regulate inflammation from becoming chronic. Release of factors such as Arg1 (used in promotion of neuroprotection) and tissue repair are also released by these cells. Adapted from Tang and Lee, 2015. 1.1.3.2 Iba-1
Iba-1, a 147 amino acid EF hand protein, is expressed by microglia and macrophages and is involved in cellular shape changing. Influenced by Ca2+ presence, Iba-1 causes shape change by binding to and manipulating filamentous actin (F-Actin) on the cytoskeleton of the cells (Sasaki et al., 2001). As such Iba-1 is a popular marker used for the detection of cells in immunohistochemistry as well as determining the state the cells are in (e.g. ramified or amoebic). The cytoskeletons of microglial cells are composed of actin and undergo membrane ruffling once Iba-1 is bound via Ca2+ stimulation (Sasaki et al., 2001). Membrane ruffling allows for the membrane to alter its phenotype, with examples including moving pseudopodia or the change of microglia from ramified to amoebic (Imai et al., 2000). As discussed previously, this change to amoebic allows for the cell to become more effective at phagocytosis and disposal of threats such as PAMPS and DAMPS. Rac GTPase may also influence F-Actin without the use of Iba-1. Activation of Rac GTPase is illustrated on Figure 5 below. It is brought about when M-CSF binds to the Fms-receptor on the microglia, which causes PI3-K to become active (Imai and Kohsaka, 2002). It then acts on Rac GTPase to lead to its activation. 11
Phospholipase-Cγ1 is also thought to work in conjunction with M-CSF-Fms activation (Kanazawa, 2002 ; Imai and Kohsaka, 2002). Other works have shown that Iba-1 activation correlates with Rac1 activation in microglial cells, perhaps enhancing the response. Iba-1 has also been shown to bind and influence an actin-cross-linking protein called L-Fimbrin, assembling F-Actin into bundles so as to allow for crosslinking and thus manipulation of shape to take place (Ohsawa et al., 2004).
Figure 5 – Interactions of Iba-1 with the cytoskeleton
As seen on the figure above, M-CSF along with PLCγ and PI3-K, as well as Ca2+ and Iba-1 act
upon Rac-GTPase to signal for the restructuring of the cytoskeleton of microglial cells. Activation of Rac-GTPase leads to actin restricting on the cytoskeleton as well as additional nuclear signalling to help promote this process. Production of radicals via NADPH Oxidase also occurs via the hydrolization of GTP. Adapted from Imai and Kohsaka, 2002.
1.1.3.3 Microglia in MSA
Another feature of MSA which further distinguishes it from PD and DLB is gliosis, the activation of glial cells such as microglia and astrocytes. Gliosis is still observed in both PD and DLB however it is not as widespread nor are the microglia seen to be as chronically active as the ones displayed in MSA (Nagatsu and Sawada, 2005; Tufekci et al., 2012) . Microglia have been shown to be highly active in MSA brain regions where α-syn and GCI‟s are in high abundance (Fellner and Stefanova, 2012; Politis, 2012). Considering both pathological α-syn and GCI‟s are classified as DAMPS due to their ability to disable oligodendrocytes and degrade other cells such as neurons and astroglia via oxidative stress and apoptosis, this activation is warranted. Even when activated however microglia are ineffective in the degradation and clearance of fibrillar α-syn (Tanik et al., 2013). It isn‟t known why this is, however there is speculation that it may be in relation to impairment within the lysosomal pathway, a major
12 process involved in phagocytic degradation. Whilst there is no current literature explicitly stating that lysosomes in microglia are non-functional, such evidence has been shown for neurons in PD explaining the high presence of LB‟s and α-syn in infected cells (Alvarez-Erviti et al., 2011; Chu et al., 2009). α-syn binding to TLR4‟s on microglia not only signals the cells activation, but also leads to the production of the free radical nitric oxide, which can lead to microglial death via apoptosis and can negatively affect lysosomes and their degradation ability (Butler and Bahr, 2006; Wakabayashi and Takahashi, 2006). Additionally aggregates of α-syn have been shown to inhibit lysosomal enzymes such as β-galactosidase, hexosaminidase, cathepsin B and β-glucocerebrosidase in addition to disrupting hydrolase trafficking, both integral components to the function of lysosomes and the autophagosome pathway (Mazzulli et al., 2016).
Aggregated α-syn has also been demonstrated to lead to increased free radical production in microglia through various mechanisms, for example the activation of NADPH oxidase (Zhang et al., 2005). NADPH oxidase transfers electrons across the wall of the phagosome into the - vacuole were they then interact with O2 to form O2 or superoxide (Segal, 2008). Superoxide is a reactive oxygen species (ROS) which then acts on the PAMP/DAMP in the phagosome to degrade it. If not disposed of correctly, ROS can cause dysfunction of mitochondria and lead to apoptosis/necrosis of the cell (Morgan, Kim and Liu, 2008). As part of the complex required for NAPDH oxidase activation, the presence of Rac GTPase suggests that long-term activation of microglia for the combat of pathological α-syn may lead to lysosome dysfunction (Imai and Kohsaka, 2002; Hordijk, 2006). This ineffective degradation is thought to be associated with the high levels of pro-inflammatory factors seen in α-syn/GCI affected regions, leading to the chronic inflammation observed in MSA and contributing to the degeneration observed in those regions (Kaufman et al., 2013; Wenning and Stefanova, 2009 ; Koga et al., 2015). Activation and expression of matrix metalloproteinases such as MMP-1, MMP-3 and MMP-9, which leads to pro-inflammatory factor release and ROS production, have been shown to be caused by interactions with mutated α-syn (Lee et al., 2010). This interaction further highlights how α-syn prolongs inflammation within MSA. α-syn is also a known chemoattractant of microglia, recruiting microglia to these sites further contributing to the gliosis within MSA (Wang et al., 2015).
A theory currently gaining some momentum is the notion that senescence is partially responsible for the reduced microglial pathogen clearing capacity within synucleinopathies. As microglial cells undergo senescence, their ability to degrade internalised DAMPS and PAMPS are lessened. This presence of dystrophic microglia is thought to be insignificant in the early stages of MSA however as the disease progresses the quantity of this microglial phenotype increases. At what point this presence becomes significant is unknown. Lysosomes are one of the key organelles primarily affected by this process, shown to have greater difficulty in 13 material degradation when microglia are dystrophic than when they are reactive (Spittau, 2017). Interestingly clearance of myelin fragments is a process undertaken by microglia which significantly drops once the cells become dystrophic (Safaiyan et al., 2016). This decreased clearance and reduction of phagocytosis is thought to be correlated with the overall reduction in phagocytic activity of microglia due to the abundance of undegraded myelin within the cytoplasm (Safaiyan et al., 2016). Whether this is also implicated in MSA or other synucleinopathies is unclear as no similar result has been reported as of yet. Outside of this finding, work with regards to microglial senescence in neurodegeneration has predominantly been focussed on AD, revealing contrasting information as to whether it is a main component associated with neurodegeneration. Work by Streit et al. (2009) via analysis of a variety of AD brains at various stages of the disease demonstrated that dystrophic microglia have a significant presence within all stages of AD (Streit et al.,2009). This is antithetical to the current belief in AD, PD and MSA whereby dystrophic microglia only make significant presence in the late stages of the diseases. Increased presence of dystrophic microglia saw with it increased presence of β-amyloid plaques as phagocytosis by dystrophic microglia appeared impaired or completely inhibited. As time passed less microglial cells were present which led to the hypothesis that neurodegeneration in AD is due more to loss of microglia than the pro-inflammatory activity of reactive microglia. Microglia act as a neuroprotective barrier against neurodegeneration and thus reduced presence equates to greater loss of tissue. Senescence is a non-reversible effect of aging. Conversely work by Krabbe et al (2013) reveals that senescent microglia are not present in all stages of AD and only becomes primarily significant in the later stages (Krabbe et al.,2013).
In this study phagocytosis profiles of normal and AD brains within mice were examined to understand the differences in microglial activity and how this may relate to β-amyloid plaque presence, the GCI/LB equivalent in AD. Much like in both MSA and PD, microglia engulf β- Amyloid plaques but appear to be highly resistant to degradation. The main finding was that over time microglia‟s phagocytosis profile lessened, which saw a reduction in both expression and enzymes involved in this process and factors involved in phagocytotic degradation. This loss of phagocytic activity could be reversed however by β-amyloid vaccine in a mouse model not too dissimilar to the vaccine used to treat PD and αsyn presented by Masliah et al.(2005). Much like in Krabbe et al., vaccine treatment targeting human α-syn saw reduction of the protein in transgenic mouse brains expressing for it, with clearance propagated by lysosomal action (Masliah et al., 2005). Whilst these vaccines will not be able to reverse or aid in the reversion of impaired phagocytosis present within senescent cells, it appears to be a potential therapeutic target for earlier stages in both AD and PD, potentially even MSA. Work presented in this study also mirrors the current notion surrounding microglial clearance in synucleinopathies, in which earlier stages excel at DAMP/PAMP clearance whilst at later
14 stages when the abundance of pathological proteins is significantly greater, the system governing phagocytosis becomes overwhelmed and thus its action is reduced (Safaiyan et al., 2016). Worth noting however is that in this particular mouse model senescence was not the primary factor being investigated. Whilst dystrophic microglia were present and their effect was investigated, this was more as a supplemental factor than a major focus and thus may not be completely indicative of what occurs in aged AD and potentially MSA brains. Given that PD and MSA both predominantly occur later in the human life cycle, it is more than likely that senescence and thus dystrophic microglia have some role to play within the diseases and α-syn clearance. Currently there is little to no literature investigating the phenomena of senescence within MSA or other related synucleinopathies, however given what is currently understood about microglial activity such a notion does warrant consideration. Microglia do not exhibit an all-or-nothing response, not all microglia in MSA are chronically activated so it would stand to reason that there would be a presence of senescent microglial cells within the brain and in regions α-syn/GCIs are in high abundance. Whether this presence is significant or a plays a significant role in α-syn and GCI abundance is unknown.
1.1.3.4 Toll-Like Receptor 4
Activation of microglia is typically triggered as a PAMP or DAMP makes contact with the cell. What this refers to is not contact with the plasma membrane but with specific receptors, in many cases those receptors being the TLRs. TLRs exist on the surface of a variety of cells involved in the innate immune response, including macrophages and dendritic cells in addition to microglia. These receptors are typically activated by LPS, the PAMPs, present on the wall of bacterial organisms. This activation will then signal for the release of cytokines and pro-inflammatories such as TNF-α, IL-1β and IL-6 for the recruitment of additional immune cells for clearance and degradation. DAMPs, such as Heat Shock Protein (Hsp) 60, may also bind to TLRs to signal for activation of immune responses (Cohen-Sfady et al., 2005). One such TLR integral to the microglial immune response is TLR4. TLR4 is a leucine rich receptor and works in conjunction with a variety of other components in order to efficiently carry out the immune response, which includes CD14, MD-2, TIRAP, TRAM and MyD88 to name a few. TLR4 possesses a domain termed the Toll/Interleukin-1 Receptor (TIR) which is essential for TLR4 to signal for activation (Takeda, Kaisho and Akira, 2003). TLR4 exist as singular receptors prior to detection of ligands and will undergo dimerization with another TLR4 once an activating ligand (PAMP or DAMP) has bound. MD-2 and CD14 also bind to TLR4 to allow for dimerisation and to aid in signalling (Visintin et al., 2001; Zanoni et al., 2011). Lack of MD-2 has been shown to prevent TLR4 dimerisation (Schromm et al., 2001; Visintin et al., 2001).
Signalling for the production of and release of cytokines can occur via one of two pathways, the MyD88 dependent pathway or the MyD88 independent pathway. In the MyD88 dependent
15 pathway, an adaptor molecule named TIR domain-containing adaptor protein (TIRAP) on the cytosol of the immune cell binds to the TIR domain of the dimerized TLR4 complex on the plasma membrane (Horng et al., 2002). This is followed by the binding of MyD88 which then leads to the further recruitment of the protein IRAK-4 which phosphorylases IRAK-1 (Takeda, Kaisho and Akira, 2003). IRAK-1 then associates with TRAF-6 so as to activate IκB kinase (IKK) complex to induce phosphorylation and degradation of IκB (Takeda and Akira, 2005; Takeda, Kaisho and Akira, 2003). This degradation leads to the transcription of NF-κB leading to the production and release of pro-inflammatory cytokines such as tumour necrosis factor-α (Latz et al., 2003). In the MyD88-independent pathway, also termed TRIF-dependent pathway, instead of associating with TIRAP, the TLR4 complexes TIR domain binds to TRIF-related adaptor molecule (TRAM) (Fitzgerald et al., 2003). TRIF then binds to TRAM and will activate TRAF3 and TRAF6. TBK1 activation occurs from binding to TRAF3 which in turn leads to the activation of IRF3 for the induction of IFNβ (Fitzgerald et al., 2003). This will signal for the production of antiviral factors. Pro-inflammatory cytokines are also reduced in the TRIF- dependent pathway, revealing that MyD88 is a major factor involved in their production and release (Kawai et al., 1999; Takeda and Akira, 2005). TRAF-6 activation in the TRIF-dependent pathway does lead to NF-κB transcription and in turn production of some anti-inflammatories, however the response is delayed and less effective (Kawai et al., 1999). The TRIF-dependent pathway appears to be more focussed on antiviral threats as opposed to the MyD88 pathway which is more suited towards bacterial and DAMP protein threats.
1.1.3.5 Toll-Like Receptor 4 and Its role in Alpha Synucleinopathy
TLRs have been revealed to combat pathological proteins such as α-syn. Classified as a DAMP, interactions between α-syn and TLR4 have been shown to exist (Stefanova et al., 2011; Roodveldt et al., 2013; Fellner et al., 2012). Many works discuss TLR4 as the main receptor involved in activation of microglia against α-syn, however other TLRs such as TLR2, have also been shown to elicit a response when bound to oligomeric α-syn (Kim et al., 2013; Fellner et al., 2012). The responses however appear to be less reactive than TLR4, perhaps suggesting that they are more of a supplemental or alternative activation pathway in the case of TLR4 dysfunction or lack of its presence on immune cells. Activation of TLR4 has shown increased clearance of the protein in PD from the extracellular environment (Stefanova et al., 2011). This clearance was shown to help promote survival of nigral dopaminergic neurons as spread of protein existing in the extracellular means was reduced. Worth noting however is that this clearance was not performed in an aged brain model and thus may not be fully indicative of what occurs in later stage PD. Given that gliosis is not a major factor in PD as it is in MSA, it is difficult to determine whether increased TLR4 activation may lead to less spread. There is the possibility that increased activation may lead to a more aggressive inflammatory reaction, further contributing to the degeneration of the neurons and oligodendrocytes. Inflammation 16 carried out by glial cells does contribute to the loss of dopaminergic neurons in both PD and MSA. The NLRP3 Inflammasome, a by-product of TLR4 activation and involved in the synthesis and release of IL-1β, is a contributing factor in inflammation based neurodegeneration present within PD and MSA (Li et al., 2018; von Herrmann et al., 2018). Absence of TLR4 sees reduced inflammation and loss of dopaminergic neurons by the NLRP3 inflammasome, suggesting that counteracting pro-inflammatory reactive microglia is beneficial in neuroprotection (Campolo et al., 2019). Additionally decreased number of α-syn positive neurons were observed as well, suggesting TLR4 aids in some fashion to α-syn spread (Campolo et al., 2019). This mechanism is still unclear.
Resartovid or TAK-242 is a signal transduction inhibitor which inhibits the actions of TLR4 pro-inflammatory factor release. The drug does not bind to the activation site where ligands bind but instead the TIR domain of the TLR4 internalised within the cell, more specifically to Cys747 (Matsunaga et al., 2010, Takashima et al., 2009). The TIR domain is composed of a central 5-strand β-sheet (βA through to βE), 5 α-helices (αA through to αE) and connecting loops, and it is predicted that Cys747 is located on αC (Takashima et al., 2009). By binding to the TIR domain, the recruitment, binding and activation of both TIRAP and TRAM is prevented. TAK-242 has been shown to be successful in lowering inflammatory reactions in both mouse models and in vitro models of endotoxin shock (Sha et al., 2007). Cells used for microglia in vitro such as THP-1 were shown to release fewer inflammatory cytokines (Zhou et al., 2013). In PD, increasing activation of TLR4 has been a focus of research as a potential mechanism of preventing spread of α-syn whilst for MSA such an option would be risky due to the chronic gliosis presented by the disease. Currently it appears that TAK-242 has yet to be tested on microglia with regards to synucleinopathies and if it still promotes clearance without excessive pro-inflammatory factor release. Perhaps treatment with a TLR4 agonist may prove beneficial in early stage MSA/PD when microglial cells are still seemingly capable of α-syn degradation. In later stages as microglial cells become less effective at degradation and are only serving to prolong inflammation, possibly due to senescence, TAK-242 treatment may prove to be a potential remedy. This way pro-inflammatory factor release is lessened as may potential α- syn spread. α-Syn has been shown to internalise within cells via a variety of methods (discussed further 1.4.2) and as such may still activate microglia through a TLR4-independent fashion such as interacting with NADPH oxidase within microglia (Zhang et al., 2005).
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Figure 6 – Pathways of TLR4 activation
The figure opposite illustrates the activation of the
TLR4 receptor. Binding of ligand leads to dimerization of TLR4 in addition to the binding of MD2 and CD14 which help reinforce this dimerized
structure and allow for the activation of either the
MyD88- or TRIF-dependent pathway. The TRIF- pathway is better suited for antiviral threats whilst the MyD88 pathway is more tailored towards
combatting bacterial and DAMP related threats. TAK-242 binds to the TIR domain of TLR4, not preventing ligand binding but instead preventing further signalling down either the MyD88 or TRIF
pathway. Adapted from Matsunaga et al., 2010. 1.1.4 Transport
1.1.4.1 Extracellular Transport of Alpha Synuclein
Currently the model for transportation of α-syn within the CNS for synucleinopathies is under debate. In MSA in particular, there are still many gaps as to how transportation of α-syn/GCIs may occur and if they are similar or not to other neurodegenerative diseases. As discussed earlier, monomeric/tetrameric α-syn is found in high concentration within presynaptic sites in neurons and have been shown to be synthesised within neuronal cells (Asi et al., 2014). Oligodendrocytes have also been shown to express α-syn under normal conditions, however synthesis of the protein is low and considerably lower than the amount expressed in neurons. In MSA, whilst the expression of α-syn does increase, it isn‟t significantly greater than that of normal conditions suggesting that the large concentration of fibrillar α-syn found in infected oligodendrocytes originate and are transported from an external source i.e. neurons (Asi et al., 2014; Djelloul et al., 2015). Coupling this with the recent finding by Sekiya et al., it appears increasingly likely that the source of oligomeric α-syn are neurons and are later transported to oligodendrocytes for GCI formation (Sekiya et al., 2019). Oligomeric α-syn has been shown to be released at neuronal synapses via Hsp70 chaperone protein in conjunction with DnaJ, providing a possible transportation method (Danzer et al., 2011; Fontaine et al., 2016). This also supports research revealing that higher levels of oligomeric α-syn exist extracellularly in the CSF of patients with synucleinopathies than those without (El-Agnaf et al., 2006). This increase in oligomeric α-syn may also be attributed to release via cell death or apoptosis of affected cells. The issue of transport in MSA is important for a few reasons. As mentioned before the rate of spread of GCIs and fibrillar α-syn in MSA is greater than that of PD. Unlike PD, there are no 18 conventional ways to treat MSA via therapeutic intervention, therefore the importance of determining how transportation of α-syn occurs is that it may provide a possible therapeutic treatment and be able to reduce the rate of spread and degeneration. Whilst spread of some α- syn in MSA may be attributed to release via cell death or chaperone proteins, these release systems are not comprehensive enough to work alone to produce the rate of spread displayed in the disease.
1.1.4.2 Exosomes and Endocytosis
A commonly observed type of transport that is utilised by cells in all tissues, including the CNS, is exosomes. Exosomes are 50-100 nm vesicles which are released by cells into the extracellular environment with the general intention of transport or export of toxic/waste materials for degradation by an immune cell (Théry, Ostrowski and Segura, 2009; Thery, Zitgovel and Amigorena, 2002). An example of the toxic material export was previously stated in the microglia section, whereby microglia engulf and degrade exosomes released by oligodendrocytes (Fitzner et al., 2011). Exosomes are composed of a lipid bilayer, with several structures existing on the outside such as integrins for signalling cells or MHC class molecules, acting as antigen-presenting cells for immune cells (Keller et al., 2006; Fedele et al., 2015; Buschow et al., 2010). The lipid bilayer is typically composed of raft lipids such as cholesterol. Synthesis of exosomes can occur via one of two methods. The first is direct shedding, whereby exosomes are released by budding off the plasma membrane itself. The cellular membrane of the sender cell begins to form a small bubble-like structure on the plasma membrane whereby cargo is then packed before the bubble buds off the membrane into the extracellular environment (Lancaster & Febbraio, 2005). In the second method, cargo is packed in an endosome termed a multi-vesicular body which then pack the cargo into exosomes. The multi- vesicular body then fuses with the plasma membrane, releasing the vesicle/s (Beaudoin & Grondin, 1991). Cargo within exosomes can vary, with some reporting transport of heat shock proteins such as Hsp70 and Hsp90, whilst others have also shown that RNA structures such as mRNA‟s also utilise this system of transport (Valadi et al., 2007; Gibbings et al., 2009; Lancaster & Febbraio, 2005).
One of the proposed normal roles for α-syn was vesicle recycling in the presynaptic terminal of neurons and is no surprise that they have also been seen utilising exosomes as a form of transport in synucleinopathies (Figure 7). Research into α-syn utilising exosomes for transport has predominantly focussed on PD and as such it is difficult to determine whether the same results would be observed in MSA. Oligomeric α-syn has been revealed to utilise exosomes for transport in vitro in both cell lines and primary neuronal cells (Fitzner et al., 2011; Frühbeis et al., 2013a; 2013b). Also revealed was that exosomes containing oligomeric α-syn were more readily taken up by target/neighbouring cells than α-syn fibrils (Danzer et al., 2012). The
19 suggested reason for this was due to the fibrils larger size and thus increased difficulty for cells to effectively endocytose them. The oligomers were also shown to either appear on the inner or outer membrane of the travelling exosomes. Impaired autophagy has also been shown to lead to increased exosome expulsion in SH-SY5Y cells, further supporting the theory of impaired degradation systems in synucleinopathies (Alvarez-Erviti et al., 2011).
Figure 7 – Visual illustration of both forms of exosome synthesis and how α-syn can take advantage of this system for spread
Both forms of exosome synthesis are depicted, the shedding from the membrane (top right) as well as synthesis and release via multi-vesicular bodies. Cargo is internalised into an early endosome before it fuses with a Multi-vesicular body (MVB). Cargo is internalised in the MVB
in a vesicle and will either be released via exocytosis in the vesicle, thus being released in an
exosome or the MVB will fuse with a lysosome. Fusing with the lysosome leads to the degradation of all contents previously within the MVB. Current works describe pathological α- syn as resistant to lysosomal degradation which may help explain how they utilise exosome
release through shedding and multivescular bodies for intercellular spread (Spencer et al., 2016) Entry of α-syn into target cells can occur via a variety of methods. Clathrin-dynamin-1 mediated endocytosis is currently the most fleshed out model. In this model, Clathrin coats small invaginations on the outside of the cellular membrane that allow entry into the cell once exosomes have made contact with it (Robinson, 1994). The invaginations then begin to push deeper into the target cell in an attempt to form an endosome (Figure 7). Dynamin-1 then coils at the end section of the forming endosome, constricting it to allow for the budding of the complex into the cell and to prevent the membrane from leaking into the extracellular environment (Schmid, 1997; Robinson, 1994). This method has been shown to be utilised by
20 neurons, oligodendrocytes and microglia in vitro, and is regularly used for the degradation of material via the fusion of the endosome with the lysosome (Reyes et al., 2013; Desplats et al., 2009; Liu et al., 2007; Lee et al., 2008a). Curiously inhibition of this pathway does not halt entry of α-syn exosomes into target cells, suggesting that it can enter via a variety of methods (Konno et al., 2012). Heparan sulfate proteoglycans on the surface of target cells may also mediate entry of fibrillar α-syn and exosomes via macropinocytosis with the intention of degradation (Holmes et al., 2013). Immunoglobulin proteins such as lymphocyte activating gene-3 are also utilised by neurons as an entry method for exosomes, further complicating how to effectively block entry of α-syn through therapeutic intervention (Mao et al., 2016). Components of endosomes have even been implicated in the entry of the protein. TM9SF2 is a nonaspanin transmembrane protein located on endosomes shown to cause entry of α-syn into target cells (Schimmöller et al., 1998; Wadman, 2016). Entry via the endosome itself may play a role in the notion of lysosomal impairment in synucleinopathies as endocytosis delivers contents to the lysosome (Usenovic et al., 2012; Siebert, Sidransky & Westbroek, 2014). How α-syn may be related to this impairment and if its pathological effects such as oxidative stress exacerbate this issue are still unknown. Exosomes have been shown to be long distance transporters, in some cases transporting contents to different organs and as such this is one of the probable causes of rapid spread of α-syn in MSA. However given that exosomes are shown to also occur in PD, it is unlikely that exosomes function alone as the only model of transport.
1.1.4.3 Tunnelling Nanotubes
Tunnelling Nanotubes (TnTs) are another mechanism used for the transportation of materials into neighbouring cells. TnTs are small tunnel-like protrusions which extend from the host cell and fuse to the plasma membrane of neighbouring cells. Predominantly composed of actin, TnTs have 2 classifications based on length and thickness, each utilised for the transportation of different sized cargo. Thin TnTs are 15-60 µm long with a diameter of 150-200 nm composed solely of F-actin used for small molecule exchange such as transfer of Ca2+(Onfeldt et al., 2004; Agnati & Fuxe, 2014). These TnTs appear to be predominantly used for rapid signalling to neighbouring cells such as the case with neuronal immune cells transferring Ca2+ to stimulate activation (Watkins and Salter, 2005). In contrast thick TnTs, which have a length of 30-140 µm and a diameter greater than 700 nm, are composed of both actin and microtubules, allowing for the transportation of larger cargo such as vesicles, mitochondria and endosomes to name a few (Onfeldt et al., 2004; Agnati & Fuxe, 2014; Koyanagi et al., 2005; Figure 8). Thick TnTs function appears to be more rescue based, transferring essential organelles or cargo to neighbouring cells when in need. An example is mitochondrial rescue, whereby neighbouring donor cells will transfer healthy mitochondria to an acceptor cell with damaged mitochondria in the hope to save them through mitochondrial fusion (Rostami et al., 2017). Transfer through TnTs is mediated via actin-based motor proteins such as Myosin-X and Myosin-Va due to their 21 presence within in vitro models of TnT transfer (Rustom et al., 2004; Gousset et al., 2013; Tardivel et al., 2016). These same factors are also thought to be involved in TnT synthesis, with Myosin-X and –Va absence being accompanied with TnT absence as well.
