An Essential Role for Sphingosine 1-Phosphate in Oligodendrocyte Survival and Remyelination

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An essential role for sphingosine 1-phosphate in oligodendrocyte survival and remyelination

Huitong Song

A thesis submitted in fulfilment of the requirements for the degree of

Master of Philosophy (Medicine)

Centenary Institute

Faculty of Medicine and Health

The University of Sydney

04/02/2021

Author Attribution

The work contained in the body of this thesis, except otherwise acknowledged, is the result of my own investigations.

In Chapter 2, lipid quantification by liquid chromatography-tandem mass spectrometry was conducted and analysed by Holly McEwen. The preliminary study on the role of SphK2 in oligodendrocyte survival in vitro was designed and conducted with help from Yanfei Qi and

Long Chung.

In Chapter 3, blood was drawn with the help of Jonathan Teo and an animal welfare veterinarian from The University of Sydney, and flow cytometry on blood cells was conducted by Quintin Lee. Measurement of drug levels was performed by Holly McEwen and Anthony

Don.

Jonathan Teo assisted with culling of all animals. Myelination scoring of LFB/CV staining in both chapters was performed with help from Jonathan Teo, Collin Tran and Anthony Don.

Thesis statement of originality

This thesis has not been submitted for any other degree or purpose. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged.

Huitong Song | 15th October 2020

As supervisor for the candidature upon which this thesis is bases, I can confirm that the authorship attribution statements above are correct.

Anthony Don | 15th October 2020 Abstract

Abstract

Oligodendrocytes (OLs) are the myelinating cells of the central nervous system. Therapeutics that promote OL survival and remyelination are needed to restore neurological function in multiple sclerosis (MS). Sphingosine 1-phosphate (S1P) is a lipid metabolite that signals through five receptors, S1PR1-5. The MS drug Fingolimod is an S1PR agonist that suppresses neuroinflammation. A key question in current research is whether S1PR agonists also directly protect OLs and promote remyelination. However, the role for endogenous S1P, synthesized by sphingosine kinase 2 (SphK2), in OL survival and myelination has not been established.

This thesis investigated the importance of SphK2 in OL survival and remyelination using the cuprizone mouse model of acute demyelination, followed by spontaneous remyelination. OL density and myelin protein levels did not differ between untreated wild-type (WT) and SphK2 knockout (SphK2−/−) mice. However, after cuprizone treatment, significantly greater demyelination and loss of mature OLs were observed in the corpus callosum and cerebral cortex of SphK2−/− compared to WT mice. Spontaneous remyelination occurred in WT but not

SphK2−/− mice following cuprizone withdrawal, despite restoration of mature OL numbers in

SphK2−/− mice. These results indicate that SphK2 is not necessary for proliferation and maturation of OL progenitor cells but is necessary for the synthesis of new myelin by mature

OLs.

Since S1PR5 is expressed exclusively by OLs, we also investigated whether S1PR1/5 agonist

Siponimod and S1PR5-selective agonist A-971432 protect against myelin and OL loss in WT mice. Siponimod but not A-971432 protected against cuprizone-mediated OL loss, demyelination, astrogliosis, and microgliosis. iii

Abstract

Overall, this thesis demonstrates the importance of SphK2 for OL stress resistance and spontaneous remyelination. Future work will determine whether the S1P produced by SphK2 promotes remyelination through autocrine stimulus of OL S1P receptors.

Acknowledgements

This thesis and the work contained within, would not have been possible without the tremendous guidance and support of my supervisor Anthony Don. Anthony, I am honoured and grateful to be your student. I owe a big thank to you for taking me on as a student in the first place. Your relentless support and the opportunities that you have provided benefit me a lot in this journey. I’ll always cherish the helps you gave me when I first came to this foreign country. Your patience and understanding to me, sometimes even with my crazy ideas, help me to accomplish everything I want to learn from my Master degree. Your energy and dedication to work are what I need to learn in my future career path continually. I am proud to have you as my supervisor.

Additional thanks to:

Jon, I couldn’t have asked for a better co-supervisor, lab mate and friend. You always treat me with your kindness and patience despite my unintentional mistakes in experiments. I’ll remember all the happy time we had and will have together, particularly down in the animal house. It’s always awesome to chat with you because you are such a good listener with interesting opinions. Every time we share thoughts about Chinese culture with each other reminds me of how proud I should be as Chinese. You play a big part in my life journey, and I would love to continue all of these in the future.

Jacob Qi, Collin Tran and Mona Lei, your selfless advice, especially in my experiments and career development, help me to make a lot of progress in two years. Thank you! It’s my pleasure to work with you guys. v

Acknowledgements

I would also like to thank Jun Yup Lee, Holly McEwen, Tim Couttas, Long Chung and Quintin

Lee for their help in my thesis. Thanks to my friends Jinfeng Lin, Sige Liu, Yu Huang, Ni Duan and other people at Charles Perkins Centre, as well as other friends that I met in Australia for helping me get through the first year and be there for friendship.

Special thanks to my lifetime friends Xuyang Yuan, Hanchi Ruan, Meng Wang and Xin Zhang, as well as my idols Rainie Yang, GAI, Taylor Swift, etc. I thank you for your companies during my hard time in research and life. I’m very grateful to have all of you guys in my life.

Lastly, I appreciate the significant support from my family, especially my parents Changying

Hou and Ligang Song. I dedicate this thesis to them. I could never finish this without your help in spiritual and financial throughout these years. You always encourage me to be myself and be passionate about what I love. As I promised, I will keep perusing my dream in scientific research and be a better version of myself in the future.

Table of Contents

An essential role for sphingosine 1-phosphate in oligodendrocyte survival and remyelination 1

Author Attribution ...... i

Thesis statement of originality ...... ii

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vii

List of Figures ...... xi

List of Tables ...... xiii

Abbreviations ...... xiv

Chapter 1 Literature Review ...... 1

1.1 Multiple Sclerosis (MS) ...... 1

1.1.1 Introduction to MS ...... 1

1.1.2 Pathogenesis and pathophysiology of MS...... 3

1.1.2.1 Myelin and oligodendrocytes in the CNS...... 3

1.1.2.2 Introduction to MS lesions ...... 7

1.1.2.3 Demyelination, remyelination and oligodendrocyte pathology of MS lesions ...... 8

1.1.2.4 Inflammation and glial cell activation in MS lesions ...... 10

1.1.3 Clinical biomarkers, diagnosis and therapy for MS ...... 13

1.2 Cuprizone model for demyelination in MS research ...... 16

1.2.1 Introduction to animal demyelination models ...... 16

1.2.2 Cuprizone and cuprizone demyelination model ...... 17

vii

Table of Contents

1.2.3 Oligodendrocyte cell culture models for studies on MS ...... 19

1.3 Sphingolipids in myelin ...... 20

1.3.1 Introduction to sphingolipids ...... 20

1.3.2 Sphingolipids in oligodendrocytes and demyelination ...... 23

1.4 S1P and S1P receptors (S1PRs) in the CNS ...... 24

1.5 S1PR agonists ...... 30

1.5.1 Fingolimod ...... 30

1.5.2 Siponimod ...... 32

1.5.3 Ozanimod ...... 33

1.5.4 A-971432 ...... 33

1.5.5 Other S1PR agonists ...... 34

1.6 Sphingosine kinases (SphKs) ...... 34

1.6.1 SphKs and S1P ...... 34

1.6.2 The role of SphKs in cancer ...... 35

1.6.3 SphKs and S1P in the CNS and neurodegenerative diseases ...... 36

1.7 Aims ...... 38

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination...... 40

2.1 Introduction ...... 40

2.2 Aims ...... 41

2.3 Materials and Methods ...... 42

2.3.1 Transgenic mice and cuprizone administration ...... 42

2.3.2 Immunohistochemistry and immunofluorescence microscopy ...... 43

2.3.2.1 Immunofluorescence and volumetric analysis ...... 43

2.3.2.2 Luxol Fast Blue (LFB)/Crystal Violet (CV) staining and analysis ...... 46

viii

Table of Contents

2.3.3 Western blotting ...... 49

2.3.4 Lipid quantification by liquid chromatography-tandem mass spectrometry (LC- MS/MS) ...... 50

2.3.5 Cell culture ...... 51

2.3.5.1 Cell culture conditions ...... 51

2.3.5.2 Cell viability assessment by MTS assay...... 52

2.3.5.3 Seahorse assay ...... 53

2.3.6 Statistical analysis ...... 55

2.4 Results ...... 56

2.4.1 SphK2 protects against loss of mature oligodendrocytes induced with cuprizone ...... 56

2.4.2 SphK2 is necessary for remyelination following cuprizone withdrawal ...... 60

2.4.3 SphK2 is essential for long-term remyelination in cuprizone model ...... 64

2.4.4 Loss of SphK2 increases levels of pro-apoptotic sphingosine and ceramide during and after cuprizone feeding...... 69

2.4.5 Preliminary investigation into the role of SphK2 in oligodendrocyte survival in vitro ...... 73

2.5 Discussion ...... 78

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo ...... 87

3.1 Introduction ...... 87

3.2 Aims ...... 89

3.3 Materials and Methods ...... 89

3.3.1 Compounds ...... 89

3.3.2 Cuprizone treatment and drug administration in vivo ...... 90

3.3.3 Flow cytometry on white blood cells ...... 90

3.3.4 Immunohistochemistry and immunofluorescence microscopy ...... 91

Table of Contents

3.3.4.1 Immunofluorescence and volumetric analysis ...... 91

3.3.4.2 LFB/CV staining and analysis ...... 92

3.3.5 Western blotting ...... 92

3.3.6 Drug quantification ...... 92

3.3.7 Statistical analysis ...... 93

3.4 Results ...... 94

3.4.1 Lymphopenia is induced with S1PR1/5 agonist Siponimod ...... 94

3.4.2 Effects of Siponimod and A-971432 on cuprizone-mediated demyelination ..... 96

3.4.3 Effects of Siponimod and A-971432 on cuprizone-mediated astro- and microgliosis ...... 103

3.4.4 Siponimod accumulates to a much greater extent than A-971432 in the brain. 106

3.5 Discussion ...... 106

References ...... 114

List of Figures

Figure 1.1 Oligodendroglial differentiation lineage and markers for different developmental stages (Revised from [70])...... 6

Figure 1.2 Cells and molecules involved in MS pathogenesis and subsequent pathophysiology.

...... 11

Figure 1.3 Schematic overview of the sphingolipid metabolic pathway...... 22

Figure 1.4 Autocrine and paracrine signalling of S1P through S1PRs...... 26

Figure 1.5 The mRNA expression profile for receptors in different CNS cell types...... 28

Figure 2.1 Sagittal mouse brain atlas and scoring example images...... 48

Figure 2.2 Outline of drug mechanisms and evaluated mitochondrial parameters in seahorse assay...... 54

Figure 2.3 Loss of SphK2 sensitizes to oligodendrocyte loss induced by cuprizone...... 57

Figure 2.4 Effect of SphK2 deletion on myelin and oligodendrocyte markers with CPZ treatment...... 62

Figure 2.5 Myelin content does not recover in SphK2−/− mice following cuprizone withdrawal.

...... 67

Figure 2.6 Altered myelin content levels in cuprizone-induced demyelination and remyelination...... 70

Figure 2.7 The role of SphK2 in vitro...... 76

Figure 3.1 Effect of S1PR agonists on blood lymphocytes...... 95

Figure 3.2 Siponimod protects against cuprizone-mediated loss of mature oligodendrocytes.

...... 97

List of Figures

Figure 3.3 S1PR agonists do not protect against cuprizone-mediated loss of myelin protein markers...... 100

Figure 3.4 Siponimod protects against cuprizone-mediated demyelination...... 102

Figure 3.5 Effect of S1PR5 and S1PR1/5 agonists on astro- and microgliosis...... 105

xii

List of Tables

Table 1.1 Medications approved for treatments of MS (Revised from [173, 174])...... 15

Table 2.1 List of antibodies used for immunofluorescence (IF) and western blotting (WB). . 45

xiii

Abbreviations

Alzheimer’s disease AD

Aspartoacylase ASPA

B cell lymphoma 2 Bcl-2

Blood brain barrier BBB

Bovine serum albumin BSA

Brain-derived neurotrophic factor BDNF

Central nervous system CNS

Ceramide synthases CerS

Cerebrospinal fluid CSF

Collision energy CE

Corpus callosum CC

Crystal Violet CV

Dihydroceramide dhCer

Experimental autoimmune encephalomyelitis EAE

Fatty acid synthase FAS

Fingolimod-phosphate FTY720-P

Galactocerebroside GalC

Galactosylceramide GalCer

Glial cell-derived neurotrophic factor GDNF

Glial fibrillary acidic protein GFAP

Glucosylceramide GluCer

Glucosylceramide synthase GCS

xiv

Abbreviations

Histone deacetylases 1 and 2 HDAC1/HDAC2

Huntington’s disease HD

Interferon IFN

Interleukin 1β IL-1β

Ionized calcium binding adaptor molecule 1 Iba-1

Leukaemia inhibitory factor LIF liquid chromatography-tandem mass spectrometry LC-MS/MS

Luxol Fast Blue LFB

Lymphotoxin LT

Magnetic resonance imaging MRI

Methyl-tert-butyl ether MTBE

Multiple Sclerosis MS

Myelin associated glycoprotein MAG

Myelin basic protein MBP

Myelin-oligodendrocyte glycoprotein MOG

Natural killer NK

Neurofilament H NF-H

Neurotrophin-3 NT-3

Oligoclonal bands OCBs

Oligodendrocyte precursor cells OPCs

Oxygen Consumption Rate OCR

Parkinson 's disease PD

Peripheral nervous system PNS

Phosphatidylinositol 3-kinase PI3K

Platelet derived growth factor PDGF

Abbreviations

Poly ADP-ribose polymerase PARP

Primary progressive MS PPMS

Proteins myelin proteolipid protein PLP

Receptor of platelet-derived growth factor α PDGFRα

Regions of interest ROIs

Relapsing-remitting MS RRMS

S1P receptor S1PR

Short hairpin RNA shRNA

Sphingosine 1-phosphate S1P

Sphingosine kinase SphKs

Tris-buffered saline containing 0.1% Tween 20 TBST

Tumour necrosis factor TNF

Wild-type WT

2,3-cyclic-nucleotide 3-phosphodiesterase CNPase or CNP

xvi

Chapter 1 Literature Review

Literature Review

Multiple Sclerosis (MS)

Introduction to MS

MS is a demyelinating neurodegenerative disease of the central nervous system (CNS), driven by autoimmune inflammation. Autoimmune lymphocytes target myelin, the fatty substance that surrounds and insulates neuronal axons, breaking down the myelin sheaths, and causing demyelination and neuroinflammation. The basis for the autoimmune demyelination of MS has not been discovered. Some genetic [1, 2] (e.g. immune genes [3]) and environmental (e.g. smoking [4], obesity [5], stress [6], lack of sunlight exposure [7], etc.) factors have been identified as risk factors for MS. People with MS are mainly female (women: men= 2:1) between the ages of 20 and 40 [8].

Relapse and remission are two major clinical patterns in MS. A relapse is defined by the occurrence of neurological dysfunction over 24 h, and a remission refers to a period of complete or temporary end to the initial relapse phase. Progression is a phase of continuous deterioration for over one year. About 85% of clinical cases feature periodic relapses followed by remission, termed relapsing-remitting MS (RRMS) (in the current thesis, unless otherwise specified, MS refers to RRMS) [8]. The majority of these RRMS cases eventually transition into secondary progressive MS, characterised by progressive deterioration of neurological function without

Chapter 1 Literature Review remission. A minority of MS cases are primary progressive MS (PPMS), progressive relapsing

MS, and secondary progressive MS [9]. PPMS is characterised by a remission-free progressive disease from the start [9].

RRMS is the most studied and common course in MS. Axonal injury, gliosis, and inflammation become progressively worse as relapses develop [10]. Axonal injury, which is one of the neuropathological characteristics of relapsing MS, is believed to be related to the degree of inflammation [11, 12]. This neurodegeneration, together with inflammation, is believed to cause clinical deficits and contribute to accumulated disability [13, 14]. The symptoms of MS are variable, depending on the location of demyelinating lesions [15-17] which often occur in corpus callosum (CC) [18, 19], cerebellum [20], hypothalamus [21] and brain stem [22], and spinal cord [23]. People with MS commonly suffer from fatigue, disability or problems with coordination, altered sensation, memory and cognition deficits and blurred vision. Along with the development of diagnostic methods and criteria, a number of treatments focused on suppressing inflammatory disease activity have been developed in the past few years [8, 24].

However, these therapeutics do not reverse myelin and axon degeneration caused by chronic inflammation [25]; thus, therapies that promote the protection and repair of myelin and neuronal axons are a current focus in MS research.

Chapter 1 Literature Review

Pathogenesis and pathophysiology of MS

Myelin and oligodendrocytes in the CNS

Oligodendrocytes produce myelin in the CNS, whereas Schwann cells carry out this function in the peripheral nervous system (PNS). Myelin is an extended oligodendrocyte and Schwann cell plasma membrane, which wraps around neuronal axons in a spiral fashion, facilitating the efficient propagation of action potentials along axons [26]. Myelin is comprised of 70%–85%

(by dry weight) lipids and only 15%–30% protein [26,27]. The high proportion of lipids in myelin confers a high degree of hydrophobicity, restricting the passage of ions across the neuronal membrane to specific gaps in the myelin sheaths called nodes of Ranvier [27-29]. Of the three major classes of membrane lipids (cholesterol, phospholipids, and glycosphingolipids), cholesterol is the most abundant in myelin [30] and an essential prerequisite for myelin sheath formation and stabilisation [31]. Other neural lipids synthesised from fatty acids [32, 33], and interactions between lipids and proteins [34, 35], are also essential for synthesis and integrity of the myelin membrane. In the CNS, compacted myelin sheaths protect the integrity and function of axons [36, 37], including the provision of metabolic support, via the supply of metabolic substrates glucose and lactose, to neuronal axons [12, 38,

39].

Oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs), developed from different ventral and dorsal origins in the brain and spinal cord (e.g. dorsal developing forebrain, ventricular germinal zone of the brain and spinal cord) [40]. OPCs migrate to sites of myelination, where they differentiate into oligodendrocytes [41]. OPCs are distributed

Chapter 1 Literature Review throughout the brain but are concentrated in the CC [41], and are more proliferative and mature in white than grey matter [42, 43]. The initiation of axon myelination begins with OPC migration to white matter in early embryonic development [44, 45], followed by proliferation, then maturation and differentiation of OPCs. This is followed by the rapid elaboration of myelin [26, 46]. Mature oligodendrocytes then migrate along blood vessels and extend cell process to identify axons for myelination [47, 48].

Evidence suggests that oligodendrocytes also promote neuronal survival via the production of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3

(NT-3) [49-52]. Studies indicate that the immunomodulatory functions of OPCs, for example, migrating to damaged sites in response to cytokines like IL-17 [47], can, in turn, promote immune cell death in vitro [53] and in vivo [54].

