Investigating the role of dual 1-phosphate receptor signalling in neuroprotection

Collin Tran

A thesis submitted for the fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) School of Medical Science Faculty of Medicine University of New South Wales February 2020

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname/Family Name : Tran Given Name/s : Collin Abbreviation for degree as give in the University calendar : PhD Faculty : Faculty of Medicine School : School of Medical Sciences

Investigating the role of dual sphingosine 1-phosphate receptor Thesis Title : signalling in neuroprotection

Abstract (347 words): Sphingosine 1-phosphate (S1P) is a signalling lipid that mediates biological processes through five G- coupled receptors (S1PR1-5). S1P synthesis occurs through the phosphorylation of sphingosine by 1 (SphK1) or SphK2. S1P levels and SphK activity are reduced in multiple neurodegenerative paradigms including Alzheimer’s disease (AD). Pharmacological targeting of S1PRs is neuroprotective in animal models of these diseases. This thesis investigates the cellular and molecular mechanisms behind S1P-mediated neuroprotection. S1P up-regulates four neurotrophic (BDNF, PDGFB, HBEGF, and LIF) in primary , but not neurons. Induction of these genes is mainly driven by S1PR2 signalling, with minor contributions from S1PR1. Phosphoproteomic analysis showed time-dependent activation of canonical pathways such as the mitogen-activated protein kinase and RhoA pathways, and phosphorylation of cJUN and Yes associated protein (YAP). Immediate early genes (IEGs) were also induced with S1P. Transcription start sites of these neurotrophic factors show predicted binding sites for cJUN and YAP, and the IEGs. While distinct phosphosites were regulated by S1PR1 and S1PR2 signalling, a large subset was regulated by both. However, the RhoA-YAP pathway was exclusively activated by S1PR2. The clinical drug (FTY720) is a sphingosine analogue that is phosphorylated in vivo by SphK2 to form the S1P structural analogue FTY720- P. Previous reports detailing the neuroprotective properties of S1PRs used FTY720-P to investigate the neurotrophic response. Unlike S1P, FTY720-P does not activate the S1PR2-RhoA-YAP pathway in astrocytes. Accordingly, S1P was a much more potent inducer of IEGs and neurotrophic expression than FTY720-P. Additionally, S1P but not FTY720-P significantly attenuated excitotoxic neuronal cell death in vitro when co-cultured with glia. Neuroprotection was ablated upon incubation with a LIF neutralising antibody, but not with antagonists to BDNF or HBEGF signalling. I have therefore established a novel neuroprotective pathway in which S1P stimulates secretion of the LIF by astrocytes, which protects neurons against excitotoxic cell death. This pathway requires dual S1PR1 and S1PR2 signalling in astrocytes, thus explaining the inefficacy of FTY720- P. Current S1PR therapies do not target S1PR2 and targeting this receptor may be a novel therapeutic option for AD and other neurodegenerative conditions.

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UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure. Publications can be used in their thesis in lieu of a Chapter if: • The student contributed greater than 50% of the content in the publication and is the “primary author”, ie. the student was responsible primarily for the planning, execution and preparation of the work for publication • The student has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis Please indicate whether this thesis contains published material or not. This thesis contains no publications, either published or submitted for publication ☐ (if this box is checked, you may delete all the material on page 2) Some of the work described in this thesis has been published and it has been documented in the relevant Chapters with acknowledgement (if this box is ☒ checked, you may delete all the material on page 2)

This thesis has publications (either published or submitted for publication) ☐ incorporated into it in lieu of a chapter and the details are presented below

CANDIDATE’S DECLARATION I declare that:

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Name Signature Date (dd/mm/yy)

Collin Tran

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Abstract

Sphingosine 1-phosphate (S1P) is a signalling lipid that mediates biological processes through five

G-protein coupled receptors (S1PR1-5). S1P synthesis occurs through the phosphorylation of sphingosine by (SphK1) or SphK2. S1P levels and SphK activity are reduced in multiple neurodegenerative paradigms including Alzheimer’s disease (AD). Pharmacological targeting of S1PRs is neuroprotective in animal models of these diseases.

This thesis investigates the cellular and molecular mechanisms behind S1P-mediated neuroprotection. S1P up-regulates four neurotrophic genes (BDNF, PDGFB, HBEGF, and LIF) in primary astrocytes, but not neurons. Induction of these genes is mainly driven by S1PR2 signalling, with minor contributions from S1PR1. Phosphoproteomic analysis showed time-dependent activation of canonical pathways such as the mitogen-activated protein kinase and RhoA pathways, and phosphorylation of cJUN and Yes associated protein (YAP). Immediate early genes (IEGs) were also induced with S1P. Transcription start sites of these neurotrophic factors show predicted binding sites for cJUN and YAP, and the IEGs. While distinct phosphosites were regulated by

S1PR1 and S1PR2 signalling, a large subset was regulated by both. However, the RhoA-YAP pathway was exclusively activated by S1PR2.

The clinical drug Fingolimod (FTY720) is a sphingosine analogue that is phosphorylated in vivo by SphK2 to form the S1P structural analogue FTY720-P. Previous reports detailing the neuroprotective properties of S1PRs used FTY720-P to investigate the neurotrophic response.

Unlike S1P, FTY720-P does not activate the S1PR2-RhoA-YAP pathway in astrocytes.

Accordingly, S1P was a much more potent inducer of IEGs and neurotrophic than

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FTY720-P. Additionally, S1P but not FTY720-P significantly attenuated excitotoxic neuronal cell death in vitro when co-cultured with glia. Neuroprotection was ablated upon incubation with a LIF neutralising antibody, but not with antagonists to BDNF or HBEGF signalling.

I have therefore established a novel neuroprotective pathway in which S1P stimulates secretion of the growth factor LIF by astrocytes, which protects neurons against excitotoxic cell death.

This pathway requires dual S1PR1 and S1PR2 signalling in astrocytes, thus explaining the inefficacy of FTY720-P. Current S1PR therapies do not target S1PR2 and targeting this receptor may be a novel therapeutic option for AD and other neurodegenerative conditions.

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Publications and presentations arising from this candidature

Publications:

• Couttas, T.A., Kain, N., Tran, C., Chatterton, Z., Kwok, J.B. and Don, A.S., 2018. Age-

dependent changes to sphingolipid balance in the human hippocampus are gender-specific

and may sensitize to neurodegeneration. Journal of Alzheimer's Disease, 63(2), pp.503-

514.

• Tonkin, R.S., Bowles, C., Perera, C.J., Keating, B.A., Makker, P.G., Duffy, S.S., Lees,

J.G., Tran, C., Don, A.S., Fath, T. and Liu, L., 2018. Attenuation of mechanical pain

hypersensitivity by treatment with Peptide5, a connexin-43 mimetic , involves

inhibition of NLRP3 inflammasome in nerve-injured mice. Experimental neurology, 300,

pp.1-12.

• Tran, C., Heng, B., Teo, J.D., Humphrey, S.J., Qi, Y., Couttas, T.A., Stefen, H., Brettle,

M., Fath, T., Guillemin, G.J. and Don, A.S., 2019. Sphingosine 1‐phosphate but not

Fingolimod protects neurons against excitotoxic cell death by inducing neurotrophic gene

expression in astrocytes. Journal of neurochemistry, p.e14917.

• Aji, G., Huang, Y., Ng, M.L., Wang, W., Lan, T., Li, M., Li, Y., Chen, Q., Li, R., Yan, S.,

Tran, C., Burchfield, J.G., Couttas, T.A., Chen, J., Chung, L.H., Liu, D., Wadham, C.,

Hogg, P.J., Gao, X., Vadas, M.A., Gamble, J.R., Don, A.S., Xia, P., Qi, Y. (2020)

Regulation of hepatic signalling and glucose metabolism by sphingosine kinase 2.

(under review)

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Presentations:

• Tran, C., Heng, B., Guillemin, G.J.,Don, A.S. (2016) Title: Neuroprotective signalling

pathways activated by sphingosine phosphate and its pharmacological agonists. 3rd

Australian Lipid Meeting, 21st – 22nd November, Melbourne, VIC, Australia (oral

presentation)

• Tran, C., Heng, B., T.A., Stefen, H., Brettle, M., Fath, T., Guillemin, G.J.,Don, A.S.

(2018) Title: Dual S1PR1 and S1PR2 pathways are crucial to neuroprotection. Australasian

Neuroscience Society Conference, 3rd – 6th December, Brisbane, QLD, Australia (oral

presentation)

• Tran, C., Heng, B., T.A., Stefen, H., Brettle, M., Fath, T., Guillemin, G.J.,Don, A.S.

(2019) Title: Parallel S1P receptor signalling synergise to induce neuroprotective

signalling. International Society for Neurochemistry – American Society for

Neurochemistry Conference, 31st – 4th July/August, Esterel, QC, Canada (oral

presentation)

• Tran, C., Heng, B., T.A., Stefen, H., Brettle, M., Fath, T., Guillemin, G.J.,Don, A.S.

(2019) Title: Parallel S1P receptor signalling synergise to induce neuroprotective

signalling. International Society for Neurochemistry – American Society for

Neurochemistry Conference, 4th – 8th August, Montreal, QC, Canada (poster

presentation)

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Acknowledgements

This thesis is dedicated to my family. Without their sacrifices and love I would not be where I am.

To my grandfather Vinh, you are no longer with us, but you inspired me to choose a project relevant to Alzheimer’s. I will never forget you just like you never forget me. To my grandmother, your love and support is second to none. To my parents, thank you for everything you have done for me. The unconditional love you both have for me is more than I deserve. Mum, you have always believed in me and supported my decision to choose this path. Dad, you are quiet and don’t express much of your thoughts, but you’ve always told me you’re proud of me. For everyone else in my family, I love you. To my girlfriend, thank you for supporting me and being my side. Your love and support through this few years has been amazing.

My greatest thanks go to my primary supervisor Anthony Don. You have been patient, supported me and guided me down this long path. These last few years have been a long ride and I’m very thankful that you’ve taught me how to be a good scientist. To the other primary supervisors, I have had over the years Thomas Fath and Pascal Carrive, thank you for your support as I transitioned from UNSW to The University of Sydney. Your kindness and support are greatly appreciated. To

Jacob Qi, thank you for being my co-supervisor and friend. Your knowledge of benchwork is outstanding and I would still be stuck if it wasn’t for you. To Timothy Couttas, thank you for always being there for help and advice.

To everyone who has helped me throughout this candidature, thank you for everything. Special thanks to Benjamin Heng for supplying me with primary cells, Holly Stefen and Merryn Brettle for the mouse cells, Sean Humphrey for helping me with the phosphoproteomnics, Jonathan Teo for helping with the staining. To the rest of the Don group: Mona, Oana, Dagny, Jun, Laura, Anna,

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and Holly, it has been a pleasure to work with you all. A very special thank you is reserved to

Amy, for being a friend and a teacher. To all my other friends: the Holst group, the Naylor group, the Wong group, the Hesson group and the Hogg group, thank you for all the support, the chats and the lunches.

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List of Figures

Figure 1. 1 APP processing via the amyloidgenic and non-amyloidgenic pathways...... 3

Figure 1.2 Mechanism of excitotoxic cell death in the presence of Aβ...... 9

Figure 1.3. Simplified sphingolipid metabolic pathway showing S1P synthesis and degradation...... 14

Figure 1.4 Autocrine and paracrine signalling by S1P...... 16

Figure 1.5 Summary of S1P actions on CNS cells...... 27

Figure 1.6. Mechanism of FTY720-P in MS...... 28

Figure 3.1 S1P induces neurotrophic gene expression in astrocytes ...... 54

Figure 3.2. Neurotrophic gene expression in primary human neurons following S1P treatment. 56

Figure 3.3 Basal expression of neurotrophic factors is higher in astrocytes compared to neurons...... 57

Figure 3.4 S1P does not induce neurotrophic gene expression in BV-2 cells...... 58

Figure 3.5 S1P receptor expression in CNS cells ...... 60

Figure 3.6 SphK1 and SphK2 gene expression correlates with neurotrophic factors in the human hippocampus...... 62

Figure 3.7 SPHK2 is necessary for basal PDGFB and LIF expression in U251 cells...... 64

Figure 3.8 Activation of S1PR2 is required for induction of neurotrophic factors ...... 66

Figure 3.9 Neurotrophic gene induction by S1P after inhibition of two S1P receptors...... 68

Figure 3.10 Induction of neurotrophic genes is dependent on Gai signalling...... 70

Figure 3.11 Ga13 not signalling Ga12 is required for induction of neurotrophic genes ...... 71

Figure 3.12 S1P activation of RhoA is required for induction neurotrophic genes ...... 73

Figure 3.13 Induction of neurotrophic genes occurs via a Rho-kinase independent mechanism. 74

Figure 3.14 RhoA and Gai contribute to the secretion of LIF protein ...... 75

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Figure 3.15 Summary diagram of the results section ...... 77

Figure 4.1 Analysis of the phosphoproteome following S1P stimulus...... 87

Figure 4.2 Volcano plots showing significant changes to phosphosites over time following S1P stimulus...... 88

Figure 4.3 S1P induces immediate early gene expression in astrocytes ...... 89

Figure 4.4 Binding sites for AP-1, EGR1 and TEAD transcription factors on BDNF, PDGFB, HBEGF, and LIF genes...... 91

Figure 4.5 Inhibition of MEK reduces induction of immediate early genes and neurotrophic genes...... 94

Figure 4.6 Phosphoproteomic analysis of pathways downstream of S1PR1 and S1PR2...... 98

Figure 4.7 Phosphoproteomic analysis of temporal changes to transcription factors downstream of S1PR1 and S1PR2...... 99

Figure 4.8. S1PR2 is required for Egr-1 and Fos induction, Jun phosphorylation, and YAP dephosphorylation...... 101

Figure 4.9 Gai and RhoA contribute to ERK signalling and activation of transcription factors...... 103

Figure 5.1 S1P is a more potent inducer of neurotrophic genes than FTY720-P...... 114

Figure 5.2 Dose response curves for neurotrophic gene induction by S1P and FTY720-P ...... 115

Figure 5.3 FTY720-P does not activate S1PR2-dependent transcription factors ...... 117

Figure 5.4 Supplementing FTY720-P with an S1PR2 agonists boosts neurotrophic gene expression...... 118

Figure 5.5 Effect of S1P and FTY720-P on the phosphorylation of ERK ...... 119

Figure 5.6 Hippocampal neurons cultured in the presence of cortical support are protected from excitotoxic cell death in an S1P dependent manner ...... 121

Figure 5.7 Stimulating neurons with S1P does not induce neuroprotective gene expression or protect from excitotoxic cell death...... 122

5.3.7 Ablating LIF reduces neuroprotection in the co-culture model ...... 123

Figure 5.8 Blocking the action of neurotrophic factors promotes excitotoxic cell death ...... 124

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List of Tables

Table 2.1. List of primers used for qPCR ...... 44

Table 2.2 List of primary antibodies used for Western blotting ...... 47

Table A1. Statistically significant phosphoproteins regulated by W146 only ...... 140

Table A.2 Statistically significant phosphoproteins regulated by JTE013 only ...... 140

Table A.3 Statistically significant phosphoproteins regulated by both W146 and JTE013 ...... 147

Table A.4 Statistically significant phosphoproteins regulated by neither W146 or JTE013 ...... 148

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Table of Contents

Thesis/Dissertation Sheet ...... I

ORIGINALITY STATEMENT ...... II

INCLUSION OF PUBLICATIONS STATEMENT ...... III

Abstract ...... IV

Publications and presentations arising from this candidature ...... VI

Acknowledgements ...... VIII

List of Figures ...... X

List of Tables ...... XII

Table of Contents ...... XIII

Abbreviations ...... XVII

Chapter 1: Literature Review ...... 1 1.1 Alzheimer’s disease ...... 1 1.2 AD Pathology ...... 2 1.2.1 Amyloid cascade hypothesis ...... 2 1.2.2 Tau hyperphosphorylation ...... 4 1.2.3 Neuroinflammation in AD ...... 5 1.2.4 Excitotoxicity in AD ...... 7 1.2.5 Current AD therapy ...... 10 1.2.6 Genetics and the risk for AD ...... 10 1.3 Sphingolipids ...... 12 1.4 Sphingosine 1-Phosphate ...... 13 1.4.1 Extracellular S1P signalling ...... 15 1.4.2 S1PR1 ...... 17 1.4.3 S1PR2 ...... 18 1.4.4 S1PR3 ...... 18 1.4.5 S1PR4 ...... 19 1.4.6 S1PR5 ...... 19 1.4.7 Intracellular S1P effects ...... 20 1.5 S1P in the CNS ...... 21

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1.5.1 Neurons and S1P signalling ...... 21 1.5.2 Astrocytes and S1P signalling ...... 23 1.5.3 S1P signalling in and Oligodendrocytes ...... 24 1.6 S1P therapeutics: the first clinical drug ...... 27 1.7 S1P in Alzheimer’s disease ...... 29 1.8 ...... 30 1.9 S1P receptor agonists in the context of AD ...... 32 1.9.1 New generation S1P receptor agonists in clinical trials ...... 34 1.9.2 S1P receptor agonists for biological studies ...... 34 1.9.3 S1P receptor antagonists ...... 36 1.10 Aims of this thesis ...... 36

Chapter 2: Methods ...... 38

2.1 Cell culture methods ...... 38 2.1.1 Cell culture conditions ...... 38 2.1.2 Immortalised cell lines ...... 38 2.1.3 Primary human cells ...... 39 2.1.4 Primary mouse cells ...... 39 2.2 Compounds ...... 41 2.3 siRNA transfections ...... 41 2.4 Human Hippocampus Tissue samples ...... 42 2.5 qPCR ...... 42 2.6 Neuronal excitotoxicity assays ...... 45 2.7 ELISA ...... 45 2.8 Western blot ...... 46 2.9 Active RhoA Assay ...... 48 2.10 Phosphoproteomic analysis ...... 48 2.11 Phosphoproteomic analysis ...... 49 2.12 Statistical analysis ...... 49

Chapter 3: Investigating the S1P-induced neurotrophic gene response in CNS cells ...... 51 3.1 Introduction ...... 51 3.2 Aims ...... 52 3.3 Results ...... 53

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3.3.1 Confirming neurotrophic gene expression in astrocytes ...... 53 3.3.2 Effect of S1P on neurotrophic factor gene expression in neurons ...... 55 3.3.3 Effect of S1P on neurotrophic gene expression in microglial cells ...... 58 3.4.4 S1P receptor expression in astrocytes and neurons ...... 59 3.3.5 Correlation of SphKs and S1P to neurotrophic factors in the human hippocampus ... 61 3.3.6 Effect of SphK silencing on neurotrophic factors ...... 63 3.3.7 Effect of S1PR antagonists on neurotrophic factor gene expression ...... 65 3.2.8 Effect of dual S1PR antagonists on neurotrophic factors ...... 67 3.2.9 Targeting G- coupled to S1P receptors ...... 69 3.3.11 Effect of RhoA and ROCK inhibition on neurotrophic factor expression ...... 72

3.3.12 Effect of Gai and RhoA on LIF protein secretion ...... 75 3.4 Discussion ...... 76

Chapter 4: Characterisation of the signalling pathways activated by individual S1P receptors...... 83 4.1 Introduction ...... 83 4.2 Aims ...... 84 4.3 Results ...... 85 4.3.1 Time course phosphoproteomics ...... 85 4.3.2 Transcription factor analysis ...... 89 4.3.3 Effects of antagonising canonical pathways on neurotrophic and transcription factors ...... 92 4.3.4 Antagonist phosphoproteomics ...... 95 4.3.5 Effect of S1PR antagonists on ...... 100

4.3.6 Effect of Gai and Ga13 inhibition on signal transduction pathways ...... 102 4.4 Discussion ...... 104

Chapter 5: Comparison of the neuroprotective properties of S1P and Fingolimod ...... 111 5.1 Introduction ...... 111 5.2 Aims ...... 112 5.3 Results ...... 113 5.3.1 Comparison of neurotrophic gene induction by S1P and FTY720-P ...... 113 5.3.2 S1PR2 signalling pathways are not activated in FTY720-P treated cells ...... 116 5.3.4 Effect of combining FTY-P with S1PR2 agonist on neurotrophic gene expression .... 118 5.3.5 FTY720-P does not phosphorylate ERK to the same extent as S1P ...... 119 5.3.6 S1P but not FTY720-P protects hippocampal neurons against excitotoxic cell death 120 5.4 Discussion ...... 125

Chapter 6: Summary and Future Directions ...... 130

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6.1 Potential limitations and future directions ...... 131 6.2 Targeting S1P receptors and sphingolipid metabolism for neurodegenerative disease therapy ...... 135

Appendix ...... 140

References: ...... 160

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Abbreviations

Ab Amyloid-b ABCA ATP-binding cassette subfamily A ACh Acetylcholine AChE Acetylcholinesterase AD Alzheimer’s disease ADAMs A disintegrin and metalloproteinase Akt APP Amyloid Precursor Protein APOE Apolipoprotein E APOJ Apolipoprotein J/Clusterin BACE1 b-site amyloid precursor protein cleaving BBB Blood barrier BCA Bicinchoninic acid BDNF Brain-derived neurotrophic factor BSA Bovine serum albumin CCL Chemokine cDNA Complimentary DNA CNS CSF Cerebrospinal fluid dhS1P dihydrosphingosine 1-phosphate DMEM Dulbecco’s Modified Essential Media DMSO Dimethyl sulfoxide D-PBS Dulbecco’s phosphate buffered saline EAE Experimental autoimmune encephalomyelitis EDG Endothelial differentiation gene

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EGF ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum ERK Extracellular signal-regulated kinase FAD Familial Alzheimer’s Disease FBS Foetal bovine serum FPKM Fragments per kilobase of transcript per million mapped reads FTY720 Fingolimod GDNF Glial-derived neurotrophic factor GPCR G-protein coupled receptor HBEGF Heparin-binding epidermal growth factor-like growth factor HD Huntington’s disease HDAC Histone deacetylase IEG Immediate early gene IFN IL IPA Ingenuity Pathway Analysis LIF Leukaemia inhibitory factor LPS Lipopolysaccharide LTP Long-term potentiation MAPK Mitogen-activated protein kinase MEK Mitogen-activated protein kinase kinase mHTT Mutant huntingtin MS NFkB Nuclear factor k-light-chain-enhancer of activated B-cells NFTs Neurofibrillary tangles NGF NMDA N-methyl-D-aspartate

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NSAIDs Non-steroidal anti-inflammatory drugs NT-3 3 OPC Oligodendrocyte precursor cell PD Parkinson’s disease PDGFB Platelet-derived growth factor PDL Poly-D-lysine PHFs Paired helical filaments PI Propidium iodide PI3K Phosphatidylinositol 3-kinase PLCG2 Cg2 PS1 Presenilin-1 PTX Pertussis toxin qPCR Quatitative real-time polymerase chain reaction ROS Reactive oxygen species RPMI Roswell Park Memorial Institute RT Room temperature S1P Sphingosine 1-phosphate S1PR Sphingosine 1-phosphate receptor SDC Sodium deoxycholate siRNA Short-interfering RNA SNPs Single nucleotide polymophisms SphK Sphingosine kinase TBST Tris buffered saline containing Tween 20 TEAD TEA domain family member TNFa Tumour necrosis factor a TREM2 Triggering receptor expressed on myeloid cells 2 TrK Tyrosine receptor kinase UCSC University of California Santa Cruz

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VEGF Vascular endothelial growth factor YAP Yes associated protein

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Chapter 1

Chapter 1: Literature Review

1.1 Alzheimer’s disease

Alzheimer’s disease (AD) is the leading cause of dementia with no available treatments that significantly alter the course of the disease. Globally, it is estimated that approximately 43 million individuals have dementia and this number is expected to increase significantly in the next decade due to a global ageing population [1, 2] . The Alzheimer’s Association reported over 18.4 billion hours were dedicated to caring for individuals with AD or other dementias, with this costing more than $232 billion dollars [2]. With an ageing population and no effective treatments available, this presents a significant challenge to the public health system.