Figure 8 – Visual representation of materials that utilise TnTs for interceullar spread
Depicted are the different modes by which cargo can be transferred along TnTs. Thin TnTs are
usually reserved for propagation of smaller cellular materials such as RNA or proteins. Thick TnTs are reserved for larger complexes such as mitochondria, which utilise a Motor Adaptor
Complex migrating along microtubule tracks powered by either Kinesin or Dynein. Other transporters such as vesicles may also utilise TnT for intercellular cargo exchange (Ariazi et al.,
2017; Drab et al., 2019).
There has been evidence of prion proteins hijacking TnTs in the brain with the intention of further prion spread (Gousset et al., 2009; Goedert, Clavaguera and Tolnay, 2010). Whilst this prion disease was not MSA, it may be possible that the α-syn strain in MSA may too be capable of performing such an action. To further give credence to this claim, fibrillar α-syn has been shown in vitro to use TnTs to spread between neuronal cells with the intention of degradation via lysosomes (Abounit et al., 2016a). Considering lysosomal dysfunction is a common factor in all inclusion-based neurodegenerative diseases, degradation via this pathway is unlikely. This spread also led to the aggregation of the normal cytosolic protein within the target cell. Studies from the last few years have only corroborated this finding and further reinforce the notion that TnTs are involved in α-syn propagation within synucleinopathies. One such example utilised neuronal cell line SH-SY5Y, which when inoculated with aggregated α-syn displayed TnT- based transfer to primary pericyte cells (Dieriks et al., 2017). Pericytes work along with astrocytes and endothelial cells in the CNS to sustain the blood-brain barrier. Whether α-syn was bound to the actin motor proteins or was bound to an organelle utilising this transfer system
22 was not investigated and is thus unclear. In another study, α-syn was observed to be propagated via TnTs between astrocytes, demonstrating TnT-based α-syn transfer is not neuron exclusive (Rostami et al., 2017). In this instance both lysosomes and mitochondria were dysfunctional due to the presence of α-syn aggregates. Healthy astrocytes formed TnTs to α-syn affected astrocytes, sending healthy mitochondria with the intention to save the damaged ones via mitochondrial fusion. In this same instance aggregates of α-syn spread to the healthy astrocytes which lead to the occurrence of lysosomal and mitochondrial dysfunction (Rostami et al., 2017). In this instance aggregates of α-syn in TnTs were not found to be bound to lysosomes and inhibition of actin polymerization saw reduction of spread. With evidence existing of non- neuronal spread in PD models of disease, it may be possible that this also extends to MSA. TnTs may be used by neurons to directly spread α-syn to oligodendrocytes or perhaps are used by microglia and/or oligodendrocytes with the intention of acquiring the protein for degradation. Evidence does exist of immune cells such as macrophages utilizing TnTs for spread of both cargo and pathological molecules in diseases such as HIV for example (Onfeldt et al., 2006; Eugenin, Gaskill & Berman, 2009; Jackson et al., 2016). Whether such methods also extend to microglia in both normal and disease states is still unknown. TnTs are still a relatively new and unexplored method of transport and whilst their role in synucleinopathies is still currently being fleshed out, it appears increasingly likely they are a factor in the spread of α-syn or GCIs to neighbouring cells.
1.1.4.4 Microglia as a Possible Transporter
A possible consideration is that microglia themselves may act as potential transporters of α-syn in MSA. Microglia themselves possess many factors which would be advantageous as a transporter for α-syn (Kettenmann et al., 2011). As discussed on numerous occasions, microglia‟s difficulty in degrading α-syn fibrils, possibly due to impairment of lysosomes, allows for α-syn to be engulfed by the cells and remain inside uncontested whilst it travels around (Tanik et al., 2013). Microglia exist and survey throughout the brain, meaning that if this does occur then microglia themselves may be unintentional transporters of α-syn in MSA. Mouse model experiments reported in Radford et al. (2015) found that activation of microglial cells not only occurred at the site where aggregated α-syn was inoculated but that it was also observed in more distal sites as well (Radford et al., 2015). Aggregated α-syn was present in these sites, potentially due to microglia transportation. Recently it has been shown that increased microglial activity does promote increased presence of human α-syn in mouse models, with increased protein found within grafted neuronal sites (George et al., 2019). As touched upon previously, senescence is also a factor that may indirectly aid in microglia‟s ability as a transporter due to impaired degradation. A potential result of senescence may be microglial cells engulfing α-syn/GCIs with the intention of degradation but failing thus possessing an internalised DAMP that may be easily transportable. There have also been reports in AD 23 whereby depletion of microglia in conjunction with suppression of exosome release led to significantly less deposition of tau protein in nearby cells (Asai et al., 2015). In this instance an animal model was used and presents evidence that microglia can act as transporters of pathological proteins in disease. Worth noting is that tau protein is a component of GCIs and thus if transportation is exclusive to tau, perhaps similar results may occur from GCIs. Tau is not present in LB and given that a majority of research on synucleinopathies has focussed predominantly on PD due to its prevalence, it may explain why the concept hasn‟t been proposed before. It may also explain the widespread chronic gliosis present in MSA. Another characteristic of microglia that would be beneficial is their plasticity, aiding in transport to different regions of the brain. This change of shape is not only aided by actin but another component in the cytoskeleton, microtubules.
(Figure Description on Next Page)
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Figure 9 – Summary of transportation mechanisms
Details of several methods of α-syn spread, beginning with aggregation witin neurons (I). Spread can occur via a variety of methods such as TnTs (II), exosome release and leakage via necrosis/apoptosis. Depending on which cell receives the released aggregated protein, reactions and methods of spread begin to differ. Oligodendrocytes predominantly appear to receive
aggregated α-syn via endocytosis (III) release aggregates of α-syn via macropinocytosis, in which microglia will later engulf to potentially begin microglial based α-syn spread. Microglia may receive aggregates of α-syn from a variety of sources such as those found within exosomes and those existing in the extracellular environment from necrotic/apoptotic leakage. How
microglia can potentially spread α-syn once acquired is unknown. Astrocytes may also be activated via aggregate α-syn acquisition (vesicles, free, TnT) and can signal for activation of
ramified microglia. Additionally it is thought that spread via the glymphatic system is also a possibility however this is purely speculative at this point (Valdinocci et al., 2017).
1.1.4.5 Microtubules
As a component of the cytoskeleton, microtubules are highly versatile and involved in a multitude of functions including migration, cell division and cell shape control and provide lines of transport for intracellular systems within the cell (Akhmanova and Steinmetz, 2015). They are also found as components of thicker TnTs, associated as tools for intercellular transport as well. They are composed of polymerized α- and β-tubulin dimers and form a hollow tunnel-like structure that can extend as far 50µm, considerably larger than other tunnel-like structures such as TnTs (Kimura and Mizuta, 1994). All microtubules in the cell originate from the microtubule organising centre (MTOC), a structure found exclusively in eukaryotic organisms (Borisy et al., 2016). From here microtubules are nucleated, allowing for their polymerisation in order to form effectively (Moritz and Agard, 2001). As depicted on Figure 10, microtubules have a plus and minus end; the plus end signifying the section that is extending whilst the minus end is anchored to the MTOC (Akhmanova and Steinmetz, 2015). In microglial cells, microtubules are intimately involved in a number of the cells processes. The change of shape observed when microglia alter their state is an example of microtubules involved in a key process, with their structures found to highly acetated and de-tyrosinated in ramified state (Ilschner and Brandt, 1996). Amoeboid microglia are shown to possess lesser levels of post-translational modified microtubules compared to ramified, leading to the structure to be less stable as a result. Presence and treatment of microglia with inflammatory factors such as tumour necrosis factor-α and interleukin-1β have also shown to interact with microtubules, causing the change seen in cell phenotype when microglia become active (Abd-El-Basset, Prashanth and Ananth Lakshmi, 2004). The migration capabilities of the cell are also in part due to the microtubules. Typically in migration the MTOC will position itself opposite the direction the cell is travelling in beside 25 the nucleus however for microglia and other immune cells such as T-cells the opposite was shown (Lively and Schlichter, 2013). Orientation change of the MTOC enhances the migration capabilities of the cell, and this change may be the leading factor as to microglial cells long distance migration when recruited for protection (Dibaj et al., 2010).
1.1.4.6 Epothilones
Epothilones are macrolides which act as microtubule inhibitors via the stabilization of microtubules (Bollag et al., 1995). Microtubules typically exist in a state termed dynamic instability whereby microtubules can either undergo polymerization or depolymerisation quickly and dynamically. This is dependent on whether the α- and β-tubulins in the αβ- heterodimers are bound to GTP or not. Binding of GTP leads to polymerisation and thus growth of microtubules from the MTOC whilst hydrolysation of GTP to GDP leads to shrinkage (Mitchison and Kirschner, 1984; Akhmanova and Steinmetz, 2015). It is dynamic instability which allows for the dynamic nature of microtubules and allows them to perform their roles such as cell shape change, motility and cell division to occur. Epothilones inhibit microtubule activity by polymerising α- and β-tubulin, thus preventing dynamic instability and instead stabilising the microtubule structure (Goodin, Kane and Rubin, 2004). Other chemical compounds such as paclitaxel, a taxol, also work via the same mechanism of action however have difficulty crossing the blood-brain barrier whilst epothilones do not (Fellner et al., 2002; Brunden et al., 2010; Hoffmann et al., 2008). Research on epothilones have predominantly focussed on its role as an anticancer drug, due to its ability to cause mitotic arrest and lead to apoptosis of rapidly dividing cells, however work has also been performed on investigating if it has any beneficial roles in neurodegeneration (Lee et al., 2007; Goodin, Kane and Rubin, 2004).
Figure 10 – Microtubules and MTOC
Figure of MTOC (red circle) within a non-descript
cell. Extending off the MTOC are the microtubules, polymerized via the addition of more α- and β-
tubulins and GTP at the plus end of the structure. Shrinking microtubules are retracting towards the
minus end, with α- and β-tubulins being removed with hydrolised GDP on the plus ends. This allows
for the actions such as plasticity and mobilisation. Adapted from Schaller et al., 2010.
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Epothilone D (EpoD) has been investigated in AD, whereby microtubules in neurons are typically depolymerised due to lack of tau acting on them. Tau protein is a stabilizing agent and in AD is one of two proteins involved in the formation of the diseases inclusion bodies. EpoD displayed promising signs of re-establishing microtubule stability and axonal integrity in affected neurons in transgenic mouse studies, pointing towards a potential agent used for the therapeutic treatment of the disease (Zhang et al., 2012; Brunden et al., 2010). Much like other epothilones, EpoD has also been shown to cross the blood-brain barrier with much more relative ease than other similar acting agents such as taxols (Brunden et al., 2010). Like other pharmaceutical products however, EpoD is not free from side-effects. Low-moderate doses of the drug can be quite cytotoxic to cell cultures in vitro and it can cause grade 3 neurotoxicity leading to symptoms such as nausea, diarrhoea, fatigue, dizziness, abdominal pain (Cheng, Bradley and Budman, 2008; Chou et al., 1998). KOS-1584, an EpoD derivative, does not cause significant neurotoxicity however patients can still present with the same additional symptoms (Cheng, Bradley and Budman, 2008). Interest in EpoD is due to its microtubule-stabilizing properties and the effects it may have on microglia and their ability to engulf as well as how it may affect their motility. Currently there is a lack of literature on the effects EpoD has on immunological cells let alone for microglia. The possibility exists that it will exert no effect on microglia and will not affect the transport capabilities nor the plasticity of the cell, however such claims cannot be substantiated without further evidence.
1.1.5 Mitochondria
1.1.5.1 Mitochondria Normal Role
Mitochondria are an essential component of all eukaryotic cells and are commonly taught and described as the powerhouse of the cell. The mitochondrion is involved in many essential components of eukaryotic cellular function, the most commonly known of which are adenosine tri-phosphate (ATP) production, cell differentiation and regulation of cell death. As an organelle, mitochondria are both structurally and genetically different than others found within the cell which naturally aid in its unique function. Mitochondria possess a double-membrane and this serves a variety of functions, for instance, the critical selection of the materials that are allowed to enter the cell itself. Mitochondria possess over 1,000 internal proteins however can only express <1% thus requiring cytosolic importation (Harbauer et al., 2014). The outer membrane of the mitochondrion (OMM) is dotted with a variety of differing receptors, each of which is responsible for allowing specific precursor proteins and RNA materials to enter the cell to prevent disruption or potential damage to the organelle from unwanted materials. An example of such a receptor is Translocase of Outer Membrane Receptor 20 (Tom20), which will work in conjunction with Tom22 to transport precursor proteins to the TOM40 translocase pore for the import of materials into the mitochondrion (Abe et al., 2000; Kanaji et al., 2000; Wiedemann,
27
Frazier and Pfanner, 2004). Precursor proteins that bind to Tom20 express specific signals termed presequences on their N-Terminus which allows for their identification for importation by said receptors (Harbauer et al., 2014; Neupert, 1997; Saitoh et al., 2007; Wiedemann, Frazier and Pfanner, 2004). Entrance of unwanted materials into mitochondria would disrupt the delicate conditions setup within the organelle to be able to regulate its function. The double membrane also serves to prevent the leakage of proteins and ROS from within the mitochondrion into the cellular cytosol. Leakage of ROS such as superoxide will damage other nearby organelles and ultimately lead to cell death. Mitochondria can allow for such a process to occur intentionally as part of its role in regulation of cellular death, releasing additional factors such as inner membrane molecule cytochrome C to signal for apoptosis (Redza-Dutordoir and Averill-Bates, 2016).
Once materials pass the outer membrane they enter the intermembrane space, the region that sits between the 2 mitochondrion membranes. Entry through the inner membrane to the mitochondrial matrix is similar to that of the outer membrane, with Translocase of Inner Membrane Receptors (TIM) lining the surface. Composed of subunits Tim 17, Tim 21 and Tim 23, the TIM23 Complex is one of the main translocase pores allowing access to the matrix (Demishtein-Zohary and Azem, 2016; Banerjee et al., 2015). OMM is permeable to proteins up to 5 kDa in size whilst the inner mitochondrial membrane (IMM) is impermeable to preserve the electro-chemical gradient required for ATP synthesis and normal matrix function (Redza- Dutordoir and Averill-Bates, 2016). Precursor proteins are folded prior to translocation into the mitochondrial matrix performed by mitochondrial chaperones such as mtHsp70 in conjunction with Tim44 prior to transportation through TIM23 into the mitochondrial matrix (Banerjee et al., 2015). Once within the mitochondrial matrix the protein pre-sequence is cut by mitochondrial protein peptidase (MPP) allowing protein expression to occur (Agarraberes and Dice, 2001; Wickner, 2005). Each of these processes from outer to inner membrane transport, precursor protein folding and cutting require energy in the form of ATP to function. Lining the IMM are 5 complexes making up the electron transport chain which is primarily involved in ATP synthesis, a process which generates ROS as a by-product. These complexes are NADPH Dehydrogenase Reductase, Succinate Dehydrogenase Reductase, Cytochrome C Reductase, Cytochrome C Oxidase and ATP Synthase making up Complex I-V respectively (Guo et al., 2018; Kühlbrandt, 2015). Complex I+II work to reduce molecules such as NADH or succinate generating electrons which serve to power Complex I-IV (Hirst, 2013; Signes and Fernandez- Vizarra, 2018). This generation of electrons also serves as the source of ROS within mitochondria. Complexes I, III and IV pump protons (H+) from the mitochondrial matrix to the intermembrane space creating an electro-chemical gradient required for ATP synthesis, with the intermembrane space possessing a net positive charge whilst the matrix a negative (Hirst, 2013). This dependency on electrons for function has led to Complex I-IV being collectively referred
28 to as the Electron Transport Chain. F0F1 ATP Synthase (Complex V) converts ADP to ATP via proton exchange from the intermembrane space to the matrix, creating an energy source that can be unitized by both transporter channels outside the mitochondrion and the surrounding organelles within the eukaryotic cell (Kühlbrandt, 2015; Signes and Fernandez-Vizarra, 2018). If the IMM was permeable or if Complexes I-IV ceases to function, the electrochemical gradient is lost and thus Complex V‟s ATP generation ceases. This is one of multiple processes involved in cellular respiration and energy generation performed by mitochondria.
Mitochondria are dynamic in terms of structure, possessing the ability to replicate and divide in addition to fusing together to form a larger mitochondrion. These processes, termed mitochondrial fission and fusion, help regulate the function of mitochondria and act as preventatives to them endangering the cell and assist in increased energy generation respectively. Both of these functions are performed by GTPases Dynamin-related Protein-1 (Drp1), Mitofusin 1+2 (Mfn 1+2) and Optic Atrophy Protein 1 (OPA1). Driving fission is Drp1 located within the cytosol and are recruited to the mitochondria by factors such as Fission-1 and Mitochondrial Fission Factor, both of which are on the OMM surface (Otera et al., 2010; Yoon et al., 2003). Drp1 encircle the mitochondrion in a 120 nm wide spiral which when constricted, causes the budding of a single mitochondrion into 2 or more smaller mitochondrial bodies (Ingerman et al., 2005; Youle and van der Bliek, 2012). Unlike mitochondrial fusion, no factors are currently known to assist fission for the inner compartments of the mitochondria which may help explain the discrepancy in width between Drp1 and the 50nm structures observed by dynamin-1 (van der Bliek, Shen and Kawajiri, 2013; Mears et al., 2010). Mitochondrial fission serves several purposes, one of which is to separate and segment damaged areas of the mitochondrial into smaller bodies for degradation (Scott and Youle, 2010; Westermann, 2010). Mitochondrial damaged typically presents in singular areas as opposed to throughout thus sectioning off problematic areas for degradation saves from having to degrade the entire organelle, especially considering if it is mostly fully functional. Additionally mitochondrial fission works in conjunction with cellular division, segmenting mitochondrion into smaller mitochondrial bodies to provide newly replicated cells an energy source (Cantó, 2018; Westermann, 2010).
Conversely mitochondrial fusion is the process of fusing smaller mitochondrion bodies to create a larger one. Driven by OPA1 on the IMM in addition to Mfn1+2 on the OMM, this process allows for greater energy production due to the increased area and capacity of mitochondria to store materials for ATP synthesis (Song et al., 2009). Mfn 1+2 drive the fusion of outer membranes of separate mitochondrial bodies, however whilst both are required for fusion to occur, each appear to perform this function differently. For instance Mfn1 is more highly dependent on GTP than Mfn2 insinuating that Mfn1 will fuse mitochondria under circumstances whereby GTP is plentiful whilst Mfn2 in others when it is not (Ishihara, 2004). Mfn‟s tether to 29 begin fusion, requiring that both mitochondrial bodies in question have Mfn present on the OMM (Hoppins and Nunnari, 2009). Loss of either leads to several deficiencies such as loss of mitochondrial networks with Mfn1 or development of diseases such as Charcot-Marie-Tooth disease type 2A, AD, endoplasmic reticulum stress and more with Mf2 loss (Santel et al., 2003; Park et al., 2015; Ngoh, Papanicolaou and Walsh, 2012; El Fissi et al., 2018; Chen, Chomyn and Chan, 2005). Additionally OPA1 is highly reliant on Mfn1 for fusion of inner membrane of connecting mitochondria, loss of which leading to dysfunction in ATP synthesis efficiency (Cipolat et al., 2004; Olichon et al., 2003). In addition to fusing IMM‟s, OPA1 regulates the shape of the mitochondrial bodies preventing it from forming irregular structures (Cipolat et al., 2004). In addition to creating larger mitochondria to satisfy energy demands, mitochondrial fusion acts as a rescue mechanism for damaged mitochondria. Through the same OPA1 and Mfn1+2 fusion process, healthy mitochondria will fuse with a damaged one attempting to rescue it from potential eruption (Twig and Shirihai, 2011). Damage to mitochondria occurs over time as a consequence of aging, mitochondrial DNA is more sensitive than other forms and exposure overtime to ROS leads to mutations (Shokolenko et al., 2009). Rescue by mitochondrial fusion attempts to mitigate this and prevent potential release of matrix ROS into the intermembrane space and beyond. In instances where damaged mitochondria cannot be rescued by mitochondrial fusion, a regulated degradation process termed mitophagy takes place.
Given the materials internalized within mitochondria such as ROS, damaged mitochondrion are at risk of leaking said materials into the cellular cytosol leading to potential cellular necrosis, hence the degradation of mitochondria require regulation to prevent this. The process of mitophagy is typically undertaken on damaged mitochondria, those that are unable to be rescued via mitochondrial fusion. A variety of differing regulators exist to control this process, one of main concern for PD and possibly MSA are Parkin and PTEN-Induced Kinase 1 (PINK1). Both of these proteins belong to the same degradation pathway, with the actions of PINK1 influencing if Parkin will interact with mitochondria. PINK1 functions as the initial signaller for mitophagy by acting as a damage sensor of healthy mitochondrial activity (Narendra et al., 2010a). PINK1 is imported into the mitochondria via TOM and TIM pores, and once imported will be cleaved by mitochondrial processing peptidase (MPP) and presenilin-associated rhomboid-like protein (PARL) (Greene et al., 2012; Jin et al., 2010; Meissner et al., 2011). This cleavage process however will only occur in normally functioning mitochondria thus cleaved PINK1 acts as a signal that the mitochondria is healthy. If PINK1 is unable to be imported into the IMM it will instead accumulate on the OMM in its uncleaved form which in turn signals for the recruitment of Parkin (Okatsu et al., 2013; Lazarou et al., 2012). Located in the cytosol, Parkin is a ubiquitin ligase, binding ubiquitin to cellular materials such as Mfn1+2 thus marking them for degradation via autophagy (Gegg et al., 2013). In relation to mitochondria, Parkin will ubiquitase them when detecting significant differences in their membrane potential, signalling
30 the damaged mitochondria for degradation via mitophagy (Narendra et al., 2008). Inactive Parkin is recruited to mitochondria based on PINK1 accumulation on damaged/dysfunctional mitochondria, from which PINK1 then phosphorylases Ser65 on Parkin to activate its ubiquitinase activity (Matsuda et al., 2010; McWilliams et al., 2018). Ubiquitin chain accumulation on mitochondria signals for factors involved in autophagosome recruitment such as p62 for the degradation of the dysfunctional mitochondria (Narendra et al., 2010b). Both of these regulators are affected in PD, leading to a dysfunction of mitophagy not too dissimilar to that occurring within lysosomal based autophagy. In dopaminergic neurons under PD conditions the presence of damaged mitochondrial bodies is increased, thought to be due to a loss or impairment of mitophagy. This is further discussed in 1.5.4.
1.1.5.2 Role of Mitochondria in Neurons, Microglia and Astrocytes
Neurons, much like other cells, are highly dependent on mitochondria for both survival and conservation of proper cellular function. As briefly discussed in section 1.2.2, neurons fire regular action potentials along myelinated axons which in turn signal for vesicular packaging and neurotransmitter release at the presynaptic terminal. Along the axon at the Nodes of Ranvier are ion-gate channels for Na+, K+ and Ca2+, all of which require ATP to function (Poliak and Peles, 2003). Firing of an action potential causes internalisation of Na+ and Ca2+ into the neuronal cytosol whilst simultaneously releasing K+ into the extracellular environment and it is this reaction that allows for the propagation of the signal to reach the synapse (Bean, 2007). Without ATP the ion-gated ATPases cease to function and thus action potential signals are either reduced or cannot occur. These ATPases are also involved in governing the ionic gradient of the cell, preventing Na+/Ca2+ accumulation or loss of K+ to retain neuronal excitability (Bean, 2007). This has been described to account for roughly 60-80% of ATP consumption within the CNS (Kann and Kovács, 2007). Mitochondria also serve to reduce cytosolic concentration of Ca2+ via import into the organelle. The voltage-dependent anion channels (VDAC) on the mitochondrial outer membrane regulate Ca2+ entry into the inter-membrane space whilst the Mitochondrial Calcium Uniporter (MCU) is the main channel associated with entry past the inner membrane (Gunter and Sheu, 2009; Shoshan-Barmatz et al., 2006; Shoshan-Barmatz and Gincel, 2003; Vos, Lauwers and Verstreken, 2010). Additionally ATP consumption occurs at presynaptic terminals for the process of neurotransmission which accounts for both the synthesis of the neurotransmitters as well as their packaging within vesicles for synaptic release (David and Barrett, 2003). Increased presence of mitochondria is also thought to aid in synaptic plasticity via long-term size increases, strengthening the synapses allowing for greater function (Mattson, Gleichmann and Cheng, 2008). Synaptic transmissions see an increase in the amount of Ca2+ that enters the synapse, preventing rapid synaptic signalling from occurring. In normal conditions export of Ca2+ to normal levels would be required for signalling to re-occur however higher concentration of mitochondria in synapses are shown to internalise Ca2+ to allow for a 31 more rapid recovery from synaptic signalling and thus allow for increased neurotransmitter release (Billups and Forsythe, 2002; David and Barrett, 2003; Talbot, David and Barrett, 2003).