Oligodendrocyte pre-progenitor cells, OPCs, pre-myelinating oligodendrocytes, non- myelinating mature oligodendrocytes, and myelinating mature oligodendrocytes can be identified by different myelin and oligodendrocyte marker proteins (Fig. 1.1). The receptor of platelet-derived growth factor α (PDGFRα) is the best-characterised marker for OPCs and is essential for OPC survival [55]. In addition, A2B5 antibody [56] and the NG2 proteoglycan

[57] can specifically identify OPCs; however, cells positive for these markers can differentiate into both oligodendrocytes and astrocytes. During differentiation, pre-myelinating oligodendrocytes can be identified by 2,3-cyclic-nucleotide 3-phosphodiesterase (CNPase or

CNP) [58], and the cell surface marker O4 [59]. For mature myelinating oligodendrocytes,

CNP, myelin basic protein (MBP), myelin-oligodendrocyte glycoprotein (MOG) and aspartoacylase (ASPA) are good markers. MBP appears in the first few days post-natally, on the cytoplasmic surface of the plasma membrane of oligodendrocytes in mice [60], whereas

Chapter 1 Literature Review

MOG appears later, on the surface of the myelin sheath [61]. ASPA is particularly useful for marking mature oligodendrocytes, as it stains the oligodendrocyte cell body rather than myelin

[62]. The transmembrane proteins myelin proteolipid protein (PLP) [63] and myelin associated glycoprotein (MAG) [64], and membrane lipid galactocerebroside (GalC) [65] are also markers for mature oligodendrocytes. MAG, specifically, is a myelin antigen located in the most peri- axonal oligodendrocyte processes [66]. Transcription factors [67], including Olig2 [68] and

SOX10 [69], participate in oligodendrocyte development, thus are present throughout the oligodendroglial lineage.

Chapter 1 Literature Review

Oligodendrocyte Oligodendrocyte Pre-myelinating Non-myelinating Myelinating

pre-progenitor progenitor cells oligodendrocytes mature mature

cells oligodendrocytes oligodendrocytes

PDGFRα SOX10 O4 PLP MBP PSA-NCAM A2B5 CNP GalC MOG Nestin NG2 SOX10 O4 MAG Olig2 Olig2 CNP PLP PDGFαR SOX10 GalC Nestin Olig2 O4 CNP SOX10 Olig2

Figure 1.1 Oligodendroglial differentiation lineage and markers for different developmental stages (Revised from [70]).

Chapter 1 Literature Review

Introduction to MS lesions

MS lesions are focal, demyelinated areas found throughout the CNS, and are not limited to the white matter [71, 72]. In particular, subpial cortical lesions are unique for MS, and are therefore crucial for clinical diagnosis [73]. White-matter lesions are classified as active (active inflammation with abundant infiltrated microglia/macrophages) and inactive lesions (inactive inflammatory with low or no activated microglia/macrophages), as previously described by

Brück et al. [74]. Active white matter lesions are identified as acute characterised by myelin loss and perivascular infiltration of lymphocytes and blood-derived macrophages [75], whereas almost complete depletion of mature oligodendrocytes and lower density or absence of monocytes and microglia/macrophages are seen in inactive lesions [74, 76]. In clinical practice, classic active lesions appear in people with acute MS or RRMS [77]. The proportion of late active lesions (i.e. mixed active/inactive lesions), where macrophages contain more myelin proteins than in early active lesions, represents the degree of ongoing innate demyelinating and inflammatory responses that contribute to neurodegeneration [77]. A high percentage of mixed active/inactive lesions is most common in people with progressive MS [78, 79]. Furthermore, hypocellular inactive lesions become the dominant type in the brains of people with MS from

15 years of disease onward, or secondary progressive MS [77].

Lucchinetti et al. further divided active and early demyelinating lesions into four apparent inflammation types, based on their different pathological characteristics (the pattern of demyelination, myelin and oligodendrocyte loss, immune cell infiltrates) [80]. Pattern I and II

(B cell-mediated) lesions show primary demyelination caused by T-cell-mediated autoimmunity (i.e. activated macrophages/microglia), with involvement of antibodies; whilst

Chapter 1 Literature Review pattern III and IV display primary oligodendrocyte defects such as apoptotic oligodendrocyte death and non-apoptotic oligodendrocyte degeneration, instead of autoimmunity.

Overall, MS lesions are characterized by infiltration of inflammatory cells [81, 82], demyelination and remyelination [83, 84], oligodendrocyte dystrophy [85, 86], reactive astrocytes [87, 88], and microglia/macrophages [74, 89]. Aside from CNS lesions, focal inflammatory demyelination and axonal degeneration are also good biomarkers for spinal cord lesions, which can ultimately lead to clinical disability [90].

Demyelination, remyelination and oligodendrocyte pathology of MS lesions

One of the most important pathological features of MS lesions is demyelination. There are many possible mechanisms for direct or indirect damage to myelin. Release of cytotoxic cytokines or other toxic mediators from T cells or microglia/macrophages can directly affect oligodendrocyte viability and destroy the myelin sheath [91], as can excitotoxicity caused by the direct binding of T cells to myelin antigens [92, 93]. Antibodies from infiltrating B cells can induce the reaction of other immune cells causing the cytotoxicity in the periphery, or lead to myelin destruction [94, 95]. Importantly, demyelination is also characterised by oligodendrocyte death, which is an indirect marker for myelin destruction [96]. The number of oligodendrocytes in pattern I demyelinating lesions is partially restored, while in pattern III it is greatly reduced due to apoptosis [80, 97]. In contrast, non-apoptotic cell loss occurs in pattern

IV [80]. The initial myelin injury seems to be responsible for activation of an apoptotic cascade

[98] and the increased sensitivity of oligodendrocytes to immune/toxic factors in the early

Chapter 1 Literature Review stages of MS lesion formation [80]. As the most energetically-demanding cells of the CNS, oligodendrocytes are particularly dependent on oxidative phosphorylation to meet the intense energy requirements for myelination and maintenance of membrane resting potential [99, 100].

Therefore, impaired energy metabolism could lead to apoptosis in MS lesions [101, 102].

Remyelination, in which new myelin sheaths are generated, can follow the acute demyelination process in the CNS. Endogenous remyelination appears in active MS lesions [103]. Enhanced expression of cell death inhibitors like B cell lymphoma 2 (Bcl-2) [104], restored density of oligodendrocytes and myelin proteins, and myelin protein mRNA expression has been observed during remyelination in MS [105, 106]. Some lipid pathways in oligodendrocytes have also been reported to increase during remyelination [107, 108]. For example, cholesterol- synthesis is upregulated in oligodendrocytes during remyelination, and is essential for promoting OPC survival via the Akt signalling pathway [109, 110]. Thus, remyelination can be improved by increasing myelin synthesis by existing oligodendrocytes, or by restoring OPCs with the potential to differentiate into myelinating oligodendrocytes, for example, transplanting

OPCs into the lesions [111]. Demyelinating lesions can also be characterised by axonal damage due to oligodendrocyte death [112, 113] or inflammation [12, 114]. Protecting oligodendrocyte and OPC maturation may help axon remyelination [86] via preserving axon-derived signals such as ATP [115, 116] since OPC differentiation is highly dependent on mitochondria. This provides a therapeutic approach for MS by repairing axons [117].

Lucchinetti and colleagues reported that 30% of people with MS show almost no remyelination in lesions [85]. The primary mechanism of remyelination failure is believed to be the activated demyelination micro-environment around the damaged myelin, which impairs OPC differentiation to myelinating oligodendrocytes [118, 119]. Factors that contribute to the

Chapter 1 Literature Review limited remyelination capacity within active MS lesions are still not fully understood, but include factors such as LINGO that are released by damaged cells and inhibit myelination [120,

121]. Evidence of lesion repair in younger people suggests that age might affect the remyelination process [122, 123], however it is not elucidated whether MS lesions can still remyelinate after years, and whether remyelinated lesions can be demyelinated again.

Inflammation and glial cell activation in MS lesions

A complex interplay between the adaptive and innate immune system, glia (oligodendrocytes, microglia and astrocytes), neurons and their axons contributes to new lesion formation and ultimately neurodegeneration in MS (Fig. 1.2). Inflammation is present in all clinical courses of MS [77], and is thought to be the reason for oligodendrocyte death in many studies [124-

126]. Oligodendrocyte loss has been found after CD4+ T cell infiltration into the CNS in an oligodendrocyte-depleted mouse model, inducing inflammatory responses and phagocytosis of myelin by macrophages [127, 128]. However, oligodendrocyte apoptosis has been observed prior to infiltration of macrophages and T cells in some people with MS [129, 130]. Thus, whether inflammation initiates the neurodegenerative process in MS is still debatable.

Inflammation occurs after primary oligodendroglial dystrophy in type III and IV lesions [80], showing that inflammation can be caused by dysfunctional oligodendrocytes.

Chapter 1 Literature Review

Figure 1.2 Cells and molecules involved in MS pathogenesis and subsequent pathophysiology.

Diagram (Revised from [131]) shows the major cells types in the CNS and peripheral immune system that participate in the pathogenesis of MS within white matter lesions. Autoimmunity induced by activated immune cells (CD4, CD8, B cell, NK cell, etc.), triggers a neuroinflammatory attack on the

CNS, leading to MS. Detailed descriptions can be found in the text.

Chapter 1 Literature Review

Evidence from genetic data suggests that MS has the same pathogenic features (e.g. peripheral inflammation responses) as other non-CNS autoimmune diseases [132]. Adaptive immunity is essential for new white matter lesions in MS, and both T cell subsets (CD4+, CD8+, NK, T regulatory cells, etc.) and B cells are involved [133]. T cells are autoactivated by some primary signals and against unknown myelin antigens, then differentiate into CD4+, CD8+ (cytotoxic) and helper T cells [134]. In particular, myelin specific CD4+ T cells that are mostly located in the perivascular spaces can trigger subsequent inflammation in the CNS [135]. Helper T cells can also synthesize cytotoxic cytokines (e.g. tumour necrosis factor (TNF)-β, interferon (IFN)-

γ to induce inflammation [136]. These neuroinflammatory events, including innate and adaptive infiltrating immune and CNS cells, allow the migration of T cells into the brain to generate new lesions and cause demyelination [137]. Drugs can restrict T-cell access to the

CNS, which decreases the formation of new MS lesions [138, 139]. Rapid clinical response, i.e. a reduction in new MS lesions can happen after B-cell depletion [140]. It is also suggested that other functions of B cells, such as cytokine production are relevant to MS [141, 142].

Microglia play an important role in inflammation in MS [143, 144]. Both pathogenic and protective functions have been found in the activated microglia and macrophages within MS lesions. Activated microglia/macrophages are present at the earliest stage of MS lesions [143,

144], and could mediate oligodendrocyte death by affecting their mitochondrial activity [145,

146]. In contrast, microglia mediate the clearance of myelin debris containing inhibitors of

OPC differentiation, which helps with initiating remyelination [147, 148]. Microglia and macrophages can also directly contribute to remyelination by producing signalling molecules

(e.g. growth factor activin-A) that promote OPC proliferation, differentiation and migration

[53, 149, 150].

Chapter 1 Literature Review

A widely accepted role of astrocytes following their activation is the formation of a glial scar, which acts as a barrier to remyelination [87]. However, astrocytes could also act as a balancer in MS lesions. In response to myelin damage, astrocytes can be activated. Activated astrocytes not only limit the destructive effects of demyelination by suppressing inflammation [151], but also increase inflammation and tissue damage by expressing toxic cytokines (e.g. interleukin

1β ― IL-1β) [152-154] or releasing components that can drive synapse degeneration [155,

156].

Clinical biomarkers, diagnosis and therapy for MS

MS cannot be diagnosed by a single test. Currently, neurologists rely on the McDonald Criteria for MS diagnosis [157], using neurological assessment and magnetic resonance imaging (MRI)

[158], aided by evaluation of immunoglobulin in cerebrospinal fluid (CSF) biochemistry [159].

Quantification of neurological impairments and relapses is used as a method to evaluate disease activity and severity [8]. MRI is an important diagnostic test [157] and a useful tool to assess the efficiency of therapeutics on suppression of relapses [160], as it can capture clinical lesion changes throughout the entire CNS [161, 162]. Additional biomarkers for diagnosis include neurofilament light chains [163] and oligoclonal bands (OCBs) [164] in the CSF, which correlates with neurodegeneration. Although the presence of OCBs in the CSF is not a direct evidence of neurodegeneration, and is no longer a criterion for the diagnosis of RRMS [165].

It is still an essential tool for MS pre-diagnosis and screening [159, 166]. However, biomarker development has been hampered by the impracticality of tissue biopsies and long-term clinical monitoring of the slow progression process [8].

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Early MS-modifying medications interferon-β [167, 168] and glatiramer acetate [169, 170] are believed to target the innate immune system; however, the specific mechanisms remain unclear because of the complex response of immune cells in MS [171, 172]. In recent years, safer and better-tolerated oral medications have been developed. There are currently fourteen disease- modifying drugs approved by the FDA (USA) to treat RRMS under the immunotherapy strategy alone [173, 174] (Table 1.1). Some of them, such as ocrelizumab [175], laquinimod

[176], and Fingolimod (i.e. FTY720) [177, 178] were also trialled as treatments for PPMS.

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Table 1.1 Medications approved for treatments of MS (Revised from [173, 174]).

Name Targets (proposed mechanisms) Administration method Dimethyl fumarate Changes cytokine balance Oral Fingolimod Sphingosine-1-phosphate receptor modulator, Oral reduces lymphocyte trafficking by blocking egress from lymph nodes Siponimod Sphingosine-1-phosphate receptor modulator (1 Oral and 5) Teriflunomide Mitochondrial dihydro-orotate dehydrogenase Oral inhibitor, no evidence for access to CNS Tecfidera Modulation of NF-kB regulated cytokine - production, inhibits pro-inflammatory molecules Cladribine Synthetic deoxyadenosine analogue, long lasting Oral lymphocyte depletion Glatiramer acetate (GA) Promotes anti-inflammatory response, no evidence Injectable for access to CNS Alemtuzumab Monoclonal antibody, anti-CD52, long lasting Infusion lymphocyte depletion, no evidence for access to CNS Mitoxantrone Immunosuppressant (topoisomerase inhibitor) Infusion Natalizumab Monoclonal antibody, limits T cell transmigration Infusion through the blood brain barrier (BBB) to CNS (via VLA-4 blockade) Ocrelizumab Monoclonal antibody, anti-CD20, depletes B cells Infusion Interferon β Mx A regulated gene induction, inhibits immune Injectable cell migration to CNS (via MMP inhibition)

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At present, most potential treatments have focused on tissue repair and myelin regeneration.

Many drugs that were originally used for non-CNS targets have been tested for their remyelination capability by increasing oligodendrocyte numbers or protecting against cell death. Histamine receptor 1 antagonist Clemastine can enhance myelination [179] via uninhibited differentiation of oligodendrocytes [180, 181]. Antibody rHIgM22 can prevent apoptosis by binding to the oligodendrocyte surface [182-184], however it seems to have no effects on patients in clinical trials [185]. Other strategies aim to promote remyelination by modifying the microenvironment for OPC differentiation and myelination by oligodendrocytes.

Nogo-A co-receptor LINGO-1 has been established as a good target for enhancing remyelination in animal models, since it is an endogenous inhibitor of oligodendrocyte differentiation and myelination [120, 186, 187]. Anti-LINGO-1 antibodies may increase OPC differentiation and myelination [120, 188]. However, the robustness and feasibility of stimulating remyelination is mainly challenged by the difficulty in the absence of clinical biomarkers for remyelination, and whether remyelination attenuates clinical symptoms [189].

Overall, none of the current therapies achieve “no evident disease activity” (i.e. no relapses,

MS lesions and symptoms) in people with MS [190].

Cuprizone model for demyelination in MS research

Introduction to animal demyelination models

There are three primary, well-developed animal models that are commonly used in MS research and present different features of the disease to address different research questions:

Chapter 1 Literature Review experimental autoimmune encephalomyelitis (EAE), virus-induced demyelination, and toxin- induced demyelination.

EAE is the most widely used autoimmune experimental model, focusing on autoimmune demyelination and inflammatory responses that are characteristic of MS [191, 192]. In contrast, virus-induced demyelination models (e.g. Theiler's murine encephalomyelitis virus model) are mainly used for studying the progression process [193, 194] and viral contributions in MS [195,

196]. The lysolecithin and cuprizone toxin models focus more on the mechanisms and changes in the demyelination and subsequent remyelination process. Injection of lysolecithin into the spinal cord or brain results in short-term demyelination and immune system responses [197,

198]. Cuprizone administration is better for studies concentrating on remyelination processes and the regulation of remyelination without the involvement of the adaptive immune system.

The cuprizone model can partially mimic RRMS [199, 200], particularly the pathology of type

III lesions [201-203]. It is characterised by similar de- and re-myelination features including axon damage, oligodendrocyte apoptosis, astrocyte and microglia activation, and modified myelin lipid metabolism (e.g. glycosphingolipids and cholesterol) [204].

Cuprizone and cuprizone demyelination model

Cuprizone is a copper chelator that induces rapid death of mature oligodendrocytes, causing reversible demyelination that is followed by rapid remyelination after cuprizone withdrawal

[205, 206]. Factors such as the mouse strain [207], gender [208, 209], age [210, 211], exposure time [212, 213] and cuprizone dosage [214] have been related to differences in demyelination and associated behavioural deficits in this model. For example, although SJL/J female mice did

Chapter 1 Literature Review not demyelinate to the same extent as males [209], the C57BL/6 mouse strain showed no difference between the genders [208]. Despite demyelination observed with different doses of cuprizone (0.2%-0.6% by weight in mouse chow), 0.2% is considered the best concentration for achieving reproducible demyelination with the least side effects, as higher cuprizone doses cause increased mortality [214]. In the standard protocol for this model [206], C57BL/6 mice fed 0.2% cuprizone in the diet develop acute demyelination in the CNS, especially in the white matter such as CC [215] and other regions including the cerebellum [216] and hippocampus

[217]. This is associated with a reduction in the levels of myelin and oligodendrocyte proteins

[206, 214, 215]. Remyelination occurs within 3-4 weeks after removing cuprizone from the diet.

Recent evidence suggests that oligodendrocyte apoptosis begins in the first few days after cuprizone treatment, and demyelination is evident after 3 weeks [218, 219]. This is followed by massive accumulation of migrated OPCs, after 3-5 weeks of cuprizone treatment [218, 219].

The rate of mature oligodendrocyte death is highest in the first 3 weeks and tapers off by the

5th week, [220, 221], at which point terminal differentiation of OPCs and restoration of myelin sheaths begins to occur.

The precise mechanism of mature oligodendrocyte death in the cuprizone model has not been elucidated. Cuprizone causes inactivation of copper-dependent cytochrome oxidase and reduced oxidative phosphorylation. The high bioenergetic demands and low glutathione levels in oligodendrocytes could therefore make them particularly sensitive to cuprizone intoxication

[222-225]. Mature oligodendrocyte apoptosis in this model is believed to be mediated initially by caspase-3 [226, 227], and later by a poly ADP-ribose polymerase (PARP)-dependent mechanism [228].

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Initial activated microglia can be observed in the first 2 weeks of cuprizone administration, prior to demyelination [229]. Activated and rapidly proliferating microglia start surrounding the demyelinating lesions [229], and become most apparent between 3 and 5 weeks of intoxication [152], but persist at a low level up to 12 weeks [111, 230]. In contrast, activated microglia and reactivated astrocyte appears after demyelination, possibly in response to oligodendrocyte death [203, 216, 231]. Cytokines produced by activated microglia and astrocytes may also increase OPC accumulation in demyelinating areas in this model [215, 218,

232].

Oligodendrocyte cell culture models for studies on MS

In vitro cell culture models involving primary oligodendrocytes or cell lines help to define molecular and cellular mechanisms. Primary oligodendrocytes can be obtained through different methods [233], including the differentiation of stem cells [234, 235], and the isolation of mixed glial progenitor cells and OPCs derived from neonatal mice or rats.