There are two forms of AD: familial AD (FAD) and sporadic or late-onset AD. These are essentially the same disease, differing only in age at onset and genetic cause. FAD is defined as diagnosis below the age of 61 and three generations of diagnoses. Development of FAD is attributed to mutations in genes associated with the accelerated development and deposition of amyloid b (Ab) plaques: amyloid precursor protein (APP), presenilin-1 (PS1) or presenilin-2

(PS2). However, FAD accounts for less than 1% of all AD cases [2]. The greatest risk factor for developing late-onset AD is ageing while the strongest genetic risk factor is the differential allelic expression of the three isoforms of apolipoprotein E (APOE), which will be discussed further below. Individuals with AD experience symptoms associated with a loss of synapses with increasing neuronal death. These symptoms include short term memory loss, progressing to mild cognitive impairment where individuals will experience difficulty with temporal and spatial relationships, neuropsychiatric symptoms and severe behavioural issues [2]. With time, the loss of

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Chapter 1 neurons ultimately results in incapacitation with death often resulting from infections attributed to an inability to move or swallow [3].

1.2 AD Pathology

AD presents with multiple pathological hallmarks, some of which are: an abnormal extracellular accumulation of Aβ plaques [4], hyper-phosphorylation of the cytoskeletal protein tau leading to intracellular neurofibrillary tangles (NFTs) [5], a loss of cholinergic neurons [6], a loss of synapses

[7], astrogliosis [8], alterations to lipid metabolism [9] and demyelination [10-12]. The accumulation of plaques and tangles is believed to result in neuronal cell death, however loss of synapses correlates most closely with cognitive decline [7].

1.2.1 Amyloid cascade hypothesis

The amyloid cascade hypothesis postulates that the accumulation of Aβ plaques is the trigger for

AD pathogenesis [13]. Aβ plaques are formed by aggregation of Aβ derived by proteolytic cleavage of APP. This occurs in the endosomes firstly through β-secretase or β-site APP cleaving enzyme (BACE1) followed by γ -secretase [14, 15]. In the non-amyloidgenic pathway, cleavage of APP occurs first by α-secretase at the plasma membrane followed by γ-secretase (Figure 1.1).

The resultant peptide is a truncated form of APP termed p3, which is thought to be pathologically irrelevant due to less stability [16, 17].

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Chapter 1

Figure 1. 1 APP processing via the amyloidgenic and non-amyloidgenic pathways.

APP is synthesised in the ER and transported to the Golgi before further trafficking to the plasma membrane. Here, it can be endocytosed as full-length APP or as the C83 fragment following cleavage by a-secretase. Full-length APP can be cleaved by b-secretase in the endosomes to release sAPPb and the C99 fragment. sAPPb can be exocytosed and the C99 fragment can be trafficked to the late endosomes or the Golgi where g-secretase is localised and cleaves the C99 fragment to release Ab. Ab can then be released into the extracellular space to form plaques or be shuttled to the lysosome from the late endosomes.

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Chapter 1

While amyloid plaque deposition is a notable pathological event in the development of AD, it does not necessarily result in dementia. Aβ plaques are observed in the of non-demented individuals, with Price et al demonstrating up to 40% of non-demented brains displaying Aβ plaque pathology [18]. Large clinical trials have shown that interrupting plaque formation with inhibitors targeting amyloid formation fail to improve cognition while indeed successfully alleviating Aβ plaque load in the brain [19-21]. Several anti-amyloid antibodies have also been developed as an alternative but have been unsuccessful at alleviating cognitive deficits [22-24]. The lack of efficacy of anti-amyloid therapy puts the amyloid hypothesis into question. Thus, if Ab is indeed a driving influence for AD, then it is likely that additional sensitizing factors are needed to unmask the neurotoxicity of Ab. One study found that abnormalities in the endocytic pathway occurred prior to Ab deposition in AD brains, with apolipoprotein e4 expression resulting in enlargement of the early endosomes in preclinical AD [25]. Apolipoprotein e4 expression was also found to reduce endosomal pH, Ab clearance, and increase lysosomal pH relative to the e3 isoform in astrocytes

[26]. Restoring the pH balance ameliorated the defective Ab clearance by astrocytes [26, 27].

Additionally, new research suggests that mutations in APP and the presenilin proteases that cause

FAD accelerate the disruption to endosomal and lysosomal functions that characterises late onset

AD [28, 29], further suggesting that endolysomal dysfunction may be the cause of Ab pathophysiology in AD.

1.2.2 Tau hyperphosphorylation

The microtubule-associated protein tau is most abundantly expressed in neurons and involved in the formation and stabilisation of the microtubule network [30]. Microtubules play a role in the extension of neuronal processes as well as facilitating intracellular transport of organelles and proteins [31, 32]. Phosphorylation of tau, a normal cellular event hypothesised to occur via 4

Chapter 1 multiple including glycogen synthase kinase 3β (GSK3β), affects its ability to bind and stabilise microtubules [33, 34]. In AD, aberrant hyper-phosphorylation of tau forms fibrils and secondary structures termed paired helical filaments (PHFs) [35]. PHFs are the main component of NFTs and aggregate in neuronal perikaryon [36, 37].

Multiple studies have indicated the tau and Ab-mediated toxicity are intricately linked. Studies in rhesus monkeys found that injection of Ab resulted in the phosphorylation of tau [38], while injections of Ab into tauopathy mouse models amplified NFT pathology [39]. Reducing endogenous tau ameliorated Ab-mediated behavioural deficits in the hAPP transgenic mice [40], and tau-deficient neurons were protected from Ab-mediated toxicity in vitro [41]. Tau protein kinase I was also found to be involved in Aβ-mediated cell death of hippocampal neurons [42].

Interestingly, it was shown that phosphorylation of tau at the threonine 205 residue by the γ- isoform of p38 kinase antagonises the neurotoxicity induced by Aβ in transgenic mice expressing human mutant APP [43]. This suggests that initial tau phosphorylation may be a protective mechanism in stressed neurons. However, tau hyperphosphorylation and formation of NFTs are pathological events. The mechanism by which NFTs induce cell death is yet to be fully elucidated.

1.2.3 Neuroinflammation in AD

Microglia, the resident of the central nervous system (CNS), undertake immune surveillance and are sensitive to minute changes indicating possible pathological states [44]. When the CNS undergoes changes reflecting a disease state, microglia become activated via receptors such as the lipopolysaccharide (LPS) receptor as occurs during microbial invasion [45]. Although not classically thought of as being involved in the pathogenesis of AD, there is growing evidence supporting the direct involvement of microglia and the inflammatory response in exacerbating AD

5

Chapter 1 pathology. Aβ is able to bind to the collagen-like domain of complement factor C1 leading to immune recognition by microglia and activation of the complement system [46, 47]. Aβ is also known to interact with the LPS receptor CD14 on microglia to induce [48, 49].

Resulting neuronal damage occurs due to microglial activation and attacking plaque-damaged neurons in a CD14-dependent manner [50].

Astrocytes also play a role in maintaining CNS homeostasis. Under non-pathological conditions, astrocytes are known to have roles in regulating metabolism at synapses, the blood brain barrier (BBB), and more recently synaptogenesis [51-53]. When the CNS undergoes pathologic changes including trauma and neurodegeneration, astrocytes undergo a phenotypic change known as reactive astrogliosis resulting in scarring to limit potential further damage [54].

Recently it has been found that there are two distinct phenotypes of reactive astrocytes that have opposing effects, A1 and A2. The A1 phenotype was shown to induce genes associated with the complement cascade while the A2 phenotype up-regulated multiple neurotrophic factors. The A1 phenotype was also induced by activated microglia that were primed by LPS treatment leading to a secretion of pro-inflammatory factors including tumour necrosis factor (TNF) and their presence is increased in the hippocampus and prefrontal cortex in AD [55].

With neuroinflammation present in the CNS, it would seem that anti-inflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs) would be a viable option for therapeutic treatment. However, in-depth analyses of the use of NSAIDs has provided conflicting results. One study suggests that there is an adverse effect in the later stages of AD pathogenesis, but lower incidence in asymptomatic individuals who have been prescribed NSAIDs for two to three years

6

Chapter 1

[56]. A recent meta-review noted that only observational studies supported the use of NSAIDs in

AD while randomized controlled trials did not [57].

1.2.4 Excitotoxicity in AD

Communication between neurons occurs with the pre-synaptic release of neurotransmitters such as the excitatory amino acid glutamate, which act on receptors of post-synaptic neurons. Glutamate can activate two types of receptors: ionotropic or metabotropic. Ionotropic receptors are ion channels that when bound with glutamate results in the displacement of a magnesium ion from the channel pore to cause an influx of cations such as calcium to activate intracellular signalling mechanisms, while metabotropic glutamate receptors are G-protein coupled receptors (GPCRs) that activate signalling pathways via G-proteins [58, 59]. The N-methyl-D-aspartate (NMDA) receptor is one of three ionotropic glutamate receptors, of which the other two are AMPA and kainite receptors. Expression of the NMDA receptor occurs on neurons throughout the CNS and is made up of different subunits containing the NR1 subunit and any of the other 4 (NR2A-D) [60].

Excessive and persistent activation of the NMDA receptor is known as excitotoxicity and is cytotoxic. Excitotoxicity occurs through ionic imbalance, especially of calcium and sodium. As a result there is cellular swelling due to ionic imbalance ultimately leading to rupture of cell membranes [61], and activation of a variety of enzymes such as proteases and lipases that mediate endoplasmic reticulum stress and mitochondrial dysfunction [62-65] (Figure 1.2). In AD, the accumulation of Aβ sensitises neurons to glutamate-induced excitotoxicity [66, 67]. The mechanism by which Ab causes excitotoxic cell death has been postulated to require the involvement of post-synaptic tau. In mouse models of AD, the Src kinase Fyn was found to localise at the post-synapse in a tau-dependent manner where it phosphorylated the NR2b subunit of the

NMDA receptor resulting in excitotoxic cell death [68, 69]. Furthermore, tau-deficient neurons

7

Chapter 1 treated with NMDA were resistant to excitotoxic cell death [70], while site-specific phosphorylation of tau by p38g mitogen-activated protein kinase (MAPK) uncoupled the tau/Fyn/NMDA receptor complex and reduced Ab mediated excitotoxicity [43].

8

Chapter 1

Figure 1.2 Mechanism of excitotoxic cell death in the presence of Aβ.

The interaction of Aβ plaques and excess glutamate results in aberrant activation of the NMDA receptor. This leads to a large influx of calcium ions into neurons causing mitochondrial dysfunction, accumulation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. This ultimately leads to cell death.

9

Chapter 1

1.2.5 Current AD therapy Presently, therapies only target two of the pathological features of AD: loss of cholinergic neurons and excitotoxicity. To compensate for the loss of cholinergic neurons, drugs such as donepezil and rivastigamine aim to increase the concentration of acetylcholine (ACh) by inhibiting acetylcholinesterase (AChE), which is the enzyme that hydrolyses ACh [71, 72]. To limit the excitotoxic damage in AD, the NMDA receptor antagonist memantine has been used [72]. It should be noted however, that these treatments provide only transient symptomatic relief for AD patients but do not significantly alter the course of the disease.

Although there are multiple aspects to the pathophysiology of AD, there has been an enormous focus from pharmaceutical companies and basic research to decipher the pathological role of Aβ and develop Aβ-centric therapies including monoclonal antibodies and inhibitors targeting the amyloidogenic processing pathway of APP. As discussed above, these large clinical studies show no improvements in cognitive function, despite significant reductions in amyloid load [20-23, 73].

It can be argued that disease intervention targeting amyloid deposition needs to be tested before mild cognitive impairment presents. However, that in itself is difficult to achieve as while there are many studies identifying potential biomarkers, reproducibility and reliability remain a problem.

Thus, a shift in focus for potential AD therapy from amyloid load relieving compounds to other drug targets is inevitable.

1.2.6 Genetics and the risk for AD There is strong evidence from genome-wide association studies indicating abnormal lipid metabolism as having a significant role in the pathogenesis of AD [74-76]. Small nucleotide polymorphisms (SNPs) in both Clusterin (ApoJ) and ATP-binding cassette subfamily A Member

7 (ABCA7) have been shown to increase the risk of developing AD [75, 77-79]. ApoJ is an

10

Chapter 1 apolipoprotein while ABCA7 is a lipid transporter that is involved in the release of cellular cholesterol [80, 81]. In AD, loss of function ABCA7 variants have been detected and conferred an increased risk of developing AD with one study also showing ABCA7 deficiency accelerating Ab generation and AD pathology in vivo [82, 83]. SNPs associated with Triggering Receptor

Expressed on Myeloid cells 2 (TREM2) which is a receptor expressed on microglia and binds anionic ligands including lipids [84-86] also increases the risk of developing AD. Genome-wide sequencing identified a R47H substitution resulting in an increased risk of developing AD [87, 88] which functionally impairs lipid sensing and increased autophagy in microglia [84, 89]. Mutations in other lipid metabolic genes have also been implicated including phospholipase Cg2 (PLCG2).

Interestingly, the mutation in PLCG2 was found to be protective and the SNP was found to increase enzyme activity [90, 91]. It is therefore evident that lipid metabolism is intricately linked with AD pathogenesis.

The greatest genetic risk factor for developing late-onset AD however is being a carrier of the ε4 allele of the apolipoprotein E gene (ApoE) [92]. ApoE is the primary apolipoprotein in the CNS

[93]. There are three allelic variants of ApoE (ε2, ε3, and ε4) each conferring a different level of risk in developing AD. The frequency of these alleles in the general population has been estimated at 8%, 78%, and 14% for the ε2, ε3, and ε4 alleles, respectively [94]. Individuals with one ε4 allele are at a three- to four-fold increased risk of developing AD compared to individuals without the

ε4, while those who are homozygous carriers of the ε4 have a twelve-fold increase in risk [92].

The ε2 allele however is thought to be protective against the development of AD with carriers shown to confer a lower risk of developing AD [95, 96]. The mechanism by which the ε2 allele reduces AD risk is not well understood although one study found that the protein abundance for each allelic variant is isoform-dependent [97]. Signalling between ApoE isoforms was also found

11

Chapter 1 to be different with the ε4 variant being the most potent in enhancing APP synthesis [98]. On the other hand, the ε2 variant was found to occur with lower cyclophilin A expression, which controls

BBB integrity – another impairment thought to occur in AD [97, 99-101].

As an extracellular lipid chaperone, ApoE has been demonstrated to be secreted in conjunction with lipids such as sphingosine 1-phosphate (S1P), and S1P is present on lipoprotein particles in the cerebrospinal fluid (CSF) [102, 103]. S1P is a sphingolipid with cytoprotective properties and its role is discussed further below. Moreover, the balance of sphingolipids and cholesterol are crucial to the endocytosis of APP and secretion of Ab, which as discussed above is thought to be impaired in AD [104-106]. What is noteworthy is that multiple research groups and our own group have identified a significant reduction of S1P, particularly in the early pre-clinical stages of AD pathogenesis [107-109]. As will be described in further detail below, S1P is an essential molecule to mammalian physiology with well-explored pharmacology and is a current clinical drug target.

Pharmacological restoration of S1P signalling may provide a new avenue for therapeutic treatment in AD, which is the subject of this thesis.

1.3 Sphingolipids

Sphingolipids are a class of lipids defined by their sphingoid base backbone, which serves as the building block upon which more complex lipids are synthesised. Entry into the pathway begins with de novo synthesis of 3-ketodihydrosphingosine through the condensation of and palmitoyl CoA by serine palmitoyltransferase [110, 111]. 3-ketodihydrosphingosine is reduced in to form dihydrosphingosine – the first branching point in the sphingolipid pathway – which can be phosphorylated to form dihydrosphingosine 1-phosphate (dhS1P), the reduced form of S1P.

Dihydrosphingosine can be further reduced to form dihydroceramide and . Complex sphingolipids can be synthesised from ceramide, however for the purpose of this thesis, the focus

12

Chapter 1 will be on S1P metabolism. Ceramide can be converted to sphingosine by before phosphorylation to S1P (Figure 1.3). Exiting the pathway involves irreversible degradation of S1P or dhS1P by S1P lyase to phosphoethanolamine and hexadecanal, which can then be utilised in recycling back into the de novo synthesis pathway as palmitoyl-CoA.

1.4 Sphingosine 1-Phosphate

This review and thesis will focus on S1P. S1P is synthesised by phosphorylation of the membrane lipid sphingosine, by the enzymes sphingosine kinase 1 or 2 (SphK1/2). Its structure consists of a chain with a polar amino-phosphate head group attached [112]. S1P is an essential molecule in mammalian physiology that has roles in neural and vascular development. Mice with a double deletion of SphK1 and 2, thus lacking S1P, die in utero due to major haemorrhaging and neural tube defects [113].

The pro-survival effects of S1P are well documented. Early work showed that in HL-60 and U937 cell lines, cells in the absence of serum were protected from following S1P treatment

[114]. Additionally, overexpression of SphK1 resulted in proliferation of HEK293 and NIH/3T3 cells even in the absence of serum [115]. More recently, S1P has been shown to promote proliferation and survival of primary cells, including neurons [116, 117], astrocytes [118, 119], oligodendrocytes [120], and neural stem cells [121].

13

Chapter 1

Figure 1.3. Simplified sphingolipid metabolic pathway showing S1P synthesis and degradation.

Abbreviations: SPT: serine palmitoyl transferase, 3KDSR: 3-ketodihydrosphingosine reductase,

CerS: ceramide synthase, CDase: ceramidase, DEGS: Delta 4-desaturase sphingolipid, SphK1/2: sphingosine kinase, SGPP: sphingosine phosphate phosphatase, SGPL: sphingosine 1-phosphate lyase, HD: hexadecanal, PE: phosphoethanolamine. The exit point to the sphingolipid pathway exists with irreversible S1P degradation. S1P is degraded into hexadecanal and phosphoethanolamine. Adapted from [122].

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Chapter 1

1.4.1 Extracellular S1P signalling S1P has both extracellular and intracellular signalling capabilities. For its extracellular functions,

S1P is transported from the cytoplasm to the extracellular space by lipid transporters such as

SPNS2 [123], MFSD2B [124], and ABCA1 where it can act through autocrine or paracrine signalling [103]. There S1P can signal with nanomolar potency through a family of five GPCRs,

S1PR1-5 (Figure 1.4) [112]. S1P receptors are ubiquitously expressed across multiple organ systems including the immune, cardiovascular, renal and CNS among others, although the expression of individual receptors varies between cell types. Each receptor is able to couple to different combinations of heterotrimeric G-proteins, with S1PR1 coupling exclusively to Gai,

S1PR2 and S1PR3 coupling with Gai, Ga12/13, and Gaq/11, and S1PR4 and S1PR5 with Gai and

Ga12/13 [125]. This will be discussed further below. Through the binding of the G-proteins, the activation of subsequent signal transduction pathways produces a variety of downstream effects including the inhibition of adenylate cyclase, modulation of intracellular calcium concentrations, and the phosphorylation of proteins such as protein kinase B (Akt), and the extracellular signal- regulated kinases 1 and 2 (ERK1/2).

15

Chapter 1

Figure 1.4 Autocrine and paracrine signalling by S1P.

S1P is formed by phosphorylation of the plasma membrane component sphingosine by SphK1 or

2. It can then be transported out of the cell by specific transporters to act in an autocrine or paracrine fashion to activate a plethora of signalling pathways including ERK, Protein Kinase B

(Akt) and others as shown above. Each S1P receptor subtype can couple to different G-proteins.