The role of mitochondria in microglia within normal neurological conditions is not well understood. It is known that ATP and proper mitochondrial respiration is a requirement for the cells survival, however this is a requirement of most eukaryotic cells and thus not unique to microglia. As previously discussed in sections 1.3.4 and 1.3.5, PAMP/DAMP activation of TLR4 receptors induces synthesis and release of pro-inflammatory factors. This activation in macrophages however is accompanied by ROS generation in the mitochondria due to TRAF6 (Chandel et al., 2000; Chandel, Schumacker and Arch, 2001; West et al., 2011). Activation of TLR4 leads to TRAF6 translocating to the mitochondria to bind with Evolutionarily Conserved Signalling Intermediate in Toll pathways (ECSIT) which in turn then binds to Complex I on the inner mitochondrial membrane to stimulate increased ROS production (West et al., 2011). This ROS production is then thought to aid in further synthesis of additional pro-inflammatory factors. This result however was described in macrophages and thus is currently unknown if a similar reaction occurs within microglia (Weinberg, Sena and Chandel, 2015). Whilst microglia and macrophages both share many similarities including being the immediate barrier of defence in their respective systems as part of innate immunity, without confirmation increased ROS generation through activation is only speculative. Recently work has shown that reactive microglia can shorten their mitochondria when energy demands of the cell require it. Stimulation with LPS caused increased activation of Drp1 involved in mitochondrial fission which in turn allowed for greater ROS generation in the shortened mitochondria (Katoh et al., 2017).Whilst it doesn‟t outright confirm that the same process that occurs in macrophage activation is consistent in microglia, it does reveal that mitochondria or at the very least their dynamics do have some role to play in activated microglial cells. Additionally regulation of activation appears to be controlled via mitophagy as lack thereof led to chronic activation of microglial cells (Ye et al., 2017).
Similar to microglia, mitochondrial functions within astrocytes are still largely unknown. Mitochondria are found in the fine processes exuding from the main cellular body not too dissimilar to those seen in oligodendrocytes or dendrites in neurons (Derouiche, Haseleu and Korf, 2015; Rinholm et al., 2016). The amount of mitochondrial movement to these processes is lesser than those in dendrites, insinuating lesser dependence on ATP processes in these regions based on channel energy requirements compared to dendrites (Jackson et al., 2014; Stephen et al., 2015). One of the normal functions of astrocytes is the regulation of glutamate, an excess of which can cause degeneration of neurons. Dysfunction of mitochondria has been shown to lead to increased levels of glutamate in the extracellular environment and a reduction of uptake by astrocytes (Murru et al., 2019; Fiebig et al., 2019). Astrocytic processes contain glutamate channels for uptake implying mitochondrial dependency to function. Additionally astrocytes 32 have been shown to donate mitochondria following stroke, acting as a safety-net of sorts to prevent neuronal cell death (Hayakawa et al., 2016). Based on these reports it would appear that functioning mitochondria are required to maintain the neuroprotective activities of astrocytes.
1.1.5.3 Mitochondrial Transport
Migration of mitochondria is a regularly occurring function within the CNS. As previously discussed, increased presence of mitochondria at the presynaptic terminal greatly aids in more rapid and continuous neurotransmitter release. This mitochondrial presence however is not constant, and is common to find mitochondria shifting between the soma, dendrites and presynaptic terminal depending of which is in greater need of energy (van Spronsen et al., 2013). Mitochondria themselves do not possess the ability to migrate and thus rely on a motor adaptor complex to shift them. The current most understood motor complex is trafficking kinesin protein (TRAK)/Milton which belong to humans and drosophila respectively. These complexes will slightly differ on antero- or retrograde migration. The anterograde motor complex is dependent on kinesin for transportation and is primarily composed of TRAK1 (OIP106) bound to KIF5 and/or KIF1B, heavy chain units of the motor protein kinesin-1 (Randall, Moores and Stephenson, 2013). This motor complex itself is then bound to microtubules and will shift the mitochondria toward the presynaptic terminal, the plus end of the microtubule (Brickley and Stephenson, 2011; van Spronsen et al., 2013). Alternatively the retrograde motor complex is dependent on Dynein motors for transportation and sees TRAK2 (OIP98/Grif-1) bind to dynein through KIF5A to shift mitochondrial migration toward the minus end of the microtubule, toward the soma (Brickley and Stephenson, 2011; Onodera et al., 2018; Randall, Moores and Stephenson, 2013). This same dynein motor system is also involved in mitochondrial migration toward dendrites as well (van Spronsen et al., 2013). The α-helices of the TRAK1/2 N-Terminal exist in a coiled coil motif which allows it to easily bind to the HAP1 domain found in the kinesin and dynein motor complexes. Transportation of mitochondria along actin is also possible and is the main transportation system witnessed in TnT based transport of mitochondria. For the most part however actin-based transport is this is typically utilised for short distance (Thick TnT = 30-140 µm) migration whilst microtubule is specialised for longer distance (Axon = 1000 µm-1 m). A specific adaptor protein in the form of Miro1 however is required for mitochondria to bind to the TRAK1/Kinesin or TRAK2/Dynein Motor complexes.
Miro1, also known as RHOT1, is a major adaptor protein located on the surface of the outer mitochondrial membrane. More specifically Miro1 is a Ras GTPase belonging to the Rho subgroup. 618 amino acids in length, it includes 2 GTPase domains in addition to EF hands whose co-ordination is dependent on Ca2+ (Fransson, Ruusala and Aspenström, 2006). The Miro1 C-Terminal is embedded within the mitochondrial transmembrane while its N-terminal,
33 its GTPase domain, exposed for binding. Miro1 is the bridge that connects mitochondria to the TRAK-kinesin/dynein motor complexes, acting as a tether thus allowing for the transportation of the organelle to occur (Fransson, Ruusala and Aspenström, 2006; MacAskill et al., 2009). Without it presynaptic terminals would not meet the energy requirements for neurotransmitter release nor would damaged mitochondria be able to return to the soma for controlled mitophagy or rescue via fusion. Miro1 is shown to be capable of binding to either TRAK1/2 or directly to the kinesin/dynein motor protein itself (MacAskill et al., 2009). The site at which Miro1 binds to is reliant on cytosolic Ca2+ and regulates whether transportation of the mitochondrion can occur (Saotome et al., 2008). High cytosolic concentrations of Ca2+ reveals Miro1 to directly bind to the motor protein and this interaction prevents the connection between kinesin/dynein and microtubules from occurring (Wang and Schwarz, 2009). Conversely in lower Ca2+ concentrations Miro1 binds to TRAK1/2 instead, promoting transportation to occur. This co- ordination by the Ca2+ regulated EF hands has been termed inactive and active states respectively (Wang and Schwarz, 2009). Mitochondrial docking along the axon is also partially regulated by Miro, a process beneficial to provide ATP ion-gate channels at the Nodes of Ranvier in addition to as a mechanism of mitochondrial transport control. Syntaphillin, docking protein located along neuronal axons, interacts with kinesin-1 on the motor/adaptor complex pending on Miro1 binding state to it (Kang et al., 2008; Chen and Sheng, 2013). Presence of Ca2+ drives Miro1 to relinquish hold on the motor complex, allowing for syntaphillin to bind to kinesin-1 to act as a brake, halting anterograde movement and Ca2+ levels are normalised once more.
Miro1‟s role in migration is not restricted to anterograde for synaptic energy management. Mitochondrial retrograde movement in neurons is also utilised as a rescue method, returning any damaged mitochondria present from along the axon or synaptic regions for repair via mitochondrial fusion. As previously mentioned, mitochondrial fusion is regulated by both Mitofusin1 and 2 on the outer mitochondrial membrane and Opa1 on the inner membrane, with the intention of rescuing damaged mitochondria through fusion with healthier mitochondrial bodies (Hoppins and Nunnari, 2009). Mitofusin2, in addition to Miro1 is implicated in axonal transport of mitochondria as down-regulation or lack thereof is associated with disproportionate mitochondrial distribution along the axon (Misko et al., 2010). Mitofusin2 is associated with the Miro1/Trak motor-adaptor complex, found to bind to it as aids in facilitation of proper axonal transportation. Miro1 appears to also be associated in fashion to mitochondrial homeostasis. Overexpression of Miro1 leads to presence of both condensed and elongated mitochondria whilst silencing promoted mitochondrial fragmentation (Saotome et al., 2008). The elongation and fragmentation appeared to be based on interactions with Drp1, acting to supress it and thus mitochondrial fission. The presence of condensed mitochondria is unexplained and the authors
34 speculate it may be a confirmation that Miro1 interact with other homeostasis regulating enzymes outside Drp1 (Saotome et al., 2008).
Whilst the bulk of information on Miro1 is based on its role within neurons, its presence in the CNS appears to not be exclusive to them. In astrocytes for example Miro1 aids in the transportation of mitochondria to processes for regulation of neuronal synaptic activity (Stephen et al., 2015). In contrast to neurons however, the number of mobile mitochondria and the speed at which they travel is greatly reduced compared to neurons (Jackson et al., 2014; Stephen et al., 2015). Comparative information on oligodendrocytes and microglia however is absent. Mitochondria are present within both the cellular body and myelin sheaths of oligodendrocytes but how they are transported to them, is currently unknown (Rinholm et al., 2016). As previously mentioned information regarding microglial function in microglia is scant, a trait that further carries into the role of Miro1 and mitochondrial transport. In addition to its role as an adaptor and regulator of mitochondrial axonal transport, Miro1 is implicated in TnT based transport as well. Presence of Miro1 has been shown within in vitro cultures whereby mitochondria were transported via TnTs to for rescue. The most recent of examples showed mesenchymal multipotent stromal cells (MMSCs) forming TnTs with astrocytes that had suffered an ischemic attack. This attack elevated the amount of ROS generated within the astrocytes leading to mitochondrial damage (Babenko et al., 2018). Mitochondria guided by Miro1 migrated along TnTs from the MMSCs to affected astrocytes in an attempt to rescue the damaged mitochondria from respiration based damage. A similar process was previously reported by authors when co-culturing MMSCs with neurons under the same ischaemic attack based circumstances (Babenko et al., 2015). MMSC mitochondria bound to TnTs transport complex through Miro1 migrated toward the neurons for attempted rescue. Both these circumstances implicate Miro1 as a major factor in the facilitation of mitochondrial transport, both intracellularly and intercellularly.
1.1.5.4 Role of Mitochondria in synucleinopathies
The interactions of α-syn with mitochondria help shed some light on the presentation of certain characteristics of synucleinopathies (Figure 11). For instance α-syn has been demonstrated to bind to and inhibit the actions of TOM20, preventing it to co-localise with TOM22 and thus reducing the efficiency of protein import via the TOM40 pore (Di Maio et al., 2016). Furthermore this characteristic was recently revealed to be performed by only pathological α- syn species (Wang et al., 2019). In combination with α-syn‟s ability to inhibit Complex I activity, these factors lead to a compromise in mitochondrial respiration contributing towards a decline in ATP generation (Chinta et al., 2010; Reeve et al., 2015). Given how crucial ATP is towards maintaining neuronal signalling, reductions in energy generation have consequential effects on the efficiency of the signal. In PD alone this effect is disastrous especially in later
35 stages of the disease when fewer neurons are able to synthesize and release DA. However in MSA this decline in neuronal signalling in combination with a reduction in axonal signalling insulation by loss of myelin elevates this to almost catastrophic. Whilst the generation of α-syn inclusion bodies is almost exclusive to oligodendrocytes, pathological α-syn species are still present within neurons driving degeneration (Sekiya et al., 2019). α-Syn‟s role in oligodendrocytes within MSA is still unknown. There have been reports of aberrations of mitochondria in presence of GCIs as well as elevations in the level of oxidative stress however details beyond this are scant (Shults et al., 2005; Stefanova et al., 2005). With regards to neurons, it‟s interactions with α-syn appear universal between MSA and PD.
α-Syn appears to influence mitochondrial dynamics due to increased prevalence of fragmented mitochondrial bodies (Pozo Devoto and Falzone, 2017). A53T α-syn reduced the presence and expression of Mfn1+2 up to 50% within the 12 months post-injection, leading to smaller mitochondrial bodies and thus significantly compromising energy generation (Xie and Chung, 2012). Whilst a reduction in Drp1 was also observed in this model, this reduction only materialised once α-syn presence had reached the terminal stage, implying that it was more of a consequence of the mice dying as opposed to α-syn itself. α-Syn can directly bind to mitochondrial lipids such as sodium dodecyl sulfate causing curvatures on the organelle which is thought to reduce the efficiency of mitochondrial fusion however this is still unconfirmed (Braun et al., 2012; ; Perlmutter, Braun and Sachs, 2009; Pozo Devoto and Falzone, 2017). The combination of α-syn inhibiting factors involved in ATP generation in addition promoting fission over fusion both fit the characteristics displayed in Parkinsonian cases. Mitochondrial elongation is triggered with knockout of α-syn, with factors such as PINK-1 and Parkin shown to rescue the mitochondria and promote fusion once α-syn is gone in Caenorhabditis Elegans (C. Elegans) models revealing that α-syn actions are reversible (Kamp et al., 2010).
Mitophagy, much like other degradation pathways observed in other cells, is impaired in presence of α-syn. Understanding of α-syn‟s role in relation to mitophagy is still quite limited given that most models used to study this lack in vivo examination or corroboration, however a general trend of mitophagy inhibition is consistent throughout literature. Several factors seem to coalesce for this to occur. Autosomal recessive forms of familial onset PD occur when Parkin and PINK-1 are mutated (Valente et al., 2001; Mata et al., 2004; Kumar et al., 2017). PINK-1 mutants in Drosophila PD models prevents Parkin-mediated mitophagy from occurring due to Parkin‟s reliance on PINK-1 to phosphorylate both itself and ubiquitin (Clark et al., 2006). Whilst this effect could be reversed when upregulating Parkin, this dependency reveals the ease in which this degradation system can fail. Additionally PINK-1 knockouts in models such as SHSY-5Y neuroblastoma cells had increased difficulty in respiration operations leading to increased presence of ROS and cytosolic Ca2+ (Gandhi et al., 2009; Wood-Kaczmar et al., 2008). Parkin mutants in Drosophila models presented morphological changes to dopaminergic 36 neurons, likely due to changes in energy generation from mitochondria (Greene et al., 2003; Cha et al., 2005). PINK-1 and Parkin mutations within fibroblasts revealed the importance of Mfn1+2 ubiquination for the commencement of mitophagy, with lack thereof generating similar results as to all the previous ones discussed (Rakovic et al., 2011). A recent hypothesis has proposed that loss-of-function mutations in conjunction with α-syn presence as a potential cause of PD and other Parkinsonian diseases (Cooper et al., 2018). Tested using C.Elegans, recessive loss of function mutation effects were tested such as PINK-1 both in presence of and absence of wild-type α-syn, with α-syn exacerbating the response produced. Considering PINK-1‟s role as an α-syn clearance mechanism when it‟s bound to mitochondria, such a theory does contain merit (Liu et al., 2017). These loss-of-function mutations are likely a consequence of aging which further explains the prevalence of Parkinsonian diseases in older populations. Parkin and Pink-1 independent forms of mitophagy appear impaired by α-syn presence such as the recently revealed mitospheres. Formed by Drp1 fission, mitospheres are round mitochondrial bodies formed prior to Caspase-3 activation when mitochondria produce excess ROS (Menges et al., 2017). Formation of these mitospheres appears to be due for degradation which is independent of Parkin (Menges et al., 2017). α-syn prevented the formation of mitospheres which the authors curiously described as a protective mechanism. The reasons for this claim are due to α-syn rescuing the cell from apoptosis due to inhibition of mitosphere formation. α-Syn‟s interactions with mitochondria within Parkinsonian diseases have shown it to inhibit mitophagy for the continued generation of ROS and release of calcium in PINK-1 and Parkin dependent mitophagy pathways. Mitospheres formation is dependent on Drp1 and previous work by Xie and Chung in 2012 revealed Drp1 reduction in α-syn presence, however it was proposed to be a consequence of the organism dying as opposed to an effect elicited by α-syn itself (Xie and Chung, 2012). Whether there is a potential correlative link between these 2 factors is currently unknown. Perhaps α-syn does act in a protective manner prior to misfolding which may further illustrate the further complexities of α-syn‟s role within the CNS both before and during disease.
Mitochondrial migration and transportation complexes are other factors with reduced functionality once α-syn becomes pathological. Prior to axonal degeneration, both anterograde and retrograde mitochondrial traffic is arrested within neurons with increased α-syn presence (O'Donnell et al., 2014). Within nigral dopaminergic neurons affected by sporadic PD, anterograde trafficking is affected in the early stages followed by retrograde in the later stages (Chu et al., 2012). This type of degeneration pattern has been described as a “dying back” pattern. As previously mentioned, the trafficking of mitochondria is vital to retain the electrochemical balance required for action potential generation in addition to providing energy for channels releasing neurotransmitters at the axonal terminals. Various methods have been demonstrated as to how α-syn disrupts this trafficking such as microtubule fragmentation, increasing tau expression or severing the connection between kinesin-1 motor complex and
37 microtubules directly (Melo et al., 2017; Prots et al., 2013; 2018). Interestingly many of these methods described do not appear to work in conjunction with each other. For example Melo and colleagues describe α-syn oligomers fragmenting microtubules structure however from a retrograde to anterograde direction, a “dying forward” pattern as opposed to the “dying back” described by Chu et al. (Melo et al., 2017; Chu et al., 2012). This may perhaps allude to the differences in trafficking arrest methods performed by differing α-syn species, with Melo et al., reporting their findings based on α-syn oligomer action whilst Chu et al., on Lewy Neurites and α-syn positive inclusion bodies. This may perhaps shed a light on the differences in disease profiles between synucleinopathies, serving as another potential criterion to further distinguish them. In addition to α-syn, other Parkinsonian proteins negatively affect mitochondrial trafficking for instance 1-methyl-4-phenylpyridinium (MPP+, not to be confused with Mitochondrial Protein Peptidase, MPP). MPP+ is a neurotoxin present in PD inhibiting the actions of Complex I however appears to negatively affect kinesin-1 transport whilst promoting retrograde transport based on differences of observed mitochondrial migration rates and released neurotransmitter concentrations (Kim-Han, Antenor-Dorsey and O'Malley, 2011; Morfini et al., 2007). Interestingly both papers observed a “dying back” degeneration pattern in their models. Once more this highlights the growing complexity in Parkinsonian disease treatment, with an alarming number of factors to consider for future potential therapies.
Another factor which highlights the complexities of α-syn interactions is Miro1. As expected, the role of Miro1 as a bridge between mitochondria and the motor transport is compromised once levels of pathological α-syn begin to rise. In addition to interactions with mitochondria leading to excess ROS generation, α-syn will also interact with the endoplasmic reticulum (ER) which is a key proponent in controlling the Ca2+ homeostasis within the mitochondria and the cellular cytosol (Calì et al., 2012; Parihar et al., 2008). Ca2+ is a key proponent in α-syn aggregation and inclusion body formation thus α-syn‟s interactions with the ER promote release of Ca2+ into the cytosol and uptake by mitochondria (Calì et al., 2012). Miro1 function in transport is highly dependent on cytosolic Ca2+ concentrations, with Ca2+ eliciting an inhibitory effect at higher concentrations (Saotome et al., 2008; MacAskill et al., 2009; Wang and Schwarz, 2009). Higher concentrations see Miro1 directly bind to motor protein thus arresting all mitochondrion trafficking. Interestingly however is Miro1‟s unexpected upregulation in the presence of α-syn, likely as the proteins secondary role as a mitophagy inhibitor. It has been recently revealed that Miro1 protects mitochondria from mitophagy thus allowing unregulated ROS generation to occur (Shaltouki et al., 2018). In normal conditions, Miro1 is phosphorylated by PINK-1 to commence degradation however in synucleinopathies with loss of PINK-1 function this degradation is unlikely to occur, further supported by reports of increased Miro1- dependent activity such as trafficking in PINK-1 knockout models (Liu et al., 2012; Shlevkov et al., 2016; Wang et al., 2011). A possible explanation for Miro1‟s increased traffic activity may
38 perhaps be disease stage related. Miro1‟s loss of trafficking is dependent on cytosolic Ca2+ concentrations however α-syn presence does not immediately give rise to a significant increase in this concentration. It seems likely that Miro1‟s role as mitochondrial transporter is unaffected in the earlier stages of pathological α-syn presence however as the disease progresses, intracellular Ca2+ rises acting as the catalyst of Miro1‟s role change. Whilst monomeric α-syn can cause changes in intracellular Ca2+ concentrations, oligomeric α-syn was found to enable it consistently, insinuating it as a synucleinopathy disease mechanism (Angelova et al., 2016).
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Figure 11 – Summary of mitochondrial dynamics within neurons in synucleinopathies
Figure summarises various different dynamics of mitochondria in neurons within the CNS. Top right depicts the fission/fusion of mitochondria, whereby presence of α-syn oligomers promotes mitochondrial fission whilst fusion is down-regulated. This raises production of ROS within mitochondrial bodies, which in normal conditions would begin the signalling process for mitophagy however α-syn pathological species inhibit this function. This is partially due to reduction in PINK1 and Parkin signalling, depicted top left, from occurring once more due to interference of pathological α-syn. Oligomers and post-translational mutants in turn bind to and inhibit Tom20, further raising ROS production within mitochondria. Additionally α-syn oligomers also affect migration of mitochondria. Oligomers can directly interact with microtubule tracks utilised by motor proteins, disassembling them thus preventing kinesin/dynein motors from mobilising mitochondria. Presence of pathological α-syn sees rise of intracellular [Ca2+] whereby Miro1 can then directly interact with motor protein to inhibit their activity. This same action can also inhibit activity of mitophagy promoters such as PINK1, further complicating α-syn’s relationship with mitochondria. Lastly on the left is a proposed method of intercellular α-syn transport whereby pathological α-syn species bind to migrating mitochondria within TnTs. Based on previous reports of α-syn high jacking other organelles for intercellular transport through TnTs such as lysosomes, the potential for such a method may exist (Valdinocci et al., 2019).
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1.2 Project Aims Overview
Currently there are still gaps in our understanding of how α-syn may propagate within the brain in typical and atypical PD. Depending on the type of synucleinopathy, factors including rate of spread, site of origin, rate of degeneration and sites of infection influence disease propagation. The overall aim of this project was to investigate potential mechanisms of α-syn spread in order to develop novel targets to slow disease progression. Specific aims were to:
- Investigate microglia as potential transporters of pathological α-syn. As highlighted within the literature review, microglia possess many traits which could enable them to function as transporters in atypical Parkinson‟s such as MSA in which α-syn inclusion body pathology and neurodegeneration is accompanied by widespread chronic neuroinflammation. These traits include a high degree of plasticity aiding in their mobility, inability to effectively degrade the protein once engulfed and inflammatory reactant release aiding in the recruitment of more cells. Evidence of microglia acting as a transporter in MSA has not been reported previously, however animal studies have shown that microglia could play a role in the transportation and propagation of pathological protein species such as tau in AD. For this aim, covered in Chapters 2 and 3, studies using post-mortem MSA tissue and in vitro cell culture investigate interactions between α-syn and microglia and whether they could promote for α-syn migration.
- Investigate TnTs as a mechanism of α-syn propagation, focussing on the potential role of mitochondria. Prion proteins have been reported in the past within other neurodegenerative diseases to hijack processes that can promote their propagation and transfer. One such mechanism utilises TnTs, with recent reports of α-syn bound to migrating organelles such as lysosomes within these structures. Mitochondria have been described in various reports to employ TnTs in healthy conditions for exchange between cells. Studies on the role of mitochondria in synucleinopathies has previously focussed on the contribution to neurodegeneration through increased ROS generation and calcium regulation. As pathological α-syn can transfer to adjacent cells by being chaperoned by lysosomes migrating through TnTs, whether mitochondria can mediate cell-to-cell transfer of a-syn was investigated (Chapter 4) using a variety of neural cell models.
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1.3 Specific Aims and Hypotheses
1.3.1 Chapter 2 - Epothilone D Inhibits Microglial-Mediated Spread of Alpha Synuclein Aggregates
1.3.1.1 Post-Mortem MSA Tissue Analysis
Examination of fluorescently stained post-mortem brain samples of MSA patients to closely study for any interactions between microglia and oligodendrocytes, primarily those with GCIs. This study investigated whether any information can be gleamed about these two cell types with relationship to α-syn transfer and migration. Additionally, sections were examined for any evidence in the tissue of microglial cells migrating with internalised α-syn distal to GCI-affected oligodendrocytes. Fluorescently stained for Iba-1, nuclei and α-syn.
Alternate Hypothesis 1a - There is evidence to suggest that microglia and oligodendrocytes interact with each other in the presence of α-syn. Additionally there is evidence to suggest microglia can act as a transporter α-syn within MSA.
Null Hypothesis 1a – There is no evidence suggesting microglia and oligodendrocytes interact in the presence of α-syn. No evidence of microglia acting as transporters of α-syn in tissue.
1.3.1.2 Alpha-Synuclein Mobilization Assay
To investigate whether microglial cells possessed the ability to migrate with α-syn, an assay was designed in which the α-syn displacement from a single region could be measured when exposed to cells. Additionally this assay was designed to consider the measurement of the amount of cells with internalised α-syn at the sites most distal to the α-syn region. In this assay, α-syn was spotted and dried on the centre of a coverslip before placed in a 24-well plate with cellular media, whereby cells were then loaded into the wells containing coverslips. Following a 48-hour incubation period the samples coverslips were removed to be fixed and fluorescently stained. This assayed quantified two factors. The first was the amount of residual α-syn in the centre of the coverslip based on the fluorescent intensity generated by the spot following staining. Higher fluorescent intensity denoted by mean gray value (MGV) meant higher residual protein amount and vice versa. The second factor quantified was the proportion of cells at specific regions at the coverslip edge with internalised α-syn. The coverslip edge was chosen as it is the region most distal from the centre of the coverslip, the same location α-syn was spotted. If a reduction in MGV occurred at the central region it indicated removal of α-syn from the spot by exposed cells. If these cells possessed the ability to migrate with internalised α-syn, their presence would be determined via the proportion of α-syn positive bearing cells at the distal edges. Thus a higher MGV indicates less removal by cells and in turn a lower proportion of α-
42 syn positive cells at the coverslip edge and vice versa. Fluorescently stained for Iba-1, nuclei and α-syn.
Different conditions were tested using this assay including the spotting of different α-syn types (monomer and aggregated) and loading of different cells types. The purpose of spotting different α-syn types was for examination of differences to cell load response, that is if cells reacted differently pending to the type of α-syn they were exposed to. It was hypothesised that wells bearing aggregated α-syn would show lower MGV and higher cellular proportions at the coverslip edge due to aggregated α-syn being the pathological form found in synucleinopathies such as MSA and PD, unlike monomer which is the normal form. Different cell load conditions were also tested which included THP-1 monocytes, differentiated THP-1 and highly aggressive proliferating immortalised (HAPI) rat microglial cells to examine for differences pending on the different types of protein exposed to. THP-1 were differentiated using Phorbol-12-Myristate-13- Acetate (PMA), changing them to a more microglial-like cell. It was predicted the more microglial-like cells would show lower MGV and higher edge proportions when exposed to aggregated α-syn than other groups. Use of HAPI cells was to confirm that differentiated THP-1 were acting microglial-like.