In vitro primary culture models have established some growth factors are involved in the regulation of cellular functions and survival of OPCs, maturation of oligodendrocytes and myelin production. In response to platelet derived growth factor (PDGF) and NT-3, cultured

OPCs proliferate continuously [236-239]. PDGF signalling has also been associated with OPC survival [240] and differentiation [241, 242]. However, PDGF shows an inhibitory effect on myelination in oligodendrocyte co-cultures with dorsal root ganglion neurons [237], despite the relatively high susceptibility of OPCs to remyelination than previously myelinating oligodendrocytes [243]. In addition, primary oligodendrocyte cultures are good models for

Chapter 1 Literature Review studying the mechanisms of oligodendrocyte apoptosis, for example, cultured OPCs undergo increased cell death under stress conditions (withdrawal of serum/glucose and/or oxidant stimulus) [243, 244].

Sphingolipids in myelin

Introduction to sphingolipids

There are two main groups of sphingolipids, with different types of polar head group, specifically phosphosphingolipids (e.g. sphingomyelin) and glycosphingolipids

(glucosphingolipids, galactosphingolipids, globosides and gangliosides) [245]. Ceramide is the central sphingolipid metabolite, formed by the addition of a fatty acid to the free amine of the mono-acyl lipid sphingosine. De novo ceramide synthesis takes place in the ER (Fig. 1.3).

Ceramide synthases (CerS1-6) mediate the production of dihydroceramide (dhCer), which is then desaturated to produce ceramide. Ceramide and sphingosine can also be obtained from salvage pathways through catabolism of complex sphingolipids. Alternatively, ceramide can be obtained by sphingomyelin hydrolysis at the plasma membrane or in the lysosome.

Ceramides are the lipid precursor for synthesis of a diverse range of complex sphingolipids in the Golgi apparatus [246, 247]. Glucosylation of ceramides by glucosylceramide synthase

(GCS) produces glucosylceramide (GluCer), which is the precursor for the ganglioside family.

GluCer is essential for embryonic development [248]. Likewise, Galactosylceramide (GalCer) is synthesised from ceramides and considered a marker of oligodendrocytes [249, 250]. GalCer is essential for oligodendrocyte differentiation, myelin stability, and neuronal action potential

Chapter 1 Literature Review propagation [32, 33]. Alternatively, ceramide is converted to the most abundant sphingolipid, sphingomyelin, by sphingomyelin synthases [251].

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Figure 1.3 Schematic overview of the sphingolipid metabolic pathway.

Overview of the sphingolipid pathway (Adapted from [252]). Ceramides can be obtained from the different classes of sphingolipids: sphingomyelin (SM), hexosylceramide (HexCer) and sulfatide. De novo synthesis in the endoplasmic reticulum begins with the synthesis of sphinganine, then dihydroceramide from serine and palmitoyl coenzyme (CoA). Dihydroceramides are rapidly desaturated to produce ceramides (Cer), which are transferred to the Golgi for synthesis of sphingomyelin (SM) and glycosphingolipids. Ceramides may be degraded by ceramidases to produce sphingosine, which can be phosphorylated by sphingosine kinases (SphK1 and 2) to form sphingosine

1-phosphate (S1P). S1P can be broken down in the endoplasmic reticulum by S1P lyase (SPL).

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Ceramides can also be deacylated to form sphingosine, which is phosphorylated by sphingosine kinases to yield the signalling molecule sphingosine 1-phosphate (S1P). S1P can either be released from the cell, where it acts as a signalling molecule, or degraded by the enzyme S1P lyase, yielding palmitoyl-CoA that can be recycled for new lipid synthesis [246, 247]. In addition to sphingolipid biosynthesis and breakdown, ceramides are crucial regulators of multiple cell signalling pathways involved in apoptosis, synaptic function and inflammation

[253-255].

Overall, sphingolipids contribute to diverse cellular functions, including cell proliferation [256,

257], differentiation [258, 259] and apoptosis [260, 261]. Disruption of sphingolipid balance leads to neuronal dysfunction including neurotransmission deficiency and impaired ion permeability [33]. For example, the pro-apoptotic function of sphingosine and ceramides is highly relevant to neuronal stress and survival [253, 262, 263]. Sphingolipid levels change in brains with age-associated neurodegenerative diseases [264, 265]. For example, the myelin lipid sulfatide and ceramide synthase 2 enzyme activity are reduced in the early stages of

Alzheimer’s disease (AD) pathogenesis [266, 267], whereas levels of sphingosine and ceramides are increased, possibly contributing to inflammation and cell death in the AD brain

[268, 269].

Sphingolipids in oligodendrocytes and demyelination

Sphingolipids are one of major lipid components of myelin [270]. GalC is the most abundant lipid in myelin. GalC and its sulfated form sulfatide are enriched in oligodendrocytes. It has been suggested that these lipids stimulate the differentiation of proliferative pre-

Chapter 1 Literature Review oligodendrocytes to non-proliferative immature oligodendrocytes (Fig. 1.1), indicating their importance in oligodendrocyte differentiation [271].

Disturbed sphingolipid content leads to destabilization of myelin structure. Both GalC and its sulfatide derivative sulfatide are essential myelin stability and function, although not for myelin formation [32, 33, 272]. Genetic deficiency of sphingomyelinase, which degrades the lipid sphingomyelin, repaired myelin and promoted oligodendrocyte survival after two weeks of remyelination in the cuprizone model [273, 274], suggesting an important role for sphingomyelin in myelin regeneration.

Overall, there is very little known regarding to the changes in sphingolipid levels in MS. GalC is decreased in myelin from normal appearing white matter in MS [275], whereas hydroxylated sulfatide is increased in the damaged white matter [276]. Increased levels of sphingosine were observed in normal appearing white matter of people with MS [277] and increased ceramide levels were observed in MS lesions [278-280].

S1P and S1P receptors (S1PRs) in the CNS

S1P is a bioactive metabolite that is critical for cell migration, proliferation and survival in different cell types [246, 281, 282]. S1P is present at a high level in plasma, where red blood cells are considered the main source [283-285]. This signalling molecule is generated by the phosphorylation of sphingosine, by sphingosine kinase 1 (SphK1) and 2 (SphK2) (Fig. 1.3).

After its synthesis, S1P is then transported out of the cells by specific S1P transporters Spns2

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[286, 287] and Mfsd2b [288], where it signals through a family of five extracellular-facing G- protein coupled receptors, S1PR1-S1PR5 [289, 290].

Each S1PR couples to different heterotrimeric G-proteins, leading to activation of different signalling pathways depending on the particular receptor sub-type (Fig. 1.4). S1PR1 couples selectively to Gαi, whereas S1PR3 couples to Gαi, Gα12/13, and Gαq. Both receptors stimulate

Ras/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, which can promote cell proliferation [291-293]. In contrast, S1PR2 coupling to Gα12/13 induces Rho pathway activation and inhibits cell proliferation via suppression of Rac and Akt pathways [294, 295]. S1PR4 also couples to Gα12/13 and signals via the Rho/ROCK pathway [296, 297], as does S1PR5, which couples to both Gαi and Gα12/13 [298].

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Figure 1.4 Autocrine and paracrine signalling of S1P through S1PRs.

S1P can be transported out of the cell by specific transporters [286-288] acting in an autocrine or paracrine fashion through S1PRs to activate a plethora of signalling pathways including ERK, Protein

Kinase B (Akt), Rho GTPase, IP3, and PKC. Each S1P receptor subtype can couple to different G- proteins. Abbreviations: DAG: Diacylglycerol; ERK: Extracellular Regulated Kinase; IP3: Inositol triphosphate; PI3K: Phosphatidylinositol 3’ kinase; PLC: phospholipase C; PKC: Protein kinase C;

ROCK: Rho-associated protein kinase; SRF: Serum response factor. Revised from [299].

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S1PR1-5 distribution and expression vary in different tissues and cells. S1PR1-3 are ubiquitously-expressed in most cell types [300, 301], whilst S1PR4 and S1PR5 are restricted to specific cell types and tissues. S1PR4 is specific to immune cells, lymphocytic and hematopoietic tissue [296], leading to regulation of cytokine secretion and proliferation of T- cells [302]. S1PR5 is expressed by very few cell types, specifically oligodendrocytes [303, 304] and natural killer (NK) T-cells [305, 306]. S1PR1 is an essential regulator of immune cell trafficking [307], endothelial barrier function [308] and vascular tone [309], as well as neurogenesis [310]. S1PR3 plays a key role in vascular endothelial function and inflammation

[311, 312].

S1P levels are also high in the CNS [313, 314], promoting oligodendrocyte survival [303] and differentiation [303, 315], protecting neuronal cells [316] and neural tube closure during development [317], and stimulating neurotransmitter release from pre-synaptic terminals [318-

321]. Different CNS cell types express S1PRs with different levels and this changes with development of the cells [322, 323] (Fig. 1.5). Neurons express S1PR1-3 and S1PR5 [323].

Astrocytes show very high levels of S1PR1 expression, with some expression of S1PR2 and

S1PR3 [324]. Similarly, OPCs express S1PR1 most highly, with some expression of S1PR2 and S1PR3. Myelinating oligodendrocytes express very high levels of S1PR5, and low levels of S1PR1 [303, 304, 325]. Accordingly, S1P has been shown to promote survival, proliferation, migration and differentiation of different types of brain cells via S1PR signalling [324, 326-

328].

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Astrocyte

S1PR5 Neuron S1PR4 S1PR3 S1PR2 OPC S1PR1

Myelinating Oligodendrocytes

Microglia

Endothelial

0.125 0.5 2 8 32 128 512 FPKM

Figure 1.5 The mRNA expression profile for receptors in different CNS cell types. mRNA expression level of S1PR1-5 in cells from the mouse brain, as determined by RNA sequencing

[322]. FPKM: fragments per kilobase per million mapped reads.

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In neuronal cells, S1PR2 is essential for the mediation of neuronal excitability [329, 330]. A study shows that S1PR2 binds to Nogo-A and its inhibition elevates plasticity, indicating the supressing function of S1PR2 on synaptic plasticity [331]. S1PR2 may also be related to the suppression on the S1P-mediated neural progenitor migration as pharmacologic or genetic inhibition of S1PR2 significantly promotes progenitors releasing to the ischemic region independent of proliferation and differentiation [332]. In addition, activation of S1PR1 promotes neuron production and ameliorates neurological symptoms in animal models [333,

334].

Both S1PR1 and 3 are associated with the regulation of astrocytes. Increased expression of

S1PR1 and 3 is found in reactive astrocytes in MS lesions [335], as well as in vitro under proinflammatory condition [336]. In contrast, S1PR1 levels are reduced in isolated microglia, with no change in S1PR3 [337]. Functional antagonism of S1PR1 on microglia and astrocytes has been shown to inhibit neuroinflammation in the CNS and protects against demyelination in the EAE model [338]. Studies recently report that S1PR1 and 3 are also related to the microglial polarization in the ischemic brain [339-341].

In oligodendrocytes, the effects of S1P signalling through S1PRs depends on the developmental stage of the cells [303, 315, 342, 343]. PDGF stimulus, which promotes OPC proliferation, causes S1PR1 upregulation and S1PR5 downregulation in OPCs [303, 342]. S1P promotes PDGF-dependent proliferation of OPCs through S1PR1 signalling, as well as OPC differentiation [315, 342]. S1PR5 activation promotes the survival of mature oligodendrocytes, mediated through Akt phosphorylation, whereas S1PR5 activation in immature oligodendrocytes leads to retraction of oligodendroglial processes [303, 343]. However, no gross defects in developmental myelination were observed in S1PR5-deficient mice [303].

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Interestingly, besides the well characterised extracellular functions of S1P, endogenous S1P can promote cell proliferation and inhibit death even independent of its receptor signalling pathways [344, 345]. Different neurogenesis defects were apparent between S1PR1 and

SphK1/SphK2 null animal models [317]. Severe cell death and reduced proliferation had been observed in SphK1 and SphK2 double knockout embryos, however these effects were milder in S1PR1−/− embryos. Intracellular S1P generated by overexpression of SphK1 increases cell survival in mice with S1PR defects, suggesting that endogenous S1P stimulating cell growth may not be through S1PRs [346]. These results indicate that intracellular S1P can regulate cell survival and apoptosis independent of S1PRs. In regards, S1P is known to mediate effects through a range of intracellular targets including HDAC1/2 [347] and prohibitin [348].

S1PR agonists

The unique signalling mechanisms and tissue-specific properties of S1PRs make them good targets for MS treatment, thus, S1PR-selective agonists have been developed as a new family of therapeutics [349]. This began with development of the oral drug Fingolimod, approved by the FDA for the treatment of RRMS in 2010 [350].

Fingolimod

The primary effect of Fingolimod in MS treatment is immunosuppression caused by induction of peripheral lymphopenia [281, 351]. Fingolimod is an analogue of sphingosine that is phosphorylated by SphK2 in vivo [351], forming the active drug Fingolimod-phosphate

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(FTY720-P) [352]. FTY720-P is an agonist of all S1PRs except S1PR2, inducing S1PR activation [281, 351, 353]. Immunosuppression is mediated through the drug’s action on

S1PR1.

Under normal physiological conditions, lymphocytes egress from lymph nodes into the circulation in response to high S1P levels in the blood. This migratory response is dependent on activation of lymphocyte S1PR1 [307]. FTY720-P is a super-agonist of S1PR1, inducing internalisation and proteasomal degradation of the receptor on lymphocytes [338, 353]. Thus, the drug is described as a functional antagonist of S1PR1, as it causes hyper-activation and then down-regulation of the receptor on lymphocytes. This induces lymphopenia by inhibiting lymphocyte egress from lymph nodes into the circulation [354], thereby leading to a pronounced decrease in circulating lymphocytes and inflammatory cell infiltration in the CNS.

In addition to its effect on lymphocytes, Fingolimod readily crosses the BBB and exerts direct actions in the CNS [279, 338, 350, 355], including binding to oligodendrocyte S1PRs.

Fingolimod protects mature human oligodendrocytes against cell death in vitro [303, 356]. The drug had no effects on myelination under basal conditions, but protected against mature oligodendrocyte apoptosis and demyelination in the cuprizone model [357-359]. Fingolimod administration did not enhance spontaneous remyelination when administered after cuprizone withdrawal [359-361], although Fingolimod was reported to promote remyelination in an in vitro demyelination model [362, 363], as well as in the EAE model [364, 365], possibly through

S1PR3 and S1PR5 on OPCs and mature oligodendrocytes. People treated with Fingolimod for one year show enhanced myelin integrity by MRI imaging [366]. Furthermore, Fingolimod protects OPCs against apoptosis induced by inflammatory cytokines in vitro [363, 367], but inhibits OPC migration [315]; in contrast, its role in OPC differentiation is dose-dependent

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[363, 367]. Differentiation of rat OPCs into mature oligodendrocytes is promoted by low doses of Fingolimod [342], and inhibited by higher doses [367]; however, others have shown that both low and high doses inhibit differentiation of human OPCs in vitro [363].

Fingolimod supresses the neuroinflammatory phenotype of human and murine astrocytes, microglia and CNS-infiltrating proinflammatory monocytes [368-370], particularly mediated through S1PR1 [338]. In contrast, the drug promotes expression of neurotrophic genes such as

BDNF, glial cell-derived neurotrophic factor (GDNF), and leukaemia inhibitory factor (LIF) by astrocytes [370-372].

Fingolimod also demonstrates neuroprotective effects, suppresses neuroinflammation, and reduces symptoms in experimental models of stroke [373], Huntington’s disease (HD) [374],

Parkinson 's disease (PD) [375], and AD [369].

Siponimod

Fingolimod can also cause serious side-effects such as transient bradycardia and susceptibility to severe lung infections [281, 376]. Second-generation S1PR modulators, aimed at more specific targeting of S1PR1, have been developed. The dual S1PR1/S1PR5 agonist Siponimod was first approved for treatment of RRMS and secondary progressive MS in 2019 (2020 in

Australia) [377]. Compared to Fingolimod, Siponimod’s shorter half-life promotes more rapid recovery of peripheral lymphocytes [378, 379]. Siponimod ameliorates degenerative brain events such as gliosis and neuronal loss, probably via regulation of glial cell functions [380], highlighting this drug as a potential therapy in demyelinating disorders other than MS. For

Chapter 1 Literature Review instance, synaptic degeneration was attenuated by Siponimod in an MS model [358, 381], potentially mediated through increased GABAergic inhibitory transmission [377].

Ozanimod

Ozanimod was approved in 2020 for the treatment of RRMS by the FDA (USA) and TGA

(Australia) [382]. Like Siponimod, this oral administrated S1PR agonist is selective for S1PR1 and S1PR5, and reduces lymphocyte infiltration and inflammation in the EAE model, whilst promoting the protective microglial phenotype [383, 384].

A-971432

As mentioned above, S1PR5 is very highly expressed on oligodendrocytes, and both

Siponimod and Ozanimod are agonists of S1PR5 as well as S1PR1. A selective agonist of

S1PR5, A-971432, has been developed [385] and studied in AD and HD mouse models [386,

387]. A-971432 was found to protect against permeabilization of the BBB [385, 386, 388]. In these studies, it was suggested that S1PR5 is expressed on brain endothelial cells, mediating the effects of A-971432 on the BBB [383]. Moreover, A-971432 activates neuroprotective pathways such as ERK and Akt, rescued motor deficits and an age-dependent memory deficit in the T-maze [385]. The effects of A-971432 on oligodendrocyte lineage cells have not been determined, despite very high S1PR5 expression in these cells.

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Other S1PR agonists

An increasing number of other S1PR1 agonists have been developed, e.g. Ponesimod,

Amiselimod, SEW2871, CYM5442 [389]. All of these are orally-available and inhibit inflammatory responses in lymphocyte-mediated inflammatory diseases [389]. Recently, the

S1PR1-selective agonist CYM5442 was shown to suppress oligodendrocyte apoptosis and subsequent reactive gliosis and demyelination in the cuprizone model [358], raising the possibility that S1PR1 agonism alone is sufficient to protect against demyelination through direct actions in the CNS, as well as protecting through the induction of lymphopenia. S1PR2 agonist CYM5520 [390] and S1PR3 agonist CYM5541 [391] were also developed for receptor signalling studies.

Sphingosine kinases (SphKs)

SphKs and S1P

The two human SphK isoforms share 80% similarity and 45% sequence identity, leading to five shared conserved regions within their sequence (termed C1-5) [392]. C1-3 form the catalytic domain, and C2 is the region associated with ATP binding. Compared to SphK1,

SphK2 has an extended N-terminus and an additional central proline-rich region [393]. The

SphK2 N-terminal region is required for its interaction with phosphoinositide and consequently promotes its localisation at internal membranes [394], thus, is important for the catalytic activity of SphK2 [395].

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SphK1 is more abundant in the lungs, spleen and leukocytes, whereas SphK2 is highly expressed in the CNS [396]. SphK1 is localised to the cytosol and can be recruited to the plasma membrane by ERK1/2-mediated phosphorylation [396, 397]. In contrast, SphK2 is localised more broadly in cells, including in the nucleus [398], mitochondria [399] and on internal membranes [390, 394], which allows the enzyme to access a distinctive substrate pool from

SphK1 and exert different biological functions [400]. S1P produced by SphK2 in the nucleus directly binds and inhibits the activity of histone deacetylases 1 and 2 (HDAC1/HDAC2) [401-

403], which regulates gene expression. The localisation of SphK2 between nucleus and cytosol is regulated by its phosphorylation [404].