Abbreviations: Ca2+: Calcium ion; CDase: Ceramidase; Cer: Ceramide; CerS: Ceramide

Synthase; DAG: Diacylglycerol; ERK: Extracellular Regulated Kinase; IP3: Inositol triphosphate;

PI3K: Phosphatidylinositol 3’ kinase; PLC: phospholipase C; PKC: Protein kinase C; Rho: Rho

GTPase; ROCK: Rho-associated protein kinase; S1PR1-5: S1P receptor 1-5; Sph: Sphingosine;

SphK1: Sphingosine kinase 1; SRF: Serum response factor; Adapted from [122] 16

Chapter 1

1.4.2 S1PR1 S1PR1 was first identified in 1990, in primary human endothelial cells treated with phorbol 12- myristate 13 acetate, and termed endothelial differentiation gene 1 (EDG1) [126]. Originally an orphaned receptor, S1P was discovered to be a ligand for EDG1 in 1998 [127]. Expression of

S1PR1 is quite ubiquitous across all tissue types in humans with high protein expression in multiple organs including the adrenal gland, brain, appendix and kidney [128]. Deletion of S1PR1 is lethal to mice as they do not survive past E14.5 [129]. As mentioned above, unlike other S1P receptors S1PR1 exclusively couples to Gai/o to inhibit adenylate cyclase and reduce cyclic- adenosine monophosphate. S1PR1 activation stimulates the Ras/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways [130, 131]. The structure of S1PR1 was resolved by X-ray crystallography and showed defining similarities to class A GPCRs, including

7 transmembrane helical domains connected by three extracellular loops linking helix II and III,

IV and V, and VI and VII [132]. S1PR1 can be post-translationally modified through phosphorylation by the heterotrimeric GPCR kinase-2 (GRK2) [133] and is internalised in a clathrin-mediated pathway involving recruitment of clathrin and its adaptor AP2 to the membrane, as well as engagement of β-arrestins [134]. Phosphorylation seems to be essential for internalisation of the receptor. Chavez et al. demonstrated that phosphorylation of S1PR1 on Y143 mediated internalisation on endothelial cells, as a phospho-defective mutant failed to internalise

[135]. It has also been shown that a deletion in the carboxyl terminal of S1PR1 prevents receptor phosphorylation and internalisation [136]. Interestingly, internalisation of S1PR1 following agonist activation can maintain a low level of ERK phosphorylation for five hours after initial stimulation [137].

17

Chapter 1

Clinically, S1PR1 is of great interest due to its role in promoting the egress of out of lymph nodes and into the circulation, in response to a chemotactic gradient of high S1P in the blood and much lower concentrations in lymph nodes [138]. Due to this effect, S1PR1 is a clinical drug for autoimmune diseases, as discussed further below.

1.4.3 S1PR2 S1PR2 and S1PR3 were first discovered to be receptors for lysosphingolipids, particularly S1P, in a study in 1997 and originally termed EDG5 and EDG3, respectively [139]. Unlike S1PR1, protein expression of S1PR2 is mainly localised to the gastrointestinal tract and kidneys in humans with low expression in other tissues like the CNS [128]. Apart from coupling to Gai/o, S1PR2 can also couple to Gaq, or Ga12/13, resulting in it theoretically controlling a wide variety of intracellular cascades. In vivo studies have identified a role for S1PR2 signalling in regulating vascular in the kidney through activation of the Ga12/13-Rho/ROCK-NFκB pathway [140] and vascular permeability via S1PR2-ROCK-PTEN [141]. S1PR2 also regulates bile acid-induced cortisol secretion through a S1PR2-ERK-dependent pathway [142] and has been shown to activate

Gaq-Rho to inhibit migration of vascular smooth muscle [143].

1.4.4 S1PR3

Like S1PR2, S1PR3 can also couple to all three of Gai, Ga12/13 and Gaq. Current data from the

Human Protein Atlas does not have any details on protein expression of S1PR3 in any tissue. RNA- sequencing data, however, has identified that S1PR3 mRNA is present in most organs including the brain, endocrine tissues and haematopoietic systems, but is highest in the smooth muscle of the gastrointestinal tract [128]. S1PR3 is a key regulator of inflammation. In a mouse model of lung injury, knocking out S1PR3 attenuated inflammation and fibrosis [144]. Inflammatory

18

Chapter 1 markers such as (IL-6), vascular endothelial growth factor A (VEGFA) and cyclooxygenase-2 were induced in a S1PR3-RhoA-dependent manner in astrocytes [145].

Moreover S1PR3 regulates the migration of macrophages and monocytes in response to a S1P gradient in vitro [146] and S1PR3 knockout mice are more susceptible to bacterial sepsis [147].

S1PR3 has also been shown to regulate leukocyte rolling through activation of Gaq and phospholipase C-dependent mobilisation of intracellular calcium levels [148].

1.4.5 S1PR4

S1PR4 is able to couple to Gai/o and Ga12/13 but not Gaq/11 [149]. Unlike S1PR1-3, which are more ubiquitously expressed, expression of S1PR4 is limited mainly to immune cells [150]. The Human

Protein Atlas also does not provide any details for protein expression of S1PR4, however RNA sequencing data shows high expression in , lymph nodes and appendix [128]. S1PR4 was first identified as a receptor for S1P in the year 2000 with initial studies in HEK293 showing

S1PR4 activating an ERK pathway that was pertussis toxin sensitive indicating Gai-coupling properties of the receptor [151]. Functionally, S1PR4 has been demonstrated to be involved in cytokine secretion with pathogenic interferon a (IFNa) production in dendritic cells reduced as

S1PR4 signalling prevented the internalisation of the ILT7 via the RhoA-

ROCK pathway [152]. S1PR4 is also involved in dendritic cell differentiation, while also demonstrated to regulate cytokine secretion and proliferation of T-cells but not migration [153,

154].

1.4.6 S1PR5

Similar to S1PR4, S1PR5 also couples to Gai/o and Ga12/13. Expression of S1PR5 is more or less limited to CNS although there are also reports detailing expression in immune cells. Studies in

19

Chapter 1 immune cells indicate that S1PR5 is involved in migratory responses. In one study, knockout of

S1PR5 significantly affected the ability of monocytes to migrate out of the bone marrow into the periphery [155]. While there were less patrolling monocytes in the periphery, Debien et al established this to be independent of S1P as S1P was not a chemoattractant for monocytes [155].

The pro-migratory effects of S1PR5 were also detected in natural killer cells, where studies have shown that S1PR5 regulates natural killer cell trafficking towards an S1P gradient [156, 157].

Aside from immune cells, S1PR5 has also been shown to play an important role in regulating the

BBB and endothelial tight junctions [158]. Targeting S1PR5 with the specific agonist A-971432 could significantly ameliorate motor dysfunction in a mouse model of Huntington’s disease (HD), which was attributed to a rescue of protein expression specific for tight junctions, such as Claudin-

5 and Occludin [99].

1.4.7 Intracellular S1P effects While not as well characterised as its extracellular functions, emerging evidence suggests that S1P plays a role independent of its receptor signalling capabilities. This is dependent on the cellular localisation of sphingosine and the sphingosine kinases. For example, one study found isolated nuclei from MCF-7 cells treated with S1P reversed the decreased histone acetylation on H3-K9,

H4-K5, and H2B-K12 residues when SphK2 was depleted with short interfering RNA (siRNA).

This effect could be explained by the fact that the study also found that S1P was able to bind histone deacetylases 1 (HDAC1) and HDAC2 to inhibit their activity [159]. The ability of S1P to inhibit HDACs was further validated in a later study showing FTY720-phosphate (FTY720-P), which is an S1P structural analogue, could also inhibit class 1 HDACs and enhance histone acetylation [160]. This inhibition of HDACs resulted in rescue of memory deficits indicating a role for FTY720-P independent of its immunosuppressive effects through S1P receptors, which is 20

Chapter 1 discussed below. Other intracellular effects of S1P have been reported that differ from its role in the nucleus, mainly hypothesised as a protein binding partner in the cytoplasm. S1P was found to be a co-factor for the E3 ubiquitin ligase TNF receptor-associated factor 2 and as binding partner for prohibitin 2, which is a protein that localises to the mitochondria and regulates mitochondrial assembly and function [161, 162]. Binding of S1P to TRAF2 resulted in polyubiquitination of receptor interacting protein 1, which plays a role in nuclear factor k-light chain-enhancer of activated B-cell (NFkB) signalling [161], while binding of S1P to prohibitin 2 was found to be crucial to cytochrome-c assembly and regulation of mitochondrial respiration [162]. These results highlight a role for S1P produced in the nucleus.

1.5 S1P in the CNS

Within the CNS, S1P receptor expression is cell-type specific. Publicly available RNA sequencing data from the Barres Lab has demonstrated that both neurons and astrocytes express S1PR1-3, although the levels of S1PR1 and S1PR3 are significantly higher in astrocytes compared to neurons

[163-166]. Generally, S1PR4 expression is limited to lymphoid tissue and plays a role in immune cell function and inflammation, however expression has been detected in murine but not human microglia [163, 164, 167, 168]. Oligodendrocytes express both S1PR1 and S1PR5, although

S1PR5 is the dominant receptor in these cells [163, 164, 169] .

1.5.1 Neurons and S1P signalling In the CNS, neurons represent a significant proportion of cells and are the most affected by AD pathology. In pathology-free conditions, S1P plays a major role in neuronal function that is dependent on S1P receptor signalling mainly through S1PR1 and S1PR3. S1P has been shown to regulate synapsin I to enhance glutamate secretion from pre-synaptic termini, which is a

21

Chapter 1 prerequisite for forming hippocampal-dependent spatial memory, a key impairment in AD [170-

172]. When the hippocampal slices were pre-treated with an S1PR3 antagonist, the effect was abrogated. In the same study, it was found that long term potentiation (LTP) was dependent on

SphK1 activity in the CA3 region. Subsequent tests found that SphK1 knockout mice performed poorly in the Morris water maze, which is a spatial memory test where mice that are able to induce

LTP perform well. LTP is the process which is believed to promote memory formation and consolidation through activation of the NMDA receptor resulting in strengthening of synaptic connections [173, 174]. Additionally, one study also found that SphK1 in Caenorhabditis elegans was able to regulate the release of ACh, which may be important given reduced cholinergic signalling is present in AD [175].

Neuritogenesis is a complex and tightly regulated process controlling neuronal process elongation and retraction that can also be regulated by S1P. Quarta and colleagues have shown that dorsal root ganglia neurons exhibited retraction in response to S1P in an S1PR3-RhoA dependent manner. Furthermore, a sphingosine mimetic was able to enhance neurite elongation [176]. S1P signalling has also been implicated in neurogenesis. Neural progenitor cells exposed to S1P began to proliferate and differentiate in an ERK and Rho-dependent manner respectively [121]. This could also be achieved in the presence of S1P-primed astrocytes suggesting a paracrine signalling event between glial cells and neurons [177]. In a rat model of traumatic brain injury, treatment with an S1PR1 specific agonist (SEW2871) resulted in increased neurogenesis following insult.

This was inhibited with an antagonist, but also with an ERK inhibitor. This suggests that ERK signalling is downstream of S1PR1 and plays an important role. Ye and colleagues also found that

S1PR1-ERK signalling is required for spatial memory [178]. This and the above evidence implicate a role for both S1PR1 and S1PR3 signalling in the hippocampus and memory formation.

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Chapter 1

Recently, a novel S1P-independent function of the S1PR2 receptor has been identified whereby it was found that it bound the extracellular domain of the membrane associated protein Noga A:

Nogo-A-Δ20. Interestingly, NogoA-Δ20 was found to bind a site distinct from the S1P binding pocket to signal through Ga13-RhoGEF-LARG-RhoA [179]. This signalling event was determined to regulate synaptic plasticity, as antagonising S1PR2 enhanced LTP in wild-type but not in Nogo-

A-/- mice. Furthermore, deleting S1PR2 abrogated Nogo-A-Δ20 induced neurite outgrowth. This suggests that S1PR2 is able to manipulate synaptic plasticity and neurite outgrowth in a S1P- independent manner as well as function through S1P-dependent pathways.

1.5.2 Astrocytes and S1P signalling Within the glial population of the CNS, astrocytes are the most abundant cell type and play a significant role in maintaining neuronal health and homeostasis. Astrocytes also contribute to disease states through the process of astrogliosis, and the formation of glial scars. Spatially, astrocytes reside in close proximity to neurons and have a mutually dependent relationship to maintain metabolic homeostasis, as well as promoting neuronal functions. One example of this is the -dependent recycling of the neurotransmitter glutamate, where release of glutamate from the pre-synaptic neuron can be recycled into glutamine through uptake via the cell surface excitatory amino acid transporter on astrocytes [180].

As mentioned previously, astrocytes also express S1PR1 and 3, with very low expression of S1PR2

[118, 163, 164] . Studies have found that S1P regulates proliferation in astrocytes. In vitro stimulation of striatal astrocytes with S1P resulted in proliferation that is not observed with a similar -derived signalling molecule [118]. However, a separate study found that both S1P and lysophosphatidic acid promote proliferation of astrocytes

23

Chapter 1 through the MAPKs and Rho, and increase astrocyte expression of glial fibrillary acidic protein

(GFAP) [181]. Along with regulating proliferation, S1P signalling through S1PR1 was shown to promote astrocyte migration [165].

S1P signalling specifically via S1PR1 on astrocytes has been shown to be an essential regulator of neuroprotection [182, 183]. The neurotoxic A1 astrocyte phenotype was found to be significantly higher in AD, whereas treatment of astrocytes with S1P induces the neuroprotective A2 phenotype

[55]. Moreover a previous study found that cuprizone-induced demyelination occurred in tandem to activation of astrocytes and astrogliosis, while treatment with the S1PR1-specific agonist

CYM5442 or general S1P receptor agonist FTY720 resulted in suppression of proinflammatory markers in astrocytes such as IL-6 [182] as well as the suppression of reactive astrocyte activation

[183]. An independent study found neuroprotection through FTY720 has also been proposed to occur via the blockade of nitric oxide production in astrocytes [184]. The aforementioned studies have clinical relevance as it has been demonstrated that S1PR3 is up-regulated on astrocytes in an inflammatory setting [185]. There is a significant up-regulation of both S1PR1 and S1PR3 in astrocytes of multiple sclerosis (MS) patients [186].

1.5.3 S1P signalling in Microglia and Oligodendrocytes Microglia and oligodendrocytes make up the rest of the glial population and play an important role in maintaining CNS homeostasis, with their roles involving immune surveillance and myelinating neurons, respectively. As mentioned above, both cell types express S1P receptors [163, 164, 168,

169]. S1P receptors on microglia have been demonstrated to regulate the immune response. As the

CNS-resident immune cell, the number of activated microglia is significantly increased in MS lesions and experimental autoimmune encephalomyelitis (EAE) [187, 188]. Inhibition of S1PR1 24

Chapter 1 on astrocytes reduced this activation and this may indicate a role for astrocyte-microglia interaction through S1PR1 activation. Transition to a pro-inflammatory phenotype is associate with S1PR2 activation in the context of cerebral ischaemia, as an S1PR2 antagonist attenuates the proliferation and expression of proinflammatory cytokines such as TNF-a and IL-6 [189]. In a separate study investigating transient focal cerebral ischaemia, pharmacological inhibition or genetic deletion of

S1PR3 reduced microglial expression of pro-inflammatory markers such as TNF-α and IL-1β, which are characteristic of the phenotype associated with acute inflammatory responses [190].

Oligodendrocytes are the cell type responsible for myelinating neurons to promote efficient electrical conduction along . Unlike the other cell types in the CNS, oligodendrocyte S1PR expression is primarily to S1PR5, with limited expression of the other receptors [163, 164, 169].

In fact, S1PR5 is unique to oligodendrocytes in the CNS, and the majority of studies have focused primarily on the role of S1PR5 on oligodendrocytes. However, there appears to be expression of both S1PR1 and S1PR5 in oligodendrocyte precursor cells (OPCs) [191]. OPCs treated with S1P or FTY720 were found to promote OPC differentiation to mature oligodendrocytes and promoted myelination of neurons [191, 192], while S1PR1 was found to promote platelet-derived growth factor induced mitogenesis of OPCs [191]. Other studies identified a dual role for S1PR5 in the induction of process retraction and cell survival. In immature pre-oligodendrocytes, S1P treatment induced process retraction through a Rho kinase dependent pathway. Conversely, the pro-survival effect of S1P was only present in mature oligodendrocytes and not in premature cells and this was mediated through a pertussis toxin sensitive pathway. Interestingly, these results indicate distinct signalling pathways that are dependent on the stage of development [120].

25

Chapter 1

Survival of oligodendrocytes mediated through S1P has also been demonstrated. Jaillard and colleagues demonstrated that serum withdrawal negatively impacted oligodendrocyte survival, while treatment with 100 nM S1P protected oligodendrocytes in a S1PR5-Gai sensitive manner

[120]. An independent study also found that treatment with FTY720 protected oligodendrocyte progenitors from the cytotoxic effects TNFa and IFNg [193]. Other research groups have identified a role for S1P receptors in the myelination process by investigating the effect of FTY720 in disease models. In one study, remyelination following lysolecithin-induced demyelination of organotypic slice cultures was improved with FTY720 treatment [194], while a separate study using the cuprizone model of demyelination showed reduced demyelination with FTY720, without specifically identifying the receptor subtype mediating this response [195]. FTY720 treatment also promotes neural stem cell differentiation towards an oligodendrocyte lineage and enhanced myelination in a non-inflammatory model of MS, when co-injected with neural stem cells [196].

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Chapter 1

Figure 1.5 Summary of S1P actions on CNS cells.

Summary of the distribution and function of S1P receptors, which are expressed on the cell types in the CNS and thus regulates multiple functions on the various cell types.

1.6 S1P therapeutics: the first clinical drug

MS is an autoimmune disease characterised by demyelination due to auto-reactive T-lymphocytes attacking the that surrounds axons [197]. In the immune system, S1P controls the egress of

T-lymphocytes from lymphoid tissue through S1PR1 signalling in response to an S1P gradient

[138, 198]. The Food and Drug Administration approved FTY720 as the first oral treatment option for relapsing MS in 2010 although the drug itself was first discovered in the mid 1990s [199]. Due to its immunosuppressive effects, FTY720 was initially trialled for organ transplant to prevent organ graft rejection [200-202]. Further studies demonstrated that FTY720 treatment effectively ameliorated EAE [203-205]. FTY720 is phosphorylated in vivo exclusively by SphK2 [206-208] to become the active drug FTY720-P, which mediates the lymphopaenia response [209]. FTY720-

P acts by stimulating internalisation and degradation of S1PR1 receptors [210],

27

Chapter 1 preventing the chemotactic response to S1P in the blood. This causes sequestration of lymphocytes within lymph nodes, thus preventing their autoreactive phenotype towards myelin and limiting neurodegeneration (Figure 1.5).

Figure 1.6. Mechanism of FTY720-P in MS.

FTY720-P is a structural analogue of S1P. Normally, lymphocytes react to the inflammatory cytokines produced by astrocytes and cause demyelination. In MS, FTY720-P is known to act as a super-agonist of S1PR1 receptors on lymphocytes, causing S1PR1 internalisation and degradation that inhibits lymphocyte migration from lymph nodes into the blood stream, thus inhibiting demyelination. FTY720-P can also act on the S1PR1 receptors on astrocytes, suppressing their inflammatory response in MS [188].

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Chapter 1

FTY720-P is an agonist for all five S1P receptors except S1PR2, but is especially potent in activating S1PR1 [208]. Activation of S1PR1 by FTY720-P causes momentary signalling, before recruitment of GPCR internalisation- and degradation-associated proteins [211]. This mechanism has been termed functional antagonism as long-term treatment results in S1PR1 desensitisation.

However, recent evidence has indicated that FTY720-P can also act as an agonist of S1P receptors and this is functionally important to its neuroprotective effect [137]. S1PR1 activation by FTY720-

P on astrocytes is necessary to confer protection in EAE, a mouse model of MS [188]. Moreover, a short-acting S1PR1-specific agonist that accumulates in the CNS and does not induce long-term lymphopenia as seen with FTY720-P also attenuated demyelination in EAE [212]. This suggests that the mechanism behind FTY720-P may not entirely be dependent on systemic immunosuppression and involves its agonist properties on S1P receptors.

1.7 S1P in Alzheimer’s disease

Although much research has gone into the effects FTY720 has on AD pathology, there is a lack of research into the role of S1P in AD pathology. Our research group demonstrated a decrease in S1P levels with increasing Braak pathology in AD [109]. Loss of S1P was significant in the pre-clinical

Braak III and IV stages of the disease pathogenesis. Interestingly, the loss of S1P was most pronounced in areas of the brain – the hippocampus and temporal cortex - that are heavily affected by AD pathology. Reduction of SphK1 expression and an up-regulation of the S1P degrading enzyme S1P lyase in AD brains have also been demonstrated [107, 109]. The intracellular compartmentalisation of SphK2 is also altered in AD brains with a shift from cytoplasmic to nuclear localisation [213].

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A recent study from our group, using post-mortem hippocampal tissue from neurologically normal subjects with no significant neuropathology at time of death, found age-dependent changes to brain sphingolipid metabolism in both male and female subjects. Surprisingly, gender played a significant role in the sphingolipid metabolic alterations, with males showing increased

(C16:0, 22:1, 24:1), sphingomyelin (C16:0, 16:1, 22:1), and sulfatides (C22:0, 22:1, 24:0, 24:1,

24:1 –OH) with increasing age, whereas in females, only a decline in S1P with increasing age was observed [214]. Interestingly, females also have a higher incidence of AD, even after accounting for their longer lifespan [215]. These results suggest that S1P may play a role in regulating neuronal homeostasis, whereby the loss of S1P in pre-clinical stages of AD sensitises neurons to cytotoxic insults. The MS drug FTY720-P is an agonist at four of the five S1P receptors and this raises the question of whether pharmacological restoration of S1P signalling in AD brains would be neuroprotective.