Alternate Hypothesis 1b – Microglial-like cells exposed to aggregated α-syn have significantly lower MGV and higher proportion of α-syn positive bearing cells at the coverslip edge than both their same cell group exposed to α-syn monomer and all monocyte groups.
Null Hypothesis 1b – Microglial-like cells exposed to aggregatde α-syn do not have significantly lower MGV and higher proportion of α-syn positive bearing cells at the coverslip edge than both their same cell group exposed to α-syn monomer and all monocyte groups.
Additionally the control experiment had cells exposed to Bovine Serum Albumin (BSA) in lieu of α-syn to confirm if findings were due to exposure to α-syn or not.
Alternate Hypothesis 1c – Significant differences in MGV and protein proportions at the coverslip edge existed between BSA spotted control group coverslips and α-syn spotted coverslips.
Null Hypothesis 1c – Significant differences in MGV and protein proportions at the coverslip edge did not exist between BSA spotted control group coverslips and α-syn spotted coverslips.
1.3.1.3 Epothilone D Treatment
Utilising the same mobility assay, cells were treated with 100nM EpoD in the 48 hour incubation period. EpoD functions by disrupting dynamic instability in microtubules, instead stabilising this process thus preventing them from performing the functions required for cellular motility. EpoD treatment was investigated as a potential therapeutic intervention to determine if 43 inhibiting cellular motility of cells exposed to α-syn has a significant effect on both the amount of removed residual α-syn and the proportion of protein positive cells at the coverslip edge.
Alternate Hypothesis 1d – EpoD treatment has a significant effect on both MGV and proportion of protein positive cells at the coverslip edge compared to non-treated groups.
Null Hypothesis 1d – EpoD treatment does not have a significant effect on both MGV and proportion of protein positive cells at the coverslip edge compared to non-treated groups.
1.3.2 Chapter 3 - TAK-242 Has No Effect on Microglial Mediated Transport of Alpha Synuclein Species
1.3.2.1 TAK-242 Treatment
Utilising the previously described mobility assay, cells were instead be treated with 5µM TAK- 242, a TLR4 inhibitor, with the intention of investigating whether the prevention of inflammatory activation has an effect on α-syn migration by exposed cells.
Alternate Hypothesis 2 – TAK-242 treatment has a significant effect on both MGV and the proportion of protein positive cells at the coverslip edge compared to non-treated groups
Null Hypothesis 2 – TAK-242 treatment has a significant effect on both MGV and the proportion of protein positive cells at the coverslip edge compared to non-treated groups
1.3.3 Chapter 4 - Mitochondria mediate cell-to-cell transfer of alpha-synuclein aggregates via Tunnelling Nanotubes
1.3.3.1 Confocal Immunofluorescence Examination of α-syn Transfer via TnT
To investigate for α-syn along TnTs, 1321N1 and Differentiated THP-1 cells were co-cultured whereby one of the cells types was pre-exposed to α-syn prior to incubation of cells together. Cells were incubated together for a period of 48 hrs before fixed and fluorescently stained. Intention of pre-exposure in only one cell type instead of both prior to co-culturing was to assess for transfer of α-syn from one cell type to another. If α-syn was present in non-exposed cells it indicated exchange from pre-exposed cells. Cells were exposed to α-syn for 48 hrs after which they were washed twice prior to co-culturing. 1321N1 and differentiated THP-1 were chosen for co-culturing due to easily distinguishable phenotypes based on size making identification possible without use of specific markers. Additionally structures resembling TnTs were examined for in co-cultures to determine if they may have been a potential mechanism utilised for the transfer of α-syn.
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Alternate Hypothesis 3a – Qualitative evidence confirming transfer of α-syn from pre-exposed group to non-exposed group. Additional evidence present of TnT-like structure formation, implicating it as a potential mechanism in this transfer.
Null Hypothesis 3a – No evidence of α-syn transfer from pre-exposed group to non-exposed group in the co-culture. No evidence of TnT-like structure formation.
Similar to the 1321N1 and THP-1 co-culture, SH-SY5Y were examined for formation of TnT- like structures and the transfer of α-syn within them to adjacent cells. Unlike 1321N1 or differentiated THP-1, SH-SY5Y natively express for monomer α-syn which can be made to aggregate with brief exposure to KCl. Staining of OMM receptor Tom20 was also performed for SH-SY5Y mono-cultures to determine if α-syn was bound to migrating mitochondria within TnT-like structures.
Alternate Hypothesis 3b – Qualitative evidence implicating α-syn bound mitochondrial migration along TnT-like structures as a potential mechanism of α-syn propagation.
Null Hypothesis 3b – No evidence of α-syn based migration along TnT-like structures in SH- SY5Y mono-culture. Independent from mitochondrial based migration.
1.3.3.2 Stimulated Emission Depletion Microscopy Examinations of TnT-based α-syn transfer in SH-SY5Y Monocultures
Stimulated Emission Depletion (STED) microscopy was employed for further examination of TnT-like structures and to definitively determine if α-syn could transfer from one cell to another by binding to migrating mitochondria within these structures. As a form of super-resolution microscopy, STED is specialised for the detection and examination of fine structures such as TnTs due to the depletion of excess light from stimulated fluorophores. Fluorescent confocal microscopy can detect and analyse structures at a clear resolution only at the µm scale whilst STED instruments can analyse structures at the nm scale. Once more SH-SY5Y cells were treated with KCl prior to fixation and staining. Fluorescently stained for Tom20, nucleus and α- syn.
Alternate Hypothesis 3c – Qualitative evidence confirming α-syn bind to migrating mitochondria within TnT-like structures as a mechanism of spread to adjacent cells.
Null Hypothesis 3c – No evidence to suggest α-syn bind to migrating mitochondria within TnT-like structures for propagation to other adjacent cells.
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1.3.3.3 Stimulated Emission Depletion Microscopy Examinations of TnT-based α-syn transfer in 1321N1 and Differentiated THP-1 Mono and Co-Cultures
STED microscopy was employed for 1321N1 and differentiated THP-1 mono- and co-cultures for closer examination of TnT-like structures and to determine if α-syn is bound to migrating mitochondria within them. In co-culture conditions one cell group was pre-exposed to α-syn prior to culturing with other group similar to what was performed for initial confocal analysis. Fluorescently stained for Tom20, nucleus and α-syn.
Alternate Hypothesis 3d - Qualitative evidence confirming α-syn bind to migrating mitochondria within TnT-like structures as a mechanism of spread to adjacent cells both in mono-culture and co-cultures.
Null Hypothesis 3d – No evidence to suggest α-syn bind to migrating mitochondria within TnT-like structures for propagation to other adjacent cells.
1.3.3.4 Miro1 siRNA Treatment
In addition to incubation with α-syn prior to co-culturing with differentiated THP-1, 1321N1 were treated with a small interfering RNA (siRNA) targeting Miro1. Miro1 is a critical component bridging the mitochondria to the motor adaptor complex responsible for the organelles intercellular migration. It was predicted that silencing of Miro1 would prevent mitochondrial migration along TnT-like structures and thus prevent α-syn via this method.
Alternate Hypothesis 3e - Miro1 has a significant reduction on the amount of mitochondria migrating along TnT-like structures, thus reducing α-syn spread via this mechanism.
Null Hypothesis 3e – Miro1 has no effect on TnT-like structure-based mitochondrial migration. Additionally has no effect on α-syn binding to migrating mitochondria within TnT-like structures.
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Chapter 2
Epothilone D Inhibits Microglial- Mediated Spread of Alpha Synuclein Aggregates
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2.0 STATEMENT OF CONTRIBUTION TO CO-AUTHORED PUBLISHED PAPER
This chapter includes a co-authored paper. The bibliographic details of the co-authored paper, including all authors, are:
Valdinocci, D., Grant, G., Dickson, T. and Pountney, D. (2018). Epothilone D inhibits microglia-mediated spread of alpha-synuclein aggregates. Molecular and Cellular Neuroscience, 89, pp.80-94.
My contribution to the paper involved:
- All experiment conduction, data analysis, manuscript preparation, writing and revision.
Signed: Date: December 2019 Dario Valdinocci
Signed: Date: December 2019 Dean Pountney (Co-Author)
Signed: Date: December 2019 Dean Pountney (Principal Supervisor)
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Chapter 3
TAK-242 Has No Effect on Microglial Mediated Transport of Alpha Synuclein Species
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3.0 Abstract
Synucleinopathies are characterised by both widespread α-synuclein inclusions in varying neuronal cell types and neuroinflammation throughout the central nervous system. Previous work revealed that the role of microglia in these diseases may not simply be relegated to inflammation, but perhaps pathological α-syn propagation as well. Inhibition of factors associated with migration such as microtubules proved effective in preventing the potential migration mechanism however acted more as an intervention than a preventative. Factors such as Toll-Like Receptor 4 are critical in the activation of microglial cells providing neuroprotection however overstimulation these same receptors can lead to chronic inflammation and thus degeneration over time. Whether this same factor is also involved in the propagation on pathological α-syn within microglial cells is unknown. Using the previously established α- synuclein mobilisation assay, Toll-like receptor 4 was investigated as a potential factor in the proteins propagation through the receptors inhibition by TAK-242 treatment (5 µM). Quantitative immunofluorescence revealed that TAK-242 had no significant effect on any group with regards to either residual α-synuclein content or the proportion of α-synuclein bearing cells on the coverslip edge. This result indicates that the factors involved in microglial activation are not the same ones involved in microglia-based α-synuclein migration.
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3.1 Introduction
Parkinsonian diseases are characterized by the presence of inclusion bodies in neuronal cells, the formation of which leads to the neurodegeneration present within these diseases. A common link between each of these diseases is the presence of the misfolded aggregate form of the protein α- α-syn, with each diseases inclusion body possessing α-syn in its composition. PD in addition to Multiple MSA and Dementia with Lewy Bodies (DLB) are examples of such diseases containing α-syn composed inclusion bodies. Interestingly the phenotypical symptoms of each disease are remarkably similar which include ataxia, bradykinesia and tremors even whilst different cellular sites are affected by inclusion body formation (Longo, Fanciulli and Wenning, 2015). For instance Lewy Bodies and Lewy Neurites prevalent in PD predominantly affect dopaminergic neurons and their ability to synthesize and release DA at the axonal terminals to signal for musculoskeletal innervation. The MSA inclusion body termed GCIs however exclusively forms within oligodendrocytes, leading to reductions in myelin synthesis for neuronal axons thus affecting neuronal signalling through action potentials for DA release at axonal terminals (Miki et al., 2010; Ahmed et al., 2012; Wong, Halliday and Kim, 2014; Don et al., 2014). The common link between each disease is the presence of aggregate α-syn, implicating it as a major factor in symptom formation and in turn the neurodegeneration taking place within. Various mechanisms of propagation have been proposed for α-syn transport around the central nervous system (CNS), including exocytosis, tunnelling nanotubes and vesicle release as select examples, all of which paints the complexity in tackling its prevention (Fitzner et al., 2011; Abounit et la., 2016a).
Microglia are innate immune cells predominantly involved in the clearance of damaging and/or pathological materials within the CNS (Fu et al., 2014; Kettenmann et al., 2011). Microglia typically exist in a ramified phenotype denoting their surveillant state, extending cellular processes to search for any materials which may pose a threat to the CNS. Stimulation of TLRs such as TLR2 and TLR4 or scavenger receptors like CD36 cause the cell to undergo change from ramified to amoeboid, denoting the shift from surveillant to activated state (Stefanova et al., 2011; Kim et al., 2013; Fellner et al., 2013; Tang and Le, 2015). When activated microglia are involved in both clearance of damaging/pathological materials and the regulation of the inflammatory response through release of factors such as IL-1β and TNF-α for activation and recruitment of nearby microglia. Gliosis, an overstimulation of the inflammatory response, is a major factor contributing to the degeneration within MSA and to lesser extent both PD and DLB, due to inability of microglia to degrade aggregate α-syn (Nagatsu and Sawada, 2005; Tufekci et al., 2012). Failure in the lysosomal degradation process is the current hypothesis due to factors such as cellular senescence in addition to similar observations exhibited by other immune cells such as astrocytes (Alvarez-Erviti et al., 2011; Chu et al., 2009). Recent works have proposed microglia as potential transporters of pathological materials including α-syn 66 based on observations in a variety of experimental conditions including migration with ingested protein in vitro (Valdinocci et al., 2018; Asai et al., 2015). Given microglia‟s migration capabilities and its ability to engulf α-syn even whilst not able to degrade, it may be possible that they may become unintentional transporters of α-syn in Parkinsonian diseases.
Toll-like receptors are major factors involved in the activation of various innate immune cells including microglia. TLR4‟s detection and interaction with pathological α-syn species has been implicated as the main proponent of the inflammatory response within Parkinsonian diseases (Stefanova et al., 2011; Roodveldt et al., 2013; Fellner et al., 2012). Other TLRs such as TLR2 also illicit a response to α-syn however tend to be generally lesser than TLR4‟s, indicating they may perhaps be more of an alternative or supplemental response as opposed to the primary (Kim et al., 2013; Fellner et al., 2012). The nature of TLR4‟s response in Parkinsonian diseases, whether beneficial or not is still in question. Examples of it relieving degeneration come in the form increased extracellular clearance of pathological α-syn in PD models when promoting TLR4 activation (Stefanova et al., 2011). Conversely a by-product of TLR4 stimulation is the NLRP3 Inflammasome, involved in IL-1β synthesis and release, whose activation is attributed to the degeneration of dopaminergic neurons in PD and MSA (Lee et al., 2018; Li et al., 2018; von Herrmann et al., 2018). TLR4 absence saw reductions in the NLRP3 response, neuronal degeneration and α-syn spread. These opposing responses highlight the complexity of the microglial TLR4 response to α-syn, with differences existing perhaps to stage of disease progression or stage of the microglial cells life cycle. Based on this it was investigated if suppression of TLR4 inflammatory response through the use of the signal transducer inhibitor TAK-242 had any response on α-syn mobilisation within microglial culture models. Also referred to as Resartovid, TAK-242 inhibits TLR4‟s ability to signal for the synthesis and release of pro-inflammatory factors by binding to Cys747 domain of the Toll/Interleukin-1 Receptor located on TLR4 itself (Matsunaga et al., 2010). TIRAP recruitment, a crucial factor in pro-inflammatory factor synthesis, is significantly reduced. The only reported off-target effect of TAK-242 is a report in which TAK-242 treatment saw increased protection in rats against both acute and chronic fat-induced insulin resistance (Zhang et al., 2015).
3.2 Materials and Methods
3.2.1 Cell culturing – THP-1 and HAPI
THP-1 cells, a human monocyte cell line derived from an acute monocytic leukaemia patient, were grown in media comprised of RPMI 1640 supplemented with L-Glutamine, 10% foetal bovine serum, 0.05mM β-Mercaptoethanol in addition to penicillin/streptomycin antibiotic solution. Media replacement occurred every 2-3 days and closely examined to prevent growth exceeding 1x106 cells/mL. Incubator settings consisted of 20% oxygen, 5% carbon dioxide and a temperature of 37 °C to maintain consistent cellular growth. Phorbol-12-Myristate-13-Acetate 67
(PMA), a phorbol ester required for the differentiation of THP-1 cells into a more microglial- like state, was utilised at a concentration of 5ng/mL and exposed for a period of 72 hours followed by media replacement omitting PMA.
Rat Highly Aggressive Proliferating Immortalised (HAPI) microglial cells were grown in Low Glucose DMEM medium (1g/L D-Glucose) with 5% foetal bovine serum and penicillin/streptomycin at the same incubator settings as the THP-1 cells. Media replacement occurred every 3 days and were split 1:7-1:9 every 7 days. Further treatment of HAPI cells for differentiation to more microglial-like state was not required.
3.2.2 Alpha-Synuclein Mobilization Assay
Monomeric α-Syn was expressed in Escherichia Coli and purified by anion exchange and gel permeation chromatography (see Appendix Item IV), with aggregate α-syn was produced by overnight incubation (4 ˚C) with 1mM H2O2 and 100µM of Ca2+ as previously described (Goodwin et al., 2013). Utilizing the same mobilization assay as previously described in Valdinocci et al., 2018, monomeric or aggregate α-syn (50µM, 3µL) was applied and dried to the centre of a sterilised 12mm coverslip (Valdinocci et al., 2018). Coverslips were then placed in 24-well plates and 1mL of medium containing 50,000 cells per well comprising of either THP-1, differentiated THP-1 or HAPI cells. The plate was incubated for 48 h at 37 °C, 20% oxygen and 5% CO2 followed by fixation (methanol:acetone, 4:1; 10 mins, 4 °C), blocking (20 % normal horse serum, PBS), staining with primary and secondary antibodies for immunofluorescence followed by mounting on slides using Pro Long ® Gold anti fade mountant with DAPI (Life Technologies).Primary antibodies utilised comprised of mouse anti- α-syn (LB509) (Invitrogen) and rabbit anti-Iba1 (WAKO) both at 1:200 dilution. Secondary antibodies were Alexa Fluor ® 488 (AF488) Goat anti-mouse IgG (Life Technologies) and Alexa Fluor ® 568 (AF568) Donkey anti-rabbit IgG (Life Technologies) at 1:200 dilution. Additional control protein experiments were performed against Bovine Serum Albumin (BSA) utilising Invitrogen Bovine Serum Polyclonal Antibody (Product#A11133) for the primary antibody and Alexa Fluor ® 568 (AF568) Donkey anti-rabbit IgG (Life Technologies) for secondary both at 1:200 dilution.
Select samples were treated with 5µM of TAK-242, a TLR4 signalling inhibitor, following incubation with protein group in the well. Concentration was chosen based on previous reports by Zhou and colleagues describing this concentration as sufficient in altering the inflammatory profile of THP-1 cells (Zhou et al., 2013).
3.2.3 Confocal Microscope
All images were captured using an Olympus Fluoview 1000 laser scanning confocal microscope, with slides imaged using the 10x or 20x lenses with numerical apertures of 0.4 and 68
0.45 respectively. To prevent fluorescent cross-excitation, channels were each imaged sequentially all at a resolution of 1024x1024 pixels. Olympus FV-ASW 3.2 Viewer was utilised for image false-colouring and exportation for analysis.
3.2.4 Data Acquisition – Fluorescent Intensity and Cell Counting
As in Valdinocci et al., 2018 the same methods for quantifying data were employed: mean fluorescent intensity of the residual immobilized α-syn spot and cell counts performed at select regions of the coverslip edge (Valdinocci et al., 2018). For the fluorescent intensity analysis, images were acquired at 10x magnification at a 1024x1024 resolution at a scanning rate of 10.0us/pixel of the centre, left and right sections of the immobilized spot. Mean Gray value of 5 specific 100x100 pixels sub-regions within each section image was acquired through the use of ImageJ. The co-ordinates of these sub-regions are identical to those described in Valdinocci et al., 2018. Once the fluorescent readings for each of the 5 sub-regions of each sample section were collected, they were averaged to determine the mean fluorescent intensity of the spot. For cell counts, images were acquired at 20x magnification at the same 8 coverslip edge regions with total cell count determined by DAPI-stained cells and α-syn positive bearing cells as those with AF488 score readings greater than twice the background. The regions analysed included: bottom, bottom left, bottom right, left, right, top, top left and top right.
3.2.5 Statistical analysis methods – Student t-test and ANOVA utilised
IBM SPSS Statistics 25 was utilised for both cell counts and fluorescent intensity data analysis, which included the use of independent t-tests and ANOVA tests. ANOVA was utilised in order to effectively perform the comparisons between multiple cell groups, all of which employed the LSD Post-Hoc test. Significance was set at p=0.05 for each test.
3.3 Results
3.3.1 No significant reduction in protein uptake
Analysis of residual immobilised protein in the untreated groups confirmed the previous observations in which oligomeric aggregate α-syn exposed to microglial-like cells (differentiated THP-1 and HAPI) had greater uptake than those exposed to the purified recombinant monomeric form (Figure 12a). Uptake of residual immobilized protein, both monomer and aggregate, was based on fluorescent intensity emitted by the central spots, with lower MGVs denoting lower residual protein content in the region and thus greater uptake. The reduction in fluorescent intensity likely indicates removal of protein from the central residual spot region. No significant differences in uptake arose between recombinant monomer groups nor in the comparisons between different α-syn forms exposed to undifferentiated THP-1 cells. BSA control experiments produced comparable results to previous findings, with significantly
69 lesser uptake performed by differentiated THP-1 (p=0.001) and HAPI cells (P=0.001) exposed to oligomeric aggregates. Surprisingly no significant differences in MGV was observed in any group following TAK-242 treatment when compared to their untreated counterparts. It was hypothesized that inhibition of TLR4 through TAK-242 would lead to an increase in the fluorescent intensity indicating reduced uptake of immobilised aggregate α-syn groups. Whilst MGV of treated differentiated THP-1 (Figure 12b, p=0.237) and HAPI cells (Figure 12c, p=0.085) groups saw an increase, it did not indicate that a significant reduction in uptake by the cells had occurred following treatment. This result indicates that mobilisation of α-syn, at least from the central spotted region, is not due to the actions of TLR4. An ANOVA analysis of the TAK-242 treated groups (Figure 12d) produced much of a similar result as the untreated, giving further credence to the notion that uptake and mobilisation of α-syn is likely not responsible on a singular uptake receptor such as TLR4.
Figure 12 – TAK-242 treatment has no significant effect on alpha-synuclein mobilization
(a) ANOVA of the Mean Gray Value of all protein groups exposed to untreated undifferentiated
THP-1, differentiated THP-1 and HAPI Cells (n=3, ±SEM). α-Syn exposed to the more microglial-like cells, differentiated THP-1 and HAPI cells, showed significant reductions in
fluorescent intensity from the central spot indicating uptake and mobilisation of the protein by cells from the region. Both differentiated THP-1 and HAPI cells showed significant differences of p=<0.001 to their monomer α-syn exposed counterparts respectively. (b) Independent t-test of control and TAK-242 treated differentiated THP-1 cells exposed to aggregate α-syn (n=3, ±SEM). Result was insignificant (p=0.237). (c) Independent t-test of control and TAK-242 treated HAPI cells exposed to aggregate α-syn (n=3, ±SEM). Result was insignificant
(p=0.085). (d) ANOVA of the Mean Gray Value of all TAK-242 treated cell groups exposed to monomeric and aggregate α-syn, in addition to BSA (n=3, ±SEM). Results were reflective of what was observed in the untreated groups due to lack of effect following TAK-242 treatment to uptake and mobilisation of the protein. Once more significant differences occurred only between monomer and aggregate groups for differentiated THP-1 and HAPI cells and the BSA comparisons (p=0.001 for all groups).