The intrinsic catalytic activity of both SphKs is very important not only for producing S1P, but also for controlling the levels of sphingosine and ceramide, which accumulate in the absence of SphK1 or SphK2 activity (Fig. 1.3) [252, 405-407]. External stimuli such as growth factors

(e.g. PDGF and NT-3) [408, 409] and neurotransmitters (e.g. glutamate and acetylcholine)

[410, 411] that are important for oligodendrocyte and neural cell functions potently regulate

SphK activity.

The role of SphKs in cancer

A number of studies revealed an association between SphKs and various diseases [412], including cancer [413]. Extensive research has shown that high levels of SphK1 and SphK2 activity are involved in tumour growth, chemotherapeutic resistance and patient mortality in human cancers [414-417]. In fact, SphK1 has been suggested as a functional indicator for oncogenic tolerance and chemotherapeutic resistance [418, 419]. In tumour cells, SphK1

Chapter 1 Literature Review promotes proliferation, survival [420-422], migration and invasion [423-425]. SphK2 also displays anti-apoptotic properties in different cancer cell types [426-428], despite its pro- apoptotic functions in few study [429]. These findings have raised the possibility for SphK1

[430, 431] and SphK2 [432, 433] inhibitors to be used in cancer therapy, however the promise of sphingosine kinase inhibitors for cancer therapy has not yet translated to clinical trials [434-

437]

In addition, SphK1 and SphK2 activity can be increased by pro-inflammatory cytokines such as TNF-α and IL-1β [438, 439]. SphK2, in turn, can affect the inflammatory response. SphK2 acts as an anti-inflammatory modulator decreasing pro-inflammatory cytokine/chemokine production, infiltration of monocytes and apoptosis, mostly in cancer models [440-442].

SphKs and S1P in the CNS and neurodegenerative diseases

Knockout of either SphK1 or SphK2 does not produce overt phenotypes, but mice lacking both enzymes, and thus unable to synthesize S1P, die in utero, with defects in vasculogenesis, neurogenesis, and neural tube closure [443]. The lethal vascular defect results from the failure to recruit vascular support cells (pericytes) to the developing vasculature. These findings suggest that the functions of SphK1 and SphK2 are at-least partially overlapping [443], but subtle phenotypes become evident in the SphK2 knockout mice with physiological or pathological stimuli. Mice lacking SphK2 show an abnormal fear response [444, 445], which was proposed to result from the endogenous disinhibition of HDAC2 due to the absence of nuclear S1P. HDAC2 supresses the expression of memory- and learning- related genes [445].

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S1P produced by SphK1 is involved in the release of neurotransmitters from pre-synaptic termini. Chan et al. showed that SphK1 stimulates acetylcholine release from presynaptic terminals in the worm C.elegans mediated by a novel muscarinic signalling pathway ― presynaptic signalling pathway. This pathway, in turn, requires SphK1 activity and abundance to modulate synaptic transmission [411]. In mice, SphK1 was shown to promote the release of pre-synaptic glutamate and was therefore required for long-term potentiation [321, 446]

Levels of S1P were reduced by 85%-90% in brain tissue of SphK2 knockout mice [393, 447].

SphK2/S1P signalling plays an important neuroprotective role in neurodegenerative disease models including in vitro models of PD [448] and HD [449], and in vivo models of stroke [450] and AD [406]. Notably, reduced SphK2 activity may be an important mediator of mitochondrial deficiency in PD, partly attributed to the predominant mitochondrial localisation of SphK2 and interaction with the mitochondrial prohibitin [451].

A previous study from our group uncovered significant loss of S1P and SphK2 activity in the early stages of AD pathogenesis [410]. The enzymatic basis for loss of SphK2 and S1P in AD remains unknown; however, our group presented evidence that this may be related to lysosomal dysfunction, which is a key characteristic of AD [406]. In contrast, a prior study had reported that SphK2 activity was upregulated in brain tissue from people with AD [452], although in this study SphK2 activity was measured only in the Tris-soluble fraction, whereas SphK2 is a membrane-associated enzyme. Interestingly, these authors demonstrated that S1P produced by

SphK2 activates the enzyme BACE1, which is one of two proteases that cleaves the amyloid precursor protein to release Aβ peptides [452]. This role for SphK2, demonstrated in vitro by

Takasugi et al, was subsequently confirmed by our group in vivo [406]. Another study reported

Chapter 1 Literature Review reduced SphK1 expression in AD, and an inverse correlation between Aβ plaque density/number and SphK1 expression [453].

In recent work, we have also observed marked hypomyelination in SphK2−/− mice crossed with an AD mouse model [406], providing initial evidence that the SphK2-S1P signalling system is essential for myelin synthesis and/or maintenance. Oligodendrocyte cell number was reduced in an age-dependent manner, in SphK2−/− mice expressing an amyloidogenic transgene [406].

SphK2−/− mice have functional myelin and normal motor functions under normal physiological conditions. However, in unpublished studies we have found that myelin thickness decreases significantly with ageing in SphK2−/− mice [454]. These observations led us to investigate whether SphK2 is an essential mediator of oligodendrocyte survival and remyelination using the cuprizone model.

Aims

In summary, S1P is an established regulator of immune cell migration/trafficking and astrocyte activation, and our recent data suggests an important role for SphK2 ― the major isoform catalysing S1P synthesis in the CNS ― in oligodendrocyte survival and myelination. Such a role is supported by the high expression of S1PR5 in oligodendrocytes, although the physiological functions of this receptor have barely been investigated. An oligodendrocyte- protective role for S1PRs is further indicated by studies demonstrating that Fingolimod protects against demyelination in the cuprizone model, although the drug did not enhance spontaneous remyelination following cuprizone withdrawal.

Chapter 1 Literature Review

In this thesis, I firstly investigated the role of SphK2 in cuprizone-induced oligodendrocyte loss and remyelination (Chapter 2). The results of this study inspired me to further explore

S1P signalling in myelination, by investigating the effect of selective agonists of S1PR1 and

S1PR5 on cuprizone-induced demyelination (Chapter 3). This work lays the foundation for future studies investigating the role of endogenous S1P in oligodendrocyte survival and remyelination, via S1PR1 and S1PR5 signalling.

The specific aims of this thesis were:

Determine the requirement for endogenous S1P produced by SphK2 in promoting oligodendrocyte survival and remyelination in the cuprizone mouse model.

Determine if S1PR agonists (S1PR1 and/or S1PR5) promote oligodendrocyte survival and protect against demyelination in the cuprizone mouse model.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

SphK2 protects oligodendrocytes and is required for remyelination.

Introduction

As one of the two defining pathological features of MS, demyelination caused by oligodendrocyte death in the CNS leads to loss of neurological functions. Effective remyelination requires preservation and/or restoration of proper functioning of oligodendrocytes after tissue damage, especially the ability of OPCs to differentiate into myelin forming cells and for those differentiated oligodendrocytes to re-myelinate denuded axons.

Current therapeutics for MS like Fingolimod attenuate demyelination mainly via suppressing neuroinflammation, but their capacity to directly stimulate oligodendrocyte protection and remyelination need to be investigated. Therefore, remyelinating therapies are often regarded as the next frontier in demyelinating and neurodegenerative conditions such as MS [189].

The lipid signalling molecule S1P is an important physiological regulator of neurological functions in the CNS. S1P, signalling through S1PR5, has been demonstrated to provide trophic support for mature oligodendrocytes in vitro [303], whilst S1PR1 agonists were shown to protect against demyelination in vivo [358]. These findings suggest a requirement for S1P in oligodendrocyte survival and protection against demyelination in diseases such as MS and provide a potential therapeutic target for promoting remyelination in MS. Recently, an in vitro study using slice cultures indicated that Fingolimod may directly stimulate remyelination [362].

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

However, the role for the endogenous ligand S1P in oligodendrocyte survival and remyelination has not been established.

S1P is synthesised predominately by SphK2 in the CNS. We recently demonstrated that SphK2 protects against oligodendrocyte loss and demyelination in an AD mouse model [406].

However, the mechanism of protection against demyelination by SphK2, and the specific role of SphK2 in oligodendrocyte function and myelination are unknown. In this Chapter, we employed the oligodendrocyte toxin cuprizone to determine if endogenous SphK2 protects against demyelination and/or is necessary for remyelination following a demyelinating insult.

Specifically, oligodendrocyte number and myelin content were assessed in SphK2 knockout

(SphK2−/−) mice and congenic wild-type (WT) C57BL/6 mice treated for 6 weeks with cuprizone, and at 2 weeks after cuprizone withdrawal.

Aims

1. To investigate if endogenous SphK2 is necessary to protect oligodendrocytes and prevent demyelination in mice administered cuprizone;

2. To determine whether endogenous SphK2 is required for remyelination following cuprizone treatment.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Materials and Methods

Transgenic mice and cuprizone administration

SphK2 knockout mice (SphK2−/−) were obtained under MTA with Prof Richard Prioia, NIDDK,

Bethesda, USA [317]. SphK2−/− mice were crossed to C57BL/6J mice for more than 10 generations, creating heterozygous SphK2+/− mice. SphK2−/− and WT control mice (SphK2+/+) were derived from SphK2+/− breeders. They were bred in independently ventilated cages at

Australian Bioresources (ABR, Mossvale, NSW) and genotyped as described previously [317].

Mice were monitored and weighed three times a week. Experimental protocols were approved by the University of Sydney animal ethics committee (approval #2017/1284), and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Three-month old male SphK2−/− and WT mice were used for all experiments. Mice were acclimated to the lab conditions, with food and water provided ad libitum and a 12 h light/dark schedule, for 2 weeks prior to treatment. They were then randomly allocated to the treated or untreated groups. Mice of both genotypes (n = 6 mice/genotype) were fed a 0.2% (w/w) cuprizone diet or normal chow. Cuprizone purchased from Sigma Aldrich was incorporated into normal chow pellets by Specialty Feeds, WA, AU. A third group of 6 mice from each genotype was fed cuprizone pellets for 6 weeks, after which the diet was changed to normal chow for 2 weeks, to assess remyelination. The experiment therefore included six group: untreated WT, cuprizone-treated WT, WT remyelination (i.e. 6 weeks cuprizone treatment

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination followed by 2 weeks remyelination after cuprizone withdrawal), untreated SphK2−/−, cuprizone-treated SphK2−/−, and SphK2−/− remyelination.

In a follow-up experiment, groups of 6 SphK2−/− mice (3 months old) were fed a 0.2% cuprizone or normal chow diet for 6 weeks, or fed cuprizone for 6 weeks followed by a 4 week remyelination period on normal chow.

Immunohistochemistry and immunofluorescence microscopy

Mice were perfused transcardially with sterile saline. Brains were removed and divided sagittally into halves. Half brains were postfixed overnight with 4% paraformaldehyde (Sigma-

Aldrich, #28908) in PBS, then transferred to 30% sucrose and frozen at −80 °C. Tissue was sectioned at 30 μm using a ThermoFisher Scientific Cyrotome FSE Cryostat and stored at 30 °C in cryoprotectant (25% glycerol, 25% ethylene glycol in PBS).

Immunofluorescence and volumetric analysis

Free-floating sections were incubated at 70 °C for 10 min in 10 mM sodium citrate, pH 6.0, with 0.01% Tween 20, followed by 3 washes with Phosphate-Buffered Saline with 0.1% Tween

20 (PBST). Sections then were incubated in blocking solution (5% goat serum, 0.1% BSA, 0.1%

Triton X-100 in PBS) for 1 h at RT, and overnight at 4 °C in primary antibodies (Table 2.1) diluted in blocking solution. One section was incubated in blocking buffer without primary antibody as a control for non-specific binding of secondary antibodies. Sections were then

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination washed and incubated with a fluorescent-dye conjugated secondary antibody for 2 h at RT in the dark. After 3 washes with PBST, sections were stained with 1 µg/mL diamidino-2- phenylindole dihydrochloride (DAPI) for 7 min. Sections were washed 2 times with PBST and mounted on poly-L-Lysine coated slides using ProLong Gold anti-fade (Life Technologies,

#P36935). Slides were imaged using an Olympus Virtual Slide microscope VS1200, 20×. In all cases, the experimental operator and analyser remained blinded to the treatment groups until after quantification.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Table 2.1 List of antibodies used for immunofluorescence (IF) and western blotting (WB).

Primary Source Primary Catalogue Secondary Catalogue Secondary antibody antibody number antibody number antibody dilution dilution (in blocking solution) Rabbit Anti-Iba1 Abcam 1:1000 ab178847 Anti-rabbit Cell 1:1000 (IF) IgG (H+L), Signalling F(ab')2 Technolog Fragment , #4414; (Alexa (IF) Fluor® 647 Conjugate) Rabbit anti- Abcam 1:1000 ab223269 Anti-rabbit Cell 1:1000 (IF) ASPA (IF) IgG (H+L), Signalling Rabbit anti-MBP Abcam 1:1000 ab40390 F(ab')2 Technolog 1:5000 (WB) (WB/IF) Fragment y, #4412; Rabbit anti- Abcam 1:1000 ab32760 (Alexa (IF) MOG (WB) Fluor® 488 Rabbit anti-PLP Abcam 1:1000 ab28486 Conjugate); Cell (WB) (IF) Signalling Rabbit anti- Cell 1:1000 32346 Technolog SphK2 Signalling (WB) Anti-rabbit y, #7074S; Technology IgG HRP (WB) Rabbit anti- Cell 1:1000 4060 linked phospho-Akt Signalling (WB) antibody; (Ser473) (D9E) Technology (WB) Rabbit anti-Akt Cell 1:1000 4691 (pan) (C67E7) Signalling (WB) Technology Rabbit anti-β- Abcam 1:5000 ab8227 actin Mouse anti- Abcam 1:1000 ab6319 Anti-mouse Cell 1:5000 (WB) CNPase [11-5B] (WB) IgG HRP- Signalling linked Technolog 1:1000 (IF) antibody; y, #7076S; (WB) (WB) Mouse anti- βIII- BioLegend 1:1000 TUBB3 tubulin (WB) Anti-mouse Cell IgG (H+L), Signalling F(ab')2 Technolog Mouse anti- Cell 1:1000 3670S Fragment y, #4408; GFAP (GA5) Signalling (IF) (Alexa (IF) Technology Fluor® 488 Conjugate); (IF) Chicken anti- Abcam 1:106 ab4680 Goat anti- Abcam, 1:5000 neurofilament H (WB) chicken #ab6877 (NF-H) IgY (H+L) HRP

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Quantification of ASPA-positive cells. Images were first processed using automatic brightness/contrast function, and different regions of interest (ROIs) in images (CC, cortex and hippocampus) were then selected using ImageJ (v. 1.53b). The ASPA-positive area for each region of each mouse was measured, and ASPA-positive cell number was counted using the particle analysis function in ImageJ, based on size (7.50-100.00 μm2) and circularity (0.25-

1.00). Cell density was then calculated as cell number divided by area.

Luxol Fast Blue (LFB)/Crystal Violet (CV) staining and analysis

For LFB/CV staining [455-457], three sections were selected from each group, and mounted on poly-L-lysine-coated microscope slides (ProSciTec, #G312P-W) to dry at 37 °C for 3 h.

Sections were defatted for 5 h in 1:1 alcohol/chloroform, then soaked in 95% ethanol for 30 min and incubated for 16 h at 60 °C using 1 mg/ml LFB (Sigma-Aldrich, #S3382) in 95% ethanol. Sections were rinsed with 80% ethanol followed by water until clear, and differentiated in 0.05% Li2CO3 (Sigma-Aldrich, #255823) solution for 30 min, then rinsed in 70% ethanol and water. Sections were counterstained with CV (1 mg/mL cresyl violet and 0.05 mg/mL oxalic acid), and rinsed successively in water, 70% ethanol, 95% ethanol, 100% ethanol and xylene. DPX mountant (Sigma-Aldrich, #06522) was used for mounting, and slides were dried overnight.

The splenium, body and genu of the CC (Fig. 2.1A) were imaged on an Olympus Virtual Slide microscope VS1200, using a 20× objective. For analysis, standard images of different CC parts were selected as previous described [458]. A semiquantitative scoring system (0-3) [455] was used to quantify myelin (Fig. 2.1B), with a score of 3 indicating intact myelin, whereas 0 was

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination defined as complete demyelination of the CC. 2 and 1 represent intermediate levels of myelin

(2/3 and 1/3 of full myelination, respectively). Staining was scored in a blind manner by 3 investigators independently. The average myelination score in the CC was calculated as the mean of three parts of the CC.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

B Score 3 Score 2 Score 1 Score 0

a b c d

Splenium

e f g h

Bodygenu and

Figure 2.1 Sagittal mouse brain atlas and scoring example images.

(A) CC in sagittal view, adapted from Fig. 2A in a previous publication [459]. (B) Example images for blind scoring of LFB-CV stained sections based on the blue staining changes in splenium (a-d), body and genu (e-h) of the CC. Fully myelinated was defined as score 3 (a, e), and complete demyelination as score 0 (d, h), with 2 (b, f), 1 (c, g) representing intermediate levels of myelin (2/3 and 1/3 of full myelination, respectively). Arrows show sites of myelin loss. Scale bar, 300μm.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Western blotting

Half brains were dissected into different regions, including the CC and cortex. Tissue samples

(10-20 mg) were homogenized in 0.4 mL radio-immunoprecipitation assay buffer (10 mM pH

7.4 Tris, 100 mM NaCl, 100 mM sodium pyrophosphate, 0.5% sodium deoxycholate, 1%

Triton-X, 10% glycerol, 1 mM EDTA, 1 mM NaF, 0.1% SDS) supplemented with Complete

Mini, EDTA-free Protease Inhibitor Cocktail (Sigma, #11836170001; 1 tablet/10 mL) and phosphatase inhibitors (5 mM β-glycerophosphate, 2 mM sodium orthovanadate, and 5 mM

NaF) using a Biospec mini bead beater at 4 °C with acid-washed glass beads (425-600 μm).

Extracts were centrifuged (1000 g, 15 min, 4 °C) and transferred to a new tube. Protein concentration was measured by bicinchoninic acid assay (BCA assay; ThermoFisher Scientific,

#23225). Protein lysates were resolved on Bolt™ 4-12% Bis-Tris Plus gels (Life Technologies), transferred to polyvinylidene fluoride (PVDF) membranes that were blocked for 1 h at RT with

5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). Membranes were washed three times with TBST and incubated with primary antibodies (Table 2.1) in 3% BSA-

TBST at 4 °C overnight. Membranes were washed three times with TBST, incubated with the appropriate secondary antibodies in blocking buffer for 1 h at RT (Table 2.1), and washed again before imaging. Signal was developed with ECL chemiluminescence reagent (EMD

Millipore), and images were captured on a Bio-Rad ChemiDoc Touch. Only when necessary, membranes were stripped several times using mild stripping buffer (15 g/L glycine, 0.1% SDS,

1% Tween 20, pH 2.2), then incubated with secondary antibody to check for stripping efficiency and re-probed with new primary antibodies. Primary antibodies with very strong binding affinity such as rabbit anti-actin were used last. Densitometry was performed using

Bio-Rad Image Lab software Version 5.2. Protein bands were normalised to housekeeping protein β-actin bands and loading control on each gel. A common loading control sample was 49

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination included on every gel to control for the variation in relative band intensities on different blots.

Novex Sharp pre-stained protein standards (ThermoFisher, #LC5800) were used as molecular weight markers.