1.8 Neurotrophins and AD

Neurotrophins refer to the family of peptides that play a crucial role in regulating the development, maintenance, and function of the CNS. While there are many neurotrophic factors in the CNS that also provide similar support, the neurotrophins exclusively refer to neurotrophin 3 (NT-3), NT-4, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). These peptides share a high level of homology and signal through two families of receptors. These include the tyrosine receptor kinase (Trk) receptors: TrkA, TrkB and TrkC [216], and the p75 neurotrophin receptor

[217]. Each neurotrophin has a different affinity for the different receptors. For example, BDNF has a high affinity for TrkB [218], while NT-3 has a high affinity for TrkC [219]. Activation of these receptors stimulates in intracellular signal transduction pathways that result in modulation of neuronal plasticity and survival [220]. Within the CNS, resident cells have been shown to secrete

30

Chapter 1

BDNF including astrocytes and neurons [221, 222]. Upon binding to the TrkB receptor, BDNF activates classical pathways such as ERK1/2 and PI3K-Akt to exert its pro-survival effects [223].

For this review, the main focus will be on BDNF and the role it plays in AD.

BDNF also plays an important role in memory formation through the promotion of LTP. Intra- hippocampal infusion of BDNF was shown to trigger LTP through a MEK-ERK pathway [224], while mice harbouring a deletion of the coding sequence of the BDNF gene showed significant impairment of hippocampal LTP [225]. In the context of AD in vivo experiments have shown a protective effect of BDNF on transgenic mice overexpressing Aβ, and in transgenic mice with tauopathy [226, 227]. Interestingly, in both cases there were improvements to cognitive deficits without affecting either pathologies. Furthermore, studies have shown a negative correlation in

BDNF mRNA levels in the hippocampus and serum, with AD severity and rate of cognitive decline

[228-230].

Aside from AD, BDNF is of therapeutic interest to other neurodegenerative diseases. Reductions in BDNF have also been observed in HD [231, 232], Parkinson’s Disease (PD) [233, 234] and

MS [235]. In PD, a correlation between motor impairment as well as cognitive ability with serum levels of BDNF has previously been observed [236, 237]. In addition, with its well established cytoprotective effects, BDNF treatment has been demonstrated to protect substantia nigra neurons against excitotoxic cell death [238], to protect striatal neurons in mouse models of HD [239] and improved motor dysfunction in mouse models of HD [240]. In the context of MS, BDNF is known to promote not only oligodendrocyte differentiation [241, 242] but also myelination through direct actions on oligodendrocytes [243]. Given the lack of disease modifying therapies in

31

Chapter 1 neurodegenerative diseases, targeting the up-regulation of neurotrophic factors such as BDNF is an attractive alternative.

1.9 S1P receptor agonists in the context of AD

Current therapies for AD focus on minimising further neurodegeneration incurred by excitotoxicity and preserving cholinergic function caused by loss of cholinergic neurons, through inhibition of ACh degradation. Improving or rescuing cells committed to apoptosis would be a significant improvement but is yet to be achieved in AD therapy. Attempts to introduce neuroprotective peptides such as BDNF have proven challenging without a carrier molecule as it cannot cross the BBB [244]. With well characterised pharmacology, interest in repurposing and developing S1P receptor modulators for neurodegenerative diseases has become a popular research field, especially as current S1P receptor modulators are brain-penetrant [245, 246]. Aside from

AD, studies have also identified changes to the sphingosine/S1P axis in HD and PD [247, 248].

FTY720-P has been shown to be neuroprotective in a wide array of neurological disease models including EAE [188], Rett Syndrome [221], stroke [249] , HD [250], PD [251], and AD itself [252,

253]. Several of these studies have observed that following treatment with FTY720-P there is a significant up-regulation of BDNF, and this is proposed as the mechanism through which neuroprotection is achieved. Several of these studies have reported that BDNF is up-regulated in neurons through S1PR1 signalling [221, 254, 255]. In the context of AD, FTY720 was shown to protect neurons against Ab-mediated toxicity through the secretion of BDNF [255]. In contrast,

Hoffman et al also reported the up-regulation of the neurotrophic factors heparin-binding epidermal growth factor-like growth factor (HBEGF), leukaemia inhibitory factor (LIF) and IL-

11 in astrocytes following S1P and FTY720 treatment [256]. LIF is of particular interest for AD as it promotes cholinergic differentiation and activity through induction of

32

Chapter 1 acetyltransferase activity [257, 258]. The neuroprotective properties of LIF have also been described. For example, neurons in the were protected from cell death following photoreceptor injury in a LIF dependent manner and the survival of motor neurons was increased in the presence of LIF [259, 260].

In addition to the induction of neurotrophic factors FTY720 is also a potent modulator of inflammation and excitotoxicity, which are major contributors to the pathogenesis of AD. FTY720 treatment has been demonstrated to reduce nitric oxide production in astrocytes as a way to suppress inflammation as excessive nitric oxide is cytotoxic [184, 261, 262]. Suppression of pro- inflammatory cytokines has also been observed with two studies showing astrocytes treated with

FTY720-P resulted in significantly reduced expression of B-cell activating factor, chemokine interferon-g inducible protein 10kDa, nitric oxide synthase 2 and interferon-g among others [182,

256], and another study demonstrating improvement of memory in an AD mouse model [252].

Moreover, FTY720 reduced cytotoxic activation of astrocytes and microglia as measured by levels of GFAP and Iba1 immunoreactivity in a mouse model of AD [253]. Both in vitro and in vivo models of excitotoxicity also exhibited a reduction in cell death with FTY720 demonstrating protective effects of FTY720 in this paradigm [263, 264].

Aside from FTY720, both S1P and the S1PR1 agonist SEW2871 have demonstrated neuroprotective responses with reduced cell death when cultured with Ab in vitro [265, 266].

Interestingly, Ab was found to reduce expression of S1PR1 and SEW2871 reversed this effect.

Furthermore, treatment with SEW2871 also improved spatial memory impairment in rats injected with Ab [266]. In conclusion, the therapeutic potential of S1P receptor agonists should be explored as an adjunct therapy to current treatments.

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Chapter 1

1.9.1 New generation S1P receptor agonists in clinical trials Since the approval of FTY720, more selective S1P receptor agonists have been developed to reduce the clinical side effects associated with the action of FTY720-P on multiple S1P receptors.

Bradycardia is one of the clinical side effects associated with the off-target effects of FTY720 on

S1PR3 [267]. To address this issue Novartis developed the second-generation S1PR modulator

Siponimod, which has increased selectivity for S1PR1 and S1PR5 while sparing S1PR3 and

S1PR4 [268, 269]. In organotypic cultures, treatment with lysophosphatidylcholine induced demyelination and was able to attenuate this [270]. Siponimod has proven effective particularly for the secondary progressive and relapsing-remitting forms of MS in clinical trials and received FDA approval in March 2019 as the first orally-available treatment for secondary progressive MS [271-273]. Ozanimod is a S1PR1/5 dual agonist that has also shown promise in 2 independent phase 3 clinical trials for MS, and its approval by the FDA is currently under review

[274, 275]. The S1PR1-selective agonist was developed in 2010 and is currently under investigation as a novel therapy for psoriasis and MS [276-278]. Although the aforementioned molecules have displayed efficacy for diseases with an autoimmune component, their effects in other neurodegenerative diseases such as AD are yet to be investigated thoroughly.

1.9.2 S1P receptor agonists for biological studies As there is widespread distribution of S1P receptors across multiple organ systems, development of selective receptor agonists and antagonists is highly desirable to investigate the biology of these receptors, in addition to the potential therapeutic value. Initially, specific receptor agonists focused on S1PR1 to mimic the desirable effects of lymphopenia and immunosuppression essential to treating autoimmune diseases such as MS. In 2004 Sanna et al. developed the S1PR1-specific agonist SEW2871 that, unlike S1P, does not have a solubilising or charged headgroup [267].

SEW2871 displayed a 20-fold greater half maximal receptor activation (EC50) in GTP-γS binding 34

Chapter 1 assays compared to S1P, while still in the nanomolar range. In vivo studies showed the plasma

EC50 for lymphopenia was approximately 2 μM. SEW2871 was inactive at 10 µM for S1PR2-5

[267]. With the discovery that S1PR1 agonists do not require structural similarities to S1P, the

S1PR1 agonist CYM5442 was developed [279]. Similar to SEW2871, CYM5442 is also a full agonist of S1PR1 and a potent inducer of S1PR1 internalisation in vitro, and lymphopenia in vivo, while also crossing the BBB. As brain-penetrant compounds, both SEW2871 and CYM5442 affect

S1PR1-expressing cells in the CNS. For example, cortical neurons treated with SEW2871 showed significant ERK phosphorylation and increased levels of BDNF [221]. In a cuprizone model of

MS, treatment with CYM5442 abrogated demyelination [183]. This result was demonstrated with

FTY720 treatment in an independent study [280]. Additionally, treatment with CYM5442 reduced several pro-inflammatory markers such as IL-1β and IL-6. These results are significant, as unlike the experimental autoimmune encephalomyelitis model, demyelination is caused by oligodendrocyte death through the use of a copper chelator rather than depending on an autoimmune reaction. Thus, the protective effects of CYM5442 (and FTY720) are through direct modulation of S1PR1 on CNS cells.

While there has been considerable progress in developing selective agonists for S1PR1, there has been little development of S1PR2 and S1PR3 agonists. CYM5520 and CYM5541 were developed as selective agonists for S1PR2 and S1PR3, respectively [281-283]. Both small molecules are allosteric agonists, i.e. the binding pocket of these compounds is distinct from that of S1P, yet the compounds maintain full agonist properties. However, studies on these agonists have so far been limited. There are no studies investigating S1PR2/CYM5520 pharmacology in the CNS, while there are a few in vivo studies conducted in the context of osteoporosis [284, 285]. There are also limited studies in vitro with only one using the S1PR2 agonist CYM5478 [281, 286]. In this study,

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Chapter 1

Herr et al. identified an otoprotective role for CYM5478. Induction of cell death in C6 cells with cisplatin was significantly attenuated following treatment with CYM5478. Similarly, aside from the study characterising the compound, there is also limited research into CYM5541, and a majority of these studies focus on S1PR3 pharmacology in the context cardioprotection. The effects of CYM5541 in the CNS have currently been investigated in a single study, which identified S1PR3 as primarily mediating the enhanced excitability of sensory neurons, thus resulting in increased action potential firing [287]. Interestingly, the study identified the requirement of dual receptor activation involving S1PR1 and S1PR3.

1.9.3 S1P receptor antagonists In contrast to S1P receptor agonists, antagonists have been used quite widely to elucidate the roles of specific receptors. There exist specific antagonists targeting every S1P receptor. W146, JTE013,

TY52156 and CYM 50358 are selective antagonists of S1PR1, S1PR2, S1PR3, and S1PR4, respectively [288-291]. While there is one study recently describing a selective S1PR5 antagonist, it is yet to be made publicly available and the structure has not yet been divulged [156]. Through the use of antagonists progress has been made into elucidating noteworthy molecular functions involving S1P receptors including the key finding that BDNF upregulation following FTY720 treatment, and lymphopenia invoked by FTY720, are both mediated through S1PR1 signalling

[254, 288]

1.10 Aims of this thesis

To date, the majority of studies that have identified a neuroprotective role for S1P in the paradigms mentioned above have mainly employed S1P receptor agonists rather than the native ligand S1P.

There is currently one study showing direct neuroprotection of neurons treated with Ab when pre- treated with S1P [265]. Currently, the cell-type mediating the S1P-induced neuroprotective 36

Chapter 1 response has not been clearly established with some reports indicating neurons but not glia are secreting neurotrophic factors to protect neurons [255, 263], while others have noted the ability of astrocytes to secrete neurotrophic factors and protect neurons from cell death from cytotoxic insults such as glucose deprivation and EAE amongst others [119, 184, 188, 256]. Additionally, the signally pathways activated by S1P are not fully elucidated. Given the interest in repurposing

S1P receptor agonists as potential therapeutic drugs for neurodegenerative disorders it is essential to investigate the molecular mechanisms involved in S1P-mediated neuroprotection. Therefore, this thesis aimed to:

1. Investigate the effects of S1P-induced neurotrophic factor gene and protein up-regulation

in neurons, astrocytes and microglia in vitro

2. Elucidate the receptors mediating the induction of neurotrophic factors using specific

receptor antagonists

3. Delineate the signalling pathways activated by S1P through the use of mass spectrometry

4. Compare the neuroprotective properties of S1P and FTY720-P in an in vitro model of

excitotoxicity

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Chapter 2

Chapter 2: Methods

2.1 Cell culture methods

2.1.1 Cell culture conditions All cell culture reagents, unless specified were purchased from Life Technologies (VIC, Australia).

Cell culture flasks and plates were purchased from Corning Life Sciences (NY, USA). A Class II

Biosafety Cabinet was used for the maintenance and culturing of cells for experiments.

Mycoplasma testing was performed to ensure absence of contamination.

2.1.2 Immortalised cell lines The U251 glioblastoma cells (American Type Culture Collection, RRID: CVCL 0021) were cultured in Dulbecco’s Modified Esssential Media (DMEM) (11995065), supplemented with 10%

Foetal Bovine Serum (FBS) and 2 mM L-glutamine (25030-081) and maintained in a humidified atmosphere at 37 °C and 5% CO2. When confluency reached approximately 80%, cells were washed with warm Dulbecco’s Phosphate-buffered Saline (D-PBS) (14190250) before detachment with 0.05% trypsin-EDTA (15400054). Trypsin was neutralized by addition of serum containing media. Dissociated cells were collected in falcon tubes before centrifugation at 350 g for 5 min, then resuspended for viability counting. Concentration of live cells was determined by trypan blue

(15250061) exclusion with a haemocytometer and cells were sub-cultured or plated for experiments at the desired concentration with fresh media. Fresh media was replaced every two to three days. U251 cells were used up to passage number 30.

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Chapter 2

The immortalized microglial cell line BV-2 was kindly shared by Professor Gilles Guillemin. BV-

2 cells were maintained in DMEM media supplemented with 10% FBS and 2 mM L-glutamine.

Upon reaching 70-80 % confluency, cells were passaged with trypsin, centrifuged, and re-plated at desired concentrations. For experimentation, cells were plated at 105 cells/mL. Prior to treatements, cells were serum starved overnight in OptiMEM (Life Techologies 31985070).

2.1.3 Primary human cells Primary human embryonic astrocytes and neurons were isolated from the Human Fetal Tissue

Biobank at Macquarie University Hospital, under ethics approval #5201600719 as previously described [292]. Experiments with primary human cells were approved by the University of

Sydney human ethics committee (#2017/270). Primary human astrocytes and neurons were isolated by Dr Benjamin Heng. Primary human astrocytes were cultured in Roswell Park Memorial

Institute (RPMI) media (11875119), with 10% FBS and 2 mM L-glutamine. Media was refreshed every 2-3 days for astrocytes. For experimentation, cells were detached and counted as described above and seeded at a density of 105 cell/mL unless otherwise specified. Primary human neurons were cultured in Neurobasal media (21103049) with 0.5% B27 supplement (17504-044), 1%

Glutamax (35050061), 1% penicillin/streptomycin solution (15140-122), and 0.5% glucose, on plates coated with Matrigel (BD Bioscience, 352234) diluted 1:20 in Neurobasal medium. Neurons were maintained in their original media until experimentation.

2.1.4 Primary mouse cells Primary mouse neurons and astrocytes were isolated from E16.5 C57BL/6JAusb mice, the

Australian line of the Jackson C57BL/6J strain (Jackson stock 000664). Time‐mated female mice were purchased from Australian BioResources, Mossvale, Australia, and used on the day of arrival.

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Chapter 2

Pregnant females were killed by cervical dislocation, and the embryos decapitated immediately after removal from the embryonic sacs. These procedures were approved by the UNSW Sydney animal ethics committee (approval #17/113A with co-supervisor A/Prof Thomas Fath).

Primary murine hippocampal neurons were seeded and cultured by members of A/Prof Thomas

Fath’s lab as previously described [293]. In brief, a pregnant female mouse was sacrificed when pups were at E16.5 days and hippocampi were dissected from embryos. Hippocampi were digested in trypsin for 30 min at 37 °C before addition of DNase I. Tissue was allowed to settle before manual trituration with sterile glass pipettes to achieve a single cell suspension. Cells were pelleted by centrifugation at 300 g for 7 min and counted before plating. For hippocampal co-cultures, neurons were plated at a density of 7 x 104/coverslip neurons were seeded onto glass coverslips pre-coated with 0.1 mg/mL poly-D-lysine (PDL) (P6407, Sigma Aldrich) and surrounded by a ring of mixed cortical cells. Hippocampal neurons without a cortical support ring were seeded at 5 ´

105 cells/coverslip. Neurons were maintained in Neurobasal medium supplemented with B27 supplement, 2 mM L-glutamine, 5 mM glucose and 0.5 mM sodium pyruvate (11360-070) for 3 weeks before assays.

Primary murine embryonic astrocytes were isolated and cultured as previously described [294].

Cortices from embryonic mice were incubated with trypsin for 20 min in a 37 °C water bath, with gentle agitation every 5 min. Trypsin was neutralized with serum containing media and tissue was triturated manually twenty times first using a 10 mL serological pipette ten times, then a 5 mL pipette to induce single cell suspension. Dissociated tissue was centrifuged at 350 g for 5 min to collect cells and resuspended in fresh DMEM media supplemented with 10 % FBS and 2 mM L-

40

Chapter 2 glutamine. The cell suspension was plated on a PDL-coated 75 cm2 flask. Cells were maintained in a humidified incubator at 37 °C with 5% CO2, and media was refreshed every 2-3 days until the astrocyte layer reached confluence. Cells were then shaken overnight in an incubator to remove oligodendrocytes and microglia. Cortices were kindly provided by the lab of A/Prof Thomas Fath.

2.2 Compounds

All compounds apart from S1P were purchased from Cayman Chemical (MI, USA), with catalogue numbers given below in parentheses. S1P (Biomol, UK) was dissolved in methanol at 0.5 mM, then diluted in OptiMEM containing 0.01% fatty acid-free bovine serum albumin (BSA). FTY720-

P (100006408) was dissolved directly in the OptiMEM/BSA vehicle (0.01% fatty acid free BSA).

Prior to S1P or FTY720-P treatment, U251 cells were serum-starved overnight in OptiMEM, and primary astrocytes in Neurobasal medium supplemented with B27 supplement, 2 mM L- glutamine, 5 mM glucose and 0.5 mM sodium pyruvate. Unless otherwise stated, S1P was used at

100 nM, FTY720-P at 1 µM, and CYM-5520 (17638) at 1 µM. W146 (10009109), JTE013

(10009458) and TY52156 (19119) were dissolved in DMSO and diluted to 10 μM in cell culture medium. Cells were pre-treated for 30 min with these antagonists before S1P stimulus.

2.3 siRNA transfections

U251 cells were cultured in 6-well plates at 105 cells/mL in DMEM media supplemented with 10%

FBS and 2 mM L-glutamine. On the day of transfection, serum containing media was replaced with 900 µL of reduced serum OptiMEM medium. 2 µL of Lipofectamine RNAiMAX reagent

(Life Technologies, Australia) was complexed with 50 nM GNA12 (s5867) or GNA13 (s20992) siRNA (Life Technologies), or non-silencing control siRNA (#1027310, Qiagen) in 93 µL of

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Chapter 2

OptiMEM for 20 min, before drop by drop application onto cells. Cells were incubated with siRNA mixture overnight. At 24 h after transfection, the medium was changed to growth medium containing 10% FBS and 2 mM L-glutamine. Cells were serum-starved in OptiMEM overnight prior to S1P treatment.

2.4 Human Hippocampus Tissue samples

Frozen tissue samples from the hippocampus CA1 region of neurologically-normal donors aged

65 and older were obtained from the NSW Brain Tissue Resource Centre and stored at -80 °C.

These samples have been described in a recent publication [214]. A total of 40 samples from 25 males and 15 females were used for RNA isolation and quantitative real-time polymerase chain reaction (qPCR) (from ~40 mg tissue), as described below. Experiments using these tissue samples were performed under ethics approval HREC2016/801 from The University of Sydney.

2.5 qPCR

RNA was extracted using the RNA ISOLATE II (Bioline). RNA was eluted in 40 µL RNase- free water and concentrations were determined using a Nanodrop DA-1000 Spectrophotometer

(Life Technologies) before conversion to cDNA using the First Strand Synthesis Protocol with

Mu-MLV Reverse Transcriptase (New England Biolabs). A total of 10 ng of cDNA was used in a

10 µL reaction for qPCR. Each reaction contained 1 x SensiFAST SYBR reagent (Bioline), 3 pmoles each of forward and reverse primer, and water. Cycling conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 10 s at 60 °C, 20 s at 72 °C on the Roche

Lightcycler 480. Analysis of gene expression for cell culture experiments was conducted using the

DDCt method with a geometric mean of two housekeeping genes (GAPDH and ACTB) taken as

42

Chapter 2 reference [295]. Ct values greater than 35 were excluded from analysis. For human hippocampus tissue samples, a standard curve of Ct versus input cDNA was generated for each gene of interest and used to convert Ct values to relative gene expression, which was then normalized to the mean of three housekeeping genes (GAPDH, ACTB, and RPL13). Primer sequences are listed in Table

2.1.