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a ANOVA of Untreated Groups Mean Fluorescence 25
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15 * * ** **
10 Mean Grey Value Grey Mean 5
0 THP1 THP1 Differentiated Differentiated HAPI Monomer HAPI Aggregate THP1 BSA Differentiated HAPI BSA Monomer α- Aggregate α- Cells Monomer Cells Aggregate α-syn α-syn (Untreated) Cells BSA (Untreated) syn (Untreated) syn (Untreated) α-syn α-syn (Untreated) (Untreated) (Untreated) (Untreated) (Untreated) * = 0.05 with corresponding group Well Conditions ** = 0.05 with corresponding BSA group
Mean Gray Values of HAPI Groups b Mean Gray Value of Differentiated THP- c 1 Groups exposed to Aggregate α-syn exposed to Aggregate α-syn
25 25
20 20
15 15
10 10 Mean Gray Value Gray Mean
5 Value Gray Mean 5 0 0 Differentiated Cells Differentiated Cells HAPI Aggregate α- HAPI Aggregate α- Aggregate α-syn Aggregate α-syn syn (Untreated) syn (TAK-242 (Untreated) (TAK-242 Treated) Treated) Well Conditions Well Conditions
d ANOVA of TAK-242 Treated Groups Mean Fluorescence 25
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15 * * ** ** 10
5 Mean Gray Value Gray Mean
0 THP1 THP1 Differentiated Differentiated HAPI HAPI THP1 BSA Differentiated HAPI BSA (TAK- Monomer α- Aggregate α- Cells Monomer Cells Monomer α- Aggregate α- (TAK-242 Cells BSA (TAK- 242 Treated) syn (TAK-242 syn (TAK-242 α-syn (TAK-242 Aggregate α- syn (TAK-242 syn (TAK-242 Treated) 242 Treated) Treated) Treated) Treated) syn (TAK-242 Treated) Treated) Treated) * = 0.05 with corresponding group Well Conditions ** = 0.05 with corresponding BSA group71
3.3.2 No significant difference in proportion of α-syn positive bearing cells on coverslip edge
Using the proportion of α-syn positive bearing cells as an indicator of α-syn mobilisation from the central spot confirmed several findings predicted from the MGV analysis. The first was the relationship associated with MGV and protein mobilisation. In all cases a reduction in MGV saw increased presence of α-syn bearing cells at the coverslip edge indicating that the protein was being mobilised distally from the region it was engulfed (Fig 15). Untreated differentiated THP-1 and HAPI cells exposed to aggregate α-syn produced the lowest MGVs at the central spot region offset with the largest proportions of α-syn positive bearing cells at the coverslip edges. This pattern was observed in both the untreated controls (Figure 2a) and the TAK-242 treated groups as well (Figure 2b). Prior to analysis it was hypothesized that a reduction in aggregate α-syn positive bearing cells would be observed in TAK-242 treated differentiated THP-1 and HAPI cell groups as it was presumed that TLR4 would have a significant effect on α-syn uptake and thus its eventual mobilisation. Examinations at the coverslip edge regions of TAK-242 treated groups supported the previously reported observations of lack of change to the immobilized residual groups. Whilst reductions in the proportion of α-syn-positive bearing cells were observed, they were not significantly lesser than the equivalent untreated groups for neither differentiated THP-1 (Figure 2c, p=0.151) nor HAPI cells (Figure 2d, p=0.292). This result in corroboration with the MGV analysis indicates that a sole receptor site such as TLR4 is unlikely to be solely responsible for pathological α-syn migration within microglial-like cells. Figure 13 – TAK-242 treatment does not significantly affect microglia-mediated alpha- synuclein migration
(a) ANOVA of positive protein bearing cells in the cytoplasm of cells at the coverslip edge for all untreated control groups (n=3, ±SEM). The two groups that showed a significant difference in proportion were the aggregate α-syn exposed differentiated THP-1 (p=0.0) and HAPI (p=0.0) cells from their monomeric counterparts. From this information it appears that reductions in the mean gray value are due to the mobilisation of the protein distally from the spot region as observed by the increase in cells migrating with the protein. (b) ANOVA of positive protein bearing cells at the coverslip edge for the TAK-242 treated cell groups (n=3, ±SEM). Similar to the mean gray values, little change occurred in the edge proportions following treatment thus leading the same set of significant differences between monomeric and aggregate α-syn differentiated THP-1 cells (p=0.001) and HAPI cells (p=0.001) (c) Independent t-test of between untreated controls and TAK-242 treated differentiated THP-1 cells exposed to aggregate α-syn at the coverslip edge (n=3, ±SEM). Proportion of α-syn positive bearing cells was altered following treatment, with a slight reduction observed, however was not significant enough from the untreated controls (p=0.151). (d) Independent t-test of aggregate α-syn positive bearing cell at the coverslip edge between untreated controls and TAK-242 treated HAPI cells(n=3,
±SEM). Similar to the differentiated THP-1 group, TAK-242 treated showed reductions in their amount of cells observed to be migrating with internalised α-syn at the coverslip edge however was not significantly different from the untreated controls (p=0.292). 72
a ANOVA of Untreated Proportion of α-syn and BSA Positive Bearing Cells (%)
* 50 ** * ** 40
30
20
10
0 THP1 Monomer THP1 Aggregate Differentiated Differentiated HAPI Monomer HAPI Aggregate THP1 BSA Differentiated HAPI BSA Percentage of protein positive cells (%) cells positive proteinof Percentage α-syn α-syn Cells Monomer Cells Aggregate α-syn α-syn (Untreated) Cells BSA (Untreated) (Untreated) (Untreated) α-syn α-syn (Untreated) (Untreated) (Untreated) (Untreated) (Untreated) * = 0.05 with corresponding group Well Conditions ** = 0.05 with corresponding BSA group
ANOVA Proportion of TAK-242 Treated α-syn and BSA Positive Bearing Cells b (%) 50 * * ** 40 **
30 synuclein positive synuclein
- 20 cells (%) cells 10 0 THP1 Monomer THP1 Aggregate Differentiated Differentiated HAPI Monomer HAPI Aggregate THP1 BSA (TAK- Differentiated HAPI BSA (TAK- α-syn (TAK-242 α-syn (TAK-242 Cells Monomer Cells Aggregate α-syn (TAK-242 α-syn (TAK-242 242 Treated) Cells BSA (TAK- 242 Treated)
Percentage of a of Percentage Treated) Treated) α-syn (TAK-242 α-syn (TAK-242 Treated) Treated) 242 Treated) Treated) Treated) * = 0.05 with corresponding group Well Conditions ** = 0.05 with corresponding BSA group
50 50 40 40
30 30
(%) 20
(%) 20
synuclein positive cells cells positive synuclein
-
synuclein positive cells cells positive synuclein - 10 10 0 Differentiated Cells Differentiated Cells 0
Percentage of a of Percentage Aggregate α-syn Aggregate α-syn HAPI Aggregate α- HAPI Aggregate α- (Untreated) (TAK-242 Treated) a of Percentage syn (Untreated) syn (TAK-242 c d Treated) Well Conditions Well Conditions
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3.4 Discussion
The exact nature of microglia in relation to their role as either “friend or foe” within synucleinopathies remains elusive. As part of the innate immune response microglia are critical for defending the CNS from DAMPs and PAMPs through their clearance and degradation in addition to promoting immune cell activation and recruitment (Fu et al., 2014; Kettenmann et al., 2011). Factors such IL-1β, IL-6, TNF-α and IFN-γ are synthesized and released from microglia promoting this response (Lively and Schlichter, 2018; Smith et al., 2012). Conversely these same factors are major contributors to the degeneration taking place in both typical and atypical PD once the response becomes chronic (Nagatsu and Sawada, 2005; Tufekci et al., 2012). Microglial cells inability to efficiently degrade pathological α-syn variants is partially at fault for the sustainment of this response (Tanik et al., 2013). Additionally the recent reports of microglia‟s contribution to pathological protein spread, including ours detailing microglia as a potential α-syn transporter, further complicates its exact nature within this disease group (Asai et al., 2015; Valdinocci et al., 2018; Xia et al., 2019). This raises the question of whether the microglial response to α-syn is beneficial at all if it will only lead to the cells actively contributing to the degenerative process as opposed to a deterrent against it.
TLR4 is widely regarded as a major factor for promoting the microglial immune response and whose exact nature within synucleinopathies has also been questioned. Activation of TLR4 has been demonstrated to be beneficial in the clearance of α-syn in mouse models of PD, implicating it as a factor in the proteins uptake by microglia as well (Stefanova et la., 2011). Equally TLR4 is a factor in the activation of the NLRP3 inflammasome, whose over-stimulation has been detailed to contribute to nigral neurons degradation (Li et al., 2018; von Herrmann et al., 2018; Campolo et al., 2019). Absence of TLR4 saw the NLRP3‟s degenerative response lessened; implicating that inhibition of TLR4 stimulation may be beneficial in the promotion of neuroprotection (Campolo et al., 2019). Examination of TLR4 inhibition using our mobility assay was believed to have further elucidated TLR4‟s role in synucleinopathies within the context of the proteins mobilisation. It was hypothesised that its inhibition would significantly alter microglia‟s transporter profile and provide a novel therapeutic intervention against α-syn spread. If successful it may have also provided an avenue to combat degradation promoted by inflammatory factors. The lack of change following TLR4 inhibition via TAK-242 however hints at a more complex response than initially conceived.
The control experiments matched the outcomes presented in Chapter 2, in which microglial-like cells exposed to aggregate α-syn saw greater protein uptake and mobilisation from the residual immobilised spot in combination with increased distal presence of α-syn positive bearing cells. The lack of uptake and mobilisation of monomeric α-syn was primarily predicted to be due to it not classified as a DAMP unlike the aggregate variant (Béraud et al., 2011). The role of
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Differentiated THP-1 Aggregated α-syn Untreated Left Centre Right
Differentiated THP-1 Aggregated α-syn TAK-242 Treated
a
HAPI Cells Aggregated α-syn Untreated
HAPI Cells Aggregated α-syn TAK-242 Treated
Differentiated THP-1 Aggregated Differentiated THP-1 Aggregated α-syn (Untreated) Left α-syn (TAK-242) Top
DAPI Iba1 aSN
b HAPI Cells Aggregated α-syn HAPI Cells Aggregated α-syn (Untreated) Top Right (TAK-242) Bottom Right
DAPI Iba1 aSN
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Figure 14 – Immobilized central spots and coverslip regions of both differentiated THP-1 and HAPI cell controls and TAK-242 treated groups reveal TAK-242 to be ineffective in preventing α-syn engulfment and mobilisation
(a) Select untreated and TAK-242 treated spot groups of differentiated THP-1 and HAPI cells exposed to aggregated α-syn. Examples include their corresponding left, right and centre
regions analysed to acquire MGV values. (b) Select examples of coverslip edge regions of differentiated THP-1 and HAPI cells exposed to aggregated α-syn. Observed are internalised aggregated α-syn (yellow arrows) in both untreated and TAK-242 treated groups. Both groups,
untreated and TAK-242 tretaed, equate to having 40% of cells with internalisied α-syn in their corresponding cell groups (differentiated THP-1 and HAPI cells). This reveals TAK-242 to be ineffective in combatting both α-syn engulfment and mobilisation of cells with internalised α-
syn. monomeric α-syn is currently believed to be associated with vesicle recycling at neuronal terminals based on numerous studies examining the purpose of α-syn presence in the CNS (Scott and Roy, 2012; Sun et al., 2019; Vargas et al., 2014 ; Burré, 2015). Phagocytic cells such as microglia have been demonstrated to engulf and degrade α-syn monomers with little complications (Park et al., 2008). The lack of action exerted by THP-1 monocytes exposed to α- syn aggregates may be indicative of monocytes role as a supplemental factor to the immune response within the CNS (Codolo et al., 2013). Uptake and mobilisation was still observed to be performed by these cell types however the significant difference between the actions of the THP-1 monocytes the differentiated THP-1 cells indicates that if monocytes were a factor in the propagation of α-syn within the CNS it would likely be of little significance. Similarities in the uptake and the proportions of α-syn positive bearing cells between both differentiated THP-1 and HAPI cells reinforces that differentiated THP-1 are acting as a model of microglial cells in vitro. Additionally the likeness in phenotype between these results and the reports of microglia migrating with internalised α-syn in Chapter 2‟s tissue analysis further supports microglia‟s case as an α-syn transporter.
TAK-242 treatment failed to significantly alter the transporter behaviours of all cells group regardless of which form of α-syn they were exposed to. TAK-242 treatment has been previously demonstrated to reduce the inflammatory profile of THP-1 cells with significant reductions in TNF-α and IL-1β (Zhou et al., 2013). Previous reports of TLR4‟s association with immune cell clearance capabilities led to the hypothesis that the receptors inhibition would lead to a significant reduction in protein uptake and thus its migration (Stefanova et al., 2011). Slight alterations were observed in differentiated THP-1 and HAPI groups exposed to aggregate α-syn following TAK-242 treatment, with a lower proportion of α-syn positive bearing cells at distal regions. These results indicate that TAK-242 treatment does exert some action upon TLR4 to reduce α-syn engulfment however this action is not significant enough to present as a novel
76 intervention against its spread. The lack of significant action suggests TLR4 is not the factor responsible for α-syn uptake.
The primary focus on TLR4 inhibition was based on the receptors extensive coverage however other factors have been reported to be involved in microglial activation and α-syn engulfment which include both TLR2 and scavenger receptor CD36. The importance of TLR2 with regards to microglia‟s relationship with α-syn remains elusive. TLR2 has been demonstrated to be activated alongside TLR4 in a variety of animal models (mouse and rats) however, such actions were previously deemed to occur as a method to supplement the TLR4 response as opposed to driving an action of its own (Fellner et al., 2012; Drouin-Ouellet et al., 2014; Facci et al., 2014). This perspective has begun to shift the last few years as an increasing number of reports detail activation of the receptor independent of TLR4 and promoting microglial processes such as inflammatory activation via the NLRP3 inflammasome, TNF-α and IL-1β release and phagocytic engulfment of PAMPs/DAMPs (Kim et al., 2013; Codolo et al., 2013; Doorn et al., 2014; Dzamko et al., 2016). Interactions with β-amyloid fibrils have been detailed and in the context of synucleinopathies, neuronal TLR2 was predicted to drive PD pathogenesis (Chen et al., 2005; Dzamko et al., 2016). Recently TLR2 has been demonstrated in to be involved as a factor in the transmission of α-syn from neurons to astroglial cells (Kim et al., 2018). Inhibition of the receptors saw a reduction in α-syn accumulation between both cell types in addition to a marked reduction in degeneration and neuroinflammation. Inhibition of TLR4 via TAK-242 may have still seen uptake of α-syn by differentiated THP-1 and HAPI cells due to the actions of TLR2. Scavenger receptors have been shown to elicit the same behaviour as TLRs; innate immune cell activation when detecting damaging or pathological molecules. CD36 interacts with various different pathological neurodegenerative proteins such as α-syn in PD variants and β-amyloid fibrils within AD, leading to activation of microglia for protein engulfment and pro- inflammatory factor release (Su et al., 2008; Coraci et al., 2002; Sheedy et al., 2013). Interactions with Fyn kinase sees actions by CD36 not only promote for extracellular misfolded α-syn clearance, but promotion of the inflammatory response through NLRP3 inflammasome activation (Panicker et al., 2019). Ultimately failed TAK-242 treatment highlights that microglial uptake of α-syn is not dictated by a singular pathway but is multifactorial.
TAK-242 site of action is another potential reason as to the ineffectiveness of the treatment on differentiated THP-1 and HAPI cells. TAK-242 inhibits pro-inflammatory factor synthesis and release by blocking the signalling generated by the Toll/Interleukin-1 receptor domain on TLR4 (Matsunaga et al., 2010, Takashima et al., 2009). This action was hypothesised to be associated with either α-syn uptake directly or the signalling pathway for uptake. Additionally inhibiting both uptake and inflammatory action was hypothesised to lead to a significant reduction in distal α-syn positive bearing cell proportions at the coverslip edge. In synucleinopathies such as MSA where the inflammatory response driven by immune cells is both chronic and widespread 77 throughout the CNS, inhibition of TLR4 may have provided a double benefit of both reducing α-syn spread and alleviating degeneration driven by inflammation (Jellinger, 2018). Whilst such an outcome seems unlikely from TAK-242, use of other anti-inflammatory agents may instead provide this reprieve. Carvedilol and T5342126 derivatives for instance have shown action in reduction of both cardiomyocites and BV-2 microglial cell inflammatory factor release by blocking the interaction between TLR4 and MD-2, preventing dimerization of TLR4 ( Xu et al., 2018a). Recently Lovastatin has been demonstrated to inhibit TLR4 action by the same inhibitory pathway, displaying selective actions on microglia (Peng et al., 2019). With relation to the HAPI cells, a potential reason for an ineffective response to treatment may be due to TAK-242 targeting for human TLR4. Whilst HAPI cells express and present with TLR4 on the cellular membrane, incompatibility between TAK-242 and its target may exist based on the specificity of TAK-242 on the human variant of the receptor (Zheng et al., 2012).
3.5 Conclusion
Inhibition of TLR4 activity by TAK-242 had no significant effect on the mobilisation of α-syn by immune cells. What this indicates is that the process of pathological α-syn uptake is likely to be mediated by a variety of receptors and methods such as TLR2 and CD36 as opposed to a singular one as was explored here. Understanding how α-syn is able to be propagated to various other neurological sites is a crucial step in the attempt to design an effective therapeutic intervention against it. Whilst the sole targeting of TLR4 does not appear to be the answer in this particular circumstance, inhibiting or at the least suppressing the activity of TLR4 is still an alley worthy of further exploration. Chronic inflammation based neurodegeneration due to over- activation of TLR4 is still a risk and problem of great concern in α-syn based diseases such as MSA.
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Chapter 4
Mitochondria mediate cell-to-cell transfer of alpha-synuclein aggregates via Tunnelling Nanotubes
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4.0 Abstract
The interaction of α-synuclein with mitochondria in both typical and atypical Parkinson‟s Disease is a critical component of degeneration. The mechanism of propagation of pathological α-synuclein in synucleinopathies is unclear. Intercellular exchange of mitochondria along tunnelling nanotubes has been described in other diseases such as cancer however, its role in synucleinopathies is unknown. Pathological α-synuclein species have been demonstrated previously to move from cell to cell via tunnelling nanotubes. Thus this process was further explored using co-culture and monoculture systems to determine if α-synuclein binds to migrating mitochondria within tunnelling nanotubes. Qualitative analysis via stimulated emission depletion microscopy confirmed previous reports of interaction between α-synuclein with the mitochondrial outer membrane and showed the presence of alpha-synuclein bound to mitochondria in tunnelling nanotubes between various cell types (1321N1, THP-1, SH-SY5Y). siRNA knockdown of Miro1, a critical protein bridging mitochondria to the kinesin/dynein motor adaptor complexes, had no measurable effect on potential mitochondria-mediated α- synuclein transfer. The results show that α-synuclein aggregates are bound to mitochondria in intercellular tunnelling nanotubes, suggesting that mitochondria-mediated α-synuclein transfer between cells may contribute to cell-to-cell spread of α-synuclein aggregates and disease propagation.
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4.1 Introduction
Previous studies have linked the progression of PD to the transmission of α-syn aggregates from cell-to-cell, thereby mediating the spread of disease pathology to adjacent neuroanatomical regions (Steiner, Angot and Brundin, 2011; Valdinocci et al., 2017; Domert et al., 2016; Sekiya et al., 2019). The mechanism of propagation of pathological α-syn in synucleinopathies remains unclear as a variety of mechanisms have been proposed, with no clear answer as to whether a dominant transportation mode exists. Spread of α-syn aggregates via TnTs has been demonstrated, with cells dynamically forming channels with other neighbouring cells. Thick TnTs in particular possess the width to transport larger cargo like organelles which frequently include mitochondrial bodies (Agnati & Fuxe, 2014). Past studies have shown that mitochondria generally utilize this form of transport for respiration rescue of neighbouring cells (Wang and Gerdes, 2015; Shen et al., 2018; Dong et al., 2017). Recently α-syn has been revealed to utilise TnTs for spread in a variety of in vitro models which include both mono- and co-culture models. Dieriks and colleagues detailed the spread from α-syn containing SH-SY5Y neuroblastoma cells to pericytes through the use of TnTs, whilst Rostami et al. showed TnT- mediated α-syn transfer between astrocytes (Dieriks et al., 2017; Rostami et al., 2017). These findings reveal that the spread of α-syn through TnTs is not limited to one cell type and may be relevant to synucleinopathies such as MSA, perhaps accounting for the occurrence of pathological α-syn in oligodendrocytes that lack endogenous expression. Additionally, some forms of α-syn are classified as prions, such as in MSA, and prions have been shown previously to hijack TnTs to spread from cell to cell (Prusiner et al., 2015; Gousset et al., 2009). Whilst this mechanism has not previously been reported for pathological α-syn, the spread of damage– associated molecular patterns such as Herpesvirus and tau through TnTs has further elucidated TnTs as a method of spread (Panasiuk et al., 2018; Tardivel et al., 2016).
Mitochondria are an essential component for the normal function of CNS cells. They are important for the generation of ATP to regulate the electrochemical gradient required for action potential propagation. Ion channels located on the nodes of Ranvier all require ATP to mediate Na+ entry and K+ exit (Poliak and Peles, 2003; Bean, 2007). In addition, mitochondria supply ATP at the distal regions of the neuron for channels mediating neurotransmitter release (David and Barrett, 2003). Loss of mitochondria significantly hampers the effectiveness of neuronal signalling. Immune cells such as astrocytes and microglia are also highly dependent on mitochondria for activation when an immune response is required (Fiebig et al., 2019; Nakahira et al., 2010; Zhou et al., 2010). Mitochondrial ROS generation is a key component of inflammasome formation required for reactive microglia activation (Katoh et al., 2017; Culmsee et al., 2019). In astrocytes loss of components within the electron transport chain is linked to gliosis (Fiebig et al., 2019). Whilst the role of mitochondria is less fully understood within these systems compared to neuronal cells, their presence is still required for normal function. 81
Transportation of mitochondria is critical to maintain normal cellular function. In order to migrate, mitochondria rely on motor complexes to reach various locations within the cell. These motor complexes are composed of TRAK motor proteins in addition to either kinesin or dynein pending on the direction of migration (Randall, Moores and Stephenson, 2013). Kinesin serves to migrate the mitochondria in an anterograde manner, distally from the nuclei. Likewise dynein performs the opposite function, migrating it proximally in a retrograde manner toward the nuclei (Brickley and Stephenson, 2011; van Spronsen et al., 2013). An example in neurons is mitochondrial migration along the axon, whereby kinesin transports mitochondria towards the Nodes of Ranvier and axonal terminals whilst dynein return the mitochondria back toward the nuclei in the soma. Motor complexes drive transport by being bound to microtubule “tracks”, whereby kinesin/dynein “walks” along bound to the track transporting its cargo. Miro1 on the outer mitochondrial membrane acts as the bridge connecting mitochondria to the motor adaptor complex and thus regulates whether migration will or will not occur (Fransson, Ruusala and Aspenström, 2006; MacAskill et al., 2009). In addition to intracellular migration, motor complexes appear to drive intercellular exchange of mitochondria along microtubules within TnTs. Such a method is typically employed to rescue adjacent cells by providing additional energy through ATP synthesis.
The interactions of pathological α-syn with mitochondria are relevant to pathological features of both typical and atypical PD (Valdinocci et al., 2019). For example, the loss of dopaminergic neuron signalling can be traced to a variety of sources. The first is promotion of mitochondrial fission via down-regulation of fusion factors such as Mitofusin 1+2, leading to fragmented mitochondrial bodies which in turn leads to an increase in ROS generation (Xie and Chung, 2012). ROS generation through increased presence of mitochondrial fission factors is not exclusive to α-syn, however is still worth consideration with regards to potential therapeutic treatment (Yu, Robotham and Yoon, 2006; Ježek, Cooper and Strich, 2018). Increase of internalised ROS leads to damage of mitochondria and other neighbouring organelles eventually leading to cellular death. α-syn can also directly interact with Tom20 and Complex I, inhibiting their activity in ATP synthesis thus reducing the efficiency of action potential depolarisation signalling and neurotransmitter release at axonal terminals (Di Maio et al., 2016; Chinta et al., 2010; Reeve et al., 2015). Additionally transportation of mitochondria is negatively affected by α-syn presence as well, shown to arrest transport by dismantling microtubule tracks thus preventing kinesins‟s/dynein‟s ability to migrate. α-syn presence may also have an indirect effect on Miro1 through the increase of intracellular Ca2+ (O'Donnell et al., 2014; Chu et al., 2012; Melo et al., 2017; Prots et al., 2013; 2018). The interaction of Miro1 with the motor adaptor complex is dependent on intracellular Ca2+ levels, with lower levels facilitating the connection to TRAK to allow for mitochondrial migration, whilst higher concentrations cause Miro1 to directly bind to kinesin/dynein inhibiting their ability to travel (Wang and Schwarz,
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2009). Pathological α-syn can promote an increase in intracellular Ca2+ concentration so it may be possible that the mitochondrial migration arrest observed in later stages of PD and MSA may be related to this mechanism (Calì et al., 2012; Parihar et al., 2008). Proteins such as PINK-1 and Parkin monitor mitochondria for abnormalities, promoting mitophagy of dysfunctional mitochondria. However, studies have shown that down-regulation of PINK-1 with increased pathological α-syn can inhibit mitophagy (Clark et al., 2006). Moreover, Ca2+ binding to Miro1 results in a shift from motor complex bridge to mitophagy inhibitor (Shaltouki et al., 2018). Parkin normally ubiquinates Miro1 signalling for its degradation however with loss of PINK-1, the factor involved in Parkin recruitment and activation, this process is unable to occur.
It is currently unknown how pathological α-syn is able to migrate within TnTs. It was recently shown that intercellular spread of α-syn via TnTs can be mediated by lysosomes, whereby lysosomes internalise α-syn that they are unable to degrade (Abounit et al., 2016). Given the occurrence of intercellular mitochondrial transfer between cells via TnTs, mitochondria were investigated as a potential vehicle of cell-to-cell transfer of α-syn aggregates. Mono-culture studies were performed using neuron-like SH-SY5Y neuroblastoma cells with aggregation of endogenous α-syn promoted by calcium influx. Co-culture studies investigated the spread of α- syn aggregates from cells pre-loaded with α-syn aggregates to unloaded cells. 1321N1 astrocytoma cells and differentiated microglia-like THP-1 cells were utilized as donor and acceptor cells respectively to examine the potential spread of α-syn between glial cells. In both mono- and co-culture systems, α-syn was detected by super-resolution microscopy bound to mitochondria in intercellular TnTs. siRNA-mediated knockdown of Miro1 expression was not able to block mitochondria-mediated α-syn aggregate transfer in co-culture.
4.2 Materials and Methodology
4.2.1 Cell Lines Utilised and Culture Conditions
Multiple cell lines were used for in vitro experiments including THP-1, 1321N1 and SH-SY5Y. Incubator settings for cell growth were set at 20% oxygen, 5% carbon dioxide and 37 °C.
Human THP-1 monocytes were grown in media comprised of RPMI 1640 supplemented with L-Glutamine, 10% foetal bovine serum, 0.05mM β-Mercaptoethanol in addition to 1% penicillin/streptomycin antibiotic solution. Media replacement occurred every 2-3 days to allow for sufficient growth, with growth not permitted to exceed 1x106 cells/mL. Cells were differentiated to a more microglial-like state through addition of 5ng/mL of Phorbol-12- Myristate-13-Acetate (PMA) state as described by the Bowdish protocol (Bowdish, 2011). Media was replaced following the 72 hour incubation period.
1321N1 astrocytoma cells were grown in Dulbecco‟s Modified Eagle Medium (DMEM) supplemented with L-glutamine, sodium pyruvate and 4.g/L D-Glucose in addition to 10% 83 foetal bovine serum and 1% penicillin/streptomycin antibody solution. Media was replaced every 2-3 days, with growth not allowed to exceed 1x106 cells/mL. When co-culturing with THP-1 cells, 1321N1 cells were instead incubated/grown within the same RPMI 1640 medium setup utilised for THP-1 growth.
SH-SY5Y human neuroblastoma cells were cultured in the same DMEM solution described for 1321N1. Once more, cells were not allowed to exceed to 1x106 cells/mL growth to maintain the monolayer. Unlike 1321N1 and THP-1 cells, SH-SY5Y cells were not incubated with α-syn in media prior to experimentation. SH-SY5Y cells were instead exposed to 50mM KCl (30 mins) as described by Follet and colleagues to allow for rapid depolarisation and Ca2+ influx (Follett et al., 2013). Fresh media replacement omitting KCl occurred following the incubation period.