Lipid quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Lipids were extracted from RIPA lysates of the CC and cortex (~550 and 1500 µg protein, respectively) using a two-phase methyl-tert-butyl ether (MTBE):methanol:water (10:3:2.5 v/v/v) extraction procedure [460]. Internal standards (2 nmoles d18:1/17:0 ceramide, 250 pmoles d17:1 sphingosine and 250 pmoles d17:1 S1P) were added at the start of the extraction procedure. Lipids were detected by multiple reaction monitoring on a TSQ Altis mass spectrometer with Vantage HPLC (ThermoFisher Scientific), operating in positive ion mode.

The [M + H]+ mass-to-charge (m/z) ratio was used for all precursor ions. Product ions were m/z

264.3 for all lipids except d17:1 sphingosine and d17:1 S1P, for which m/z 250.2 was used.

Lipids were resolved on a 3  150 mm XDB-C8 column (5 M particle size) (Agilent) at flow rate 0.4 mL/min [252]. Mobile phase A was 0.2% formic acid, 2 mM ammonium formate in water; and B was 1% formic acid, 2 mM ammonium formate in methanol. Total run time was

24 min, starting at 80% B and holding for 2 min, increasing to 100% B from 2 - 14 min, holding at 100% until 20.5 min, then returning to 80% B at 21 min, and holding at 80% B for a further

3 min. Peaks were integrated using TraceFinder 4.1 software (ThermoFisher), and expressed as ratios to the relevant internal standard. Lipid levels were normalised to the WT control group.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Cell culture

Cell culture conditions

The MO3.13 oligodendrocyte cell line was kindly provided by Prof Brett Garner, University of Wollongong. Stable SphK2 knockdown MO3.13 cell lines (shCtrl MO3.13, shSphK2

MO3.13) were generated using short hairpin RNA (shRNA) sequences targeting SphK2

(sequence A) as we reported before [252]. All cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine under standard culture conditions (37 °C, 5% CO2). Cell culture media and other related materials such as L-glutamine and FBS were purchased from Life Technologies.

To investigate whether S1P increases oligodendrocyte viability, both shCtrl and shSphK2

MO3.13 cell lines were seeded into 96-well plates and allowed to adhere overnight. The medium was then replaced by (i) standard DMEM/10% FBS growth medium, (ii) glucose-free

DMEM medium with 10% FBS, (iii) DMEM medium without FBS, and (iv) glucose-free

DMEM medium without FBS. To achieve these conditions, glucose-free DMEM (Sigma-

Aldrich, #D5030) without phenol red was supplemented with 20 mM sodium lactate, 2 mM sodium pyruvate, 2 mM L-glutamine and 15 mM HEPES, with or without 5 g/L glucose and

10% FBS. S1P (Avanti Polar Lipids, #860492P) was dissolved in methanol, then diluted in

OptiMEM containing with 0.01% fatty acid-free bovine serum albumin (BSA) (Sigma-Aldrich,

#A4161). Cell viability was assessed by MTS assay (details are explained in 2.3.5.2) after 24 h and 48 h incubation in glucose- or FBS-free medium with or without S1P supplementation.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

For cuprizone treatment in vitro, a 2 mM cuprizone (Sigma-Aldrich, #370-81-0) stock solution was prepared fresh in 1% ethanol, and only the shCtrl MO3.13 cell line was used (n = 3 wells).

The vehicle control was 0.5% ethanol. Viability was assayed after 24 h. To test the effect of

S1P supplementation on loss of viability induced with cuprizone, shCtrl and shSphK2 MO3.13 cell lines were seeded into 96-well plates, wells were rinsed with D-PBS on the following day, and the medium was replaced by either 200 nM S1P, 1 mM cuprizone, or both, in glucose-free,

FBS-free medium supplemented with 0.1 g/L glucose. Cells were treated for 24 h with cuprizone +/− S1P prior to assaying viability.

Cell viability assessment by MTS assay

This chromogenic assay is based on reduction of tetrazolium compound 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) by viable cells. MTS assay Kit was purchased from Promega (#G3582). MTS assays were performed according to the manufacturer’s instructions. MTS solution (20 µL) and 100 µL of

FBS-free, glucose-free medium were pre-mixed and added to each well, then incubated for 1 h at 37 °C. Absorbance was read at 490 nm using an Infinite M200 PRO plate reader (37 °C, humidified, 5% CO2). The mean optical density (OD) after blank subtraction under each treatment was calculated and converted into percentages (cell viability, %) for statistical analysis.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Seahorse assay

The Seahorse assay is widely used for measuring mitochondrial function in cells. Plates, medium and related components for the seahorse assay were purchased from Agilent

Technologies, Australia. The procedure followed the manufacturer’s instructions. On the first day, an XFe96 Sensor Cartridge was hydrated with 200 µL/well pure H2O at 37 ºC w/o CO2 overnight. shCtrl MO3.13 and shSphK2 MO3.13 cells were seeded at 2×104 cells/well in

Seahorse XF96 cell culture microplates coated with collagen, in DMEM supplemented with

10% FBS. After overnight culture under standard conditions, cells were rinsed twice, and the medium was replaced with 180 µL Seahorse assay medium (Agilent, #103575100) containing

10 mM glucose, 1 mM pyruvate and 2 mM glutamine. Cells were then equilibrated at 37 ºC w/o CO2 for 2 h, and the hydrated cartridge for 45 min, before the assay. Mitostress test kit

(Agilent, #103015) was loaded into the Sensor Cartridge. Oxygen Consumption Rate (OCR) was assayed in live cells using an XFe96 Analyzer (Agilent), with sequential injections of 1

µM Oligomycin to inhibit ATP synthase (complex V), 0.5 µM FCCP, an uncoupling agent that drives maximal OCR by complex IV, and 0.5 µM Rotenone + Antimycin A to inhibit complex

I and III, shutting down mitochondrial respiration (Fig. 2.2A). OCR values were normalised to protein concentration, and the following facets of mitochondrial respiration were calculated as shown in Fig. 2.2B: Basal respiration (basal OCR-OCR values after injecting A/R) and ATP production (basal respiration-OCR values after injecting oligomycin).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Figure 2.2 Outline of drug mechanisms and evaluated mitochondrial parameters in seahorse assay.

(A) The modulators of electron transport chain (ETC) complexes used in the Seahorse mitochondrial respiration assay. (B) Key parameters of mitochondrial function. Adapted from Agilent Seahorse XF

Cell Mito Stress Test Kit, User Guide.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Statistical analysis

All statistical analysis was conducted using GraphPad Prism 8.0, with values reported as mean

± SEM. Statistical significance was accepted at p < 0.05. Data was not assessed by the normality test because of the small group sizes (n < 8) used in this study.

In most analyses, ordinary two-way ANOVA was used when two independent variables were involved (Fig. 2.3C-E, Fig. 2.4B,C, Fig. 2.6A-C, Fig. 2.7B,C and E). Sidak’s post-test was used to compare means between specific treatment groups and between two genotypes. For data from in vivo experiments (including western blotting data, ASPA staining and myelin content measurement), p values for the main effect of diet (i.e. normal chow vs cuprizone) and genotype (i.e. WT vs SphK2−/−) were reported.

Analysis of myelination score for LFB/CV staining (Fig. 2.5F) was performed using nonparametric Mann–Whitney rank test, making two comparisons of myelin content: control with cuprizone groups and cuprizone and remyelination groups.

Ordinary one-way ANOVA was used to measure the effect of cuprizone concentration on shCtrl cells in vitro (Fig. 2.7D), and to analyse western blotting results in the 4-week remyelination experiment using SphK2−/− mice (Fig. 2.5C,D). Dunnett’s post-test was applied to perform comparisons between control and other groups.

Unpaired, two-tailed t-tests were used to assess basal respiration and ATP production in the seahorse assay (Fig. 2.7G).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Results

SphK2 protects against loss of mature oligodendrocytes induced with cuprizone

Cuprizone is selectively toxic for oligodendrocytes, leading to demyelination caused by oligodendrocyte loss [461]. To determine if SphK2 is required for the protection of oligodendrocytes and myelin, SphK2−/− mice and their WT littermates were administered normal chow as controls, or chow containing 0.2% cuprizone for 6 weeks to induce demyelination and culled immediately. A third group of WT and SphK2−/− mice were administered cuprizone for 6 weeks, then switched back to a cuprizone-free diet for 2 weeks prior to culling, to asses spontaneous remyelination (Fig. 2.3A).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

A −/− Remyelination (WT & SphK2 mice) 2 weeks Demyelination, 6 weeks normal chow

Normal chow + 0.2% cuprizone Brain tissue

or Normal chow control, 6 weeks collection (n = 6)

B ASPA DAPI Ctrl CPZ Re-M

Cortex

CC WT

HIP

/−

− SphK2

C D

ASPA+ Cells of CC ASPA+ Cells of cortex 4000 WT 1000 WT -/-

SphK2-/- SphK2 2 2 800

3000

✱ 600 ✱ ✱✱✱

r e

2000 e p

s 400

e C 1000 C 200

0 0 Ctrl CPZ Re-M Ctrl CPZ Re-M

E ASPA+ Cells of hippocampus 500 WT SphK2-/-

2 400

m m

300

s 200

l e C 100

0 Ctrl CPZ Re-M

Figure 2.3 Loss of SphK2 sensitizes to oligodendrocyte loss induced by cuprizone. 57

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

(A) Schematic overview of the experimental design. (B) Representative immunofluorescence images for ASPA (green) and DAPI (blue) in WT and SphK2−/− mice fed cuprizone (CPZ) or control chow

(Ctrl) for 6 weeks, or fed CPZ for 6 weeks then normal chow for 2 weeks to assess remyelination (Re-

M). Three different regions are marked and outlined: CC, cortex and hippocampus. Scale bar, 500 μm.

Quantified ASPA-positive cell density (cells per mm2) in the CC (C), cortex (D) and hippocampus (E)

(n = 5 - 6 mice/group). Boxes show the 25th-75th percentile, and bars show the range. Statistical significance was determined by two-way ANOVA with Sidak’s post-test. p values for statistically- significant comparisons between WT and SphK2−/− mice are shown (*p < 0.05, ***p < 0.001).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

We first determined if cuprizone differentially affected oligodendrocyte number in WT versus

SphK2−/− mice, by counting cells positive for the mature oligodendrocyte marker ASPA [322,

462] (Fig. 2.3B-E). Cuprizone administration caused a pronounced depletion of ASPA- positive cells in both the CC and cerebral cortex, which was more notable in SphK2−/− compared to WT mice. Following 6 weeks of cuprizone treatment, ASPA-positive cell density was reduced 61% in the CC of WT mice, and 84% in SphK2−/− mice (Fig. 2.3C) (2-way

ANOVA, effect of genotype F = 8.2, p = 0.008; effect of treatment F = 86.7, p < 0.001; interaction F = 1.7, p = 0.19). Two weeks after cuprizone withdrawal (i.e. remyelination condition), mature oligodendrocyte density in the CC had returned to 80% of the control condition in WT mice, and 77% of the control in SphK2−/− mice. The difference in oligodendrocyte density between the cuprizone and remyelination conditions was statistically significant (p < 0.001 by Sidak’s post-test) for both genotypes. Similarly, oligodendrocyte density in the cortex was reduced by 50% in WT and 72% in SphK2−/− mice immediately after cuprizone administration (Fig. 2.3D) (2-way ANOVA, effect of genotype F = 26.7, p < 0.001; effect of treatment F = 56.1, p < 0.001; interaction F = 1.5, p = 0.25). In contrast to the CC, oligodendrocyte density in the cortex did not recover significantly at 2 weeks after cuprizone withdrawal. We therefore quantified oligodendrocyte density in the hippocampus to further test whether mature oligodendrocyte cell numbers recover in SphK2−/− mice following cuprizone withdrawal (Fig. 2.3E). Cuprizone administration produced a pronounced 92% and 96% reduction in oligodendrocyte density in the hippocampus of WT and SphK2−/− mice, respectively (2-way ANOVA, effect of genotype F = 3.0, p = 0.09; effect of treatment F = 386, p < 0.001; interaction F = 0.04, p = 0.96). At the second week after cuprizone withdrawal, oligodendrocyte density had increased 3-fold in WT mice (remyelination vs cuprizone

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination condition, p = 0.035), and 4-fold in SphK2−/− mice (remyelination vs cuprizone condition, p =

0.073).

Oligodendrocyte cell density did not differ between untreated WT and SphK2−/− mice at the experimental endpoint (5 months of age) in the CC, cortex, or hippocampus. These results indicate that loss of oligodendrocytes is more severe in SphK2−/− mice during cuprizone treatment, but the capacity for oligodendrocyte number to recover following cuprizone withdrawal is not impaired in the CC or hippocampus of SphK2−/− mice.

SphK2 is necessary for remyelination following cuprizone withdrawal

To quantify myelin content, we performed western blotting for the myelin protein markers

MBP, PLP, MOG and CNP in homogenates of dissected CC and cortex tissue (Fig. 2.4A-C).

Cuprizone administration produced a marked loss of all four myelin markers in the CC and cortex (p < 0.0001 by Sidak’s post-test comparing untreated to cuprizone-treated), which was more pronounced in SphK2−/− compared to WT mice. A statistically-significant effect of both cuprizone treatment and genotype was observed for all four myelin markers in both CC and cortex, and post-tests indicated that CNP levels were significantly lower in the CC of cuprizone-treated SphK2−/− compared to cuprizone-treated WT mice (p = 0.0009). Myelin protein levels did not differ between untreated WT and SphK2−/− mice in the CC, although PLP and MOG levels were significantly lower in the cortex of untreated SphK2−/− mice.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

A CC Cortex SphK2-/- WT SphK2-/- WT Ctrl CPZ Re-M Ctrl CPZ Re-M Ctrl CPZ Re-M Ctrl CPZ Re-M 30 kDa 30 kDa MBP 15 kDa MBP 15 kDa β-actin 40 kDa CNP 50 kDa 50 kDa CNP β-actin 40 kDa 30 kDa β-actin 40 kDa PLP 30 kDa PLP MOG 30 kDa 30 kDa MOG β-actin 40 kDa β-actin 40 kDa

MBP, CC CNP, CC 4 5 ✱✱✱✱ WT WT

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Genotype Treatment Interaction

F stat p value F stat p value F stat p value

MBP 58.0 < 0.001 214.2 < 0.001 61.3 < 0.001

CNP 104.2 < 0.001 462.2 < 0.001 38.7 < 0.001

MOG 5.9 0.022 42.9 < 0.001 0.5 0.610

PLP 7.2 0.012 47.3 < 0.001 3.3 0.052

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

MBP, Cortex CNP, Cortex

1.5 WT 1.5 WT

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F stat p value F stat p value F stat p value

MBP 13.4 0.001 44.1 < 0.001 0.9 0.420

CNP 14.0 < 0.001 99.1 < 0.001 3.6 0.040

MOG 19.2 < 0.001 84.7 < 0.001 2.2 0.140

PLP 18.0 < 0.001 73.6 < 0.001 0.6 0.583

LFB/CV Ctrl CPZ Re-M

WT HIP

/ -

SphK2

Figure 2.4 Effect of SphK2 deletion on myelin and oligodendrocyte markers with CPZ treatment.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Western blots (A) and densitometric quantification of myelin and oligodendrocyte markers in the CC

(B) and cortex (C) of WT and SphK2−/− mice fed cuprizone (CPZ) or control (Ctrl) chow for 6 weeks, or fed CPZ for 6 weeks then Ctrl chow for 2 weeks to assess remyelination (Re-M). Protein levels were normalised to β-actin in each sample, n = 5 - 6 mice/group. Statistical significance was determined by two-way ANOVA with Sidak’s post-test. Tables show overall two-way ANOVA results for levels of

MBP, CNP, MOG and PLP. Results for post-tests comparing WT and SphK2−/− mice in the three different treatment conditions (Ctrl, CPZ, and Re-M) are shown on the graphs (** p < 0.01, *** p <

0.001, ****p < 0.0001). (D) Representative LFB/CV staining in CC of both genotypes. Two different regions are marked and outlined: CC and hippocampus (HIP). Arrows show sites of myelin loss. Scale bar, 500μm.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Following cuprizone withdrawal, MBP, CNP and PLP levels increased significantly in WT mice (p < 0.0001, 0.0001 and p = 0.02 by Sidak’s post-test), whereas none of these markers increased in SphK2−/− mice after cuprizone withdrawal. There was no increase in myelin marker levels in the cortex of WT or SphK2−/− mice two weeks after cuprizone withdrawal, indicating that remyelination proceeds more rapidly in the CC than the cortex, in accordance with mature oligodendrocyte cell densities.

LFB/CV staining supported these results, showing demyelination in cuprizone-fed mice that was more pronounced in SphK2−/− mice (Fig. 2.4D). After cuprizone removal, LFB staining recovered in WT but not in SphK2−/− mice.

SphK2 is essential for long-term remyelination in cuprizone model

We next sought to determine if remyelination in the CC of SphK2−/− mice is delayed due to the greater loss of mature oligodendrocytes with cuprizone feeding. SphK2−/− mice were fed chow containing cuprizone for 6 weeks, then allowed 4 weeks for recovery on normal chow before assessing myelin content (Fig. 2.5A).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

A (SphK2−/− mice) Remyelination

Demyelination, 6 weeks 4 weeks

Normal chow + 0.2% cuprizone normal chow

or Normal chow control, 6 weeks Brain tissue

collection (n = 5) B CC Cortex Ctrl CPZ Re-M Ctrl CPZ Re-M 30 kDa 30 kDa MBP MBP 15 kDa 15 kDa 50 kDa 50 kDa CNP CNP

β-actin 40 kDa β-actin 40 kDa 30 kDa 30 kDa PLP PLP 30 kDa 30 kDa MOG MOG β-actin 40 kDa β-actin 40 kDa

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Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

MOG, CC PLP, CC 1.5 ✱✱✱ 2.5 ✱✱

2.0

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Hippocampus

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

LFB/CV staining

✱✱✱

e r

o 3

o i

t 2

l e

y 1 M

0 Ctrl CPZ Re-M

Figure 2.5 Myelin content does not recover in SphK2−/− mice following cuprizone withdrawal.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

(A) Schematic overview of the long-term remyelination experiment design. Only SphK2−/− mice were used for this experiment. Western blots (B) and densitometric quantification of myelin and oligodendrocyte markers in the CC (C) and cortex (D) of control (Ctrl), 6-week cuprizone (CPZ), and

6-week cuprizone + 4-week remyelination (Re-M) groups. Protein levels were normalised to β-actin in each sample. Graphs show mean ± SEM (n = 5). Statistical significance was determined by one-way

ANOVA with Dunnett’s post-test. Representative images (E) and myelination score (F) for LFB/CV staining of the CC. Scale bar, 500 μm. Graphs show mean ± SEM from 5 mice per group. Each data point is the mean value from 3 sections per mouse. The degree of myelination between the CPZ group and other groups was compared by nonparametric Mann–Whitney U test. p values for comparisons were two-tailed p value and reported significant in the graphs (**p < 0.01, ***p < 0.001, ****p <

0.0001).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Western blotting showed a significant decrease of MBP, CNP, MOG and PLP in both CC and cortex with cuprizone administration for 6 weeks (Fig. 2.5B,C). However, there was no notable effect of cuprizone withdrawal on these myelin markers in the CC and cortex of SphK2−/− mice, compared with cuprizone treatment. To further verify the lack of remyelination in SphK2−/− mice at 4 weeks after cuprizone withdrawal, myelin content was scored from LFB/CV images

(Fig. 2.5E,F). The significantly reduced myelination score following cuprizone feeding did not change after the 4-week normal chow diet, confirming the western blotting results. These results imply that SphK2−/− is essential for remyelination, rather than simply delaying remyelination following cuprizone withdrawal.