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Chapter 2

Table 2.1. List of primers used for qPCR Gene Species Forward Reverse

GAPDH Human TGTTGCCATCAATGACCCCTT CTCCACGACGTACTCAGCG

Mouse GCCTGGAGAAACCTGCCAAG TCATTGTCATACCAGGAAATG

ACTB Human CATGTACGTTGCTATCCAGGC CTCCTTAATGTCACGCACGAT

Mouse GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT

RPL13 Human CCGGCATTCACAAGAAGGTG CGAGCTTTCTCCTTCTTATAGACGT

BDNF Human CTACGAGACCAAGTGCAATCC AATCGCCAGCCAATTCTCTTT

Mouse TTACCTGGATGCCGCAAACAT TGACCCACTCGCTAATACTGTC

PDGFB Human CTCGATCCGCTCCTTTGATGA CGTTGGTGCGGTCTATGAG

Mouse ATTGTGCGAAAGAAGCCCATC GGGTCACTACTGTCTCACACTT

HBEGF Human ATCGTGGGGCTTCTCATGTTT TTAGTCATGCCCAACTTCACTTT

Mouse CGGGGAGTGCAGATACCTG TTCTCCACTGGTAGAGTCAGC

LIF Human CCAACGTGACGGACTTCCC TACACGACTATGCGGTACAGC

Mouse AGCTATGTGCGCCTAACATGA CGACCATCCGATACAGCTCC

GNA12 Human GGAAAGCCACCAAGGGAATTG TGGACGTGATCCCGTCGAA

GNA13 Human CCCAAGGAATGGTGGAAACAA ACCCAGTTGAAATTCTCGACG

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Chapter 2

2.6 Neuronal excitotoxicity assays

Neuronal co-cultures in 24-well plates were pre-treated with vehicle, S1P or FTY720-P for 6 h.

Half of the medium was removed, and the cells were pulsed with 100 µM NMDA (N-methyl-D- aspartate) for 15 min. The medium containing NMDA was replaced (with the half of the culture medium that was removed prior to NMDA stimulus) and cells were left overnight before assessing viability. TrkB receptor antagonist ANA-12 (100 nM) (14309, Cayman Chemical), EGF receptor inhibitor (100 nM) (11492, Cayman Chemical), and anti-LIF antibody (20 ng/mL)

(#AF449, AB_354362, R&D Systems) were added 1 h after NMDA pulse. To assess viability, coverslips were gently washed with warm D-PBS, then incubated with 1 µg/mL propidium iodide

(PI) (P4864) for 10 min and 1 µM calcein-AM (C1430) for 1 min. Calcein-AM and PI were purchased from Sigma Aldrich (NSW, Australia). Images were captured on a Zeiss Axio Vert A1 microscope and analyzed using the Fiji software. Cells stained with calcein-AM only were counted as live while cells stained with PI were counted as dead. Images were captured and cells were counted blind to treatment groups. Imaging and counting were also performed by different people.

2.7 ELISA

Primary human astrocytes were seeded at a density of 5 × 104 cells/mL in 24 well plates in RPMI media supplemented with 10% FBS and 2 mM L-glutamine. Prior to treatments, cells were serum starved in supplemented Neurobasal media. After overnight incubation with treatments, cell culture supernatant was cleared of cellular debris by centrifuging at 2000 × g and frozen at - 80

°C prior to assay. BDNF and LIF protein concentration was measured in the culture medium of astrocytes treated with vehicle, S1P or FTY720-P for 24 h using human BDNF (Abcam, ab99978)

45

Chapter 2 and human LIF (Abcam, ab100582) ELISA kits according to the manufacturer instructions. Each group contained 3 biological replicates.

2.8 Western blot

Cells were washed twice with D-PBS before lysis with 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

Protease Inhibitor Cocktail (Roche) and phosphatase inhibitors (5 mM β-glycerophosphate, 2 mM sodium orthovanadate and 5 mM NaF). Lysates were sonicated with a Q800R3 sonicator

(QSonica) for 5 min at 4 °C (30 sec on/off, 70% amplitude). Protein concentrations were determined with a bicinchoninic acid (BCA) assay (Life Technologies). Samples were resolved on

Bolt 4-12% Bis-Tris Plus gels (Life Technologies) with the Novex Sharp Pre-stained protein standard (Life Technologies) as a molecular marker. Gels were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked for 1 h at RT with 5% skim milk, then washed three times with Tris-buffered saline containing 0.1% Tween 20 (TBST). Primary antibodies were incubated overnight at 4 °C, followed by three washes with TBST, and the appropriate secondary antibody was used for 1 h at RT, again followed by three washes. Membranes were imaged with

ECL chemiluminescence reagent (EMD Millipore), and images captured on a Bio-Rad ChemiDoc

Touch. When probing for total protein after phosphoprotein blots, membranes were stripped by incubating with Restore™ PLUS Western blot Stripping Buffer (Life Technologies) for 45 min at

RT before washing for 5 min with TBST twice. Membranes were then blocked with 5 % skim milk for 1 h at RT. To ensure total stripping of primary antibody, membranes were imaged after incubation with secondary antibody and ECL. Upon confirmation that the primary antibody was

46

Chapter 2 stripped efficiently, membranes were re-probed with the appropriate primary antibody.

Densitometry was performed using Image Lab Software version 5.2. Antibodies for Western blotting were purchased from Technology (MA, United States) as listed in Table

2.2.

Table 2.2 List of primary antibodies used for Western blotting

Protein Supplier Cat. # Dilution RRID

EGR1 Cell Signaling Technology 4153 1:1000 AB_2097038 c-FOS Cell Signaling Technology 2250 1:500 AB_2247211

Phospho-JUN (S63) Cell Signaling Technology 2361 1:1000 AB_490908

Total JUN Cell Signaling Technology 9165 1:1000 AB_2130165

Phospho-YAP Cell Signaling Technology 13008 1:1000 AB_2650553

(S127)

Total YAP Cell Signaling Technology 14074 1:1000 AB_2650491

GAPDH Cell Signaling Technology 97166 1:1000 AB_2756824

Phospho-ERK Cell Signaling Technology 9101 1:1000 AB_331646

Total-ERK Cell Signaling Technology 4695 1:1000 AB_390779

Anti-rabbit Cell Signaling Technology 7074 1:5000 AB_2099233

Secondary

Anti-mouse Cell Signaling Technology 7076 1:5000 AB_330924 secondary

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2.9 Active RhoA Assay

RhoA activation was assayed with a kit from Cell Signaling Technologies (#8820), according to the manufacturer’s instructions. U251 cells were seeded onto 10 cm dishes in growth media and serum starved in OptiMEM prior to assay. Cells were treated with agonists for 2 min before media was aspirated and cells were washed with ice-cold D-PBS. Cells were scraped upon addition of ice-cold lysis buffer, which was supplied in the kit. Lysates were incubated with recombinant GST-

Rhoteckin-Rho Binding Domain fusion protein for 1 h at 4 °C. Activity was determined as the amount of active RhoA bound to GST-Rhotekin beads following immunoprecipitation. Relative activation levels were then determined via Western blot with a RhoA antibody supplied in the kit.

2.10 Phosphoproteomic analysis

U251 cells were seeded into 10 cm dishes at 1 × 105 cells/mL and grown to 80% confluency, then serum-starved overnight in OptiMEM. Cells were pre-treated with vehicle, W146 (10 µM) or

JTE013 (10 µM) for 30 min prior to a 60 min stimulus with 100 nM S1P. Phosphopeptides were analysed using the EasyPhos platform according to the recently described protocol [296]. Briefly, cells were washed 3 × with ice-cold TBS, scraped in 300 µL sodium deoxycholate (SDC) lysis buffer (4% (w/v) SDC, 100 mM Tris pH 8.5), boiled immediately (95°C, 5 min), and frozen at -

80°C for further processing. Lysates were sonicated (2 × 30 s), and protein concentration was determined by BCA assay. Samples containing 200 µg protein were reduced and alkylated in a single step using Tris(2-carboxyethyl)phosphine and 2-chloroacetamide. Protein was digested directly in a 96-well plate by the addition of Trypsin and LysC (1:100 enzyme:protein ratio) overnight at 37 °C. Following digest, phosphopeptides were enriched directly in a parallel 96-well format using TiO2 beads as described [296]. Eluted phosphopeptides were separated by high

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Chapter 2 performance liquid chromatography in a single run (without pre-fractionation) and analyzed on a

Q-Exactive HF-X mass spectrometer.

2.11 Phosphoproteomic analysis

Raw data from mass spectrometry runs were processed using MaxQuant. Data was transformed into a log2 scale for processing. Bioinformatic analysis and statistical analysis was performed using the Perseus program (version 1.6.2.2) [297]. For the both the time course and antagonist experiments, a one-way ANOVA was performed to identify significantly regulated phosphopeptides across the three time points or treatment groups, applying the Benjamani

Hochberg false discovery rate (FDR) correction. FDR was set at q = 0.05 to adjust for multiple comparisons. Significant phosphosites were filtered and a built-in post hoc analysis was applied to identify significantly regulated treatment pairs. For the antagonist experiment, phosphosites regulated by S1P were first filtered to remove phosphosites that were not regulated by S1P. Further filtration steps were applied to group phosphosites into four categories: regulated by W146 alone, regulated by JTE013 alone, regulated by both W146 and JTE013, and regulated by neither.

Canonical pathways activated by S1P and regulated by S1P receptor antagonists were investigated using QIAGEN’s Ingenuity® Pathway analysis program with significantly regulated phosphosites identified by the phosphoproteomic experiments and the respective q-values as input.

2.12 Statistical analysis

All statistical analysis was performed as a one-way ANOVA with Dunnett’s post-test for multiple comparisons using GraphPad Prism software (San Diego, USA), unless otherwise specified. P <

0.05 was considered statistically significant. Gene expression values derived from human

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Chapter 2 hippocampus tissue samples were subjected to Spearman correlation analysis, as the data was not normally distributed (D’Agostino and Pearson normality test). Hippocampal neuron protection experiments were analysed using two-way ANOVA, with NMDA as one variable and drug treatment as the other. Tukey’s post-test was then applied to compare BSA vehicle, S1P, and

FTY720-P treatment groups, or S1P treatment with S1P + ANA-12, Afatinib, or LIF antibody.

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Chapter 3: Investigating the S1P-induced neurotrophic gene

response in CNS cells

Certain results from this chapter are published in Tran et. al, 2019 - “Sphingosine 1-phosphate but not

Fingolimod protects neurons against excitotoxic cell death by inducing neurotrophic gene expression in astrocytes”. J Neurochem

3.1 Introduction

S1P is a potent lipid signalling molecule that transduces its effects through activation of a family of five GPCRs, S1PR1-5, whereby activation potentiates intracellular signalling pathways such as the MAPKs [112, 118, 156, 298]. SphK1 and 2 catalyse the phosphorylation of sphingosine to form S1P, and this process is essential to mammalian physiology [113]. We have previously shown a negative correlation between S1P levels and increasing Braak stages in the hippocampus and temporal grey matter of AD subjects, while SphK1 and 2 activity also decreased with progression of disease [109]. In the absence of neuropathology there is a gender-specific loss of S1P in females during physiological ageing [214]. Additionally our research team has very recently demonstrated that deficiency of SphK2 sensitises to neurodegeneration in an amyloidogenic AD mouse model

[299], whilst loss of SphK1 promotes a neuroinflammatory phenotype in an S1P-independent manner and impaired microglial phagocyotosis, similar to AD [300].

Multiple recent studies have demonstrated that activation of S1P receptors by pharmacological agonists results in the induction of neurotrophic factors like BDNF [221, 255, 301], whilst two studies have shown S1P receptor agonists protect neurons from excitotoxic cell death [263, 264].

Results from two of these studies have indicated that ERK signalling downstream of S1PR1 is

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Chapter 3 essential for the induction of neurotrophic genes [221, 263]. The work presented in this chapter sought to investigate how the loss of SphK and S1P may contribute to sensitising the CNS to neurodegenerative conditions and elucidate how S1P signals in glia and neurons to induce neurotrophic gene expression.

3.2 Aims

The overall aim of this chapter was to investigate the role of the sphingosine kinases and S1P receptors, and the G-proteins associated with the S1P receptors, in the induction of neurotrophic genes in neurons and astrocytes.

The specific aims addressed in this chapter are:

1. Characterise the neurotrophic gene response in astrocytes and neurons stimulated with S1P

2. Investigate the potential role of sphingosine kinases in basal neurotrophic gene expression

3. Characterise/define the role of individual S1P receptors and G-proteins in the induction of

neurotrophic genes by S1P

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3.3 Results

3.3.1 Confirming neurotrophic gene expression in astrocytes The expression of a panel of neurotrophic genes over time, following S1P treatment (100 nM) of

U251 astrocytoma cells and primary human astrocytes, was analysed by qPCR (Figure 3.1). We examined BDNF [221, 255], HBEGF and LIF expression on the basis of prior studies detailing their induction following S1P or S1P agonist treatment [256], whilst my own prior studies had shown significant induction of platelet-derived growth factor B (PDGFB) with S1P [302]. Cells were maintained in serum-free medium prior to S1P stimulus as S1P is abundant in serum [303].

U251 cells significantly up-regulated BDNF, PDGFB, HBEGF, and LIF with peak induction at 4 h after S1P stimulus (Figure 3.1a). In primary human astrocytes, S1P also significantly induced these four neurotrophic factors, with peak induction at 4 h (Figure 3.1b). For all future assays the

4 h time point was chosen when measuring growth factor gene induction.

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Figure 3.1 S1P induces neurotrophic gene expression in astrocytes

Neurotrophic gene expression was analysed in primary human astrocytes (a) and U251 astrocytoma cells (b) at the indicated time points following S1P stimulus (100 nM). Statistical significance was determined by one-way ANOVA, with Dunnett’s post-test used to compare each time point to 0 h. N=4 biological replicates * p<0.05, ** p<0.01, *** p<0.001. Graphs depict mean

± SEM.

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3.3.2 Effect of S1P on neurotrophic factor gene expression in neurons Having observed the induction of these growth factors in astrocytes, we determined if this response was also seen with neurons (Figure 3.2). Primary human neurons were treated with 100 nM S1P for the indicated times. Unlike astrocytes, primary human neurons did not up-regulate BDNF,

PDGFB or HBEGF (Figure 3.2a). Increased LIF expression was observed at 2 and 4 h but this was not statistically significant (p>0.05). A similar result was observed when comparing primary murine cells treated with S1P for 4 h, with significant induction of BDNF (p<0.0001), PDGFB

(p<0.001), HBEGF (p<0.001), and LIF (p<0.001) in astrocytes but not neurons (Figure 3.2b). To determine whether this neurotrophic gene response translates to protein expression, we measured the secretion of BDNF and LIF by primary human astrocytes and neurons (Figure 3.3). Substantial levels of secreted BDNF were detected in astrocyte conditioned medium, but these levels were not increased by S1P stimulus. S1P increased secretion of LIF by 54 % in astrocyte cultures, although this was not significant (p=0.17). Neuronal protein expression of both BDNF and LIF was below the level of detection in both vehicle and S1P-treated conditions, with the lower limit being 60 and

10 pg/mL.

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Figure 3.2. Neurotrophic gene expression in primary human neurons following S1P treatment.

(a) Relative gene expression levels of BDNF, PDGFB, HBEGF, and LIF in primary human neurons (n= 4 biological replicates) following S1P stimulus at the indicated times. Bars show mean

± SEM. Statistical significance was tested with one-way ANOVA, and Dunnett’s post-test to compare each time point to the 0 h time point. (b) Gene expression for primary murine hippocampal neurons (n=6) and astrocytes (n=4) was assessed by qPCR following 4 h treatment with S1P. Statistical significance was determined using a two-tailed t-test for each gene, comparing

S1P to the vehicle control (dotted line). *** p<0.001

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Figure 3.3 Basal expression of neurotrophic factors is higher in astrocytes compared to neurons.

Graphs depict mean ± SEM protein expression in cell culture media for (a) BDNF (n=3 biological replicates) and (b) LIF (n=7 from 2 biologically independent batches of cells) in primary human neurons and astrocytes treated with BSA vehicle (BSA) or S1P for 24 h. Statistical significance was tested with two-way ANOVA and Tukey’s multiple comparison test to compare the effect between cell types and treatment.

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3.3.3 Effect of S1P on neurotrophic gene expression in microglial cells Microglia also express S1P receptors so we utilised the immortalised murine microglia cell line

BV-2 to do a preliminary study into whether microglial cells upregulate neurotrophic genes in response to S1P (Figure 3.4). BV-2 cells were treated for 2 or 4 h and gene expression was compared to untreated cells. S1P stimulus did not significantly induce expression of any of these genes in BV-2 cells, and we did not pursue this in primary microglia.

Figure 3.4 S1P does not induce neurotrophic gene expression in BV-2 cells.

Graphs depict mean ± SEM of relative gene expression levels of BDNF, PDGFB, HBEGF, and

LIF in BV-2 microglia cells (n= 4 biological replicates) following S1P stimulus at the indicated times. Significance was tested for by one-way ANOVA with Dunnett’s post-test relative to the 0 h time point. N=4 biological replicates

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3.4.4 S1P receptor expression in astrocytes and neurons To understand the astrocyte-specific response to S1P, publicly-available RNA sequencing data

(www.brainrnaseq.org) [163, 164] was used to compared S1P receptor gene expression in astrocytes and neurons (Figure 3.5). In both human and murine CNS cells, S1P receptor expression, particularly that of S1PR1, is considerably higher in astrocytes than neurons. S1PR1 expression was measured at 46.8 Fragments Per Kilobase of transcript per Million mapped reads

(FPKM) in human astrocytes compared to only 2.175 FPKM in neurons. Expression of both

S1PR2 and S1PR3 is also higher in foetal human astrocytes compared to neurons, although expression of these receptors is considerably lower compared to S1PR1. Similar results were observed with publicly-available RNA sequencing data from mouse astrocytes and neurons

(Figure 3.5b) [163].

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Figure 3.5 S1P receptor expression in CNS cells

Publicly available RNA-seq data (www.brainrnaseq.org) for S1P receptor expression in (a) human and (b) murine astrocytes and neurons. Expression is measured in FPKM. Data is presented as means ± SEM as provided in the public datasets. Standard error is not available for human neurons as sample size is too small (n=1).

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3.3.5 Correlation of SphKs and S1P to neurotrophic factors in the human hippocampus After verifying the induction of neurotrophic factors in S1P-treated astrocytes, we sought to confirm whether there was a correlation of these factors with expression of the sphingosine kinases in human hippocampal tissue. mRNA levels of BDNF, PDGFB, HBEGF, and LIF were all positively correlated with SPHK1 (Figure 3.6a). BDNF and PDGFB expression were significantly correlated with SPHK2 suggesting that S1P may be an important endogenous regulator of these neurotrophic factors (Figure 3.6b). A positive trend was observed when examining the relationship between HBEGF and SPHK2 expression although this was not significant; and LIF expression was not correlated with SPHK2. However, of these four neurotrophic genes, only LIF expression was positively correlated with levels of S1P (p=0.035) (Figure 3.6c).

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Figure 3.6 SphK1 and SphK2 gene expression correlates with neurotrophic factors in the human hippocampus.

Correlations between mRNA expression levels for neurotrophic growth factors (BDNF, PDGFB,

HBEGF, and LIF) and (a) SPHK1, (b) SPHK2 or (c) S1P in human hippocampal samples (n=40).

Correlations were determined using Spearman correlations as data was not normally distributed as determined by a normality test (D’Agostino and Pearson). Correlation co-efficient (R) and p-value are shown on each graph. S1P data was obtained from Couttas et al (2018) [214].

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3.3.6 Effect of SphK silencing on neurotrophic factors To assess functionally whether SphK1 and SphK2 are necessary for basal expression of these neurotrophic genes, the kinases were down-regulated in U251 cells using siRNA (Figure 3.7).

Cells were maintained in serum-free medium. SphK1 siRNA treatment resulted in a 65% reduction in SPHK1 mRNA expression (p=0.065) and a compensatory 57% increase in SPHK2 expression

(Figure 3.6A). This was accompanied by a 25% reduction in PDGFB expression (p<0.05). BDNF and LIF were not affected by SphK1 siRNA, whereas HBEGF expression doubled (p<0.05).

Knockdown of SPHK2 produced a 76% reduction of SPHK2 expression (p<0.0001) coupled with a 75% reduction of PDGFB expression (p<0.0001) and a 56% reduction in LIF expression

(p<0.05). HBEGF expression was unaffected by SphK2 siRNA. Combining SphK1 and SphK2 siRNA resulted in a 28% decrease in BDNF expression (p<0.05), 69% decrease in PDGFB expression (p<0.0001), and 59% reduction in LIF (p<0.05).

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Figure 3.7 SPHK2 is necessary for basal PDGFB and LIF expression in U251 cells.

U251 cells were incubated with non-silencing (ns), SphK1, SphK2 or SphK1 and SphK2 siRNA

(SphK1/2) for 72 h before analysis of SPHK1 (a), SPHK2 (b), BDNF (c), PDGFB (d), HBEGF

(e), LIF (f) gene expression. N=8 from two independent experiments. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test comparing to the non-silencing siRNA group. * p<0.05, ** p< 0.01, *** p< 0.001. Graphs depict mean ± SEM.

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3.3.7 Effect of S1PR antagonists on neurotrophic factor gene expression Astrocytes primarily express S1PR1-3. To directly assess the role of the three S1P receptor types expressed in astrocytes, U251 cells were pre-treated with DMSO vehicle, S1PR1 antagonist W146,

S1PR2 antagonist JTE013 (JTE), or S1PR3 antagonist TY52156 (TY), before stimulation with

S1P for 4 h (Figure 3.8a). W146 and JTE reduced HBEGF expression by 72% (p < 0.001) and

70% (p = 0.002), respectively; LIF expression by 60% (p < 0.001) and 68% (p < 0.001); and

PDGFB by 59% (p < 0.001) and 69% (p < 0.001). BDNF was induced to a smaller extent than the other three factors, and this induction was not significantly affected by the S1P receptor antagonists. TY did not significantly affect the expression of these neurotrophic genes. In primary human astrocytes, HBEGF induction with S1P was suppressed 27% by W146 (p = 0.037) and 54% by JTE (p < 0.001); whilst LIF expression was 23% lower with W146 (p = 0.035) and 51% with

JTE (p < 0.001) (Figure 3.8b). TY did not affect expression of either gene. W146, JTE013 and TY reduced PDGFB expression by 23% (p = 0.054), 60% (p < 0.001) and 64% (p < 0.001), respectively. To determine if the reduced gene expression translated to reduced secretion of neurotrophic factors, we quantified secreted LIF by ELISA (Figure 3.8c). W146 pre-treatment slightly reduced secreted LIF (p<0.05), whilst JTE reduced it to baseline levels (p<0.001).