4.2.2 SDS-PAGE and Western Blotting Protocol
Samples were lysed to extract mitochondria for analysis. 1321N1 and differentiated THP-1 were cultured in 3 wells of a 6-well plate each, with THP-1 cells having undergone differentiation prior to culturing in wells. At 70-80% confluency, the plates were placed on ice to keep samples cool and underwent three phosphate buffer saline (PBS) washes prior to centrifuging (500g, 5 mins, 4°C). Supernatant was discarded and 100µL of Thermo-Fisher radioimmunoprecipitation assay (RIPA) buffer with phosphatase inhibitors were added to break the pellet before shaking on ice (30 mins). Once shaken the sample was centrifuged (16,000g, 5 mins, 4°C) from which the supernatant was taken as the sample of interest. Once extracted, quantification of protein content was performed using the Pierce™ BCA Protein Assay Kit, whereby several protein standards were prepared including 2000µg/mL, 1500µg/mL, 1000µg/mL, 750µg/mL, 500µg/mL and 250µg/mL BSA, in addition to sterile deionised H2O (dH2O) acting as both a control and sample dilutor. 5µL triplicates of each standard, including dH2O controls, and 1:4 dilution of supernatant samples were loaded in to a 96-well microplate. Working Reagent was added to each well up to 100µL to create 1:20 dilution. The microplate was placed on the plate shaker (300rpm, 37°C, 30mins) before analysis via Tecan Infinite 200 plate reader i-Control software. 35 µg was determined to be the most suitable sample concentration for SDS-PAGE.
Prior to SDS-PAGE, 15µL sample protein was mixed with 5µL 4x Laemmli sample buffer creating a 3:1 buffer solution. If the amount of protein sample was below 15µL, RIPA was added to make up 15µL. Sample solution was vortexed then boiled (99°C, 5mins) prior to loading into the SDS-PAGE gel (1.5mm width, 20µL capacity). The composition of the upper 4% stacking gel included dH2O, 0.5M Tris HCl (pH 6.8), 30% acrylamide/bis solution, 10% sodium dodecyl-sulfate (SDS), 10% ammonium persulfate and tetramethylethylenediamine. The 15% separation gel composition was identical except for the replacement of 0.5M Tris with 1.5M Tris HCl (pH 8.8), allowing for separation of proteins sized between 10-100kDa. 5µL of Bio Rad Protein All Blue Standard in select wells served as the protein ladder. Initial voltage 84 was set at 100V until samples reached the separation gel, after which it was increased to 120V. Gel electrophoresis ran until samples were 5mm from the end of gel. When electrophoresis ceased, separation gel was soaked in 1x transfer buffer. 0.2µm nitrocellulose membrane was cut to the dimensions of 8.5cm x 5cm and soaked in 1x transfer buffer*. A specialised transfer cassette was prepared stacked as such: black cloth, 2x 0.83mm thick blot paper, separation gel with dye facing upward, nitrocellulose membrane, 2x 0.83mm thick blot paper and black cloth. Each component was washed and soaked in 1x transfer buffer prior to stacking. Nitrocellulose membrane was carefully placed onto separation gel and rolled using a small roller to eliminate any potential air bubbles. The cassette was placed into the transfer container and filled with 1x transfer buffer for western blotting (4°C, 100V, 75mins).
Once transfer was complete the nitrocellulose membrane was placed in 0.05% PBS/Tween and the separation gel discarded. Ponceau was employed to confirm transfer of the protein from the separation gel to the nitrocellulose membrane and once confirmed, the membrane was washed with PBS/Tween until bands were no longer visible. The membrane was blocked in 5% skim milk (room temperature (RT), 1hr) followed by primary antibody incubation (Overnight, 4°C, 1:1000). Diluted in skim milk, primary antibodies consisted of Thermofisher Scientific RHOT1 (Miro1) monoclonal antibody (CL1083) and Santa Cruz -Tom20 primary rabbit polyclonal antibody, provided by the Molecular Therapy Group at BIOCEV in Vestec, Czech Republic. Following primary incubation, membranes underwent 3x PBS/Tween washes (10 min), before incubation with secondary antibodies (RT, 1hr, 1:10000). Membranes underwent three PBS/Tween washes (10 mins) before loading with chemical luminescent FEMTO-ECL for band stain capture using an Azure 600C Gel Imaging System. For β-actin staining, membranes were rinsed in PBS/Tween and re-blocked in 5% skim milk. The primary antibody used was Cell Signalling‟s anti-β-actin (13E5) rabbit monoclonal antibody (1:1000) provided by BIOCEV‟s Molecular Therapy Group at Vestec, Czech Republic. Secondary incubation and analysis are identical to that previously described.
4.2.3 Miro1 and Control siRNA Transfection
Utilising both a selective and control small interfering RNA (siRNA), 1321N1 cells were examined for the silencing of Miro1 expression. The protocol for siRNA transfection was slightly altered from that provided by the manufacturers. Transfection regent was prepared, composed of 9µL of Invitrogen Lipofectamine® RNAiMAX Reagent diluted in 150µL of gibco Opti-MEM® Reduced Serum Medium. 3µL of either Ambion RHOT1 (Miro1) Silencer® Select Validated siRNA or Ambion Silencer® Select Negative Control #1 siRNA was diluted in 150µL in Opti-MEM® Medium, which reduced the siRNA concentration from 10µM to 30pmol. Diluted siRNA and diluted Lipofectamine® RNAiMAX Reagent were mixed in a 1:1 ratio (150µl each) and left to incubate (RT, 30 mins). 1321N1 cells were washed with DPBS
* 10x Transfer Buffer = 30.33g TRIS BASE + 110.32g Glycine + dH2O, filling to 1L 85 1x Transfer Buffer = 100mL Transfer Buffer + 200mL Methanol + dH2O, filling to 1L and lifted with Trypsin-EDTA for cell counting. Following incubation period, 250µL of siRNA- RNAiMAX solution was spotted on a 6-well plate well to a concentration of 25pmol of siRNA and 7.5µL of Lipofectamine® RNAiMAX per well. Cells were seeded at 0.2x106 and incubated (37°C, 48 hours). Following incubation, cells were prepared for WB analysis as previously described.
4.2.4 Sample Preparation for Fixation and Fluorescent Staining
SH-SY5Y cells were incubated with 50mM KCl in media prior to staining (30 mins), after which the media was discarded and the cells washed with PBS before addition of fresh DMEM media excluding KCl as previously described by Follet and colleagues (Follet et al., 2013). Ca2+ measurement controls were not conducted. SH-SY5Y cells were removed using Trypsin-EDTA 2 hours following KCl incubation and transferred to a 6-well plate (3x105 cells/mL per well). Residing on the bottom of each well were pre-autoclaved High-Performance, ISO 8255 compliant Zeiss Cover glasses with a thickness of 170±5µm and dimensions of 18mm by 18mm. Fresh cellular media was within each of these wells for continued SH-SY5Y culturing. The 6-well plate was placed within an incubator (48 hrs) before fixation and fluorescent staining began. Untreated samples, SH-SY5Y cells unexposed to KCl, acted as controls.
Preparations for 1321N1 and THP-1 were for the most part identical with the exception of differentiating THP-1 cells with PMA prior to α-syn exposure. For co-culturing the untreated groups, those not pre-exposed to aggregated α-syn, were loaded first into a 6-well plate with pre-autoclaved Zeiss coverslips, filled with RPMI 1640 cellular growth media. 1321N1cells were loaded 2.5x105 cells/mL per well whilst differentiated THP-1 was 5x105 cells/mL per well to account for their inability to replicate. 1321N1/Differetiated THP-1 were incubated with 2µg/mL of aggregated α-syn (48 hrs) before removal of media. Cells underwent 2x PBS washes before removal with Trypsin-EDTA to the 6-well plate. For co-culture, treated cells were transferred to wells pre-loaded with non-α-syn exposed healthy cells. Culturing conditions included 1321N1 and differentiated THP-1 mono-cultures and co-cultures. Different co-culture conditions include α-syn treated and non-treated, untreated controls and Miro1 siRNA/Control siRNA treated 1321N1 (α-syn) co-cultured with differentiated THP-1 (healthy). All conditions were left to incubate for a period of 48 hours prior to commencement of fixation and staining protocol.
4.2.5 Fixation and Fluorescent Staining Protocol
Following the incubation period, media was discarded and each well underwent two rinses and a wash (5min) with 0.05% PBS/Tween. Cells underwent fixation in 1mL 4% paraformaldehyde (RT, 30 mins), permeabilization in 0.1% Triton X-100 in PBS/Tween (RT, 10 mins) and blocking in 1mL of 20% normal goat serum (NGS) in PBS solution (RT, 1hr) with 3x 0.05%
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PBS/Tween washes in between (10 mins). Wells underwent a single 0.05% PBS/Tween wash (10 min) prior to incubation with primary antibodies (RT, 60 mins). Primary antibodies used were LB509 anti-α-syn primary mouse monoclonal antibody supplied by Abcam and Anti- Tom20 primary rabbit polyclonal antibody supplied by Santa Cruz, provided generously by the Molecular Therapy Group at BIOCEV in Vestec, Czech Republic. LB509 was diluted to a concentration of 1/350 whilst anti-Tom20 1/200 in 1% NGS/PBS.
Following primary antibody incubation, coverslips underwent a triplicate wash with 0.05% PBS/Tween (10 mins) prior to incubation in the dark with secondary antibodies (60 mins). Secondary antibody solution consisted of anti-mouse IgG−Abberior® STAR 580 antibody and anti-rabbit IgG−Abberior® STAR RED antibody, both produced in goat for stimulated emission depletion (STED) application supplied by Sigma-Aldrich, provided by the Molecular Therapy Group at BIOCEV in Vestec, Czech Republic. Secondary antibodies were diluted in 1% NGS/PBS (1/100) in addition to Hoescht dye (1/500) for nuclear staining. Following secondary incubation, coverslips underwent a triplicate wash with PBS (10 mins). Coverslips were rinsed twice using dH2O for removal of any excess antibodies or saline solution and left to dry in the dark prior to mounting. Once dried, 1 drop of Aberrior Mount Liquid Antifade was spotted on a glass slide before mounting the coverslip. Excess mounting media was removed from the coverslip edges before sealing with nail polish. Glass slides were left in 4.0°C overnight prior to observation under microscopy.
Minor alterations in the staining protocol were made for β-actin staining. Fixation using 4% paraformaldehyde was ineffective in preserving the structure of the actin filaments thus cells were fixed in 100% ice-cold methanol (15 mins, -20°C) instead. For primary antibody staining, Cell Signalling‟s anti-β-actin (13E5) rabbit monoclonal primary antibody was utilised instead of anti-Tom20, diluted 1/200. Alterations were made for confocal staining as well including fixation with methanol-acetone (3:1), washing with PBS, blocking with 20% normal horse Serum (NHS)/PBS, using WAKO Iba-1 primary antibody, diluting antibodies in 1% NHS/PBS, with the use of Alexa Fluor® 488 and 568 secondary antibodies (anti-mouse and anti-rabbit respectively) and foregoing Hoescht staining for DAPI. Hoescht staining produced better clarity of nuclei when imaging in the Abberior Instruments 775 STED microscope compared to DAPI, hence its substitution for STED staining. SH-SY5Y confocal work followed this staining protocol, with the exception of using Santa Cruz anti-Tom20 primary as opposed to WAKO Iba-1. The same secondary Alexa Fluor® antibodies were utilised.
4.2.6 Microscope Analysis and Fiji Software
STED super-resolution microscopy was utilised to examine TnT structure formation in fixed fluorescent samples, specifically to observe if α-syn was bound to migrating mitochondria highlighted by Tom20. STED microscopy is a form of super-resolution laser microscopy 87 allowing for analysis of samples at the nm scale. The instrument utilised was the Abberior Instruments 775 STED at the Imaging Core Facility at BIOCEV in Vestec, Czech Republic. The 775 STED possessed a 2-colour STED far-red laser with 775nm depletion laser and pulsed 561nm and 640nm excitation lasers. The objective lens utilised for all imaging was Nikon CFI Plan Apo Lambda 60x Oil, Numerical Aperture of 1.40 and working distance of 0.13mm. The detector utilised was an Excelitas Technologies 4x Photon counting module, which included two time resolved detectors optimised for FLIM for high detection sensitivity for red light. Abberior Instruments Imspector software was utilised for capturing both overview confocal and STED images. STED microscopy functions by stimulating the fluorophore of a secondary antibody using an excitation laser and detecting the region emitting the greatest amount of excitement following stimulation. A secondary depletion laser filters out the regions generating lesser excitation, leaving only the original site excited by the laser, thus removing significant amounts of excess light generated by the focal point providing better clarity images. Whilst a vast majority of qualitative work utilised the Abberior 775 STED Microscope, an Olympus FV1000 was utilised for the confocal experiments described in section 4.3.1. The 60x immersion oil lens had a numerical aperture of 1.4, capturing images at a resolution of 1024x1024 pixels. Olympus FV-ASW 3.2 viewer was utilised for image false colouring and exportation.
(Fiji Is Just) ImageJ, commonly referred to as Fiji, was the program utilised for exportation of Abberior Instruments Imspector software files. Fiji‟s “Bio-Format Exporter” function was utilised to convert Imspector files to PNG for qualitative analysis. All post-scan additions were performed using Fiji which includes scale bar addition and false colouring. Quantitative analysis was attempted through fluorescence activated cell sorting (FACS) however this was proven to be unsuccessful. Utilising the same co-culturing method as previously designed, the aim was to investigate transfer of α-syn to the cell type not exposed to α-syn. For example investigating the proportion of THP-1 cells with α-signalling when co-cultured with 1321N1 cells pre-exposed to α-syn. Ultimately sorting of these two cell types proved unsuccessful and a poor indicator of α- syn spread via solely TnT-based methods. SPSS Version 25 was utilised for statistical analysis.
4.3 Results
4.3.1 Confocal Analysis Revealed Formation of TnTs in KCl Treated SH-SY5Y Mono- Cultures and Potential α-syn Exchange in 1321N1 and Differentiated THP-1 Co-Cultures
Transfer of α-syn from α-syn-treated to untreated cells was examined in 1321N1 and differentiated THP-1 co-cultures. As seen in Figure 15a and 1b Iba-1, a marker for macrophage and microglial cells, was used to stain the samples. Differentiated THP-1 and 1321N1 cells were distinguished based on morphology. Differentiated THP-1 cells were smaller and more round in appearance compared to 1321N1 cells, which were larger, less circular and possessed 88 protrusions extending off the cell body. Additionally the nucleus was smaller in differentiated THP-1 cells compared to 1321N1. Iba-1 staining highlighted small tunnel structures forming between the two cell types, aspects of which matched those previously been reported as thick TnTs. Given the lack of specific markers for thick TnTs, the criteria used to identify them was based on 3 categories: thickness (700nm-1.2µm), length (30µm-130µm) and cargo (organelles). Figure 15a revealed the formation of these structures in a control environment, whereby no cells were pre-exposed to α-syn prior to co-culturing. In Figure 15b, 1321N1 cells were pre-exposed to α-syn prior to co-culturing with differentiated THP-1 cells, in this example revealing that the neighbouring THP-1 cells to the 1321N1 had internalised α-syn. This confirmed that spread from one cell type to another had occurred but the method under which it did was unclear. Z- scanning of the highlighted 1321N1 and THP-1 revealed a thin structure that may have connected the 2 structures, implicating potential transfer by TnT. TnTs are fragile structures that are commonly reported to be easy to destroy during the fixation and staining process. If this structure was once a TnT then it is likely that it may have been damaged from this process. Unfortunately more information regarding these structures could not be attained due to limitations in confocal microscopy. The small size the structures formed makes it difficult to image without loss of detail and clarity.
To explore mitochondrial exchange via TnTs, SH-SY5Y neuroblastoma cells were chosen due previous reports of TnT formation in addition to the cells expressing monomeric α-syn. Treatment with KCl depolarises SH-SY5Y cells, raising intracellular Ca2+ thus causing the monomeric α-syn to aggregate (Follet et al., 2013; Sousa et al., 2013). As observed in Figure 15c, examinations produced two interesting findings. First was the formation of a structure matching the description of a TnT with potentially migrating mitochondria between cells (white arrow). TnT formation for exchange of cargo had been previously reported by Smith and colleagues in 2011 however in that case it was Ca2+ as opposed to an organelle exchange (Smith, Shuai and Parker, 2011). Recently it was reported that mitochondria could transfer via TnTs from one SH-SY5Y cell to another, further supporting this hypothesis (Dilsizoglu Senol et al., 2019). The second interesting finding was that both connected cells displayed α-syn within their cytoplasms in addition to faint signall within the TnT-like structure itself (yellow arrow). The presence of aggregated α-syn in both cells is likely due to prior KCl treatment as opposed to exchange from one cell type to another however this does not rule out the possibility of additional α-syn through TnT transfer. Red fluorescent signals signifying α-syn staining appear to be within the TnT-like structure bound to mitochondria however signals were faint, thus unable to confirm α-syn bound to mitochondria.
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1321N1 + THP-1 Control 60x Confocal
a
DAPI Iba1
30µm
1321N1 (α-syn) + THP-1 Control 60x Confocal
b
DAPI Iba1 aSN
20µm
c SH-SY5Y (KCl-Treated) 60x Confocal
DAPI Tom20 aSN
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Figure 15 – Confocal Immunofluorescence shows α-syn in TnT-like Structures between
SH-SY5Y monocultures and 1321N1 and Differentiated THP-1 Co-Cultures
(a) Confocal image captured by Olympus FV1000 at 60x magnification of 1321N1 (Green
Arrow) and differentiated THP-1 (Red Arrow) cell co-culture. Control Samples (No α-syn). Left image is the fluorescence capture whilst the image on the right is the same region under DIC. Structure matching the description of a TnT is present between both cell types visible
under both fluorescent (White Arrow) and DIC (Black Arrow) microscopy. (b) Confocal
image captured by Olympus FV1000 of α-syn pre-exposed 1321N1 and differentiated THP-1 co-culture. (Left) Close-up of 1321N1 and THP-1 cells whereby α-syn (Yellow Circle) is present in both cells indicating transfer from 1321N1 to THP-1. (Right) Z-Scan at a lower
level revealing a damaged structure that may have once been a TnT (Purple Box). Potentially
damaged during the fixation or staining process. (c) Confocal image of SH-SY5Y cells taken using an Olympus FV1000 microscope at 60x magnification. Formation of a structure matching the description of a TnT is present between 2 separate SH-SY5Y cells (white arrow).
The red and yellow boxes correspond to regions within the SH-SY5Y cells where α-syn is
present. Yellow arrow represents potential α-syn signal within TnT-like structure. 4.3.2 STED Analysis Shows Formation of TnT-like Structures Between SH-SY5Y Cells in Addition to α-syn Migrating Within These Structures Whilst Bound to Mitochondria
Repeating the SH-SY5Y experiments for analysis via STED microscopy showed the formation of TnT-like structures between SH-SY5Y cells. The TnT-like structures where highlighted by cellular autofluorescence on the α-syn channel, an unintended effect of the protocol as seen on Figures 16a +16b. These structures matched previous descriptions of TnTs with regards to thickness and length. Additionally Tom20 staining revealed the presence of mitochondrial bodies within the structures themselves. Density of mitochondria within these structures was less than those observed proximal to the nucleus and those in thicker protrusions produced by the SH-SY5Y cells. As seen in Figure 16a, formation of the TnT-like structures occurred in non-KCl treated samples, corroborating what has been previously discussed in the literature with regards to TnTs and how their formation is likely to occur as a part of normal cellular cargo exchange, not just a mechanism of spread in disease. Whilst the structure was highlighted by autofluorescence signalling, there was no indication to suggest that there was presence of α- syn anywhere within neither the structure nor the connecting cells. This is based on the strength of the α-syn signalling produced in the KCl treated cells as shown by the example in Fig 16b. The overview confocal scan reveals a multitude of small aggregated α-syn bodies in a multitude of regions surrounding SH-SY5Y cell nuclei, which is in contrast to the lack of these signals in untreated examples. The intensity of the fluorophore signal is also distinct to that produced from autofluorescence staining, with α-syn appearing as bright green in contrast to the backgrounds darker, faded appearance. Additionally STED images captured using the 775
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a Hoechst aSN Tom20 aSN Tom20
~900nm
~700nm ~700nm 2µm SH-SY5Y Untreated STED SH-SY5Y Untreated 10µm Overview
SH-SY5Y α-syn 2µm Confocal ~1100nm SH-SY5Y α-syn STED b ~3000nm
~900nm aSN Tom20 c ~900nm
Hoechst aSN aSN 2µm Tom20 SH-SY5Y α-syn Overview Tom20
Hoechst aSN 2µm d β-Actin
SH-SY5Y β-Actin α-syn STED SH-SY5Y β- Actin α-syn Overview
Hoechst ~800nm aSN β-Actin
aSN β-Actin
10µm
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Figure 16 – STED immunofluorescence Imaging of SH-SY5Y Cells Revealed The Formation of TnT-like Structures and Migration of α-syn Bound to Mitochondria
(a) (Left) Overview Capture at 60x magnification revealing the a long thin protrusion matching the description of a TnT. Structure highlighted unintentionally by auto- fluorescence. Untreated SH-SY5Y Control Sample (Right) STED Image of Tom20 in the highlighted region within the yellow box of Overview image. Multiple mitochondrial bodies were visible with clear clarity. (b) (Left) Overview capture of KCl-treated SH-SY5Y cells.
Thin TnT-like structure visible in the middle of the image (yellow arrow) whilst a thicker structure (orange arrow) too thick to be a TnT. (Right) STED image of highlighted region within overview image. α-Syn appears bound to surface of mitochondria highlighted by Tom20 (yellow arrow).(c) Example of confocal capture of same region highlighting the
difference of image and structure clarity at the nm level. (d) (Left) Overview images of β- actin stained SH-SY5Y cells. Both upper and lower left images are the same region at different Z-Scale elevations, revealing the TnT-like structure of the upper cell connecting to the axon stem of the lower cell. The white dotted line in the lower image may indicate where
the TnT-like structure potentially originated from or connected to. (Right) STED image of region highlighted in the yellow box in both overview images. Region is TnT whereby both autofluorescence and presumably α-syn aggregates are present (yellow circles).
STED microscope and Imspector software helps remove autofluorescent signalling during acquisition, aiding in distinguishing what is and is not α-syn staining. As exhibited in Fig 16b, analysis via STED of the TnT-like structure formed between two α-syn-containing cells revealed possible mitochondrial migration within these structures as previously seen in 4.3.1, and aggregated α-syn was bound to mitochondria (yellow circle). Figure 16c is a confocal capture of the same area analysed via STED in Figure 16b, highlighting the difference in image quality and clarity between the two methods of laser image acquisition.
Thick TnTs are composed of both microtubules and filamentous actin, thus it was predicted that staining for actin would highlight and reveal similar structures to those produced for α-syn staining. Due to a limitation of the 775 STED instrument, only 2 channels can be used for STED image acquisition, thus Tom20 was substituted for β-actin. α-syn staining served to confirm that staining matching that of aggregate bodies resided within the TnT-like structures highlighted by β-actin. As exhibited in Figure 16d, β-actin staining highlighted a TnT-like structure and the cytoplasms of the SH-SY5Y cells. The two different planes in the confocal overview images once more show a distinct difference in the thickness between the TnT structure on the left and the axon structure on the right. Additionally the distal section of the axon-like structure resembles an axonal terminal. The TnT-like structure connects the top cell with the axon of the bottom cell. STED imagery revealed α-syn signals denoting aggregate bodies were within the β-
93 actin structure suggesting α-syn migration thus giving further credence to the structures observed in Figures 16a and 16b to be TnTs.
4.3.3 1321N1 and THP-1 Mono-Culture Experiments Displayed Formation of TnT-like Structures and Confirmed α-syn-Mitochondria-mediated Migration As a Mechanism of Spread Within Other Cell Types
Prior to co-culture examination of 1321N1 and Differentiated THP-1 cells, each cell type was examined for TnT-like structure formation separately. Similar to the SH-SY5Y experimentsdescribed in 4.3.2, the examined conditions included untreated controls, α-syn exposed and β-actin staining, with the exception of KCl treatment which was instead substituted to 24 hour aggregate α-syn incubation due to cells inability to express α-syn. Results from the 1321N1 mono-culture experiments displayed many similarities to those produced in the SH- SY5Y work. In the untreated samples for instance TnT-like structures, once more highlighted by autofluorescence signalling, formed between separate cells thus confirming that formation of these structures can occur normally. Figure 17a not only highlights the TnT structure in question but the difference in thickness to the protrusions extending from the central cellular mass. These thicker protrusions are the same as those highlighted by Iba-1 in Figures 15a and 15b and serve to highlight not only differences in morphology from differentiated THP-1 cells, but SH-SY5Y cell as well. These protrusions are visually distinct from the axonal-like protrusions produced by the SH-SY5Y cells. In 1321N1 α-syn treated samples, the formation of TnT-like structures was observed once more. As exhibited by Figures 17b, α-syn phenotypical interactions with mitochondria are characteristically similar to both the SH-SY5Y results in section 4.3.2 (Figure 16b) and the work reported by Grassi and colleagues (Grassi et al., 2018). In 1321N1 cases, α- syn was bound to the outer membrane of the mitochondria, matching the Grassi et al. report and was possibly migrating within the TnT-like structure identically to those observed within SH- SY5Y cells. This provides more evidence for α-syn transport within TnT structures whilst bound to mitochondria not only within SH-SY5Y but other neuronal cell types, suggesting it as a potential method of α-syn spread. β-actin staining (Figure 17c) confirmed the presence of α- syn aggregates within TnT-like structures highlighted by β-actin.
Examination of differentiated THP-1 cells revealed similar features to those observed in SH- SY5Y and 1321N1 cells. In all cases, formation of TnT-like structures was observed. The morphology of THP-1 cells was circular and lacked extensive thick protrusions as observed in both SH-SY5Y and 1321N1 cells, thus making morphological distinction possible. Possible α- syn migration within the TnT- like structures whilst bound to mitochondria was observed
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1321N1 Untreated 2µm Overview
aSN Tom20
a Hoechst aSN Tom20 1321N1 Untreated STED
1321N1 α-syn Overview 1321N1 α-syn STED 2µm
b ~3000nm
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aSN Tom20
Hoechst aSN Tom20
1321N1 β-Actin α-syn 1321N1 β-Actin α-syn Overview STED c
Hoechst aSN aSN ~900nm β-Actin β-Actin
~700nm
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Figure 17 - Immunofluorescence of 1321N1 Cells monocultures via STED Revealed
The Formation of TnT-like Structures and Migration of α-syn Bound to Mitochondria in Treated Samples
(a) (Left) Overview Image of untreated 1321N1 cells. A thin protruding structure was observed that matched the description of a TnT based on thickness, length and cargo. (Right) STED image of highlighted region in overview picture. As was evident in the SH-
SY5Y cells, mitochondria are clearly highlighted by Tom20 but no α-syn is present. (b) (Left) Overview picture of an α-syn treated 1321N1 cell sample, highlighting TnT-like structure formation and potential migration of α-syn whilst bound to mitochondria within the structure. (Right) STED image of the region highlighted in the overview figure, whereby
α-syn is seen on the outer membrane surface of mitochondria whilst within the suspected TnT structure (yellow circle) similar to what was observed in KCl-treated SH-SY5Y cells. (c) (Left) Overview of β-actin stained α-syn treated 1321N1 cells. TnT-like structure highlighted by β-actin structure. TnT structure was at a higher z-scale than the rest of the cellular body.