Loss of SphK2 increases levels of pro-apoptotic sphingosine and ceramide during and after cuprizone feeding.

As a first step in determining how loss of SphK2 sensitizes to loss of mature oligodendrocytes and inhibits remyelination following cuprizone feeding, levels of S1P and the upstream metabolites sphingosine and ceramide (Fig. 1.3) were quantified in CC and cortex tissue using

LC-MS/MS. In agreement with previous results from our research group [444], S1P levels were substantially lower, and sphingosine substantially higher in cortex of untreated SphK2−/− compared to WT mice [444] (Fig. 2.6A,B). This trend was more pronounced in the CC.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

S1P in CC S1P in cortex

) WT l ) WT

l 1000

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) ) l

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Figure 2.6 Altered myelin content levels in cuprizone-induced demyelination and remyelination.

Total levels of S1P (A), sphingosine (B) and ceramide (C) in the CC and cortex of WT and SphK2−/− mice fed cuprizone (CPZ) or control (Ctrl) chow for 6 weeks, or fed CPZ for 6 weeks then Ctrl chow

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination for 2 weeks to assess remyelination (Re-M). Lipid levels were normalised to the WT control group (Ctrl) in each sample, n = 5 - 6 mice/group. Statistical significance was determined by two-way ANOVA with

Sidak’s post-test (***p < 0.001, ****p < 0.0001).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Mean S1P levels declined by 33% and 59%, respectively, in the CC and cortex of WT mice with cuprizone treatment, but increased 10-fold in the CC (2-way ANOVA, effect of genotype

F = 0.43, p = 0.52; effect of treatment F = 113.4, p < 0.001; interaction F = 2.0, p = 0.15) and

8.3-fold in cortex (2-way ANOVA, effect of genotype F = 2.4, p = 0.13; effect of treatment F

= 64.6, p < 0.001; interaction F = 2.7, p = 0.08) after cuprizone withdrawal. Remarkably, in

SphK2−/− mice total S1P levels increased 14-fold relative to the untreated condition in the CC, and 2.3-fold in the cortex, following the 6-week cuprizone treatment, and increased a further

5.7-fold in CC and 3.2-fold in cortex after cuprizone withdrawal.

Mean sphingosine levels increased 3-fold (p = 0.002) and 0.4-fold (p > 0.05) in the CC of

SphK2−/− and WT mice with cuprizone treatment, respectively (2-way ANOVA, effect of genotype F = 13.4, p < 0.001; effect of treatment F = 5.055, p = 0.012; interaction F = 3.854, p = 0.031) (Fig. 2.6B). In the 2 weeks following cuprizone withdrawal, mean sphingosine levels in the CC declined 21% in WT and 62% in SphK2−/− mice, resulting in sphingosine levels that were 2.7-fold higher in SphK2−/− compared to WT mice in the remyelination phase

(p = 0.4516). In the cortex, sphingosine levels were not significantly altered by cuprizone feeding in WT mice but increased 96% in SphK2−/− mice (p = 0.004).

No alteration of mean ceramide levels was observed in either CC or cortex of WT or SphK2−/− mice with cuprizone-induced demyelination (Fig. 2.6C). However, ceramide levels were 2- fold higher (p = 0.002) and 3-fold higher (p < 0.0001) in the CC of WT and SphK2−/− mice, respectively, two weeks after cuprizone withdrawal compared with cuprizone treatment (2-way

ANOVA, effect of genotype F = 20.92, p < 0.0001; effect of treatment F = 58.69, p < 0.0001; interaction F = 7.823, p = 0.002). Ceramide levels were 1.9-fold higher in the CC of SphK2−/− compared to WT mice (p < 0.0001 by Sidak’s post-test), 2 weeks after cuprizone withdrawal.

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

2.4.5 Preliminary investigation into the role of SphK2 in oligodendrocyte survival in vitro

The above experiments demonstrate that endogenous SphK2 protects against cuprizone- mediated oligodendrocyte loss; and is necessary for remyelination, but not generation of new mature oligodendrocytes, following cuprizone withdrawal. As a starting point for investigating the biochemical basis for these phenotypes, we used the oligodendrocyte model cell line

MO3.13 [463]. SphK2 was stably down-regulated using shRNA, with effective knock-down demonstrated by western blotting (Fig. 2.7A).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

80kDa SphK2 60kDa β-Actin 40kDa

B S1P treatment in different medium for 24 h

120 shCtrl shSphK2

100

) %

( 80

i l i ✱ b 60

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e 40 C

0 Glucose + + + - - - + + + - - - FBS + + + + + + ------100 nM S1P - + - - + - - + - - + - 200 nM S1P - - + - - + - - + - - +

S1P treatment in different medium for 48 h 120 shCtrl

100 shSphK2

) %

( 80

l i

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0 Glucose + + + - - - + + + - - - FBS + + + + + + ------100 nM S1P - + - - + - - + - - + - 200 nM S1P - - + - - + - - + - - +

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

CPZ treatment CPZ treatment in 0.1 g/L glucose medium in 1 g/L glucose medium 150 200

✱✱

)

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% 150

(

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) CPZ 1 mM

% pAkt (S473) ( S1P + CPZ

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Oligomycin FCCP Antimycin A/Roteonone )

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O t 0 K C K s C h h h h p 0 50 100 150 a p s S s S B h h Time (min) s s

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Figure 2.7 The role of SphK2 in vitro.

(A) Western blots for SphK2 protein. (B and C) The effect of (B) 24 h or (C) 48 h incubation in different media conditions with 0, 100, or 200 nM S1P on viability of shCtrl and shSphK2 cells (4 replicates per condition). Viability was determined by MTS assay, and normalised to the control growth medium

(with FBS and glucose). Statistical significance was determined by two-way ANOVA with Sidak’s post-test. (D) Effect of cuprizone (CPZ) on viability of shCtrl cells in 0.1 g/L and 1 g/L glucose medium

(n = 4). Statistical significance was assessed by one-way ANOVA with Dunnett’s post-test, comparing each treatment with vehicle control. (E) Effect of 1 mM CPZ and 200 nM S1P viability of shCtrl or shSphK2 cells in medium containing 0.1 g/L glucose (n = 5). (F) Phospho-Akt (pAkt) protein levels in untransfected, shCtrl and shSphK2 MO3.13 cell pools. Total Akt (tAkt) and β-actin were shown as loading control proteins. (G) Analysis of mitochondrial oxygen consumption rate (OCR) in shSphK2

MO3.13 cells by seahorse assay. Arrows show times for addition of oligomycin, FCCP, and antimycin

A + rotenone. Graphs show basal respiration and ATP production, as defined in section 2.3.5.3

Statistical significance was analysed by unpaired t-test (G). Statistical significance was determined by two-way ANOVA with Sidak’s post-test (B,C,E). Statistical significance was determined by one-way

ANOVA with Dunnett’s post-test (D). Graphs present mean ± SEM. p values for statistically-significant comparisons are shown (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination

Sensitisation of shSphK2 MO3.13 cells to glucose deprivation and cuprizone was then tested by MTS viability assay. We first determined if shSphK2 cells were sensitive to serum or glucose deprivation. Testing the effect of S1P in cell culture necessitates the use of serum-free medium, since S1P is abundant in serum and responsible for much of the mitogenic and pro- survival effect of serum [256, 464]. MO3.13 cells were unaffected by serum deprivation, but highly sensitive to glucose deprivation (Fig. 2.7B-C). The effect of 24 h glucose deprivation was exacerbated by serum deprivation, and further exacerbated by SphK2 knock-down (Fig.

2.7B). The sensitizing effect of SphK2 knock-down was reversed by addition of exogenous

S1P (100 - 200 nM). The effect of 48 h glucose deprivation on MO3.13 viability was pronounced even in the presence of FBS, making it difficult to assess the added effect of SphK2 deficiency at this time point (Fig. 2.7C).

The sensitivity of MO3.13 cells to cuprizone was then assayed in serum-free medium with a very low glucose level (0.1 or 1 g/L) (Fig. 2.7D). Cell viability was effectively reduced using

1 mM cuprizone in 0.1 g/L glucose medium, and this effect was reversed with addition of 200 nM S1P. shSphK2 cells were more sensitive to cuprizone than shCtrl cells (62% vs 73% of the untreated control cell viability, p = 0.0027, for shSphK2 cells vs shCtrl cells treated with cuprizone). Exogenous S1P reversed the effect of SphK2 deficiency and cuprizone on cell viability (Fig. 2.7E).

The PI3K-Akt pathway is a critically-important hub for pro-survival signalling and energy production in response to a range of growth factors. Our research team recently demonstrated an essential requirement for SphK2 in PI3K-Akt pathway activation in hepatocytes responding to insulin [465]. Analysis of Akt phosphorylation on its activation site, S473, demonstrated reduced Akt phosphorylation in the shSphK2 cell line (Fig. 2.7F). Myelination is an energy-

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination intensive process due to the high lipid content of myelin [466]. This high energy demand necessitates abundant ATP production through respiratory metabolism. The inability of

SphK2−/− mice to remyelinate, sensitivity of shSphK2 cells to glucose deprivation, and reduced basal Akt phosphorylation in shSphK2 cells, raise the possibility that mitochondrial energy production is impaired in these cells. We therefore measured the oxygen consumption rate

(OCR) of Ctrl and shSphK2 cells using a seahorse analyser (Fig. 2.7G). Basal respiration (prior to oligomycin addition), ATP production, and maximal respiration rate (refer to Fig. 2.2) were all reduced in shSphK2 compared to shCtrl cells, suggesting that SphK2 is an essential regulator of mitochondrial content and/or quality.

Discussion

The clinical success of S1PR agonists in the treatment of MS has generated great interest in the neurological functions of this signalling lipid. This study investigated the role of SphK2 in protection against cuprizone-induced demyelination, and spontaneous remyelination following cuprizone withdrawal. After 6 weeks of cuprizone feeding, western blotting, immunofluorescence and LFB staining showed greater oligodendrocyte loss and demyelination in the CC and cortex of SphK2−/− compared to WT mice. Remyelination following cuprizone withdrawal was observed in the CC of WT but not SphK2−/− mice. The absence of remyelination in SphK2−/− mice was observed even after a 4-week remyelination period, and despite a strong restoration of mature oligodendrocyte numbers in these mice following cuprizone withdrawal. These results strongly suggest that SphK2 is not necessary for proliferation and maturation of OPCs in vivo but is necessary for the synthesis of new myelin by mature oligodendrocytes. It therefore seems likely that two distinct roles for SphK2 have

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination been identified in this study: the first is as a mediator of mature oligodendrocyte survival, and the other is as an essential mediator of myelin synthesis by newly-formed oligodendrocytes.

Cuprizone-mediated loss of mature oligodendrocytes, identified with the marker ASPA, was more pronounced in SphK2−/− mice than WT littermates. This loss of mature oligodendrocytes is assumed to result from increased apoptosis, however this was not directly measured.

Cuprizone-induced oligodendrocyte death can be characterized by activation of caspase-3 and

TUNEL-staining. High levels of the cleaved, active form of caspase-3 were found in oligodendrocytes after a few days of cuprizone administration [226, 227]. Future studies will use these markers in combination with ASPA to clarify if increased apoptosis is the basis for reduced mature oligodendrocytes in SphK2−/− mice fed cuprizone. The significant loss of other markers such as CNP and MOG observed in our study make it highly likely that reduced ASPA- positive cell number reflects loss of mature oligodendrocytes. Using a second marker of mature oligodendrocytes, such as the CC1 antibody would confirm this. In addition, combining an oligodendrocyte marker like Olig2 with stress response markers such as transcription factor

ATF-3 or DNA damage response protein DDIT3 [467], would be a good approach to assess how oligodendrocytes respond to stress in WT versus SphK2−/− mice.

Original studies have suggested that SphK2 is a pro-apoptotic protein [468, 469]. However, these early findings may have resulted from gross overexpression of the protein [470], and more recent studies indicate that SphK2, like SphK1, generally promotes cell proliferation and survival [471-473]. The effect may be cell-type specific, as loss of SphK2 protects renal mesangial cells against stress [474, 475] but sensitizes other cells to apoptosis [465, 476, 477].

An important role for SphK2 in oligodendrocyte survival is supported by our recent work

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination demonstrating that loss of SphK2 enhances age-dependent loss of oligodendrocytes in an AD mouse model [406].

In vitro studies with the MO3.13 cell line further support the requirement for SphK2 in oligodendrocyte stress resistance and survival, as cells stably expressing shRNA to SphK2 were sensitized to both glucose deprivation and cuprizone. This increased sensitivity could be reversed with addition of exogenous S1P. Exogenous S1P at 100 nM can effectively activate

S1PRs [372] but cannot cross the plasma membrane, suggesting that loss of SphK2 sensitizes to cell death through a deficiency in secreted S1P that then signals in an autocrine manner through S1PRs. Prior studies have demonstrated the pro-survival functions of S1P [303] and

S1PR agonist Fingolimod [355] in oligodendrocyte lineage cells, acting through G‐protein coupled receptor signalling pathways. The next chapter investigates the protective properties of S1PR5-selective agonist A-971432 and S1PR1/5 agonist Siponimod in the cuprizone model.

Future studies must investigate whether these S1PR agonists can replicate S1P in rescuing oligodendrocyte cell viability in SphK2-deficient cells, and whether these agonists rescue the oligodendrocyte survival and remyelination deficits in SphK2−/− mice administered cuprizone.

Oligodendrocytes are particularly susceptible to oxidative stress, which is implicated as a mediator of demyelination leading to cell death in MS [126, 478]. The sensitivity of oligodendrocytes to cuprizone has been attributed to their particular requirement for mitochondrial energy production [479, 480]. Cuprizone is believed to act on Complex IV of the electron transport chain, as well as impairing copper-dependent antioxidant enzymes, leading to the deficiency of ATP production and oxidative stress [222, 223, 225, 481]. SphK2 deficiency greatly reduced respiratory ATP production in MO3.13 cells, which likely increases their sensitivity to glucose deprivation and cuprizone. Oligodendrocytes also need a large

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination amount of ATP to synthesize lipids for myelin membranes [482, 483]. Hence, the inability to produce ATP may impair myelin biosynthesis in mature SphK2−/− oligodendrocytes, providing a potential explanation for the failure of remyelination in SphK2−/− mice. Further experiments are needed to determine whether the deficiency in oxygen consumption and ATP synthesis is attributed to a reduction in overall mitochondrial content, accumulation of dysfunctional mitochondria, or a more specific requirement for SphK2 in respiratory metabolism. In this regard, S1P generated by SphK2 is reported to directly interact with the mitochondrial protein prohibitin [348]. Decreased activity of prohibitin is associated with mitochondrial dysfunction

[484]. Endogenous S1P, synthesized by SphK2, may therefore be associated with mitochondrial function in oligodendrocytes.

The PI3K-Akt pathway also feeds directly into anabolic metabolism and ATP generation by myelinating oligodendrocytes, and is an important survival hub in many cell types [485, 486].

In the EAE model, activation of the PI3K-Akt signalling pathway promotes oligodendrocyte development and stimulates myelination [487]. Reduced Akt phosphorylation in MO3.13 cells expressing SphK2 shRNA observed in my study is in agreement with our laboratory’s recent publication showing a requirement for SphK2 in PI3K-Akt pathway activation in hepatocytes

[465], and provides an important insight into the mechanistic basis through which SphK2 deficiency sensitizes to oligodendrocyte cell death. Akt-mTOR pathway activity promotes anabolic metabolism through up-regulation of enzymes such as fatty acid synthase (FAS) and p70S6K [488, 489], which regulate lipid and protein synthesis, respectively.

These in vitro studies of effects with SphK2 knockdown need to be confirmed using a second shSphK2 knock-down cell line. Although the MO3.13 cell line is useful for generating insights, results must be confirmed in primary, non-transformed oligodendrocytes. MO3.13 cells do not

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination share all the features of myelinating oligodendrocytes, including forming myelin sheaths in vitro [490]. Experiments currently underway in our laboratory are testing the requirement for

SphK2 in survival of primary oligodendrocytes derived from WT and SphK2−/− mice.

Alternatively, other in vitro models such as mature oligodendrocytes derived from induced pluripotent stem cells (iPSCs) that we are currently establishing in our lab to extend our studies of demyelination and remyelination with cuprizone treatment [491, 492]. Due to the limited time, I was unable to perform the experiments with primary oligodendrocytes but will conduct these studies in the future.

Whilst our in vitro studies indicated that S1P restores viability in SphK2−/− cells deprived of glucose or treated with cuprizone, an important caveat is our result demonstrating that S1P levels in the CC of cuprizone-treated SphK2−/− mice are equivalent to those in WT mice, despite being almost an order of magnitude lower in untreated SphK2−/− mice. Based on this in vivo result, the enhanced loss of mature oligodendrocytes in SphK2−/− mice treated with cuprizone may not be attributed solely to a bulk S1P deficiency. One important possibility is that cuprizone feeding results in down-regulation of enzymes that degrade S1P, specifically S1P- specific phosphatases Sgpp1 and 2, and S1P lyase [493]. An alternative explanation is a compensatory up-regulation of SphK1 in SphK2−/− mice treated with cuprizone. SphK1 is upregulated in the blood of mice lacking SphK2, leading to higher blood S1P levels [494]. Thus, examining the mRNA, protein, and enzyme activity levels of both SphK1 and SphK2 in CC of untreated and cuprizone-treated SphK2−/− mice will be important for future studies.

Quantification of SphK1 protein levels in this study was hampered by the lack of a suitable antibody, as all SphK1 antibodies tested failed to show loss of the putative SphK1 band in

SphK1 knockout mouse brain tissue. We very recently identified an antibody that is suitable for SphK2 detection in mouse brain by immunohistochemistry, and will use this antibody to 82

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination quantify SphK2 expression in WT mice before, during, and after cuprizone treatment. Similar to the upregulated SphK2 level in cerebral ischaemia [495], our initial tests show intense

SphK2 immunoreactivity with cuprizone treatment, suggesting that SphK2 is up-regulated as a protective response.

Previous studies have shown the increased sphingosine in cultured oligodendrocytes with the induction of stress from cytokines [496], suggesting that oligodendrocyte stress results in increased sphingosine levels. Whilst SphK2−/− mice did not show loss of S1P in the CC with cuprizone treatment, there was a highly significant accumulation of the SphK2 substrate sphingosine. Moreover, this increase was more pronounced in SphK2−/− mice with the control diet, due to the absence of sphingosine conversion to S1P by SphK2. Unlike S1P, sphingosine is a pro-apoptotic signalling molecule [497, 498] that interacts with the abundant scaffolding protein 14-3-3 [499, 500] and activates specific protein kinases (sphingosine dependent kinases)

[500, 501]. In a Krabbe disease model, the increased level of sphingosine is suggested to be associated with oligodendrocyte cell death and demyelination [502]. Our lab’s recent work directly attributed defective insulin signalling in SphK2−/− hepatocytes, including reduced

PI3K-Akt pathway activation, to increased sphingosine levels rather than a deficiency in S1PR signalling [465]. The enhanced sensitivity of SphK2−/− oligodendrocytes to cell death might therefore be attributed to sphingosine accumulation.