Surprisingly, TY also significantly reduced secretion of LIF (p<0.001).

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Figure 3.8 Activation of S1PR2 is required for induction of neurotrophic factors

Gene expression of (a) U251 cells and (b) primary human astrocytes following 30 min pre- treatment with vehicle control (Veh), W146, JTE013 (JTE), and TY52156 (TY) before stimulus with S1P for 4 h. An unstimulated BSA vehicle control for S1P was included (BSA). (c) LIF protein secretion was measured in primary human astrocytes pre-treated for 0.5 h with S1PR antagonists, then incubated for 24 h with S1P. Significance was determined by one-way ANOVA with Dunnett’s post-test comparing to the Veh+S1P condition. * p<0.05, ** p<0.01, *** p<0.001.

Graphs depict mean ± SEM.

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3.2.8 Effect of dual S1PR antagonists on neurotrophic factors To determine if blocking two of the three S1P receptors on astrocytes produces an additive effect in reducing the expression of these neurotrophic genes, primary human astrocytes were pre-treated with two antagonists in combination before S1P stimulus (Figure 3.9). For all four genes, simultaneous antagonism of S1PR1 (W146) and S1PR2 (JTE) resulted in greater inhibition relative to one antagonist alone. However, this was not statistically significantly in comparison to JTE alone, which itself produced a significant reduction in S1P-stimulated expression of all four genes.

JTE pre-treatment synergised with TY in reducing PDGFB expression but this combination resulted in higher HBEGF and LIF expression compared to JTE alone (not significant). Similarly, the combination of W146 and TY non-significantly reduced PDGFB expression relative to either antagonist alone but had no notable impact on expression of other genes in comparison to either

W146 or TY alone.

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Figure 3.9 Neurotrophic gene induction by S1P after inhibition of two S1P receptors.

Primary human astrocytes were pre-treated with vehicle control (Veh), W146 (W), JTE013 (J),

TY52156 (T), a combination of W146 and JTE (WJ), W146 and TY (WT) or JTE and TY (TY) before S1P stimulus for 4 h. An unstimulated BSA vehicle control for S1P was included (BSA). n=8 from two biological samples and experiments. Significance was determined by one-way

ANOVA with Dunnett’s post-test comparing to the Veh + S1P condition. * p<0.05, ** p<0.01,

*** p<0.001. Graphs depict mean ± SEM

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3.2.9 Targeting G-proteins coupled to S1P receptors Although antagonising S1PR2 produced the most pronounced reduction in gene expression, antagonising S1PR1 and S1PR3 also had a significant effect in primary human astrocytes. S1PR1,

S1PR2 and S1PR3 are able to couple to Gai [125]. Thus, we sought to confirm the importance of

Gai activation in activating gene transcription. Gai is encoded by 3 separate genes that represent

3 isoforms: GNAI1, GNAI2 and GNAI3. Therefore, we used pertussis toxin (PTX), which is a well- characterised inhibitor of Gai. Pre-treatment with PTX significantly suppressed induction of

PDGFB by 39% (p<0.001), HBEGF by 57% (p<0.001), and LIF by 58% (p<0.001) (Figure 3.10).

S1PR2 also couples to Ga12/13 [245]. Ga12 and Ga13 are independent proteins with overlapping function that are encoded by the GNA12 and GNA13 genes, respectively. We therefore utilised gene silencing with siRNA to assess their roles in neurotrophic gene induction. U251 cells treated with GNA13 siRNA exhibited a 70% reduction in GNA13 mRNA expression (p<0.001), while

GNA12 siRNA resulted in a 75% reduction in expression of the target gene (p<0.001) (Figure

3.11a). Knockdown of GNA13, but not GNA12, effectively ablated induction of neurotrophic genes by S1P, confirming the requirement for Ga13 (Figure 3.11b).

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Figure 3.10 Induction of neurotrophic genes is dependent on Gai signalling.

Gene expression was measured for neurotrophic genes following vehicle (Veh) or 100 ng/mL pertussis toxin (PTX) for 4 h followed by S1P stimulus for 4 h (n=6 biological replicates from two independent experiments). Statistical significance was determined by one-way ANOVA with

Dunnett’s post-test relative to the BSA control (BSA). ***p<0.001. Graphs depict mean ± SEM.

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Figure 3.11 Ga13 not signalling Ga12 is required for induction of neurotrophic genes

U251 cells were treated with siRNA targeting GNA12 (G12), GNA13 (G13), or a non-silencing control (ns). qPCR was utilised to check the efficiency of gene knockdown (a) and the effects on neurotrophic genes following S1P (b) (n=8 biological replicates from two independent experiments). Statistical significance was determined by one-way ANOVA with Dunnett’s post- test relative to the BSA control (BSA). *p<0.05, ***p<0.001. Graphs depict mean ± SEM.

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3.3.11 Effect of RhoA and ROCK inhibition on neurotrophic factor expression

Ga13 coupling to S1PR2 can mediate its effects through the activation of the small GTPase RhoA.

We therefore checked the activation of RhoA in U251 cells in response to S1P, with the S1P receptor agonist FTY720-P as a negative control that does not activate S1PR2. RhoA activation was observed in response to S1P but not FTY720-P stimulus in U251 cells (Figure 3.12a).

Inhibiting RhoA with C3 toxin reduced S1P mediated induction of PDGFB by 50% (p<0.01),

HBEGF by 35% (p<0.001), and LIF by 49% (p<0.05) (Figure 3.12b). However, the reduction in

BDNF induction was minor and not significant. The kinase Rho-associated kinase (ROCK) is required for transduction of signalling responses downstream of RhoA [304, 305]. When U251 cells were treated with the ROCK inhibitor Y-27632, neurotrophic gene expression was not affected for any of the four neurotrophic genes, strongly suggesting a ROCK-independent mechanism (Figure 3.13).

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Figure 3.12 S1P activation of RhoA is required for induction neurotrophic genes

(a) Activation of RhoA was measured in U251 cells (n=4) following BSA vehicle (BSA), S1P or

FTY720-P treatment for 2 min. (b) Gene expression following inhibition of RhoA with C3 toxin was measured by qPCR. U251 cells (n=6) were pre-treated with 1 µg/mL C3 toxin for 4 h before stimulus with S1P. Data was derived from two independent experiments. Statistical significance was measure with one-way ANOVA with Dunnett’s post-test relative to BSA (a) or Veh + S1P

(b). *p<0.05, ***p<0.001. Graphs depict mean ± SEM.

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Figure 3.13 Induction of neurotrophic genes occurs via a Rho-kinase independent mechanism.

Gene expression was measured in U251 cells that were pre-treated with 10 µM ROCK inhibitor

Y27632 for 1 h before stimulus with S1P for 2 h. Data was derived from four biological replicates.

Statistical significance was tested for with a one-way ANOVA with Dunnett’s post-test compared to Veh + S1P. Graphs depict mean ± SEM.

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3.3.12 Effect of Gai and RhoA on LIF protein secretion

To confirm the requirement for both Gai and Ga13-RhoA dependent signalling on the induction of neurotrophic genes, we measured the secretion of LIF protein following S1P stimulus in the presence of C3, PTX and the combination of both C3 and PTX (Figure 3.14). Both C3 and PTX alone reduced the secretion of LIF, however the effect was not statistically significant. The combination of C3 and PTX, significantly reduced LIF secretion below baseline levels (p=0.008), confirming the requirement for dual signalling pathways originating from independent G-proteins.

Figure 3.14 RhoA and Gai contribute to the secretion of LIF protein

Primary human astrocytes were pre-treated with vehicle control (Veh), C3, pertussis toxin (PTX), or both C3 and PTX (C+P) for 4 h before stimulus with BSA control (BSA) or S1P for 24 h.

Supernatant was collected and secreted LIF was measured by ELISA (n=3 biological replicates).

Statistical significance was determined by one-way ANOVA with Dunnett’s post-test. Graphs depict mean ± SEM.

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3.4 Discussion

S1P is recognised as an important protective molecule in the cardiovascular system, signalling through the S1PR1 and S1PR3 receptors [306-309]. The role for S1P, signalling through its receptors, in mediating neuroprotective functions is less well investigated. Studies have focused on S1P receptor agonists, particularly Fingolimod, with little investigation of the native ligand S1P itself. Three recent studies investigating protection of neurons against cell death induced with excitotoxic stimulus or amyloid beta by S1P receptor agonists have also focused on the requirement for S1PR1 [221, 255, 264]. These studies also noted the direct protective effects of

S1P receptor agonists in neurons, through induction of BDNF. While there is a study identifying a role for S1PR3 in conjunction with S1PR1 [256], to date there have only been a few studies focusing on the role of S1PR2. Here, we show that S1P induces expression of the neurotrophic growth factors LIF, HBEGF, BDNF, and PDGFB in astrocytes, but not neurons or microglia; and that this requires both S1PR1 and S1PR2 signalling, rather than being mediated through one or the other receptor alone. Investigating the G-proteins downstream of the receptors identified the involvement of Gai and Ga13 proteins, as well as RhoA, suggesting that dual S1PR1-Gai and

S1PR2-Ga13-RhoA pathways mediate induction of these genes (Figure 3.15).

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Figure 3.15 Summary diagram of the results section

Both U251 cells and primary human astrocytes utilised synergistic signalling driven by S1PR2 and

RhoA to induce the neurotrophic genes BDNF, PDGFB, HBEGF, and LIF. S1PR1 contributes to this pathway through activation of Gai, however downstream targets of both receptors are yet to be elucidated.

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In both the U251 cell line and primary human astrocytes, S1P treatment significantly induced a neurotrophic gene response that was not only cell-type specific, but also neurotrophic factor specific. While astrocytes treated with S1P upregulated BDNF, PDGFB, HBEGF, and LIF, my previous data (Honours project) showed that S1P does not induce PDGFA, VEGFA and NGF

[302]. Other studies have shown that astrocytes up-regulate several neurotrophic factors including -2 [310], glial cell line-derived neurotrophic factor (GDNF) [182, 311],

LIF [182, 256], and HBEGF [182, 256] in response to S1P and S1P receptor agonists. We also identified a novel response in astrocytes with the induction of PDGFB following S1P stimulus, although a previous study has reported that S1PR2 on hepatic myofibroblasts regulates PDGFB mRNA [254]. Many studies note an important neuroprotective role of S1P-induced BDNF [221,

255], however we found that BDNF was the least significantly induced neurotrophic factor following S1P stimulus. Published gene expression analyses show an average 2-fold induction of

BDNF expression [221, 255]. There are multiple splicing variants of the BDNF gene [312], and our primers were not specific for any individual variant. We were therefore unable to elucidate the specific BDNF mRNA isoform being up-regulated. Nonetheless, the lack of significant BDNF gene induction was substantiated with ELISA results showing a lack of protein induction. This low-level induction of BDNF may be due to the higher basal level of expression compared to the other growth factors, as levels of BDNF protein secreted by primary human astrocytes were an order of magnitude higher than LIF. Attempts to measure secreted PDGF-BB using ELISA did not yield any significant differences in S1P treated cells (data not shown). After synthesis, HBEGF is transported to the plasma membrane and requires proteolytic cleavage by metalloproteases for secretion [313, 314]. For this reason and the substantial cost of ELISA kits, we did not measure levels of secreted HBEGF protein.

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The levels of neurotrophic gene induction were higher in U251 cells compared to primary human astrocytes, most likely due to their transformed nature. In primary astrocytes S1PR2 expression is the lowest of the three S1P receptor subtypes [118, 145, 163, 164, 315], whilst multiple glioma cells lines and primary exhibit high S1PR2 expression levels [315-317]. As the results in this chapter indicate, S1PR2 is the main driver of neurotrophic gene expression and this increased expression in gliomas may explain why the levels of induction are so different between U251 cells and primary astrocytes. Nevertheless, the results obtained from U251 cells were qualitatively the same as those seen with primary astrocytes.

We did not observe any induction of neurotrophic factor gene expression in neurons with S1P stimulus, however this does not eliminate the possibility that neurons do not induce neurotrophic factors in response to S1P. The results in this chapter examined the mRNA expression of neurotrophic factors in a relatively short window of 8 h, which was sufficient for a strong gene expression response in astrocytes. Other studies have observed an increase in BDNF expression in neurons following S1P receptor agonist treatment over a longer time frame of 24 h [221, 254]. For example, Anastasiadou and colleagues showed induction of BDNF in mouse cerebellar neurons, with the level of induction peaking after 8 h [254]. We also used a hippocampal neuronal culture that is highly enriched for neurons as opposed to mixed cortical or cerebellar neuronal preparations used by others [254, 263]. It is possible that BDNF induction in these other studies was due to glial cells in the culture, and not directly derived from the neurons. Aside from astrocytes and neurons, microglia have also been shown to up-regulate BDNF and GDNF in an S1PR1 dependent manner

[168]. We found no significant increase in the expression of our panel of neurotrophic genes in the

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BV-2 microglial cell line following S1P treatment and thus we did not investigate this further in a primary cell culture model.

Gene expression analysis in hippocampal tissues from cognitively normal subjects identified positive correlations between sphingosine kinases and neurotrophic factors, particularly with

SPHK1. Although correlations in the human hippocampus identify strong correlations between

SPHK1 and neurotrophic factor expression, SphK1 siRNA treatment of U251 cells did not affect the levels of neurotrophic genes to the same extent as SphK2, producing only minor reductions in

PDGFB and LIF expression and a surprising increase in HBEGF expression. SphK2 siRNA treatment in astrocytoma cells indicated a role this enzyme in maintaining basal, homeostatic expression of BDNF, PDGFB and particularly LIF, in accordance with SphK2 being the major isoform responsible for S1P formation in the CNS [299, 300, 318, 319]. A positive correlation between S1P levels, measured by mass spectrometry, and LIF gene expression was also demonstrated in our data, suggesting that S1P production by SphK2 is functionally significant for

LIF expression in the human brain. Multiple studies have identified reductions to both sphingosine kinase isoforms in neurodegenerative paradigms such as AD [107, 109], PD [320], and HD [247].

We have previously reported loss of S1P and SphK2 activity with increasing neuropathological stage in pre-clinical and clinical AD [109], and an inverse correlation between hippocampal S1P levels and age in cognitively normal females [214]. A previous study in breast cancer cells showed activation of SphK1 by an oestrogen receptor [321], while estradiol was also able to increase intracellular levels of S1P [322]. With the rate of incidence of AD higher in females, future studies should investigate how sphingosine kinase activity and protein are regulated in the CNS and whether oestrogen regulates SphK activity.

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Although we examined the role of SphKs in astrocytes, neuronal SphK1 has also been shown to be an important mediator of CNS homeostatic function. Lee et al showed that SphK1 levels were lower in AD patient-specific neurons and loss of SphK1 in neurons was able to simulate AD-like pathology in vivo [300]. In their study they identified a non-S1P forming role for SphK1, whereby

SphK1 acetylated cyclooxygenase 2 and induced secretion of pro-resolving lipid mediators that are crucial to the phagocytic activity of microglia. Taken together, these results suggest that SphKs are crucial in maintaining homeostasis in the CNS and their dysfunction may sensitise to neurodegeneration.

Studies on S1P signalling have usually focused on an individual receptor mediating a specific signalling response. Prior reports have described the requirement for S1PR1 activation of ERK signalling in the induction of BDNF [221, 254], with others also describing the contribution of

S1PR1 and S1PR3 in mediating the induction of LIF and HBEGF [256]. There is little evidence for the role of S1PR2, with only three studies showing the neuroprotective properties of S1PR2

[286, 323, 324]. Our results certainly support the requirement of S1PR1, known to be the major

S1P receptor transducing the Gai activation in most cell types, as a key mediator of neurotrophic gene induction. However, our study identifies S1PR2 as the major essential mediator of neurotrophic gene induction in both primary astrocytes and U251 cells. Anastasiadou and colleagues showed S1PR1-dependent induction of BDNF in cerebellar neurons, although interestingly, inhibition of S1PR2 further boosted expression of BDNF suggesting S1PR1 and

S1PR2 signalling antagonise each other [254]. This is not the case in our data.

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Down-regulation of Ga13 with siRNA and RhoA inhibition with C3 toxin both ablated the neurotrophic gene expression, suggesting that S1PR2 is the major receptor driving Ga13-RhoA signalling in response to S1P. A recent publication also reported that the induction of HBEGF and

LIF in glioblastoma cells is dependent on RhoA signalling [325]. While the induction of neurotrophic genes is RhoA dependent, the downstream mediator ROCK is not involved, as pre- treatment with Y27632 did not affect gene expression. How S1P signals downstream of RhoA will be discussed further in Chapter 4. Although S1PR2 is the major driver of signalling for the up- regulation of neurotrophic genes, the effect of Gai inhibition with PTX and the significant suppression of neurotrophic gene expression with S1PR1 and/or S1PR3 antagonists support the hypothesis that dual signalling from two S1P receptors is required. This co-ordinate signalling effect by multiple receptors has also been reported to occur during embryonic [326].

Moreover, a recent publication identified the ability of S1P receptors to either homo- or heterodimerize [327]. Together with activation of independent G-protein signalling pathways by

S1PR1, S1PR2, and S1PR3, heterodimersation of these receptors could also contribute to a maximal gene expression response.

Overall, the results arising from this chapter demonstrate the role of dual S1P receptor signalling in neurotrophic gene induction in astrocytes, with S1PR2 as the principal receptor mediating this response. Greater molecular insight into the intracellular signalling mechanisms that are triggered by S1P receptor activation is presented and discussed in the following chapter.

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Chapter 4: Characterisation of the signalling pathways activated

by individual S1P receptors.

Certain results from this chapter are published in Tran et. al, 2019 - “Sphingosine 1-phosphate but not

Fingolimod protects neurons against excitotoxic cell death by inducing neurotrophic gene expression in astrocytes” J Neurochem

4.1 Introduction

In the previous chapter, we demonstrated transcriptional up-regulation of neurotrophic genes in astrocytes but not neurons following S1P stimulus and identified the specific receptor(s) driving this response. In this chapter, I investigated the intracellular signalling pathways activated by

S1PR1 and 2 that drive induction of these neurotrophic factors.

S1PR1 couples exclusively to Gαi, while S1PR2 and S1PR3 couple promiscuously to Gαi, Gαq/11, and Gα12/13 [125]. Stimulation of Gαi/o results in inhibition of adenylate cyclase (AC) activity and suppress formation of cyclic AMP (cAMP). Gαs activates AC, however S1P receptors do not couple to Gαs [125, 328, 329]. Other G-proteins that can be activated by S1P include Gαq/11, which can activate phospholipase C (PLC), leading to mobilisation of intracellular calcium [330], or

Gα12/13, resulting in activation of RhoA [331]. Studies have elucidated G-protein coupling to S1P receptors and physiological effects mediated by S1P, however there are still major gaps in the understanding of how each S1P receptor signals.

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One of the main mechanisms of intracellular signalling is the activation of kinases or phosphatases, which in turn results in phosphorylation or dephosphorylation of target proteins. In the previous chapter, we identified that S1PR2 is the major driver for the upregulation of neurotrophic genes, with involvement from S1PR1. Developments in mass spectrometry methods have allowed for quantification of system-wide changes in the phosphoproteome and signalling pathways following different ligand treatments. For instance, a recent study in mice identified the mammalian target of rapamycin (mTOR) as a key regulator of conditioned place aversion, a side effect associated with the k- agonist U-50,488H [332]. Understanding the downstream signalling pathways of each receptor may provide insight into mechanisms controlling potential side effects, as demonstrated by the aforementioned study.

4.2 Aims

The overall aim of this chapter was to delineate signalling pathways mediated by S1P in astrocytes, through the use of proteomic mass spectrometry and western blotting.

The specific aims covered in this chapter are:

1. To investigate changes to the phosphoproteome in a time dependent manner following S1P

treatment in astrocytic cells.

2. To identify the signalling pathways activated by S1PR1 and S1PR2 through

phosphoproteomics and western blotting

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4.3 Results

4.3.1 Time course phosphoproteomics To investigate the signalling pathways activated by S1P in U251 cells, we treated serum-starved cells for

0, 5, 15, and 60 min with S1P (100 nM) and performed phosphoproteomic analysis (Figure 4.1). A total of

11525 phosphopeptides were identified, of which 2777 were significantly regulated by S1P as determined by one-way ANOVA adjusted for multiple comparisons. To identify pathways significantly regulated by

S1P at each time point, an unpaired t-test was performed comparing each time point to the 0 min time point control. The top 5 significantly regulated pathways at each time point were identified following analysis with QIAGEN Ingenuity Pathway Analysis (IPA) (Figure 4.1c-e). These results confirm the activation of

RhoA signalling established in the previous chapter. When visualising changes across all 3 time points relative to the control on a volcano plot, the phosphosites on proteins showing the greatest fold change correspond to threonine 185 and tyrosine 187 (T185/Y187) on MAPK1, commonly known as ERK2 (Figure

4.2). This suggested that activation of the canonical ERK1/2 pathway is involved in driving neurotrophic gene expression.

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Figure 4.1 Analysis of the phosphoproteome following S1P stimulus.

(a) Simplified workflow for the analysis of phosphoproteins in U251 cells treated with S1P for 0,

5, 15, 60 min. (b) Changes to the phosphoproteome in U251 cells at 0, 5, 15, and 60 min of S1P stimulus, displayed as a heatmap. Heatmap shows fold-change for each replicate relative to the mean of the control (unstimulated) condition (i.e. 0 min). The top 5 canonical pathways activated following 5 min (c), 15 min (d), and 60 min (e) of S1P as determined by pathway analysis with

IPA.