(Right) STED image of region highlighted in focus figure. As observed in the SH-SY5Y β- actin stain, autofluorescence and α-syn aggregate stains (yellow circle) all within the actin structure. between differentiated THP-1 cells (Figure 18b). The β-actin stain example exhibited in Figure 18c highlights how TnTs form between neighbouring cells. In this example the THP-1 cell on the left is forming the TnT, visualized by the extending protrusion emanating from the cellular body, to the cell on the top right. As mentioned previously, TnTs are an extension of the cellular cytoskeleton, denoted by the identical components required for their construction. This specific example illustrates how the formation of these structures occurs, with one cellular body forming the protrusion until it connects with the cytoplasm of a neighbouring cell. Worth noting that in other cell β-actin examples, the bases of the structures on either end were thicker than the structure bodies. Once more α-syn aggregate staining all resided within the structure highlighted by β-actin.
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Hoechst THP-1 Untreated aSN STED Tom20 a
aSN Tom20 ~1000nm
10µm THP-1 Untreated 1µm Overview
THP-1 α-syn Overview THP-1 α-syn STED
Hoechst aSN ~900nm Tom20 aSN Tom20 b ~900nm
10µm 1µm
THP-1 β-Actin α-syn THP-1 β-Actin Overview α-syn STED c
~900nm
~1000nm
Hoechst aSN aSN β-Actin β-Actin
10µm 1µm
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Figure 18 - Imaging of Differerentiated THP-1 Cells monocultures via STED Revealed The Formation of TnT-like Structures and Migration of α-syn Bound to Mitochondria in
Treated Samples
(a) (Left) Overview picture of untreated control differentiated THP-1 cells. TnT-like structure seen connecting 2 separate cells in addition to highlighting morphology of THP-1 cells, mostly circular. (Right) STED image of region highlighted in focus figure. Once more no evidence of α-syn is present. (b) (Left) Overview picture of α-syn treated differentiated THP-1
structure. Once more image presents the morphology of the cells and TnT-like structure between them. (Right) STED image of region highlighted in focus figure. Once more evidence of α-syn potentially migrating within structure is witnessed (yellow arrows), exhibiting the same interactions in previous cells. Yellow lines serve to highlight the TnT-like structure. (c)
Left) Overview picture of β-actin stained α-syn treated differentiated THP-1 cells. TnT-like structure in this example has not yet connected to neighbouring cell. Perhaps an example of
how TnT structures may look like whilst structures are forming. β-acting staining also serves to highlight morphology. (Right) STED image of region highlighted in focus figure. Once
more α-syn staining (yellow arrows) all within the bounds of the structure highlighted by actin staining.
4.3.4 Evidence of TnT-like Structure Formation and α-syn-Mitochondria-mediated transfer between 1321N1 and Differentiated THP-1
Co-culturing of 1321N1 and differentiated THP-1 proved successful, with formation of TnT- like structures observed. Similar to the mono-culture experiments, formation of the structures occurred without α-syn presence. In the control samples potential migration of mitochondria was witnessed, placing forth the notion that mitochondrial spread is a method of exchange that can occur between cells of various types as opposed to singular cell types (Figure 19a). Similar observations were discovered with the α-syn treated co-culturing samples as well. Regardless of which cell was incubated with α-syn prior to culturing together, evidence of potential α-syn migrating within the structure was observed. As displayed in figures 19b and 19c, connecting structures match the phenotype of those presented in other samples in addition to matching the specification of a TnT-like structure. In each case internalised α-syn was present within both the cell type exposed to the protein prior to culturing and the healthy cell thus confirming transfer. Whilst α-syn can utilise a variety of spread models, given the evidence of potential mitochondrial migration, it can be hypothesised that the TnT-like structures have aided in some capacity to its spread within this model. Whether this capacity is significant is unknown.
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Hoechst aSN Tom20
1321N1 + THP-1 Untreated Overview 20µm
1321N1 (α-syn) + 1µm THP-1 Overview
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1321N1 (α-syn) + ~700nm b THP-1 STED
Hoechst aSN aSN 10µm Tom20 Tom20
c 2µm 1321N1 + THP-1 (α-syn) STED ~850nm
aSN Tom20
Hoechst aSN 1321N1 + THP-1 (α-syn) Tom20 Overview
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Figure 19 – TnT-like Structure Formation with Migrating α-syn between 1321N1 and Differentiated THP-1 cells
(a) Overview picture of untreated control of 1321N1 (orange arrow) and differentiated THP-1
(blue arrow) co-culture. TnT-like structure formed between the two cells confirming that TnT formation not only occurs in a mono-culture setting but a co-culture one as well. Presents
with many of the same characteristics as the other previously presented examples. (b) (Left) Overview picture of 1321N1 and differentiated THP-1 co-culture, wherein 1321N1 was
incubated with α-syn prior to culturing with THP-1. TnT-like structure observed similar in presentation to those observed in mono-culture and the untreated co-culture control. TnT was
connecting THP-1 and 1321N1. (Right) STED image of region highlighted in overview picture. α-syn was seen bound to mitochondria (yellow circle) within structure identical in
presentation to both previous samples and report from Grassi et al. Yellow lines serve to highlight TnT-like structure and differentiate it from 1321N1 protrusion just behind the
structure. (c) (Left) Overview picture of 1321N1 and differentiated THP-1 co-culture, wherein THP-1 was incubated with α-syn prior to culturing with 1321N1. TnT-like structure observed
similar in presentation to those observed in mono-culture and the untreated co-culture control. TnT was connecting THP-1 and 1321N1. (Right) STED image of region highlighted in overview figure. Once more TnT-structure presented with evidence of potential α-syn
migration whilst bound to mitochondria (yellow arrow). This serves to highlight that spread
via this method could be multi-directional.
4.3.5 Miro1 knockdown does not affect α-syn transfer via TnTs
Investigation into whether Miro1, the conduit which connects mitochondria to the motor transport complexes kinesin/dynein, was conducted via siRNA transfection to determine if this is a significant factor involved in mitochondrial intercellular exchange. Transfections resulted in significant reduction of Miro1 expression (p=0.024) with no reductions in Tom20 signifying that Miro1 was only targeted in transfections. Negative siRNA had no effect on either factor. Whilst reduction in Miro1 was significant, qualitatively it had no effect on potential mitochondrial migration along the TnT-like structures. Evidence of α-syn bound to mitochondria for potential migration was still observed within transfected samples, displaying the same characteristics of TnT-based spread as those observed in previous sections (Figure 20b). Negative siRNA transfection samples presented with the same phenotypes as the non- transfected controls and the Miro1 siRNA sample (Figure 20c). Attempts were made to quantify transfer from one cell type to another through the use of Flow Cytometry however these findings were unclear.
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2µm Hoechst aSN Tom20 1321N1 (α-syn) + THP-1 1321N1 (α-syn) + THP-1 Hoechst Miro1 siRNA Overview aSN Miro1 siRNA STED Tom20 1321N1 (α-syn) + THP-1 ~700nm Miro1 siRNA STED
b
10µm aSN 2µm Tom20
Hoechst 1321N1 (α-syn) + THP-1 aSN 1321N1 (α-syn) + THP-1 2µm aSN Contr. siRNA Overview Tom20 Contr. siRNA STED Tom20 1321N1 (α-syn) + THP-1 Contr. siRNA STED
c aSN Tom20 ~700nm
10µm 2µm
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Figure 20 –Miro1 siRNA knockdown does not affect Mitochondria-mediated α-Syn migration in TnT-like Structures
(a) WB results revealing siRNA was successful in reducing Miro1 expression in 1321N1. No significant reduction in Tom20 nor β-actin was observed. (b) (Left) Overview confocal capture of Miro1 siRNA treated 1321N1 and differentiated THP-1 co-culture. 1321N1 cells
were treated with aggregated α-syn prior to co-culturing and is visible in the 1321N1 cytosol. TnT-like structure with mitochondria within is evident, having formed to connect 1321N1 and the differentiated THP-1 cell. (Middle) STED image of TnT-like structure highlighted in yellow box. Similar to previous examples, α-syn appears bound to OMM within the structure
indicating possible spread via this method. Miro1 siRNA seemingly was not efficient enough to prevent mechanism of spread from occurring. (Right Upper) Focus image of purple box
region highlighted in 6b. This image highlights the α-syn present within the differentiated THP-1 (yellow circles) which, based on α-syn presence in the TnT-like structure, can be
assumed to be partially due to the connection formed between it and 1321N1 cells. (Right Lower) STED image capture of yellow box region in the focus image. STED analysis reveals
that α-syn is bound to mitochondria of THP-1 similar to other previous examples. (c) (Left) Overview confocal capture of negative siRNA treated 1321N1 and differentiated THP-1 co-
culture. Similar to Figure 4c, TnT-like structure is not connecting both cells but appears to be in the process of formation. Emanating from the 1321N1, it presents with many of the
phenotypical features of the one observed in THP-1 in 4c including the increased thickness at the end of the process. α-syn is present within both 1321N1 and THP-1 cell. (Middle) STED
image of yellow box in overview image. Highlights the end of the structure in addition to presence of mitochondrial bound α-syn (yellow circles). (Right) STED Image of purple box
region highlighted in overview image. STED image confirms TnT-like structure originates from 1321N1.
4.4 Discussion
The intersection between mitochondrial migration and α-syn transfer is a topic not previously investigated. Mitochondria are transported both intracellularly and intercellularly by motor transport complexes driven by kinesin or dynein, contributing to action potential maintenance and neurotransmitter release intracellularly and to respiration salvation in the case of intercellular transport (Kann and Kovács, 2007; Babenko et al., 2018; Dong et al., 2017). Cell- to-cell transfer of α-syn may utilize a variety of transport mechanisms, including exocytosis, leakage, transynaptic release, exosomes and even recently via gap junction proteins such as connexin-42 (Reyes et al., 2019). We have shown that α-syn aggregates have the potential to move between adjacent cells in TnTs bound to mitochondria. Much is understood about TnT structures, such as composition, role and cargo, however their significance in disease is not well described. TnTs have been shown to lead to increased propagation of pathological and 102 damaging materials such as HIV-1, tau fibrils and huntingtin to name a few (Okafo, Prevedel and Eugenin, 2017; Tardivel et al., 2016; Costanzo et al., 2013; Abounit et al., 2016b; Dieriks et al., 2017; Rostami et al., 2017). Lysosomes have been demonstrated to migrate within TnTs with attached α-syn, implicating intercellular organelle exchange as a potential disease material spread mechanism (Abounit et al., 2016a). This thus brings us back to the intersect of mitochondrial intercellular transfer and α-syn spread. The relationship between mitochondria and α-syn is well documented, with interactions between α-syn and mitochondria in synucleinopathies leading to organelle damage and eventual cell death (Chinta et al., 2010; Reeve et al., 2015; Wang et al., 2019). The interaction between α-syn and mitochondria however has not been explored previously in the context of cell-to-cell spread of α-syn aggregates.
TnT structures have been demonstrated in a variety of in vitro mono and co-culture models. We have identified via confocal microscopy structures with features corresponding to TnTs in differentiated THP-1 and 1321N1 co-cultures. THP-1 cells have been demonstrated previously to form TnT-like connections with immune cells such as dendritic cells, for the purpose of Ca2+ flux signalling to adjacent cells (Watkins and Salter, 2005). The observation of TnT-like structures between untreated control cells indicates that TnT fomation is a normal cellular process. The presence of TnT-like structures between THP-1 and 1321N1 cells bearing α-syn aggregates allows for the possibility that the transfer of α-syn aggregates from donor to acceptor cells may in part be mediated via TnTs. α-Syn has been reported to employ a variety of mechanisms in order to transfer to neighbouring cells. Thus, it seems likely that cell-to-cell spread of α-syn aggregates is multimodal. Nevertheless our current findings implicate TnT formation as providing one of multiple avenues whereby α-syn aggregates are able to move from affected to unaffected cells during disease progression. Abounit and colleagues previous report detailed intercellular lysosome migration via TnTs as a mechanism of α-syn transfer (Abounit et al., 2016a). Although TnT-mediated transfer of α-syn between connected cells has been demonstrated previously, this is the first to show α-syn aggregates bound to mitochondria within TnTs. A previous report of TnT-based α-syn migration in human strocyte cultures showed that formation of connecting TnT-like structures occurred as a method for mitochondria to be acquired from adjacent cells for respiratory rescue (Rostami et al., 2017). In this instance however mitochondria were not described to be transporting α-syn from donor to acceptor cells.
It was found that neuron-like cells are able to potentially transfer α-syn aggregates between cells in TnTs bound to mitochondria, indicating that this may provide a facile mechanism of cell-to- cell spread in PD. Much of the previous work on the role of mitochondria in synucleinopathies has focussed on interactions between α-syn and mitochondria in neurons, primarily relating to the contribution of ROS generation by dysfunctional mitochondria to cellular degeneration (Ryan et al., 2015). Neurons are the primary site within the central nervous system (CNS) where 103
α-syn is expressed and where aggregates form. Thus, TnT-mediated transfer of α-syn aggregates from neurons to adjacent cells driven actively by intercellular mitochondrial exchange may explain how α-syn aggregation initially propagates to different sites. SH-SY5Y cells have been established in previous cell culture studies to transfer mitochondria via structures matching the description of TnTs (Sartori-Rupp et al., 2019; Dilsizoglu Senol et al., 2019). Additionally they have also been demonstrated to form TnT structures for the spread of α-syn as reported by Dieriks and colleagues in which α-syn exchanged from SH-SY5Y cells to adjacent pericytes (Dieriks et al., 2017). No prior report has found that intercellular mitochondrial migration via TnTs can act as a vehicle for cell-to-cell α-syn transfer.
The confocal microscopy analysis of co-cultured cells showed structures resembling TnTs connected adjacent cells, each of which contained internalised α-syn. In addition, fluorescent signals highlighting mitochondria were detected within the proposed TnT structures, consistent with previous studies. The relatively small size of the TnT structures however made it difficult to ascertain using confocal microscopy whether α-syn was present within them. Employing STED helped overcome the limitations of the confocal analysis. Data on the untreated controls confirmed presence of mitochondria within TnTs suggesting this a normal feature of each cell type within both mono and co-culture conditions. Whether this is for potential cargo exchange is currently unknown. Whether this is for potential cargo exchange is currently unknown. Additionally, the increased resolution provided by the STED method confirmed the presence of α-syn not only within TnTs connecting SH-SY5Y cells, but also between differentiated THP-1 and 1321N1 mono and co-cultures. This finding indicates that potential spread of α-syn via TnTs is not exclusive to neurons. Recently it was revealed that α-syn interacts with outer mitochondrial membrane receptor Tom20, preventing the import of mitochondrial-targetted proteins through the mitochondrial outer membrane, which subsequently leads to excess ROS generation and eventual cell death (Di Maio et al., 2016). Additionally, the interaction of α-syn with mitochondria was recently found to be exclusive to pathological variants such as aggregates and not the normal monomeric form (Wang et al., 2019). Recent STED investigations by Grassi and colleagues have shown binding of a nonfibrillar, phosphorylated α- syn species to the outer membrane of cytosolic mitochondria in primary mouse neurons (Grassi et al., 2018). Our STED data demonstrated α-syn bound to the outer membrane of mitochondria in each of the cell culture systems investigated in both the cytoplasm of connected cells and internalised within TnT-like structures.
Additional information gained from STED examination included the confirmation of the deployment of thick TnTs for intercellular mitochondrial exchange. β-actin staining revealed that the structures with migrating mitochondria transporting α-syn matched the characteristics of thick TnTs, including thickness (700nm-1,100nm), length (30µm-130µm) and cargo (Rustom et al., 2004; Austefjord, Gerdes and Wang, 2014). Although the presence of α-syn bound to 104 mitochondria in TnT-like structures does not exclude other potential mechanisms of cell-to-cell transfer, the experimental setup of the co-culture experiments, in which only one cell type was pre-loaded with α-syn aggregate prior to co-culture, confirmed α-syn exchange from donor to acceptor cell type and indicates that possible mitochondrial-mediated α-syn tranfer via TnTs contributes to α-syn exchange. An interesting detail, shown in Figure 18c, depicts a potential method by which TnT-like structures form, with one cellular body forming and extending the protrusion until it forms a connection with the cytoplasm of a neighbouring cell. This may aid in explaining why the distal “head” of the protrusion is thicker than the body, which matches the previously detailed TnT criteria. Perhaps if the distal end of the structure fuses with the cytoplasm of the neighbouring cell, the structure would reflect what has been observed in other examples. A role for TnTs has been described previously in other neurodegenerative diseases, such as Creutzfeldt-Jacobs Disease, where TnTs were found to mediate cell-to-cell transfer of pathological proteins (Gousset et al., 2009; Eugenin, Gaskill and Berman, 2009). Indeed Abounit and colleagues remarked upon the mechanistic similarities between lysosome-mediated α-syn exchange via TnTs and prion-diseases (Abounit et al., 2016a). Given the proposed prion- like mechanism of templated misfolding and aggregation of α-syn, leading to the classification especially of atypical PD variants such as MSA as prion diseases (Prusiner et al., 2015), our findings add further evidence to support the prion hypothesis of PD propogation.
The role of Miro1 as an adaptor protein between mitochondria and the motor adaptor complex is well categorised (Kay et al., 2018; Shaltouki et al., 2018; Fransson, Ruusala and Aspenström, 2006; MacAskill et al., 2009). Whilst the majority of work focussing on Miro1 has been in the context of its role in intracellular mitochondrial migration, evidence exists of it facilitating mitochondrial connection to the kinesin/dynein motor adaptor complex for intercellular mobilisation (Ahmad et al., 2014). However, Miro1 siRNA knockdown did not block potential mitochondria-mediated intercellular α-syn exchange. Pre-loading of α-syn aggregates in donor cells (1321N1) prior to culturing with the acceptor (differentiated THP-1) allowed for qualitative measurement of the effect of Miro1 knockdown on intercellular mitochondrial exchange. The presence of α-syn bound to mitochondria within the TnT-like structures between Miro1 knockdown 1321N1 cells and differentiated THP-1 cells indicates that Miro1 may not be solely responsible for connecting mitochondria to the motor adaptor complex. The role of Miro1 as a bridge protein for mitochondria is highly dependent on intracellular [Ca2+], with increased Ca2+ leading to Miro1 binding directly to the motor protein to block microtubule-directed transport (Wang and Schwatz, 2009). Elevation of intracellular [Ca2+] can be promoted by α-syn aggregates such as by interaction with the endoplasmic reticulum calcium pump, which promotes Ca2+ release from the endoplasmic reticulum (Calì et al., 2012). Other factors than Miro1 may be associated with connecting mitochondria to the motor adaptor complex.
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Alternatively complete Miro1 knockout may be required to quantitatively eliminate intercellular migration of mitochondria.
Determining the significance of mitochondrial intercellular migration as a mechanism of α-syn propagation will require further exploration. Examination post-fixation makes it difficult to determine the mechanisms responsible for the presence of α-syn in acceptor cells and how significant the role played by potential mitochondrial-mediated α-syn migration via TnTs is. The use of live super-resolution techniques could provide further insight, allowing for the examination of TnT structure formation and a real-time analysis of mitochondrial migration within them. The use of super-resolution techniques or other techniques allowing for live analysis at the nm scale are essential, given both the relative size of these structures and the difficulty in analysis of finer details using lower resolution techniques, such as confocal microscopy. Utilisation of this approach may also shed light on the role of other organelles within TnTs involved in intercellular α-syn propagation. Expansion to include the investigation of other unexplored cells types may aide in understanding how α-syn is transported in atypical PD. In the case of MSA, oligodendrocytes are believed to acquire α-syn from an external source due to the lack of endogenous expression (Asi et al., 2014). Further exploring the current in vitro model utilising other cell types present within the CNS, such as oligodendrocytes, may help elucidate the presence of pathological α-syn. Additionally exploration of different co- culture combinations may reveal potential limitations as to which cell types can exchange mitochondria and α-syn via TnTs. Recently work by Dilsizoglu Senol and colleagues described an actin polymerization inhibitor, tolytoxin, as a promising inhibitor of TnT formation. As a cyanobacterial macrolide, tolytoxin prevents actin from forming structures such as TnTs, consequently leading to significant reduction of intercellular mitochondrial transfer (Dilsizoglu Senol et al., 2019). Additionally, tolytoxin treatment of SH-SY5Y cells was able to inhibit α-syn fibril spread (Dilsizoglu Senol et al., 2019).
Taken together, the current findings shift our current understanding of mitochondrial dynamics within synucleinopathies, as degeneration promoted by mitochondrial dysfunction no longer appears localised to a singular cellular type. The multi-cellular composition of the brain, comprising of neurons, oligodendrocytes, microglia and astrocytes as select examples, each perform their own individual functions that culminate together, allowing for the CNS to function normally. The formation of aggregated α-syn in any of these cells permanently alters the CNS from being able to function normally again (Lashuel et al., 2013). Most work on TnTs has primarily focussed on their role in vitro, thus making it difficult to ascertain their significance in an in vivo setting. This focus in vitro is primarily due to the small size of TnTs, their fragility and lack of specific markers, making detection in vivo difficult. Whilst reports exist of structures matching the description of TnTs in both mouse corneas and cancer cells in mesothelioma lung tissue, contention still exists as to whether TnTs are present in vivo or are an 106 exclusive factor of in vitro culturing (Chinnery, Pearlman and McMenamin, 2008; Lou et al., 2012; Dupont et al., 2018). If present in vivo, the reported findings both here and by previous authors, hint at the interconnectedness of the CNS as a whole and the ease by which pathological materials can spread. If TnT formation is an exclusive trait of in vitro culturing, it does not rule out the notion of mitochondria as a potential transporter for α-syn, if the organelle can still participate in intercellular exchange due to the reported interaction between α-syn and Tom20.
Mitochondrial dynamics within synucleinopathies is more complex than initially thought. The spread of pathological α-syn aggregates is now an additional factor implicated in the organelles role within both typical and atypical PD, in addition to their active role in cellular degeneration via ROS generation. Whilst further exploration is required to fully understand the significance of mitochondria-mediated α-syn intercellular exchange via TnTs, the data reported here fundamentally alters the current status of our understanding of mitochondrial dynamics in both typical and atypical PD. Interaction with pathological α-syn may change the mitochondrion from an organelle beneficial to normal cellular function to one actively participating in cell degeneration. Clearly a more complete understanding of how α-syn aggregates are able to propagate cell-to-cell is required for future therapeutic interventions.
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Chapter 5
Discussion
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Understanding the mechanisms of α-syn transfer is critical for the development of interventions aimed at reducing its effective spread within the CNS. No predominant mechanism has emerged whose inhibition ceases α-syn spread entirely, indicating that this process is likely to be a multifactorial one utilising varying mechanisms. For instance cell contact-based transfer mechanisms between adjacent cells such as TnTs and gap junctions have been reported for α- syn spread (Dieriks et al., 2017; Rostami et al., 2017; Reyes et al., 2019). Reports independent of cell contact also exist such as the utilisation of exosomal vesicles or uptake from the extracellular environment through methods such as endocytosis (Fitzner et al., 2011; Frühbeis et al., 2013a; 2013b; Konno et al., 2012). Regarding exosomes, α-helical α-syn, the non- pathological form, is a significant factor in the regulation of vesicle release at neuronal terminals (Ramezani et al., 2019). A recent report of α-syn transfer to adjacent cells in vivo via neuronal terminals may be linked to such a function, mimicking the “hijack” characteristics typically described by prion proteins (Okuzumi et al., 2018). α-Syn bears many resemblances to that of prion proteins mainly the ability of aggregates to propagate and seed in varying cellular models and its ability to transfer to grafted sites in vivo (Steiner, Quansah and Brundin, 2018; Tarutani et al., 2018; Angot et al., 2012). Various experimental models have also described, potential hijacking by α-syn in order to spread, resembling previous reports of prion proteins in Creutzfeldt-Jakob Disease (Abounit et al., 2016a; Okuzumi et al., 2018; Gousset et al., 2009). The complexity of the interactions of α-syn within these reports indicate a complete understanding of the mechanisms of how the protein is able to transfer is required to develop an effective novel therapy.
Examinations of post-mortem MSA brain tissue produced recurring evidence of both potential α-syn exchange between oligodendrocytes and microglia and microglial transporting internalised α-syn. The choice to examine MSA tissue came from a variety of unique factors believed to be better suited for exploring α-syn transmission. The first was the more rapid rate of degeneration observed within MSA, which has been hypothesized to be in relation to a faster development of GCIs compared to LB (Lipp et al., 2009). As a key component of GCIs, this would also insinuate a faster rate of α-syn spread. Unique to MSA is its classification as a prion disease and its α-syn variant displaying hallmark characteristics of prions based on reported culture seeding rates in addition its cell-cell transmissions observed in animal models (Prusiner et al., 2015; Bernis et al., 2015; Woerman et al., 2018; Yamasaki et al., 2019; Tarutani et al., 2018). Oligodendrocytes acquisition of α-syn for GCI formation is largely unknown. Oligodendrocytes express trace amounts of α-synuclein unlike neurons in PD, however, alterations in expression are minimal following inclusion body synthesis indicating acquisition from an external source (Asi et al., 2014). MSA‟s widespread chronic inflammatory profile also presented an opportunity to study microglias role in α-syn transfer. Asai and colleagues report on microglia aiding in the spread of tau fibrils, sparking an interest in investigating a similar
109 effect within synucleinopathies (Asai et al., 2015). Additionally microglial cells difficulty in both tau fibril and pathological α-syn degradation may be indicative of a similar outcome within MSA (Hopp et al., 2018; Tanik et al., 2013; Zhang et al., 2005).