Sphingosine can be converted back to pro-apoptotic ceramides by ceramide synthases. A study in vivo and using cultured OPCs showed that lymphotoxin (LT) induces stimulation of ceramide synthase enzyme activity in oligodendrocytes, and in turn, accumulated ceramides cause cell death [503]. Ceramides C16:0 and C24:0 are increased in CSF of people with RRMS, which leads to mitochondrial dysfunction in neurons [504]. Increased ceramide levels also

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination promote oxidative stress through direct effects on the mitochondrial electron transport chain

[505]. In contrast to a recent study [506], our results did not show an elevation of ceramide levels in cuprizone-treated mice. However, ceramide levels increased substantially after cuprizone withdrawal, and were acutely increased in SphK2−/− mice at this time point. This pattern of increasing ceramides during the remyelination is in agreement with the findings of

Yoo et al [506], who proposed that the increased ceramide levels cause myelin decompaction through their biophysical effect on membrane curvature. Ceramide is the precursor for synthesis of the myelin lipids sphingomyelin, GalCer, and sulfatide. Thus, increased ceramides in the CC after cuprizone withdrawal may reflect new myelin lipid synthesis by oligodendrocytes. Excessive ceramide builds up in SphK2−/− oligodendrocytes due to impaired degradation through SphK2 and S1P lyase might therefore interfere with myelin synthesis and elaboration. This will be resolved in future studies by quantification of the metabolically- related myelin lipids GalCer, sulfatide, and sphingomyelin, as well as cholesterol. Cholesterol and sphingolipids are the major constituents of myelin and essential for its stability and function [32, 507, 508].

Complete failure of remyelination in SphK2 mice following cuprizone withdrawal may be attributed to a lack of OPC differentiation and maturation, or an inability of mature oligodendrocytes to synthesize myelin and/or ensheath axons [199]. Our results strongly suggest that OPC proliferation is unimpeded in SphK2−/− mice since ASPA-positive cell number recovered rapidly in these mice after cuprizone withdrawal, yet remyelination is blocked. This will be clarified by staining for OPC markers PDGFRα or NG2, in combination with the pan-oligodendrocyte marker Olig2. NG2+ OPCs accumulate in the demyelinating lesions of mice fed cuprizone [509, 510]. ASPA is a reliable and useful marker for mature oligodendrocytes [62], but is present in the oligodendrocyte cell body rather than the myelin 84

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination sheaths [511]. Additional markers of mature, myelinating oligodendrocytes may help to clarify the role of SphK2 in maturation versus myelination. For instance, MOG is an important marker for oligodendrocyte maturation and myelin sheaths since it is expressed on the surface of oligodendrocytes at the late postnatal stage [512], whereas GST-π stains only mature myelinating oligodendrocytes [513]. Whilst the transcription factor olig2 is a marker for oligodendrocytes at all stages of differentiation, olig1 is particularly important for oligodendrocyte differentiation and remyelination [67, 109, 514]. Alternatively, gene expression analysis on flow-sorted oligodendrocytes could be used to more correctly classify the phenotype of the newly-made oligodendrocytes in SphK2−/− mice, and contrast this phenotype with the equivalent cells in WT mice. It is important to note that without physiological insult, myelination and oligodendrocyte number are normal in SphK2−/− mice at

3 months of age, indicating that developmental myelination proceeds normally in these mice.

Previous results from our lab using electron microscopy demonstrated a significant accumulation of structural deficits and thinner myelin sheaths in male SphK2−/− mice at 12-15 months, but not 2 months of age [454].

Despite the use of S1P receptor agonists in MS therapy, there have been very few studies to date examining changes to sphingosine, S1P, and ceramide in MS. In particular, elevated sphingosine was found in MS samples by Miller et al [496]. A study by Qin and colleagues showed decreased S1P [515], however their findings did not concur with a separate study, which further observed that ceramides were upregulated [516]. Enhanced ceramide levels without any change to S1P in MS lesions has also been shown by Kim et al [517], in contrast, a different study reported reduced levels [518]. The study conducted by Qin et al [515] did not provide their sample size or suitable information on experimental variance, raising serious concerns about the validity of their study. Future studies should investigate whether there is 85

Chapter 2 SphK2 protects oligodendrocytes and is required for remyelination depletion of S1P in demyelinating lesions, and examine whether there is any change in remyelinating lesions, in human brain samples.

Overall, these results identified an essential role for SphK2 in protection of mature oligodendrocytes against cuprizone-mediated cell death and demyelination; and for spontaneous remyelination following cuprizone withdrawal. Future work will clarify whether

SphK2 in oligodendrocytes or other CNS cell types is necessary for remyelination, using Cre- lox recombination to selectively ablate SphK2 in myelinating cells, neurons, or other glial cell types. Future work will also establish whether SphK2 promotes remyelination through production of S1P that stimulates S1PRs on oligodendrocytes, or through its requirement for correct metabolism of lipids in oligodendrocytes. This research is important for establishing whether S1P is itself essential for remyelination following injury, and therefore whether agonists of S1PRs, already used in MS therapy, also possess remyelinating properties.

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

The effect of S1PR1/5 and S1PR5 agonists on cuprizone- induced demyelination in vivo

Introduction

In the previous chapter, I established that SphK2 deficiency sensitizes mice to cuprizone mediated loss of mature oligodendrocytes and demyelination, indicating that endogenous S1P synthesized by SphK2 protects oligodendrocytes and is crucial for remyelination. We hypothesize that the severe hypomyelination phenotype in SphK2−/− mice results from a deficiency in both S1PR1 and S1PR5 signalling, and that activation of these receptors by pharmacological agonists will protect oligodendrocytes and promote remyelination following acute demyelination in the CNS.

S1PR1 expression is predominant in OPCs in rodents, shown to promote OPC maturation in vitro [519], whereas S1PR5 is expressed only at very low levels in OPCs and inhibits their migration [315]. S1PR1 is expressed by all cells of the CNS, and is very highly expressed by astrocytes [520]; in contrast, S1PR5 is limited to oligodendrocytes and highly expressed by myelinating oligodendrocytes [304, 325]. S1PR5 mRNA expression is reduced in demyelinated MS lesions [521]. Moreover, a recent study suggested that S1PR5 mRNA expression is down-regulated around 3-fold in cuprizone-treated mice, along with other oligodendrocyte genes, 2 days after the start of cuprizone administration [51]. In vitro studies have shown that S1PR5 activation protects against apoptosis [303].

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

The MS drug Fingolimod protects against oligodendrocyte apoptosis in the EAE model [323,

522], which is driven by autoreactive encephalitogenic T cells [356, 523]. Fingolimod can cross the BBB, and its active form FTY720-P has been demonstrated to act directly on CNS cells through S1PRs except S1PR2 [323, 351, 356]. In the cuprizone model, demyelination proceeds independently of T-cell activation [524, 525]. Studies showed that oligodendrocyte number, myelin and axon integrity can be preserved by Fingolimod in response to cuprizone-induced

CNS demyelination [526, 527]. Recent evidence suggests that Fingolimod, signalling through

S1PR1 enhances oligodendrocyte survival and protects against demyelination in vivo [358,

528], but inhibits oligodendrocyte differentiation in vitro [342]. Whether S1PR5 activation is important for protecting oligodendrocytes from cell death and/or stimulating remyelination in the cuprizone model remains to be determined.

The contribution of specific S1PRs to physiological functions in the CNS cannot be determined by Fingolimod alone. A-971432 is a recently developed selective agonist of S1PR5 [385], which was shown to protect against neurodegeneration and cognitive deficits in a HD model

[386, 529]. The effect of this agonist on oligodendrocyte survival and remyelination has not been tested. The recently-approved MS drug Siponimod is a dual selective S1PR1 and S1PR5

(S1PR1/5) agonist [378], which was shown to protect against demyelination in an organotypic slice culture model [380, 530]. Notably, this drug has not been tested for myelin-protective functions in the cuprizone model. In this chapter, we investigated the myelin-properties of these two oral S1PR agonists in the cuprizone model.

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Aims

This Chapter describes a preliminary study aiming to investigate the protective potential of

S1PR5 and S1PR1/5 agonists in WT mice with cuprizone-induced demyelination.

The specific aims for this Chapter are:

1. To determine if pharmacological agonists of S1PR1/5 and S1PR5 protect against cuprizone-mediated oligodendrocyte apoptosis and demyelination.

2. To investigate the effect of S1PR1/5 and/or S1PR5 agonists on cuprizone-induced changes to the extent of microgliosis and astrogliosis.

Materials and Methods

Compounds

A-971432 (#5766, Tocris) and Siponimod (BAF312) (#S7179, Selleckchem, Australia) stocks were prepared in sterile DMSO at 20 mg/ml, and stored at -20 ºC until use. The stocks were diluted to 0.1 mg/mL in hypromellose (HPMC, 0.2% w/v in drinking water) and warmed to 37

ºC on the day of administration. For vehicle control, the same amount of sterile DMSO was diluted in 0.2% HPMC.

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Cuprizone treatment and drug administration in vivo

Male C57BL/6J mice obtained from Australian Bioresources (ABR, Mossvale, NSW) were used for these experiments. Twenty-four mice (age 12 weeks; 25 to 30 g) were divided into 4 groups (n = 6): vehicle control (VC), cuprizone with vehicle control (CPZ + VC), cuprizone with A-971432 treatment (CPZ + A-971432) and cuprizone with Siponimod treatment (CPZ +

Siponimod). All mice were fed normal chow mixed with 0.2% cuprizone as described in

Chapter 2, except for the VC group. For drug administration, mice were weighed daily and gavaged with 0.5 mg/kg drug using gavaging needles and a 1 mL syringe. Gavaging was also performed daily for 6 weeks, and commenced at the same time as the cuprizone diet. At the end of the study, mice were culled, and brains were dissected as described in Chapter 2.

Flow cytometry on white blood cells

After 3 weeks of gavaging, ~100 µL blood samples were collected by submandibular bleeds, and transferred to EDTA-coated tubes (SARSTEDT, #41.1504.005) on ice. Samples were centrifuged to remove plasma in the upper layer (2000 rpm, 4 min, RT). ACK lysis buffer (145 mM ammonium chloride, 0.1 mM EDTA disodium, 12 mM sodium bicarbonate in MilliQ water, pH 7.35) was added to the cell pellet on ice for ~8 min, and the samples were centrifuged

(2000 rpm, 5 min, 4 ºC) to pellet the white blood cells and remove red blood cell lysate. To ensure red blood cell removal, the efficiency was assessed before proceeding. The supernatant was removed, and the pellet was washed with 1 mL FACS wash (2.5% heat-inactivated FBS,

2 mM EDTA, 0.01% sodium azide in PBS). Samples were resuspended in 200 μL FACS wash and transferred to a 96-well U-bottom plate. The plate was centrifuged (530 g, 5 min, 4 ºC) and

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo flicked to remove supernatant, after which the pellet was washed with FACS wash. Samples in each well were mixed with a cocktail of antibodies diluted in FACS wash and incubated for 1 h at 4 ºC. Antibodies were as follows: Anti-mouse CD45 (BD Horizon, #564279, 1:200 dilution), anti-mouse CD3e (BD Horizon, #562600, 1:200 dilution), anti-mouse CD45R/B220

(BD Pharmingen, # 553093, 1:100 dilution). Wells were topped up with 140 μL FACS wash, centrifuged (530 g, 5 min, 4 ºC), and washed twice more with 200 μL FACS wash. Counting beads (10 μL) and 5 μL DAPI (5 µg/mL) were added to each sample, and the samples were filtered before flow acquisition on a BD Fortessa cell analyzer.

Immunohistochemistry and immunofluorescence microscopy

Brain tissue samples were processed as described in Chapter 2 (2.3.2).

Immunofluorescence and volumetric analysis

Staining and imaging of ASPA, Glial fibrillary acidic protein (GFAP) and Ionized calcium binding adaptor molecule 1 (Iba1) (Table 2.1) were conducted as in Chapter 2, as well as the analysis of ASPA+ cell density.

Immunofluorescence staining and quantification of gliosis. GFAP and Iba1 staining were quantified with staining area-fraction, i.e. % area occupied by the stain. The average % area was calculated as the mean of splenium, body and genu parts of CC.

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

LFB/CV staining and analysis

Myelin was assessed by LFB/CV staining as described in Chapter 2.

Western blotting

Lysis of brain tissue samples and western blotting were performed as described in Chapter 2.

Drug quantification

Levels of Siponimod and A-971432 in cortical brain tissue were quantified using liquid LC-

MS/MS. Frozen cortical tissue samples (~10 mg) were transferred to 2 mL phase-lock

Eppendorf tubes, and subjected to ultrasonication in 1 mL ethyl acetate/isopropanol/water

(6:3:1) for 1 h at 37 C, then incubated for a further 1 h at 37 C. The suspension was centrifuged for 5 min at 4000 g, and the supernatant transferred to a 5 mL glass tube. The pellet was re-extracted with sonication in 1 mL ethyl acetate/isopropanol/water, and the supernatant was combined with that from the first extraction. The supernatant was dried under vacuum in a Savant SC210 SpeedVac (ThermoFisher Scientific), and the extract reconstituted in 400 µL

60% methanol/40% water/0.1% formic acid. Our recently published ceramide synthase 1 inhibitor HDTP053 [531] was added as an internal standard at the start of the extractions (20 ng per sample), due to its structural and physicochemical similarity to A-971432. Standard curves covering the range 0.002 - 20 µg/g tissue were created by spiking Siponimod and A-

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

971432 into 10 mg mouse cortex tissue samples, in triplicate, together with 20 ng per sample

HDTP053.

Reconstituted extracts (10 µL injection) were analysed on a TSQ Altis triple quadrupole mass spectrometer in positive ion mode, coupled to a Vanquish HPLC system (ThermoFisher

Scientific). Samples were resolved on a 3  150 mm XDB-C8 HPLC column (5 µM pore size)

(Agilent), using a binary gradient with solvent A comprising 0.2% formic acid/2 mM ammonium formate in deionised water, and solvent B comprising 0.2% formic acid/1 mM ammonium formate in methanol. The gradient was: 0 - 1 min, 40% A/60% B, increasing to

95% B from 1.0 - 2.8 min, holding at 95% B until 3.8 min, decreasing to 60% B from 3.8 - 4.0 min, then re-equilibrating at 60% B from 4.0 - 7.0 min. Precursor and product ion pairs, and collision energy (CE), were 517.3 and 461.2 (CE = 19) for Siponimod, 366.1 and 158.9 (CE =

23) for A-971432, and 354.1 and 158.9 (CE = 29) for P053. Cycle time was 0.6 s.

Chromatography peaks were quantified using XCalibur software (ThermoFisher Scientific).

Peak areas were normalised to the P053 peak to control for extraction efficiency and mass spectrometry performance across samples.

Statistical analysis

All statistical analysis was conducted using GraphPad Prism 8.0, and reported as means ± SEM.

Statistical significance was accepted at p < 0.05.

One-way ANOVA was used to measure the percentage of CD3 positive (CD3+) T cells (Fig.

3.1A) and B220 positive (B220+) B cells (Fig. 3.1B), as well as the effect of treatment groups

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo on live and dead CD45+ cells, respectively (Fig. 3.1C). This was also applied to compare different treatment groups for ASPA+ cell numbers (Fig. 3.2), western blotting data (Fig. 3.3), and the proportion of GFAP- and Iba1-positive areas (Fig. 3.5). Dunnett's post-test was performed to compare VC group and CPZ + VC group with other groups.

Myelination scores for LFB/CV staining were compared between CPZ + VC group and other treatment groups using the nonparametric Mann-Whitney rank test (Fig. 3.4B).

Results

Lymphopenia is induced with S1PR1/5 agonist Siponimod

Mice were administered a 0.2% cuprizone diet and gavaged daily with Siponimod, A-971432

(0.5 mg/kg/day), or vehicle control, for 6 weeks. Since S1PR1 agonists induce peripheral lymphopenia through functional antagonism of S1PR1 [281, 354], the total number of leukocytes (CD45+ cells), and proportion of CD3+ T cells and B220+ B cells in the circulation was assessed after 3 weeks of treatment, to confirm absorption and efficacy of the S1PR1/5 agonist Siponimod (Fig. 3.1). A significant reduction in the proportion of CD3+ T cells (Fig.

3.1A) (p < 0.0001) and B220+ B cells (Fig. 3.1B) (p < 0.0001) was observed with Siponimod treatment relative to the controls with cuprizone. The total number of live blood leukocytes was also significantly decreased in the CPZ + Siponimod group (p < 0.0001) compared to the

CPZ + VC group (Fig. 3.1C). In contrast, there was no effect of A-971432 on blood lymphocytes or total leukocytes, neither of cuprizone feeding.

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

A B CD3+ T Cells B220+ B Cells

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Figure 3.1 Effect of S1PR agonists on blood lymphocytes.

(A) CD3+ T cells and (B) B220+ B cells as a proportion of live CD45+ cells. (C) Number of CD45+ cells per 100 μL blood. Graphs present mean ± SEM, n = 6 mice per group. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test, comparing each group with CPZ + VC. Overall

ANOVA for CD3+ T cells, F = 35.07, p < 0.0001; B220+ B cells, F = 25.24, p < 0.0001; CD45+ cells, treatment F = 14.1, p < 0.0001. P values for statistically-significant comparisons are shown (****p <

0.0001).

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Effects of Siponimod and A-971432 on cuprizone-mediated demyelination

Following 6 weeks of cuprizone and drug administration, the effect of S1PR5-selective agonist

A-971432 and S1PR1/5 agonist Siponimod on oligodendrocytes loss was tested. The density of ASPA-positive mature oligodendrocytes was quantified in CC (Fig. 3.2B), cortex (Fig. 3.2C) and hippocampus (Fig. 3.2D). Compared to mice that did not receive cuprizone, mean oligodendrocyte density in CC was 44% lower in the CPZ + VC group (p < 0.0001), 38% lower with A-971432 (p < 0.0001), but only 22% lower with Siponimod (p = 0.0003), indicating that

Siponimod protected against cuprizone-mediated oligodendrocyte loss (p = 0.0002, compared to CPZ + VC) in the CC. Oligodendrocyte number was not affected by A-971432 treatment

(p > 0.05, compared to CPZ + VC). Neither Siponimod nor A-971432 treatment changed

ASPA+ cell density with cuprizone treatment in the cortex and hippocampus (p > 0.05, compared to CPZ + VC).

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

VC Cortex CPZ + VC

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Figure 3.2 Siponimod protects against cuprizone-mediated loss of mature oligodendrocytes.

(A) Representative images of ASPA staining (green) in CC, cortex and hippocampus (HIP), merged with DAPI (blue). Scale bar, 100 μm. Quantification of ASPA-positive cell density in CC (B), cortex

(C) and HIP (D). Graphs present mean ± SEM, n = 6 mice per group. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test comparing each group with CPZ + VC and VC, respectively. Overall ANOVA for ASPA+ cells in CC, F = 35.05, p < 0.0001; cortex, F = 26.4, p <

0.0001; HIP, F = 473, p < 0.0001. P values for comparisons (CPZ + VC vs other groups) were reported significant in the graphs (***p < 0.001, ****p < 0.0001).

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Western blotting was then conducted to obtain semi-quantitative data on myelin protein markers in the CC (Fig. 3.3A,B) and cortex (Fig. 3.3C,D). Compared with the VC group, CNP,

MOG and PLP were significantly reduced by cuprizone treatment in both regions. In contrast to the other markers, no significant loss of MBP was observed in the CC (Fig. 3.3A,B), whereas

MBP levels were significantly reduced by cuprizone in the cortex (Fig. 3.3C,D). There was no significant effect of either Siponimod or A-971432 on these myelin protein markers relative to the CPZ + VC group. In addition, cuprizone had no significant effect on levels of the neuronal marker neurofilament H (NF-H) in either region.