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Figure 4.2 Volcano plots showing significant changes to phosphosites over time following S1P stimulus.

Significant changes in the phosphoproteome are depicted as volcano plots following 5 min (a), 15 min (b), 60 min (c) S1P stimulus in U251 cells. MAPK1 (T185/Y187) is highlighted in red to illustrate the lasting phosphorylation change and to highlight the magnitude in change. Volcano plots were generated with Perseus (v1.6.2.2)

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4.3.2 Transcription factor analysis The canonical MAPK signalling pathways are known to induce the immediate early genes Early

Growth Response 1 (EGR1) and FOS [333]. Examination of the promoter regions of BDNF,

PDGFB, HBEGF and LIF using the UCSC genome browser revealed ChIP-verified binding sites for EGR1, AP1, and TEAD4 within 5 kB of the transcriptional start sites (Figure 4.4). EGR1 binding was present on all four genes while AP1 binding was present only on BDNF, HBEGF and

LIF. AP1 is a heterodimer of FOS and Jun, and this heterodimerisation is positively regulated by post-translational modification of Jun [334]. We therefore examined whether S1P stimulus will up-regulate these transcription factors. In both U251 cells and primary human astrocytes, treatment with S1P induced gene expression of both transcription factors significantly at 0.5 and 1 h (Figure

4.3).

Figure 4.3 S1P induces immediate early gene expression in astrocytes

Gene expression was analysed in U251 cells (a) and primary human astrocytes (b) following treatment with S1P at the indicated time points. N=4 biological replicates. Statistical significance was determined with one-way ANOVA with Dunnett’s post-test comparing to the 0 h time point.

*** p< 0.001. Graphs depict mean ± SEM.

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Figure 4.4 Binding sites for AP-1, EGR1 and TEAD transcription factors on BDNF, PDGFB, HBEGF, and LIF genes.

Publicly available ChIP-seq data on the UCSC genome browser showing binding sites of transcription factors at the BDNF (a), PDGFB (b), HBEGF (c), and LIF (d) genes. Transcription direction is displayed on the gene with arrows. In all four cases, transcription runs right to left.

Transcription factors displayed have been filtered to show AP-1, EGR1 and TEAD binding sites.

The shading of the box representing the transcription factor is proportional to the maximum signal observed experimentally in the publicly available database.

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4.3.3 Effects of antagonising canonical pathways on neurotrophic and transcription factors Upstream of ERK1/2 are the MAPK/ERK kinases (MEK). We therefore used a MEK inhibitor to confirm the functional relevance of ERK signalling in mediating neurotrophic gene induction.

U251 cells were pre-treated with the MEK inhibitor PD98059 for 30 min to inhibit MAPK signalling before stimulus with S1P for 30 min. Inhibition of MEK resulted in a 72% reduction in

EGR1 and 68% reduction of FOS expression following S1P stimulus, although these results were not statistically significant (Figure 4.5a). With MEK inhibited, HBEGF expression was reduced by 64% (p<0.001) and LIF expression was reduced by 43% although the latter result was not statistically significant (Figure 4.5b).

Phosphoproteomic pathway analysis of the 15 min time point identified insulin receptor signalling as one of the top five regulated pathways. We observed phosphorylation of Akt2 and Akt3 at 15 min as evidence of this (Figure 4.5c-d). PI3K is known to phosphorylate Akt, and thus we used the PI3K inhibitor LY29004 (LY) to examine the effect on the neurotrophic factors (Figure 4.5e).

Surprisingly, inhibition of PI3K resulted only in the significant suppression of PDGFB expression

(p<0.01), with a 59% reduction.

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Figure 4.5 Inhibition of MEK reduces induction of immediate early genes and neurotrophic genes.

Gene expression in U251 cells pre-treated with 10 µM of the MEK inhibitor PD98059 (PD) for 30 min before stimulus with S1P for (a) 30 min (n=7) or (b) 4 h (n=6). Expression of immediate early genes EGR1 and FOS (a) or neurotrophic factors (b) was analysed by qPCR. Results are combined data from two independent experiments. Graphical representation of changes to Akt2 (c) and Akt3

(d) as measured by phosphoproteomics. Statistical significance was determined by one-way

ANOVA with Dunnett’s post-test comparing to the Veh + S1P condition. *** p< 0.001 (a, b, e).

Graphs depict mean ± SEM.

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4.3.4 Antagonist phosphoproteomics To identify additional transcriptional regulators activated by S1PR1 and S1PR2 in astrocytes, we performed phosphoproteomic analysis of U251 cells treated for 60 min with S1P, in the presence or absence of W146 or JTE013 (Figure 4.6). Out of a total of 16351 identified phosphopeptides,

498 were significantly increased or decreased by S1P compared to the vehicle control, as determined by one-way ANOVA adjusted for multiple comparisons. Of the 498 phosphopeptides regulated by S1P, 13 (2.6%) were significantly affected by W146 but not JTE013, 157 (32%) by

JTE013 but not W146, 55 (11%) by both W146 and JTE013, and 273 (55%) by neither W146 nor

JTE013 (Appendix Table A1-4). In agreement with the role for immediate early genes, downstream of ERK1/2, in neurotrophic gene expression in astrocytes, both W146 and JTE013 impeded S1P-mediated phosphorylation of mitogen activated protein kinase 1 (MAPK1; also known as extracellular regulated kinase 2, ERK2), and p38 kinase (MAPK14) (Figure 4.6e-f and

Appendix Table A3).

Of the significantly regulated phosphoproteins, 7 were transcription factors including Jun, Nuclear factor 1B (NFIB), NFIX, Yes-associated protein (YAP), Ets domain-containing transcription factor ERF (ERF), Y-box-binding protein 3 (YBX3) and cyclic AMP-dependent transcription factor ATF2 (ATF2). However, regulation of NFIB and ATF2 were independent of W146 and

JTE013. Of the remaining transcription factors, YBX3, Jun and YAP were identified in the group of phosphoproteins regulated by JTE013 but not W146. Specifically, dephosphorylation of YAP at S127 (Fig. 4.6c) prevents degradation of this factor and allows its translocation into the nucleus, whilst phosphorylation of Jun at S63 (Fig. 4.6d) promotes transcription of target genes [335, 336].

Phosphorylated Jun heterodimerizes with FOS to form the AP1 transcription factor, whilst YAP

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Chapter 4 heterodimerizes with TEAD and other transcriptional activators. Importantly, YAP dephosphorylation is an event that occurs downstream of RhoA activation [337]. YBX3 was phosphorylated in response to S1P with peak phosphorylation occurring at 15 min and this phosphorylation was significantly reduced with JTE013 (Figure 4.7a, b). Phosphorylation of ERF was W146-dependent and also peaked at 15 min (Figure 4.7 c, d). NFIX phosphorylation was dependent on both S1PR1 and S1PR2 with significant suppression of phosphorylation following respective antagonist treatments (Figure 4.7e, f).

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Figure 4.6 Phosphoproteomic analysis of pathways downstream of S1PR1 and S1PR2.

(a) A simplified workflow of the processing of U251 cells treated with S1P receptor antagonists to analyse changes to the phosphoproteome. (b) U251 cells were subject to a 30 min pre-treatment with vehicle control (Veh), W146, JTE013 (JTE) before addition of 100 nM S1P or BSA vehicle

(BSA). Changes to the phosphoproteome are depicted as a heatmap. (c-e) Phosphoproteomic data for (c) p-YAP (S127), (d) p-Jun (S63), (e) MAPK1 (T185/Y187), (f) p38a MAPK (T180/Y182)

(g) Venn diagram showing the phosphosites regulated by JTE013 (JTE) or W146. N=4 biological replicates. Statistical significance was determined by one-way ANOVA with post-test. *p<0.05.

Graphs depict mean ± SEM.

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Figure 4.7 Phosphoproteomic analysis of temporal changes to transcription factors downstream of S1PR1 and S1PR2.

Graphical representation of phosphorylation changes to (a) ERF (S185), (c) NFIX (265), and (d)

YBX3 (S102) over time and in the phosphoproteomic antagonist experiment for (b) ERF (S161),

(d) NFIX (S265), and (f) YBX3 (S102). N=4 biological replicates. Graphs depict mean ± SEM.

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4.3.5 Effect of S1PR antagonists on signal transduction Western blotting was used to verify the phosphoproteomic results showing S1PR2-dependent YAP dephosphorylation and Jun phosphorylation (Figure 4.8). JTE but not W146 or TY significantly impeded YAP dephophosphorylation on S127 and Jun phosphorylation on S63 in both U251 cells and primary astrocytes. The effect of S1P receptor antagonists on protein levels of Egr-1 and Fos was also tested. Fos protein induction by S1P in U251 cells was reduced 70% (p=0.01) by JTE pre-treatment and 62% by TY (p=0.033), whereas W146 (58%, p=0.002) and JTE (63%, p=0.001) pre-treatment reduced Fos induction in primary astrocytes. Only JTE reduced S1P dependent

EGR1 induction in U251 cells, however there was no statistically significant of any of the antagonists on S1P-induced EGR1 protein levels in astrocytes.

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a c + 1 h S1P + 1 h S1P BSA Veh W146 JTE TY BSA Veh W146 JTE TY 40 kDA p-JUN(S63) 40 kDA p-JUN (S63) 40 kDA J UN 40 kDA J UN p-YAP (S127) p-YAP (S127) 60 kDA 60 kDA 60 kDA YAP 60 kDA YAP EGR1 EGR1 60 kDA 60 kDA 40 kDA c-FOS 40 kDA c-FOS 40 kDA GAPDH 40 kDA GAPDH

b d p-JUN (S63) p-YAP (S127) p-JUN (S63) p-YAP (S127) 4 2.0 8 1.5 * *** ** 3 ** 1.5 *** 6 * *** 1.0 ** 2 1.0 4 0.5 p-JUN/JUN pYap/YAP

p-JUN/JUN 2 1 0.5 pYap/YAP

0 0.0 0 0.0 BSA Veh W146 JTE TY BSA Veh W146 JTE TY BSA Veh W146 JTE TY BSA Veh W146 JTE TY +1 h S1P +1 h S1P +1 h S1P +1 h S1P

EGR1 c-FOS EGR1 c-FOS * 3 ** 40 ** 150 * 40 ** 30 100 2 30 20 20 50 1 10

EGR1/GAPDH 10 cFOS/GAPDH EGR1/GAPDH cFOS/GAPDH 0 0 0 0 BSA Veh W146 JTE TY BSA Veh W146 JTE TY BSA Veh W146 JTE TY BSA Veh W146 JTE TY +1 h S1P +1 h S1P +1 h S1P +1 h S1P

Figure 4.8. S1PR2 is required for Egr-1 and Fos induction, Jun phosphorylation, and YAP dephosphorylation.

(a) U251 cells and (b) primary human astrocytes were treated for 1 h with 100 nM S1P or fatty acid free BSA control (BSA), following a 30 min pre-treatment with W146, JTE013 (JTE),

TY52156 (TY), or vehicle control (Veh). Protein levels quantified by densitometry are shown below example blots (6 independent cell preparations). EGR1 and FOS were normalized to

GAPDH loading control, whilst p-Jun (S63) and p-YAP (S127) were normalized to total Jun and

YAP, respectively. Statistical analysis was determined with a one-way ANOVA with Dunnett’s

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(p < 0.05). Significance is relative to the vehicle control Veh + S1P, except for YAP phosphorylation, which was assessed relative to BSA control (as it is a dephosphorylation event).

Graphs depict mean ± SEM.

4.3.6 Effect of Gai and Ga13 inhibition on signal transduction pathways

The involvement of Gai and Ga13 in the induction of neurotrophic genes was established in Chapter 3. To test the signalling pathways activated by these G-proteins, U251 cells were pre-treated with PTX and C3 toxin to examine the effects on ERK1/2 phosphorylation and S1P-activated transcription factors (Figure

4.9). Pre-treatment with PTX significantly reduced phosphorylation of ERK1/2 in response to S1P.

Surprisingly, pre-treatment with C3 toxin also inhibited phosphorylation of ERK1/2 to similar levels, while the combination of PTX and C3 completely prevented phosphorylation of ERK1/2. As expected, with the inhibition of ERK1/2 by both C3 and PTX, EGR1 and FOS protein induction by S1P was also significantly reduced, and the combination of C3 and PTX further suppressed induction. YAP dephosphorylation was prevented only by C3 toxin, while cJun phosphorylation was not significantly affected by either C3 or PTX alone, but the combination resulted in a 43% reduction (p<0.05).

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Figure 4.9 Gai and RhoA contribute to ERK signalling and activation of transcription factors.

Activation of ERK and transcription factors in U251 cells following Gai and RhoA inhibition with

C3, PTX, and both C3 and PTX (C + P) was measured by Western blot (a) and quantified using densitometry (b) (n=4 from two independent experiments). Statistical significance was determined by one-way ANOVA with Dunnett’s post-test relative to Veh + S1P for all target proteins except

YAP, which was relative to the BSA control as it is a dephosphorylation event. *p<0.05, **p<0.01,

***p<0.001. Graphs depict mean ± SEM.

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4.4 Discussion

Genetic and pharmacological approaches have been used to investigate intracellular pathways activated by S1P in astrocytes [145, 185, 256, 286, 338-340], however given that astrocytes express three S1P receptors, there is potential for diverse signalling pathways to be activated in response to S1P. Here we applied mass spectrometry to identify system-wide changes to the phosphoproteome in a temporal and receptor specific manner following S1P stimulus in an astrocytoma cell line. We confirmed that the major signalling pathways activated by S1P are downstream targets of Ga13, which we identified as the major G-protein mediating neurotrophic gene induction in the previous chapter. The greatest magnitude change in phosphorylation following S1P treatment was phosphorylation of ERK1/2 on four phosphosites, and accordingly, the MEK/ERK cascade was found to be a major regulator of HBEGF and LIF expression.

Activation of these pathways resulted in the induction of the immediate early transcription factors

Egr-1, Fos, and Jun, as well as the RhoA/HIPPO signalling pathway factor YAP.

Applying phosphoproteomics we identified early (5 min), intermediate (15 min), and late (60 min) phosphorylation cascades activated by S1P stimulus. In U251 cells, 5 min S1P treatment resulted in activation of pathways mainly associated with the Rho family of , as the top 5 canonical pathways activated including cdc42, Rac, and RhoA signalling. Previous literature has identified that activation of these pathways results in physiological responses associated with cellular shape and motility. For example, Cdc42 signalling regulates astrocyte polarity during migration [341,

342]. Knockdown of Cdc42 in astrocytoma cell lines reduced cellular migration through decreases in filopodia formation and actin stress fibres, and altered cellular morphology to a rounder shape

[343]. The pro-migratory effect of S1P in lymphocytes and endothelial cells are also very well

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S1PR3 signalling through RhoA, p38 MAPK and PI3K [344-346]. In lymphocytes, migration is mediated through S1PR1 signalling as demonstrated by previous studies using FTY720 and the

S1PR1 agonist SEW2871 [138, 198, 267]. Our phosphoproteomic analysis did provide evidence for actin cytoskeletal remodelling, as S1P induced phosphorylation of filamin A (S2158), myosin light chain kinase (MYLK) (S1773), and formin binding protein 1 like (S501) [347-351].

The activation of RhoA signalling is in line with results from the previous chapter showing that

C3 toxin reduces neurotrophic gene expression, as well as recent literature on S1P signalling through a RhoA [286, 325, 335]. Previous literature has also identified RhoA signalling downstream of S1PR3 as an important mediator of inflammatory gene expression in astrocytes, through induction of cyclooxygenase 2 [145]. Our data also suggests that the HIPPO pathway is one of the top 5 canonical pathways activated by S1P with 11.8%, or hits for 10 out of the 85 phosphoproteins that IPA attributes to this pathway in the database. However, it received a z-score of 0, indicating equal evidence of activation and inhibition in the phosphoproteomic data. Although

RhoA and HIPPO signalling are identified in the pathway analysis, HIPPO signalling constitutes one branch of RhoA mediated signalling [352-354]. HIPPO signalling itself specifically refers to the kinase pathway consisting of the regulation of YAP through the mammalian sterile 20-related

1 and 2 kinases (MST1/2) and large tumour suppressor 1 and 2 kinases (LATS1/2) [355], whereas

RhoA signalling is far more complex with multiple downstream targets [356]. Nonetheless, activation of YAP through dephosphorylation on S127 was clearly observed in our data.

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When examining the intermediate (15 min) effects of S1P on the phosphoproteome, we were able to observe activation of several classical phosphorylation cascades. These included proteins such as Akt2 (S478) and Akt3 (S476). S1P has previously been reported to phosphorylate Akt at S473

[185, 357], which we also observed in our phosphoproteomic data. However, the functional relevance of this phosphorylation to the induction of neurotrophic factors is less clear. We observed a significant decrease in the induction of PDGFB, with a 56% reduction following pre- treatment with the PI3K inhibitor LY29004. We also established in the previous chapter that the driver of PDGFB induction is S1PR2, which upon examination of the antagonist phosphoproteimic data was found not to affect Akt phosphorylation. A possible explanation may be that the phosphorylation of Akt occurs through a PI3K-independent mechanism. Studies have identified

Akt-independent pathways downstream of PI3K [358] and that phosphorylation of Akt can be mediated in a PI3K-independent mechanism by kinases such as Cdk2 or cyclin A [359], and

TANK-binding kinase 1 [360, 361].

S1P stimulus in U251 resulted in activation of the MAPK cascade, with phosphorylation of

MAP2K2 (MEK2, S295), MAPK1 (ERK2, Y187), and MAPK3 (ERK1, Y204). S1P activation of

ERK1/2 is well established [118, 181, 185, 362]. In fact, across all three time points, MAPK1 and

MAPK3 were consistently the proteins that had the greatest increase in phosphorylation. In the previous chapter, we established that antagonism of both S1PR1 and S1PR2 resulted in significant suppression of neurotrophic gene up-regulation. Surprisingly, MAPK1 phosphorylation (T185,

Y187) was dependent on the activation of both S1PR1 and S1PR2, as were other phosphosites such as MAPK14 (p38 MAPK) (T180, Y182), and MYLK (S1773, 1776, 1779). S1PR1 coupling to Gai can activate ERK through the GTPase Ras [32, 81] whereas RhoA is reported to activate

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ERK1/2 via the upstream kinase MEK [82]. We speculated that combined S1PR1-Gai and S1PR2-

RhoA signalling pathways are required for sustained ERK phosphorylation and up-regulation of immediate early genes. In agreement with this hypothesis, pre-treatment with C3 toxin, PTX, or both suppressed S1P-mediated ERK1/2 phosphorylation and significantly reduced induction of immediate early genes. Prior work has shown the importance of AP1, which is a heterodimer of

FOS and cJUN, for induction of BDNF transcription in neurons and LIF in mammary epithelial cells; and EGR1 for induction of LIF, HBEGF, and PDGFB in mesenchymal stem cells [83-85].

The mechanism by which S1PR1 and S1PR2 can signal synergistically to enhance MEK/ERK pathway activation is yet to be elucidated. Studies have noted the ability of S1P receptors to hetero- and homodimerize [327]. This phenomenon of synergistic signalling by separate G-proteins targeting a common downstream regulator has previously been reported in the context of calcium signalling [363, 364]. Prior phosphoproteomic studies have included the shorter time points of 5,

10, 15 and 30 sec to elucidate the immediate-early signalling events that occur after receptor activation, which we have not included [365, 366]. Future studies should consider investigating earlier time points following S1PR activation to elucidate potential common targets of S1PR1 and

S1PR2 signalling.

Previous publications have noted that induction of BDNF by S1P is mediated through the activation of ERK, as are the neuroprotective effects of S1P signalling [221, 255, 367]. We found that inhibiting the MEK/ERK pathway with PD98059 resulted in a significant reduction of

HBEGF, while LIF was also reduced (not statistically significant). However, MEK inhibition had

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Chapter 4 no effect on PDGFB or BDNF expression. As discussed in the previous chapter, it has been quite difficult to measure any decreases in BDNF expression following treatment with inhibitors given the low level of induction following S1P. The lack of effect of the MEK inhibitor on PDGFB expression, the significant reduction of PDGFB expression following RhoA and PI3K inhibition, and the result following dual antagonism of either S1PR1, 2 or 3 indicate a potential S1PR3 pathway contributing to PDGFB induction that is yet to be elucidated. After filtering for phosphosites significantly regulated by W146 and JTE013, the remaining phosphosites may hint as to how S1PR3 signals in glioma cells given the S1P receptor profile expression in such cells

[315-317]. EGFR (T693) was one of the phosphosites not regulated by either W146 or JTE013.

S1P transactivation of EGFR has previously been reported in breast cancer cells, and was dependent on a S1PR3-GaI mediated mechanism [368]. This represents a potential mechanism mediating the effects of S1PR3 on neurotrophic gene expression.