Recurring observations of microglial cells with internalised α-syn proximal to GCI-affected oligodendrocytes indicated a potential exchange between both cells. Exchange commonly occurs between them for the degradation of vesicles containing antigen factors released by oligodendrocytes via macropinocytosis (Fitzner et al., 2011). Whether this mechanism was utilised is unknown, however, α-syn‟s ability to influence vesicle release and hijack exchange mechanisms in a prion-like manner warrants consideration. Mitochondrial TnT-based exchange is a potential mechanism based on both the results presented in Chapter 4 in addition to reports of microglial cells altered mitochondrial profile within synucleinopathies. Fragmentation of mitochondria attributes to the NLRP3 inflammasome response, prolonging activation and thus contributing to degeneration (Sarkar et al., 2017). Mitochondrial exchange via TnTs is a commonly observed mechanism for the rescue of damaged mitochondria within adjacent cells (Wang and Gerdes, 2015; Shen et al., 2018). Unfortunately examination for TnT-like structures is difficult in part due to these structures fragility and lack of specific identifying markers. Development of reliable methodologies for both in vivo and tissue staining analysis are still an ongoing endeavour. Whilst reports of structures matching their description have been reported in vivo, the scarcity of these observations indicates the difficulty in their confirmation outside of culture models (Chinnery, Pearlman and McMenamin, 2008; Lou et al., 2012; Dupont et al., 2018).
Microglia‟s role as a potential α-syn transporter was initially observed in tissue examinations and confirmed in the developed mobility assay. The initial recurring observation in the MSA tissue of microglia with internalised α-syn distal to GCI-affected oligodendrocytes, independent from the other previous observations, does not confirm source of acquisition. Whilst potential exchange with oligodendrocytes may have occurred for the proteins acquisition within microglia, tissue staining‟s inherent limitation of displaying information from only a singular time point, death, make exploration of this difficult. It is possible that microglial cells may have acquired it from extracellular environment DAMP clearance (Lee et al., 2008b). In the context of the previous finding however it implicates microglia‟s acquisition of α-syn from oligodendrocytes via an unknown transfer mechanism. Following this transfer it then begins to migrate distally from the initial acquisition site. Whether microglia transfers α-syn to other cellular sources or deposits it to the extracellular environment to further promote spread is unknown. Microglia have been demonstrated in vitro to release internalised pathological materials but are unable to degrade materials in the extracellular environment, such as tau fibrils (Hopp et al., 2018). If such a mechanism occurs for α-syn is unknown, however release of internalised aggregate α-syn was observed in the mobility assay via cell death. As a contributor 110 to both mitochondrial dysfunction and increased ROS generation, α-syn promoting cell death as a mechanism for release is probable (Hsu et al., 2000). Recently microglia-derived exosomes have been revealed to transfer α-syn to neurons, further strengthening the claim of microglia as a potential α-syn transporter (Xia et al., 2019).The limitations presented by the initial tissue analysis and the inability to confirm neither microglia‟s role as a potential α-syn transporter nor the mechanism of exchange between oligodendrocytes and microglia, lead to the development of the mobility assay and the exploration of mitochondria‟s role in α-syn intracellular transfer.
The mobilisation assay was designed to assess microglia capability as a transporter of α-syn in addition to verifying if this trait was exclusive to pathological variants. Lack of uptake and reduction of fluorescent intensity in all α-syn monomer exposed groups confirmed the latter possibility. Activation of microglia and its immune response is promoted by pathological variants of α-syn such as fibrils, oligomers and aggregates (Zhang et al., 2005; Ferreira and Romero-Ramos, 2018). Thus it was hypothesized that transporter behaviours would be witnessed when the THP-1 cells, differentiated to a microglial-like state, were exposed to aggregated α-syn. The significant reduction in the residual immobilised α-syn aggregate MGV confirmed this notion. Increased presence of differentiated cells with internalised α-syn at the distal edge regions further confirmed transport of protein from the immobilised region by microglial-like cells. Aggregated α-syn taken-up by microglial-like cells was transported distally from the residual immobilised spot mimicking what was proposed when examining potentially migrating microglia within MSA tissue. Further exploration of this finding through repeated experiments substituting differentiated THP-1 with HAPI cells, produced similar outcomes, implicating this as a microglial behaviour rather than one unique to differentiated THP-1 cells. Clearance of extracellular α-syn via microglial activation initially promotes neuroprotection as a method to cease further α-syn spread within the CNS (Lee et al., 2008b; Stefanova et al., 2011). Chronic inflammation induced by overstimulation of inflammatory activation pathways and perpetuated by increased pathological α-syn presence in addition to microglial cells inability to efficiently degrade the protein shifts this behaviour to become neurodegenerative (Kempuraj et al., 2016; Muroya et al., 2012). In combination with our findings, microglial cells further contribute to neurodegeneration via transportation and potential transfer of α-syn. α-Syn transfer has been reported to still occur in animal models depleted of microglial populations, reinforcing the notion of the multiple avenues in which the protein is able to spread (George et al., 2019). This raises the question of how significant microglial cells are as a singular mechanism for α-syn transfer and requires further investigation in a less controlled environmental model.
The mobility assay provided further insights into other minor factors outside of the primary investigation of microglial cells potential transporter role within synucleinopathies. The role of monocytes in α-syn propagation is one such factor. Whilst partial clearance and mobilisation of 111 aggregate protein was performed by THP-1 monocytes, the lack of significant change compared to controls and microglial-like cells supports claims of these cells role as a supplemental factor in the immune response as opposed to a significant one in α-syn clearance and propagation (Codolo et al., 2013). Additionally the lack of significant results in the monomer exposed groups indicates lack of immune activation or response by the exposed cells. Differentiated THP-1 cells displayed the greatest uptake of α-syn from the residual immobilized spot, however the low proportions internalised α-syn cells at the distal edges hints of degradation of the protein following uptake (Park et al., 2008). Recordings of α-syn positive bearing microglia in the MSA tissue analysis are thus more likely to be indicative of pathological α-syn transportation than its monomeric form. BSA control experiments further reinforced behaviours exhibited by both differentiated THP-1 and HAPI cells were as a result of exposure to pathological α-syn and its classification as a DAMP due to lack of observed uptake or mobilisation (Béraud et al., 2011). Testing of the mobility assay with 1321N1 cells, an immobile cell line, confirmed that the presence of cells with internalised α-syn at distal edge regions is due to α-syn‟s removal from the central immobilized spot and not via other potential transport mechanisms or solubility by other cells. Lack of a significant proportion of α-syn positive bearing cells in the 1321N1 exposed conditions indicates that the cells inability to migrate prevented transfer of α-syn to distal regions from the residual immoblilzed spot or at least within the 48 hour incubation period used. 1321N1 cells were still capable of internalising α-syn but their immobility prevented the proteins removal from the region, thus confirming the distal regions assessment of cells migrating capabilities.
Treatment against microglial-cell motility and the TLR4 pathway inflammatory activation were the primary interventions conducted to investigate the inhibition of microglial cells α-syn transporter capabilities. Stabilisation of microtubular activity proved effective in combatting α- syn migration, however the latter treatment against activation proved unsuccessful. Previous reports of agents targeting the dynamic instability of microtubules through their stabilisation described reduced motility of effected cells (Ganguly et al., 2012; Su et al., 2016). Additionally microtubule disruption was shown to reduce, but not completely hinder, the ability of immune cells such as T-cells from migrating implicating their significance in their mobility (Takesono et al., 2010). Whilst the effect of microtubule activity alteration on microglial-like cells mobility was unclear prior to experimentation, EpoD treatment confirmed a similar outcome. Lack of residual aggregate protein removal, in conjunction with a significantly reduced presence of aggregate α-syn positive bearing cells at distal regions by differentiated THP-1 cells corroborates the notion of impaired microglial motility through microtubule activity inhibition. EpoD capability to permeate through the blood-brain barrier makes it a prime candidate for further assessing its inhibition of microglia‟s transportation α-syn in vivo (Brunden et al., 2010). A risk of EpoD treatment in such models however is the drugs cytotoxicity (Cheng, Bradley and
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Budman, 2008; Chou et al., 1998). A recent report by Clark et al., described the effects of increased EpoD dosage on maturing cortical neuron cells in vitro, reporting that higher concentrations significantly reduced the viability of these cells pending on their stage of maturation (Clark et al., 2020). Further analysis of the effects of EpoD is required in both in vitro co-culture and in vivo settings to fully determine the wider effects of the drug upon the CNS.
Prevention of inflammatory activation was initially hypothesised to have produced a similar outcome of EpoD treatment through the reduction of residual protein uptake. TLR4 stimulation by LPS and DAMPs like α-syn promotes pro-inflammatory factors such as IL-1β and TNF-α synthesis and release for microglial recruitment and inflammatory activation (Fellner et al., 2013; Hughes et al., 2019). Previous reports of TAK-242 treatment saw significant reductions in pro-inflammatory release within THP-1 cells (Zhou et al., 2013). Additionally α-syn uptake for phagocytic degradation is implicated to be promoted by TLR4 (Stefanova et al., 2011). Taken together, it suggested an outcome in which TLR4 inhibition via TAK-242 would significantly affect microglia‟s ability to acquire α-syn for migration, however it did not. This outcome suggests either that TAK-242 is ineffective in inhibiting α-syn activation of TLR4 or that TLR4 is not solely responsible for microglial cells acquisition of α-syn. Scavenger receptors for instance, elicit similar behaviours to toll-like receptors; innate immune cell activation when detecting damaging or pathological molecules. CD36 for instance interacts with various pathological neurodegenerative proteins such as α-syn and β-amyloid fibrils, promoting microglial inflammatory activation and extracellular clearance of DAMPS (Su et al., 2008; Coraci et al., 2002; Panicker et al., 2019). Additionally other TLRs such as TLR2 also promote for microglial activation and pro-inflammatory factor release via NLRP3 inflammasome stimulation (Codolo et al., 2013; Kim et al., 2013). TLR2 was previously thought to be a supplementary factor in TLR4 inflammatory activation, but recently TLR2 inhibition has been revealed to reduce α-syn transmission in neuron and astroglial cells suggesting the opposite (Fellner et al., 2012; Kim et al., 2018). In this context it is unlikely that TAK-242 action alone would reduce α-asyn clearance. Future improvements would include a more comprehensive scope in investigating if a dominant receptor exists for α-syn‟s extracellular clearance and if a combined inhibition of TLR2, TLR4 and CD36 would lead to a reduction of such.
Observations of potential α-syn exchange between microglia and oligodendrocyte within MSA tissue were unclear as to the specific process for this transfer. Generation of TnT-like structures in both confocal and STED examinations of 1321N1 and differentiated THP-1 cells co-cultures, and SH-SY5Y monocultures for potential aggregate α-syn transmission, provides a potential elucidation. 24 hour α-syn exposure saw uptake by both 1321N1 and differentiated THP-1 cells, matching previous reports utilising similar methods (Aulić et al., 2014; Braidy et al., 2013; Hoffmann et al., 2019). Initial confocal co-culture examination was performed to assess if 113 evidence of potential α-syn would present. Formation of TnT-like structures in normal untreated conditions matched previous reported observations for cargo exchange, whilst their presence in the different α-syn treatment conditions implicated it as a mechanism for α-syn exchange (Onfeldt et al., 2004; Agnati & Fuxe, 2014; Koyanagi et al., 2005). The report by Abounit and colleagues of intercellularly migrating lysosomes contribution to α-syn propagation in culture raised the query of whether other organelles may also partake in such an action (Abounit et al., 2016a). Mitochondrial intercellular transfer has been well categorised in culture for its role in respiration rescue (Wang and Gerdes, 2015; Shen et al., 2018; Dong et al., 2017; Babenko et al., 2015). Additionally dysfunction via interactions with pathological α-syn variants has been demonstrated to actively contribute to cellular degeneration and both typical and atypical PD pathogenesis (Braidy et al., 2013, Foti et al., 2019; Monzio Compagnoni et al., 2018). Whether this dysfunction was localised to a singular cellular site or could be transferred to adjacent ones in a prion-like manner as was suggested by Abounit was, prior to STED analysis, unknown (Abounit et al., 2016b).
Employment of STED microscopy provided evidence to support that TnT-like structures promote α-syn spread to adjacent cells in addition to elucidating the role of mitochondria in the context of this transfer. Formation of TnT-like structures not only saw presence of α-syn in connected cells but internalised within the TnT-like structures themselves. The similarity in the manner in which α-syn was potentially transferred within each of the culture models hints at the potential significance such a mechanism would be for the spread of α-syn within the CNS. Both the CNS‟ cellular density and multicellular composition indicate that formation of pathological α-syn at any site places the entire system at risk (von Bartheld, Bahney and Herculano-Houzel, 2016; Keller, Erö and Markram, 2018). Intercellular mitochondrial transporting of α-syn via TnTs may be a significant factor in the cascade of dysfunction and degeneration present in both typical and atypical PD‟s. In both TnT-like structures and the cytoplasm, α-syn presence on the outer mitochondrial surface matched previously reported interactions of pathological α-syn variants with Tom20 influencing the organelles dysfunction (Di Maio et al., 2016; Wang et al., 2019). Additionally this result produced phenotypically mimicked that of Grassi and colleagues previous STED investigation on α-syn‟s influence on mitochondrial dynamics, in which α-syn was reported to be bound to Tom20 (Grassi et al., 2018). Prior to our finding, this interaction was relegated to an inhibitory mechanism of mitophagy for the promotion of excessive ROS generation. This result proposes that this interaction additionally promotes for intercellular propagation of α-syn, not dissimilar to previous reports of prion-like hijacking (Abounit et al., 2016a; Aulić et al., 2017). Figure 21 depicts the precicted method by which α-syn may spread via mitochondrial-based TnT migration. The presentation of such a mechanism in each of the culture models tested suggests that this method of α-syn transfer is not limited to cells of the same type nor to particular cells. Partial qualitative similarities with the reported MSA tissue
114 samples may suggest a potential α-syn acquisition model for oligodendrocytes within the disease although development of a reliable method for in vivo detention is required for confirmation of this possibility.
Figure 21 –Mitochondrial Intercellular Transfer of α-syn via TnTs
Inside thick TnTs, microtubules tracks exist to allow for motor adaptor complexes such as the anterograde kinesin-1 and KIF5 adaptor or the retrograde dynein and KIF5A adaptor to migrate along them. The purpose of this migration is to transport cargo for intercellular exchange. Adaptors such as TRAK1 for the anterograde motor, and TRAK2 for the retrograde motor, allow for connections to specific sites on organelles in order to initiate their migration such as Miro1 found on the OMM. This mechanism of mitochondrial migration is the same one believed to be used by pathological α-syn as discussed in chapter 4, whereby α-syn binds to Tom20 receptors situated around the OMM of migrating mitochondria. Thus α-syn is able to undergo intercellular transfer by essence of “hitch-hiking” on mobilising organelles such as mitochondria.
Silencing of Miro1 proved ineffective in the prevention of α-syn migrating along TnT-like structures whilst bound to mitochondria. Miro1 is essential for linking mitochondria to the motor-adaptor complex for intracellular migration. The significance of Miro1‟s role in intercellular migration of mitochondria is largely unknown. A few studies have shown that it bridges mitochondria to the motor adaptor complex in the same manner as described for intracellular migration (Gao et al., 2019; Babenko et al., 2018). The lack of visible effect from silencing however hints that Miro1 is not the sole factor connecting mitochondria to mobility complexes. Other GTPases, such as Miro 2, can also similarly perform the same functions 115 enacted by Miro 1 (Tang, 2015). Recently Miro2 has been described as a critical component for intercellular mitochondrial transfer via TnTs in a neuron-astrocyte co-culture model, highlighting their importance within the CNS (Gao et al., 2019). These reports have been extended to culture models outside the CNS such as cardiomyocytes, strengthening the notion of the ongoing migration in the STED samples (Cao et al., 2019). Utilisation of other motor proteins except kinesin and dynein may also explain the continued mitochondrial migration following Miro1 silencing. Myosin-XIX for instance has been demonstrated to transport mitochondria for intercellular exchange (Quintero et al., 2009; Titus, 2018; Shneyer, Usaj and Henn, 2015). Unlike kinesin and dynein, myosin motors migrate along actin polymers, a component of thick TnTs and whose presence was confirmed with β-actin staining (Onfeldt et al., 2004; Agnati & Fuxe, 2014; Koyanagi et al., 2005). Additionally both Miro1 and Miro2 have been reported as links bridging mitochondrial bodies to these motor complexes (Oeding et al., 2018). Alternatively a complete knockout of Miro1 may be required for inhibition of TnT- based mitochondrial intercellular migration. Inhibition of other lesser explored migrational mechanisms and factors may further elucidate on α-syn‟s ability to hijack and propagate via TnTs.
Further exploration of the discussed mechanisms may better illuminate their significance in spread in addition to explaining specificities of their function. Employment of live microscopy for instance, may help elucidate the rate and frequency by which α-syn spreads to adjacent cells via TnTs. Examination of fixed samples utilising STED microscopy was critical for the identification of potentially migrating mitochondria transporting α-syn within TnTs. The inherent limitation of fixation analysis however is the lack of context on the mechanism itself, failing to provide answers for queries such as how frequently TnTs form and the length for which structures last before deforming. Additionally how frequently α-syn utilise TnTs for potential spread and their rate of spread to adjacent cells are also currently unknown. α-Syn‟s ability to propagate through a variety of mechanisms makes it difficult to assess the significance of intercellular mitochondrial migration for its propagation on fixation analysis alone. Various reports exist of live TnT examination in vitro, each of which describe factors such as rate of cargo spread, thus providing a potential avenue to further explore α-syn migration via this method (Drab et al., 2019; Halász et al., 2018; Keller et al., 2017; Panasiuk et al., 2018). Worth noting is the use of super-resolution techniques in a majority of live examinations, likely to be due to TnT structure size and fragility. Additionally, employment of live microscopy may help elucidate similar queries with regards to the relevance of microglia in α-syn migration. Primarily the speed by which microglial cells migrate with internalised α-syn. Understanding how quickly microglia can mobilise once α-syn is internalised may provide potential answers for the proteins rate of spread in synucleinopathies, such as MSA, where the rate is greater than
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PD. Exploration of such a correlation may provide hints as to the significance of microglia in the spread of α-syn within the CNS.
Examinations of microglia in animal models of PD/MSA would be largely beneficial in determining their significance for α-syn spread in vivo. For instance, a mouse model with suppressed microglial expression may have a reduced widespread presence of α-syn in comparison to a control model. Alternatively a model in which chronic microglial activation is stimulated through excessive pro-inflammatory factor promotion may also present with a differing profile. Studying alterations in the microglial activation profile may provide potential insight into a method of reducing α-syn spread. Currently no reliable methods exist for the detection and examination of TnTs in post-mortem tissue, a limitation that extends to live in vivo analysis. Reports of their formation in vivo are scant and much debate currently exists to whether TnTs are an exchange mechanism exclusive to in vitro culturing. Benefits for exploring mitochondria‟s role in intercellular α-syn spread may yet exist regardless of outcome. Expanding examinations to include other untested cell types such as oligodendrocytes in addition to unexplored co-culture combinations may better inform on potential limitations of this transfer. To speculate, such limitations may include lack of TnT formation or mitochondrial exchange in specific co-culture combinations. Until the development of methods for TnT examination in vivo/post-mortem, this may be the most reliable way to simulate the CNS to examine for the role of intercellular mitochondrial migration in α-syn spread.
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Chapter 6
Conclusion
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This investigation uncovered mechanisms associated with α-syn spread in both typical and atypical PD‟s. The perception of microglia‟s role in synucleinopathies has shifted from neuroprotector to active contributor to degeneration. The primary aims of Chapters 2 were to further explore this notion in the context of whether microglia could actively contribute to α-syn spread. This investigation proved illuminating, with microglial-like cells in culture presenting with traits reminiscent in profile to that of transporters. These characteristics manifest from exposure to pathological α-syn variants, displaying protein clearance from a defined region in addition to increased presence of α-syn positive bearing cells at distal sections. Qualitative similarities presented by both α-syn positive migrating microglial-like cells and microglia in MSA brain tissue further reinforced the cells role as a propagator of pathological α-syn in the CNS. Investigations on potential inhibitors were mixed. Inhibiting microtubule dynamic instability through EpoD treatment presented with a novel therapy in reducing α-syn spread via microglia motility inhibition. Conversely the use of TLR4 inhibitor TAK-242, as described in Chapter 3, proved unsuccessful, insinuating more than just TLR4 in microglia‟s activation and promotion of α-syn uptake.
Interactions between mitochondria and pathological α-syn are directly associated with the promotion of cellular degeneration. Investigations on this relationship have predominantly focussed on local sites, thus its role in the promotion of degeneration in neighbouring cells was unknown. TnTs facilitate the exchange of cargo, with previous cases displaying spread of disease materials to adjacent connected cells. Mitochondria have been regularly demonstrated to utilise this same mechanism for organelle reparation and respiratory rescue of nearby cells. Exploring whether mitochondria could promote the intercellular spread of α-syn via TnTs became the primary focus of Chapter 4. Not only was a similar relationship observed but was displayed to not be limited to singular cell types. Observations of α-syn bound to potentially migrating mitochondria within TnTs in neuronal, astrocytic and microglial-like monocultures, in addition to microglia-astrocyte co-cultures, provides potential reasoning as to how α-syn materialize in irregular sites such as oligodendrocytes in MSA. With the suppression of Miro1 proving ineffective, the exact method promoting potential mitochondrial migration whilst transporting α-syn remains elusive. Previous reports of α-syn‟s “prion-like” hijacking of TnTs for its propagation display many qualitative similarities to our observations thus indicating it as a potential prion-like function.
Overall the propagation and formation of pathological α-syn species in differing brain regions is still a source of great uncertainty. Whilst some of the reported experiments have begun to shed some light on potential methods of propagation, it has in essence further revealed the complexity of the task in which to combat and inhibit this process. Normal cellular functions such as engulfment of pathological protein for phagocytosis or intercellular transport of cargo to neighbouring sites can seemingly be shifted to become potential pathological mechanisms on α- 119 syn form. Whilst further exploration will be required to fully grasp the significance of these mechanisms and their overall role in synucleinopathies, it has begun to elucidate our understandings of other unexplored aspects within these diseases. With microglia specifically, not only may their ineffective degradation of pathological α-syn species contribute to degeneration outside of chronic pro-inflammatory factor release, but also through α-syn mobilisation and use of mechanisms such as TnTs. Moreover, the role of mitochondria as a vehicle for the spread of degenerative material is a novel mechanism that may mediate cell-to- cell spread of α-syn via TnTs. Given this ever-growing complexity, treating for α-syn transport inhibition may require a multifactorial approach.
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79/9 79/8 77/8 77/7 73/9 73/8 77/7 76/9 78/9 78/8
Figure 22 – Gel Electrophoresis of recombinant α-syn revealed consistent molecular weight across most samples
Gel electrophoresis of 10 separate purified α-syn samples extracted from E.Coli revealed a consistent molecular weight between most of the samples. These ten samples were termed 77/8, 77/7, 73/9, 73/8, 77/7, 76/9, 779/9, 79/8, 78/9 and 78/8. Eight of the ten samples produced visible bands, each of which were slightly above the 15kDa marker, falling in range of the approximate molecular weight of α-syn (14.4kDa). Of the eight samples to produce visible bands, only 79/8 and 77/8 were utilised for the experiments within Chapter 2 and 3 both acting 2+ as monomeric α-syn, and to produce aggregates using Ca and H2O2. Additionally these same samples were aggregated for experiments in Chapter 4.
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A
*
B
Figure 23 – Dilution control tests revealing monomeric α-syn is significantly diluted in cellular media whilst aggregated α-syn is not t-tests investigating the significance of cellular media presence in the dilution of both monomeric and aggregated α-syn. Groups examined comprised of α-syn (monomeric or aggregated) exposed to cellular media and no cells (n=3; 48hrs; ±SEM) and protein exposed to no cells nor cellular media (n=3; 48hrs; ±SEM). Measured was fluorescent intensity dictated by MGV. (A) Monomeric α-syn was found to significantly dissolve in the presence of cellular media (p=<0.001). (B) Aggregated α-syn was found not to significantly dissolve in the presence of cellular media (p-0.142).
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THP-1 ‖ 1321N1 THP-1 ‖ 1321N1
a
Miro1 Β-actin
1321N1 1321N1 THP-1 THP-1 siRNA - + siRNA - +
b Β-actin Miro1
1321N1 ‖ THP-1 1321N1 ‖ THP-1
c Β-actin
Tom20
1321N1 1321N1 THP-1 siRNA - + THP - 1 siRNA - +
d Β-actin
Tom20
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Figure 24 – Whole blots of bands displayed in Figure 20
Displayed are the full blots to the selected examples used in Figure 20 to show reduction in Miro1 following siRNA treatment. Each figure is comprised of the protein of interest stain on the left, the marker label (blue) in the centre and the corresponding β-actin stain on the right. On the central markers, bands for Miro1 (red), β-actin (green) and Tom20 (purple) are shown. (a) (Left) Blots of the untreated controls featuring 1321N1 (right) and differentiated THP-1 cells (left). Miro1 bands were slightly above the 70kDa marker, falling in range of the proteins predicted molecular weight of 71kDa. (Right) Corresponding β-actin stain of the controls. Β- actin bands were slightly above the 40kDa marker, falling in range of the proteins predicted molecular weight of 42kDa. (b) (Left) Blots of the 1321N1 cells transfected with Miro1 siRNA (right) and scrambled siRNA (left). Miro1 bands were dimmer for Miro1 siRNA group. The space residing between 1321N1 and the marker were the THP-1 lanes, which as seen on the figure, were not highlighted by neither the Miro1 nor Tom20 (24d) antibody. Due to this, only 1321N1 was utilised for 1321N1 transfections. (Right) Corresponding β-actin stain of this group. Of note the THP-1 bands were highlighted, revealing THP-1 content in the lanes. (c) (Left) Blots of the untreated controls featuring 1321N1 (left) and differentiated THP-1 cells (right). Tom20 bands were slightly above the 15kDa marker, falling in range of the proteins predicted molecular weight of 16kDa. (Right) Corresponding β-actin stain of this group. (d) (Left) Blots of the 1321N1 cells transfected with Miro1 siRNA (right) and scrambled siRNA (left). (Right) Corresponding β-actin stain of this group.
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