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

A C Cortex CC VC CPZ + CPZ + CPZ + VC CPZ + CPZ + CPZ + VC A-97 Siponimod VC A-97 Siponimod 220kDa NF-H 220kDa NF-H 30kDa 30kDa MBP MBP 15kDa 15kDa β-Actin CNP 50kDa 40kDa CNP 50kDa β-Actin 40kDa β-Actin 40kDa 30kDa 30kDa PLP PLP 30kDa MOG 30kDa MOG β-Actin 40kDa β-Actin 40kDa

MBP, CC CNP, CC MOG, CC PLP, CC NF-H, CC

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Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

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Figure 3.3 S1PR agonists do not protect against cuprizone-mediated loss of myelin protein markers.

Representative western blots (A, C) and densitometric quantification (B, D) of myelin and neuronal markers in CC (A, B) and cortex (C, D). Protein levels were normalized to β-actin. Graphs present mean

± SEM, n = 6 mice per group. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test comparing each group to CPZ + VC group. Overall ANOVA for CC: MBP, F = 1.809, p =

0.1779; CNP, F = 47.74, p < 0.0001; MOG, F = 15.04, p < 0.0001; PLP, F = 32.65, p < 0.0001; NF-H,

F = 3.038, p = 0.0529. Overall ANOVA for cortex: MBP, F = 10.23, p = 0.0003; CNP, F = 7.935, p =

0.0012 ; MOG, F = 7.142, p = 0.0021; PLP, F = 4.921, p = 0.0122; NF-H, F = 1.177, p = 0.3447. P values for comparisons were reported significant in the graphs (*p < 0.05, **p < 0.01, ***p < 0.001,

****p < 0.0001).

100

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Given the negative results for protection against myelin protein loss in Siponimod-treated mice, myelin content was further checked by scoring of LFB/CV histochemistry staining (Fig.

3.4A,B). Consistent with Chapter 2, notable myelin loss was observed in the CC and cortex of cuprizone-treated mice (44% reduction of myelin score in VC + CPZ compared to VC group, p = 0.0022). Remarkably, the myelin score in the CPZ + Siponimod group was not significantly reduced compared to the no-cuprizone VC group (p = 0.0931), and was substantially improved compared to the CPZ + VC group (p = 0.0043), indicating that Siponimod exerted a protective effect on myelin levels. However, scores did not change with A-971432 treatment compared to CPZ + VC (p = 0.2739).

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Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

A B

LFB/CV LFB/CV staining VC Cortex CPZ + VC ✱✱ ✱✱

3 e

C r o

c 2

C s

o i

HI t

n i

P l

CPZ + A-971432 CPZ + Siponimod e 1

y M

0 2 C C 3 d V V 4 o + 1 7 im Z 9 n P - o C A ip + S Z + P Z C P C Figure 3.4 Siponimod protects against cuprizone-mediated demyelination.

(A) Representative LFB/CV staining of CC. Scale bar, 300 μm. (B) Myelination score for LFB/CV staining in CC. CC average was calculated as the mean of splenium, body and genu. Graphs show mean

± SEM from 6-7 mice per group, 3 sections per mouse. The degree of myelination between either the

CPZ + VC or VC group and other treatment groups was compared by nonparametric Mann–Whitney U test. p values for comparisons (CPZ + VC vs other groups) were two-tailed and significant comparisons are shown (**p < 0.01).

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Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Effects of Siponimod and A-971432 on cuprizone-mediated astro- and microgliosis

Cuprizone induces glial cell activation in multiple brain regions, including CC [532, 533], which was suppressed with S1PR agonist Fingolimod [358]. We therefore investigated whether

Siponimod and A-971432 suppress the accumulation of reactivated astrocytes and activated microglia in CC. Representative staining images for either GFAP (Fig. 3.5A) or Iba-1 (Fig.

3.5B) are shown. In cuprizone-treated mice, GFAP-positive (reactive) astrocytes and Iba1- positive microglia with activated morphology ― larger cell bodies and thicker processes ― were present, compared to the mice with no treatment. In agreement with previous studies [358,

532], the area staining of the CC covered by GFAP-positive astrocytes (Fig. 3.5C) and Iba1- positive microglia (Fig. 3.5D) was significantly increased with cuprizone treatment.

Siponimod reduced CPZ-mediated astrogliosis and microgliosis (GFAP, p = 0.009 and Iba1, p

= 0.002, CPZ + Siponimod vs CPZ + VC), whereas A-971432 had no effect. Although

Siponimod treatment reduced astrogliosis and microgliosis induced by cuprizone, both measurements were still significantly higher than in control mice that were not administered cuprizone (p = 0.0004 and p = 0.0008 compared to VC).

103

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

A VC CPZ + VC CPZ + A-971432 CPZ + Siponimod

GFAP

DAPI

B VC CPZ + VC CPZ + A-971432 CPZ + Siponimod

Iba1

DAPI

/ Iba1

104

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Iba1+ Cells C GFAP+ Cells D ✱ ✱✱ ✱✱✱ 40 40 ) ✱✱✱✱

) ✱

(

n o

i 30

o t

i 30

a 20 a

20 e

i v

i 10

10 t

P P 0 0 2 d C C 2 d C C 3 o V V 3 o V V 4 4 + 1 m + 1 im 7 i Z 7 n Z 9 n P -9 o P - o A p C A ip C i S + S + Z + Z + P Z P Z C P C P C C

Figure 3.5 Effect of S1PR5 and S1PR1/5 agonists on astro- and microgliosis.

(A, B) Representative images of GFAP and Iba1 staining. Scale bar, 20 μm. Boxed areas show enlarged images of individual cells, outlined with a dashed line. (C, D) Area of the CC (%) covered by GFAP-

(GFAP+) and Iba1-positive (Iba1+) cells. Graphs present mean ± SEM, n = 6 mice per group. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test, comparing each group with

CPZ + VC and VC respectively. Overall ANOVA for GFAP, F = 28.66, p < 0.0001; Iba1, F = 31.33, p < 0.0001. p values for comparisons (CPZ + VC vs other groups) were reported significant in the graphs (*p < 0.05, **p < 0.01, ****p < 0.0001).

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Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Siponimod accumulates to a much greater extent than A-971432 in the brain.

Levels of Siponimod and A-971432 in cortical brain tissue were measured by LC-MS/MS.

Siponimod and A-971432 levels at the 6-week experimental endpoint were 881  156 ng/g (1.7 nmoles/g) and 25  5.8 ng/g (68 pmoles/g), respectively (n = 4 - 5). These results raised the possibility that the ineffectiveness of A-971432 compared to Siponimod was due to sub- effective dosing of A-971432.

Discussion

Herein, we investigated whether S1PR5 and/or S1PR1/5 agonism suppress cuprizone-mediated oligodendrocyte apoptosis and demyelination. Administration of S1PR1/5 agonist Siponimod produced a statistically-significant protection against cuprizone-mediated oligodendrocyte loss, reactive astrogliosis, and microgliosis in the CC. However, Siponimod did not protect oligodendrocytes in the cerebral cortex or hippocampus, indicating that the responsiveness of oligodendrocytes to Siponimod might be region-specific. The basis for protective effects of

Siponimod on oligodendrocytes only in the CC remains unknown. This is potentially related to local drug concentration, as Fingolimod is known to accumulate in white matter [522].

However, we did not quantify levels of Siponimod in different brain regions in this study.

Siponimod protected against myelin loss based on semi-quantitative scoring of LFB staining, but not by western blotting. On balance, we conclude that Siponimod exerted a protective effect against cuprizone-mediated oligodendrocyte loss and demyelination. In contrast, S1PR5- selective agonist A-971432 did not suppress loss of myelin and oligodendrocytes, or glial cell 106

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo activation. However, levels of Siponimod in the cortex were over an order of magnitude higher than those of A-971432 at the conclusion of the 6-week drug administration, suggesting that a higher dose should be tested.

Siponimod was shown to protect against demyelination in the EAE model [381]. Our results demonstrating the protective effect of Siponimod in the cuprizone model are in agreement with published research showing that this drug protects against lysolethicin-induced demyelination in organotypic slice cultures [380], and demyelination induced by defective lipid catabolism in a mouse model of the lysosomal storage disorder Krabbe’s disease [534]. In vivo absorption and activity of S1PR1 agonists can be readily assayed by quantifying peripheral lymphopenia

[535, 536], however there is no such simple in vivo assay for S1PR5 agonists. A-971432 at a dose of 0.1 mg/kg/day was shown to protect against loss of BBB integrity and aggregation of mutant Huntington protein in a HD mouse model [386]. Our dose of 0.5 mg/kg/day achieved a mean level of 25 ng/g brain tissue at the conclusion of the 6-week administration, equivalent to the 28 ng/g reported by Hobson et al (0.1 mg/kg/day) [281]. The level that we measured equates to 68 pmoles/g, which is roughly equivalent to 68 nM, based on the assumption that 1 g equates to 1 mL. This is theoretically well above the EC50 for activation of S1PR5 by A-

971432 (4-6 nM) [281], but still well below the level achieved with Siponimod, at 1.7 nmoles/g tissue. Our measurements were performed using a highly accurate LC-MS/MS method with an appropriate internal standard, and a standard curve created by spiking naïve brain tissue with the compounds prior to extraction, conferring a high degree of confidence to the values that we obtained. These results raise the possibility that the levels of A-971432 were insufficient to protect oligodendrocytes.

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Although our current results suggest that S1PR5 agonism alone may not be sufficient to protect oligodendrocytes, they do not exclude the possibility that endogenous S1PR5 is important for oligodendrocyte stress resistance. Firstly, we should repeat this experiment using a higher dose of A-971432, such that the levels achieved in the brain are closer to those achieved with

Siponimod. Secondly, we should test the effect of cuprizone administration in S1PR5 knockout mice. We recently reported that both S1PR1 and S1PR2 signalling in astrocytes is necessary for protection of neurons against excitotoxic stimulus [372], and it is possible that combined signalling from both S1PR1 and S1PR5 is needed to activated protective signalling in oligodendrocytes. Interestingly, a study reported no myelination abnormalities in S1PR5−/− mice at baseline, however, there has been no comprehensive characterisation of myelin structure and integrity in these mice with stresses such as cuprizone or EAE [303]. S1PR5 is the most abundant receptor in oligodendrocytes, was shown to mediate protection of oligodendrocytes in vitro [303], and its expression is significantly affected by cuprizone treatment. Expression of S1PR1 and 5 was reported to increase in subcortical white matter with cuprizone treatment [358]. In contrast, another study reported 3-fold down-regulated of S1PR5 mRNA, together with other oligodendrocyte genes, 2 days after the start of cuprizone administration [537]. Determining whether S1PR5 agonists affect S1PR5 levels in the CC may help with ascertaining whether this receptor is activated by these agonists in vivo.

In this thesis, mature oligodendrocytes were quantified using the marker ASPA. A limitation of this work is that I did not determine how OPC cell number and proliferation is affected by

Siponimod. Cuprizone is toxic to mature oligodendrocytes but not OPCs, explaining the rapid remyelination following cuprizone withdrawal [538]. Accumulation of OPCs with cuprizone- induced demyelination has been reported in many studies [218, 219]. Upregulated S1PR1 and downregulated S1PR5 expression were observed in proliferating OPCs with PDGF stimulation 108

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

[303, 342]. OPC proliferation and differentiation can be promoted by Fingolimod in vitro [342] and in the EAE model [364, 365], possibly mediated through S1PR1 signalling [315, 342, 519].

Therefore, it would be of interest to test the effects of S1PR1 and 5 agonists on proliferation and differentiation of PDGFR-positive OPCs in vivo. This will also indicate if S1PR1 and 5 contribute to remyelination capacity, as OPC differentiation is essential for remyelination. A closely-related limitation of the current study is that we can only conclude that Siponimod protects against mature oligodendrocyte loss, rather than preventing cell death. Analysis of oligodendrocyte apoptosis may further define the oligodendrocyte-protective function of

Siponimod [539]. Studies have reported that Fingolimod and the S1PR1-selective agonist

CYM-5442 reduce oligodendrocyte apoptosis in the cuprizone model [358]. In contrast, the results of this study suggest that S1PR5 agonism alone does not protect oligodendrocyte viability, however further experiments are required to make this conclusion.

S1PR1 agonists like Fingolimod and Siponimod suppress inflammation largely by inducing peripheral lymphopenia [535, 536]. Although these drugs are S1PR1 agonists, meaning that they activate the receptors’ signalling cascades, they cause internalisation and degradation of this receptor on lymphocytes, so inhibiting the normal chemotactic response of lymphocytes to the high S1P concentration in blood. This immunosuppressive mechanism is therefore referred to as functional antagonism of S1PR1 [540]. Similarly, it has been proposed that functional antagonism of S1PR1 is the basis for the anti-inflammatory mechanism of action of these drugs acting on astrocytes [358, 540]. This hypothesis is based on the assumption that S1PR1 signalling in astrocytes is pro-inflammatory. Whilst pro-inflammatory cytokines such as TNF-

α induce S1PR1 expression on astrocytes [541] and S1P promotes astrocyte proliferation [542], there is considerable evidence indicating that S1P is an anti-inflammatory lipid [543, 544].

Furthermore, specific deletion of S1PR1 in myelinating cells was shown in one study to 109

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo enhance oligodendrocyte apoptosis and increase sensitivity to cuprizone [519], a finding that is incompatible with S1PR1 activation in the CNS playing a deleterious role in oligodendrocytes survival. Together with our finding that SphK2−/− mice show more pronounced oligodendrocyte loss in response to cuprizone, it seems more likely that activation of S1PR1, rather than functional antagonism, protects oligodendrocytes. This will be investigated in future studies by determining if Siponimod or selective S1PR1 agonists reverse the sensitivity of SphK2−/− mice to cuprizone.

In agreement with published work using Fingolimod [526], we show that the more selective

S1PR1/5 agonist Siponimod prevents extensive infiltration of microglia and reactive astrocytes into the CC in mice treated with cuprizone. Reactive astrocytes and microglia are thought to be important local inflammatory mediators of oligodendrocyte apoptosis in the cuprizone model [229, 545]. A very recent study demonstrated that S1P carried on microglial exosomes induces an anti-inflammatory, pro-myelinating phenotype in astrocytes, which promotes OPC migration, differentiation, and myelination [543]. S1PR1 is highly expressed on astrocytes and its activation induces the expression of pro-regenerative growth factors including LIF and

BDNF [358, 546]. LIF is upregulated in the first week of cuprizone treatment, and decreased during the subsequent demyelination phase, whereas BDNF showed an opposite trend [547].

S1PR1 activation might therefore protect oligodendrocytes and inhibit gliosis and demyelination induced by cuprizone [358]. However, more extensive investigation of astrocyte phenotypes both in vitro and in vivo is required to conclusively determine whether activation or functional antagonism of astrocyte S1PR1 is an important intermediary in the oligodendrocyte-protective properties of S1PR1 agonists.

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Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo

Even though Siponimod treatment produced a statistically-significant protection of oligodendrocytes and prevented demyelination measured with LFB staining, no protection was observed in western blots for any of the myelin proteins. We note, however, that MBP levels were not significantly reduced in western blots on CC, contrasting with the results in Chapter

2 (Fig. 2.4). Three additional analyses will help to resolve this quandary: Firstly, immunohistological staining for MBP presents an alternative way to quantify myelin protein changes. Secondly, we should quantify myelin lipids, particularly cholesterol and sphingolipids, using LC-MS/MS. Myelin is comprised 70-80% of lipids, which are essential for myelin stability and function [31-33]. Thirdly, neuronal stress visualised through staining for APP swellings, and measures of motor function (accelerod and balance beam) will provide additional functional measures of demyelination.

Since our results indicated that Siponimod protects against oligodendrocyte loss in the CC, but the results for myelin protection were less clear, a repeat experiment with a slightly shorter cuprizone treatment time (3 - 5 weeks) may also help to resolve clearly whether Siponimod protects against myelin loss. Quantification of apoptotic oligodendrocytes in a shorter-term cuprizone administration is also warranted, as is testing of the S1PR1-selective agonist CYM-

5542 and administration of a higher dose of A-971432, to properly resolve the effect of S1PR1 vs S1PR5 agonists. A recent publication demonstrated a protective effect of CYM-5442 in the cuprizone model [358]. Given the importance of S1PR1 agonists for clinical therapy of MS, this finding needs confirmation by another laboratory.

This study did not investigate the role for S1PR1 and S1PR5 activation in remyelination. Future studies must investigate the role of both S1PR1 and S1PR5 signalling in remyelination following cuprizone withdrawal. Fingolimod fails to stimulate remyelination following

111

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo cuprizone withdrawal in many studies [527, 548, 549] but restores myelin in a model of

Krabbe's disease [534]. Recently, a pro-myelinating effect of Siponimod was reported in a toxin-induced demyelination model using Xenopus tadpoles, and the effect of Siponimod was impaired in tadpoles lacking S1PR5 [550], implying an important role for S1PR5 signalling in remyelination. Activation of S1PR5 was also suggested to be important for remyelination in a slice culture model [362], although the compounds used were not highly specific. Whilst the cuprizone model is well suited for testing interventions that inhibit remyelination after cuprizone withdrawal, investigating pro-myelinating effects of drugs in this model is difficult, due to the very rapid spontaneous remyelination that occurs following cuprizone withdrawal.

In this regard, our capacity to now test Siponimod in SphK2−/− mice, which do not remyelinate after cuprizone withdrawal, is important for resolving whether S1PR signalling promotes, or is essential for, remyelination.

In summary, work from this Chapter supports the hypothesis that S1PR1/5 signalling promotes oligodendrocyte survival and prevents gliosis in mice fed cuprizone. Further experiments are required to determine with clarity whether the S1PR1/5 agonist Siponimod prevents demyelination. S1PR1 and 5 may act synergistically to protect oligodendrocytes and preserve myelination in the CC, or the effects of Siponimod may be attributed entirely to its action on

S1PR1 on oligodendrocytes or astrocytes. This preliminary study urges further exploration of the functions of S1PR1 and S1PR5 in oligodendrocyte protection and remyelination using mice bearing cell-specific deletion of these receptors in astrocytes or oligodendrocytes, and S1PR1- or S1PR5-selective agonists in parallel with S1PR1/5 dual agonists. In vitro systems that effectively model the interactions between oligodendrocytes, astrocytes, microglia, and neurons will also help to resolve these questions. Future research will also test the effects of

S1PR1 and S1PR5 agonists on both oligodendrocyte survival and remyelination in SphK2 112

Chapter 3 The effect of S1PR1/5 and S1PR5 agonists on cuprizone-induced demyelination in vivo knockout mice, which are deficient for endogenous S1P synthesis and were shown in the previous chapter to respond poorly to cuprizone, with enhanced loss of oligodendrocytes and myelin, and a lack of remyelination. However, we have shown in Chapter 2 that S1P levels are high in SphK2 mice during and after cuprizone treatment, suggesting that the effect of SphK2 deficiency cannot be attributed to bulk S1P deficiency alone. Nonetheless, it is possible that high levels of S1P in SphK2 mice fed cuprizone are produced by cells other than oligodendrocytes or neurons, and therefore not localised to the correct cell compartments or sub-cellular locations to compensate for loss of S1P produced by SphK2. The importance of these experiments is underscored by the growing use of S1PR agonists in the clinic, particularly with Ozanimod just approved for RRMS and Siponimod now used to treat secondary progressive MS.

113

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