The phosphoproteomic data also revealed receptor-specific activation of transcription factors that are predicted to bind on promoter regions of the neurotrophic factors of interest. When examining phosphosites affected exclusively by W146, we observed 13 significantly regulated phosphosites, of which three belong to two different transcription factors: nuclear factor 1 X-type (NFIX) (S265,

S268) and ETS domain-containing transcription factor ERF (ERF) (S161). Current literature examining the functional relevance of NFIX phosphorylation at S265 and S268 is lacking, although one study found that dephosphorylation of its homolog NFI increased transcription of target genes [369]. Similarly, the transcriptional repressor ERF is reported to be a downstream target of ERK signalling [370]. Functionally, ERF phosphorylation, particularly at S161 is reported to be involved in mediating its intracellular localisation, with phosphorylation shuttling ERF to the

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Chapter 4 cytoplasm and rendering it inactive [370, 371]. Of 158 phosphosites significantly affected by

JTE013 alone, four belong to 3 different transcription factors: Jun (S63), YAP (S127), YAP

(S138), and Y-box-binding protein 3 (YBX3) (S102). The phosphorylation of Jun at S63 is significant as this regulates heterodimerisation with its binding partner FOS[334], which was transcriptionally up-regulated after 30 min of S1P stimulus. The dephosphorylation of YAP at

S127, which occurs in response to activation of RhoA/HIPPO signalling, regulates gene transcription through its intracellular localisation. When phosphorylated, YAP is sequestered in the cytoplasm and bound to 14-3-3, which recruits an E3 ubiquitin ligase to target YAP for degradation [337, 372, 373]. S1P-mediated dephosphorylation of YAP at S127 has been reported previously [352] and its involvement in the induction of HBEGF and LIF has also been demonstrated [325, 374]. To the best of our knowledge, the role of phosphorylation on S102 is yet to be reported for YBX3. However, studies have noted that its related isoform YBX1 requires phosphorylation at S102 for nuclear translocation [375]. The UCSC genome browser also does not report any ChIP verified binding sites for YBX3 at any of the four neurotrophic factors of interest.

The functional role of all these transcription factors should be assessed in the future through overexpression, gene silencing, and chromatin immunoprecipitation.

Taken together with results from the previous chapter, we have identified signalling pathways and transcription factors activated by S1P receptors 1 and 2 that are required for the induction of neurotrophic factors. What is noteworthy is the identification of signalling pathways that require both S1PR1 and S1PR2 activation for full phosphorylation responses, as well as pathways that are selectively activated by only one receptor. It would be interesting in future studies to determine whether S1PR1 and S1PR2 feed into ERK1/2 activation and immediate early gene transcription

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Chapter 4 through independent signalling pathways that converge on the MAPK pathway, or through a common signalling complex at the plasma membrane.

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Chapter 5: Comparison of the neuroprotective properties of S1P

and Fingolimod

Certain results from this chapter are published in Tran et. al, 2019 - “Sphingosine 1-phosphate but not Fingolimod protects neurons against excitotoxic cell death by inducing neurotrophic gene expression in astrocytes” J Neurochem 5.1 Introduction

In the previous chapters, I established that S1PR2 drives the upregulation of neurotrophic genes in astrocytes, with contributions from both S1PR1 and S1PR3. Activation of the Ga13-RhoA-YAP pathway by S1PR2 is important, and both S1PR1 and S1PR2 signalling appears to converge on activation of the MAPK pathway and transcription of immediate early genes, which bind to the promoters of the neurotrophic factors of interest. In this chapter, the neuroprotective properties of

S1P are compared to the clinical S1P receptor agonist Fingolimod/FTY720.

The sphingosine mimetic FTY720 is an orally-available treatment for relapsing MS. In vivo phosphorylation by SphK2 forms the S1P analogue FTY720-phosphate (FTY720-P), which is a pharmacological agonist at all S1P receptors except S1PR2 [198, 208]. As discussed in the literature review, FTY720-P acts as a functional antagonist of S1PR1 whereby activation results in receptor internalisation, ubiquitinylation and degradation via the proteasome [165, 210, 211].

This is the primary mechanism through which FTY720 causes immunosuppression [138, 376].

Although activation of S1PR1 results in degradation of the receptor, FTY720-P activates signalling pathways such as ERK1/2 that are indicative of receptor activation rather than functional antagonism [137, 338, 340]. FTY720 has also been demonstrated to be neuroprotective in several

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Chapter 5 pathological paradigms including AD [255], HD [250] and cerebral ischaemia [249, 377, 378] and induces neurotrophic genes via S1PR1 and S1PR3 activation [221, 256].

Given the importance of S1PR2 in inducing BDNF, PDGFB, HBEGF, and LIF, it is important to compare FTY720-P with S1P to elucidate any differences in potential neuroprotective properties.

As FTY720-P is not an agonist of S1PR2, we hypothesized that FTY720-P would be less effective at inducing these genes than S1P.

5.2 Aims

I aimed to compare the signalling of S1P and FTY720-P and elucidate any neuroprotective properties of both ligands in the context of excitotoxicity in vitro.

The specific aims covered in this chapter are:

1. To compare neurotrophic gene expression in astrocytes stimulated with S1P versus

FTY720

2. To compare signalling pathways activated by S1P and FTY720

3. To elucidate whether S1P and FTY720 protect neurons against excitotoxic cell death

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5.3 Results

5.3.1 Comparison of neurotrophic gene induction by S1P and FTY720-P S1P (100 nM) produced significant induction of BDNF, PDGFB, HBEGF, and LIF mRNA expression in U251 cells, whereas FTY720-P (1 µM) did not produce a significant induction of any of these genes (Figure 5.1). In primary human astrocytes, S1P treatment induced a 2-fold increase of BDNF (p<0.001), 4.2-fold increase in PDGFB (p<0.001), 3.9-fold increase in HBEGF

(p<0.001), and 5.8-fold increase in LIF expression (p<0.001). In contrast, FTY720-P treatment produced only a 1.6-fold increase in BDNF (p<0.001), 2.1-fold in HBEGF (p<0.001), 2.7-fold in

LIF (p<0.001), and no significant induction of PDGFB. These differences were not attributed to the dose used, as dose response experiments indicated maximal induction of these genes with 100 nM S1P and 1000 nM FTY720-P (Figure 5.2). We then sought to confirm that there is also a difference in protein expression. Secreted levels of BDNF and LIF were quantified by ELISA following 24 h treatment with BSA, S1P or FTY. As expected, S1P stimulus resulted in significant secretion of BDNF and LIF compared to BSA control, but no change in either factor was observed with FTY720-P (Figure 5.1c-d).

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Figure 5.1 S1P is a more potent inducer of neurotrophic genes than FTY720-P.

U251 cells (a) and primary human astrocytes (b) were treated with BSA control (dotted line), S1P or FTY720-P (FTY) for 4 h before expression of neurotrophic genes was analysed by qPCR (n=8 biological replicates from two independent experiments). Secreted BDNF (c) and LIF (d) protein was measured by ELISA in primary human astrocytes treated with BSA, S1P or FTY for 24 h.

Significance was determined by a one-way ANOVA with Dunnett’s post-test. **p<0.01,

***p<0.001. Graphs depict mean ± SEM

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Figure 5.2 Dose response curves for neurotrophic gene induction by S1P and FTY720-P

Dose response curves for neurotrophic gene expression with S1P in primary human astrocytes (a) and FTY720-P in U251 cells (b). Significance was determined by a one-way ANOVA with

Dunnett’s post-test. **p<0.01, ***p<0.001. Graphs depict mean ± SEM.

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5.3.2 S1PR2 signalling pathways are not activated in FTY720-P treated cells S1PR2 is essential for S1P-mediated induction of neurotrophic genes (established in Chapter 3) and FTY720-P does not activate S1PR2. However, S1PR3 is also able to couple to the same G- proteins as S1PR2 [125]. We therefore examined activation of the signalling pathways identified in Chapter 4 by S1P and FTY720-P, to determine whether FTY720-P activates these pathways

(Figure 5.3). In both U251 cells and primary human astrocytes, S1P treatment resulted in significant phosphorylation of c-Jun (S63) and dephosphorylation of YAP (S127). FTY720-P treatment also increased Jun phosphorylation and dephosphorylated YAP, however the changes were smaller in magnitude than those induced by S1P, and not statistically significant. Similarly,

S1P but not FTY720-P produced significant induction of the immediate early transcription factors

EGR1 and FOS in both U251 cells and primary astrocytes.

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Figure 5.3 FTY720-P does not activate S1PR2-dependent transcription factors

U251 (a) and primary human astrocytes (b) were treated with BSA vehicle (BSA), S1P or FTY720-

P (FTY) for 1 h prior to analysis of c-Jun phosphorytion, YAP phosphorylation, EGR1 protein levels, and FOS protein levels (n=4 biological replicates from two independent experiments).

Densitometry for western blots were quantified and presented below their respective blots. EGR1 and FOS expression was normalised to GAPDH as a loading control. Phospho-Jun (p-Jun) and phospho-YAP (pYAP) were normalised to total Jun and YAP, respectively. Significance was determined by one-way ANOVA with Dunnett’s post-test relative to the BSA control. *p<0.05,

**p<0.01, ***p<0.001. Graphs depict mean ± SEM.

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5.3.4 Effect of combining FTY-P with S1PR2 agonist on neurotrophic gene expression Given the lack of efficacy of FTY720-P at S1PR2, we hypothesised that the addition of an S1PR2 agonist may boost neurotrophic gene expression by FTY720-P. U251 cells were treated with 1 µM

FTY720-P, 1 µM CYM5520, or 1 µM of both FTY720-P and CYM5520 (Figure 5.4). As expected, when the treatments were administered alone, there was no significant increase in gene expression for any of the four neurotrophic genes. However, the combination of both FTY720-P and

CYM5520 produced significantly increased expression of BDNF, PDGFB, HBEGF and LIF.

Figure 5.4 Supplementing FTY720-P with an S1PR2 agonists boosts neurotrophic gene expression.

Neurotrophic gene expression in U251 cells following treatment with BSA vehicle, 1 µM FTY720-

P (FTY), 1 µM CYM5520 (5520), or both (FTY+5520) for 4 h (n=8 from two independent experiments). Statistical significance was determined by one-way ANOVA, with Dunnett’s post- test comparing to the BSA control condition. * p<0.05. Graphs depict mean ± SEM

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5.3.5 FTY720-P does not phosphorylate ERK to the same extent as S1P Having identified the essential requirement for ERK signalling for the induction of immediate early genes, as well as HBEGF and LIF, we tested the activation of ERK1/2 over 60 min in primary human astrocytes (Figure 5.5). In agreement with prior publications, FTY720-P induced phosphorylation of ERK1/2, particularly ERK2 [137, 340]. However, S1P treatment resulted in a more pronounced and sustained phosphorylation of ERK1/2 over 60 min. After 60 min, ERK1/2 phosphorylation in FTY720-P treated cells had almost returned to basal levels.

Figure 5.5 Effect of S1P and FTY720-P on the phosphorylation of ERK

Phosphorylation of ERK was measured by western blot over the indicated times following treatment with either S1P or FTY720-P (n=3 biological replicates from 3 independent experiments). Densitometry is presented to the right of the western blots. Significance was determined by two-way ANOVA with Sidak’s post-test comparing S1P and FTY at every time point. **p<0.01, ***p<0.001. Graphs depict mean ± SEM.

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5.3.6 S1P but not FTY720-P protects hippocampal neurons against excitotoxic cell death To test the functional significance of the neurotrophic response to S1P, we used a neuronal culture system in which hippocampal neurons, grown at low density on glass coverslips, are supported by a ring of mixed cortical cells [293]. We quantified the protective effect of S1P or FTY720-P against excitotoxic cell death induced through overactivation of NMDA receptors, a mechanism of neuronal cell death that is clinically relevant to AD [379, 380]. In the absence of exogenous S1P, a 15 min pulse with 100 µM NMDA increased the percentage of non-viable neurons (assessed 18 h after NMDA pulse) from a mean of 16% to 50% (p=0.0008) (Figure 5.6). When pre-treated with

S1P for 6 h prior to NMDA pulse, a significant protective effect was observed with a reduction in cell death to 33.6% following NMDA pulse (p=0.023 compared to vehicle treated) whereas pre- treatment with FTY720-P afforded no protection. To elucidate whether the neuroprotective effect was mediated through a direct effect of S1P on the neurons, we cultured a high-density hippocampal culture on coverslips in the absence of the mixed cortical ring (Figure 5.7). In this model, pre-treatment with S1P increased the mean proportion of non-viable cells from 27% to

47% (p=0.009). FTY720-P also increased the level of cell death in the absence of NMDA

(p=0.0012). When cells were pulsed with NMDA, neither S1P nor FTY720-P afforded protection.

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Figure 5.6 Hippocampal neurons cultured in the presence of cortical support are protected from excitotoxic cell death in an S1P dependent manner

Percentage of non-viable neurons, quantified by fluorescence microscopy, 18 h after a pulse with

100 μM NMDA. Cells were incubated with 100 nM S1P, 1 μM FTY720-P, or BSA vehicle control for 6 h prior to NMDA stimulus. This experiment used low density hippocampal neurons (7 × 10

4 cells/coverslip) with a ring of mixed cortical cells around the outside of the well (4 independent cell culture experiments). Boxes indicate 25th to 75th percentiles, whiskers show minimum and maximum values, and horizontal line indicates the median. Statistical significance was determined using two-way ANOVA with Tukey’s post-test to compare treatments within NMDA or control groups.

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Figure 5.7 Stimulating neurons with S1P does not induce neuroprotective gene expression or protect from excitotoxic cell death.

Percentage of non-viable primary murine hippocampal neurons (5 × 105 /coverslip) was assessed via fluorescent microscopy the following day after a 10 min NMDA pulse (a). Hippocampal neurons (n=4) were pre-treated with BSA vehicle, S1P, or FTY720-P (FTY) for 6 h prior to

NMDA stimulus. Boxes indicate 25th to 75th percentiles, whiskers show minimum and maximum values, and horizontal line indicates the median. Statistical significance was determined using two- way ANOVA with Tukey’s post-test to compare treatments within NMDA or control groups.

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5.3.7 Ablating LIF reduces neuroprotection in the co-culture model

We next tested the functional role of BDNF, HBEGF, and LIF in mediating the neuroprotective effect of S1P. Neurons cultured in the presence of cortical support cells were incubated with 100 nM S1P for 6 h, pulsed with NMDA and then treated with a TrkB (BDNF receptor) antagonist

(ANA-12), an EGF receptor antagonist (Afatinib) to block HBEGF signalling, or a neutralising polyclonal antibody against LIF, 1 h after NMDA stimulus (Figure 5.8). Neither the EGF antagonist nor the TrkB antagonist produced a statistically significant reversal of the S1P-mediated protection of the hippocampal neurons. However, the neutralising LIF antibody significantly attenuated the neuroprotective effects of S1P (p<0.002 for NMDA + S1P vs NMDA + S1P vs LIF antibody). This suggests that secretion of LIF is necessary for S1P-mediated protection of hippocampal neurons against excitotoxic cell death.

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Figure 5.8 Blocking the action of neurotrophic factors promotes excitotoxic cell death

Hippocampal neurons with mixed cortical cells were treated with S1P or BSA control for 6 h prior to NMDA pulse, then with 100 nM ANA-12 (ANA), 100 nM Afatinib (Afat), or 20 ng/mL anti-

LIF antibody (AbLIF) 1 h after NMDA stimulus (5 independent experiments). Boxes indicate 25th to 75th percentiles, whiskers show minimum and maximum values, and horizontal line indicates the median. Statistical significance was determined using two-way ANOVA with Tukey’s post- test to compare treatments within the NMDA-treated or control groups.

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5.4 Discussion

Since its approval, multiple studies have indicated direct neuroprotective properties of FTY720 in addition to its established immunosuppressive properties as a therapeutic agent in multiple sclerosis [182, 221, 250, 255, 280, 301]. Here we show that FTY720-P is far less effective at inducing neurotrophic gene expression than the native ligand S1P, due to its lack of efficacy at activating S1PR2-mediated pathways. This translated to a lack of neuroprotection against NMDA- induced excitotoxic cell death in hippocampal neurons. Additionally, work from this chapter highlighted the importance of LIF, but not BDNF or HBEGF, in mediating S1P-induced neuroprotection against excitotoxic cell death. S1P did not protect hippocampal neurons cultured in the absence of glial support cells, pointing to a pathway in which glial cells, most likely astrocytes, secrete LIF in response to S1P, which mediates protection against excitotoxic cell death in hippocampal neurons.

Induction of LIF, BDNF, HBEGF, and PDGFB with S1P was two to three-fold higher than with

FTY720-P. We attribute this to the lack of activation of S1PR2 by FTY720, which was demonstrated to be the main receptor driving neurotrophic gene expression in Chapter 3. Hoffman et al. also showed that the induction of neurotrophic factors HBEGF, LIF and IL-11 by FTY720-

P in U373 astrocytoma cells and primary human astrocytes was lower than that seen with S1P, however this was not discussed in their paper [256]. Instead, these authors proposed a dual receptor mechanism for induction of these genes with FTY720-P, involving mainly S1PR3 activation with some contribution from S1PR1 signalling. This finding is not incongruous with our own findings.

S1PR3 is able to couple to the same G-proteins as S1PR2 and this raises the question as to whether

FTY720-mediated activation of S1PR3 is able to couple to Ga13. However, our data indicates that

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FTY720-P does not produce an appreciable induction of cJUN phosphorylation, YAP dephosphorylation, and EGR1 and FOS protein expression. This suggests that although S1PR3 is able to couple to the same G-proteins as S1PR2, functional redundancy of S1PR3 activation by

FTY720-P cannot substitute for S1PR2 activation by S1P. EGR1 and FOS are induced through canonical ERK1/2 signalling, and our phosphoproteomics data demonstrated this to be S1PR1 and

S1PR2-dependent [333, 381, 382]. In agreement with this, FTY720-mediated phosphorylation of

ERK was less efficient, with reduced magnitude and duration compared to S1P. This suggests that combined S1PR1 and S1PR2 signals drive the sustained activation of ERK1/2 signalling that is necessary for robust immediate early gene induction. Supplementing FTY720 with the S1PR2 agonist CYM5520 produced a clear increase in BDNF, PDGFB, HBEGF, and LIF gene expression compared to either compound alone. However, the signalling pathways activated by CYM5520 are not well defined. Future work should determine if the combination of CYM5520 and FTY720-

P is able to boost induction of immediate early genes, phosphorylate cJUN, and dephosphorylate

YAP.

Anastasiadou and colleagues reported that neuronal induction of BDNF was greater with FTY720 treatment than with S1P [254]. In our studies, BDNF gene expression increased when neurons were pre-treated with JTE013 before FTY720 or S1P stimulus, indicating that neuronal S1PR2 signalling may antagonise BDNF induction. Although we did not examine the induction of neurotrophic genes by FTY720 in neurons it is unlikely to be significant or functionally important given (i) the lack of neuroprotection by FTY720 in the in vitro excitotoxicity assays, and (ii) insignificant S1P-dependent induction of BDNF expression in neurons compared to astrocytes

(Chapter 3). The lack of neuroprotection with FTY720-P contrasts with previous studies showing

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Chapter 5 an increase in BDNF protein and neuronal survival following cytotoxic insults such as Ab and excitotoxic cell death [221, 255, 263]. These differences may relate to the culture system employed. We used purified astrocytes and hippocampal neuron cultures rather than mixed cortical

[221, 263] or cerebellar [254] neurons. In fact, our in vitro co-culture viability assays confirmed that cortical support cells (most likely astrocytes) protected hippocampal neurons from excitotoxic cell death, rather than this being mediated through a direct action of S1P on neuronal S1P receptors.

In the absence of the cortical support ring, both S1P and FTY720-P increased the number of non- viable neurons without addition of NMDA, suggesting a buffering mechanism whereby the cortical support cells converted the S1P signal into a protective response for neurons, via the production of neurotrophic factors. ELISA assays demonstrated a significant induction of both BDNF and LIF when astrocytes were treated with S1P but not FTY720-P, whereas in Chapter 3 we established the levels of BDNF and LIF were below the limit of detection in neuronal supernatants, even after

S1P stimulus. In the current chapter, we also observed a significant induction of both secreted

BDNF and LIF in primary human astrocytes, whereas in Figure 3.3 we observed a non-significant increase in LIF and no increase in BDNF following S1P stimulus. The primary human astrocyte cell culture supernatants collected for the ELISA experiments in this chapter were obtained from a separate biological sample to those used for the assay in Figure 3.3. The increase of secreted LIF protein with S1P stimulus was also statistically significant in Figure 3.8c. Natural biological variation in these astrocyte preparations may explain why we observed some variability in protein induction between experiments.

Interestingly, neither BDNF signalling through its receptor TrkB, nor HBEGF signalling through

EGF receptors, mediated neuroprotection in response to S1P, as neither ANA12 or Afatinib

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Chapter 5 attenuated the neuroprotective effects of S1P. BDNF is a potent neurotrophic factor that is currently a target of significant interest for neurodegenerative diseases [223, 226, 230, 383]. BDNF is involved in long term potentiation, which is required for memory formation [384]. In AD, there is a correlation between cognitive function and BDNF levels [229, 230, 385]. Given the highly established neuroprotective and neurotrophic properties of BDNF, the lack of action in mediating neuroprotection in the in vitro excitotoxic assays was unexpected. Our study is in agreement with a recent publication also describing a lack of neuroprotection by FTY720 in a mouse model of PD

[386]. However, we established a significant role for LIF in protection of neurons against excitotoxic cell death. Our study demonstrating the functional relevance of LIF is in agreement with a previous study also showing a neuroprotective role for LIF in the context of excitotoxicity

[387]. In the context of AD, LIF has been shown to promote cholinergic differentiation and function [260, 388]. Loss of cholinergic neuron function is a key impairment in AD [389, 390].

LIF has also been shown to promote myelination in vivo [391, 392], and myelin deficits have also been reported in AD [393-396]. Moreover, neural stem cell self-renewal is enhanced by LIF in the adult brain [397]. Thus, the induction of LIF secretion by S1P receptor activation may afford a means to recapitulate cholinergic signalling mechanisms and improve cognitive deficits.

Protein therapeutics cannot permeate the BBB without conjugation to carrier molecules. The induction of neuroprotective factors through S1P receptor activation provides a means to boost the levels of these factors in the CNS using brain-penetrant small molecule therapeutics. With the clinical success of FTY720, recent developments in S1P receptor agonists have increased selectivity for S1PR1 to reduce side effects associated with S1PR3 such as bradycardia [245, 267,

269]. However, the current S1P agonists in the clinic and clinical trials do not target S1PR2. In

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Chapter 5 fact, S1PR2 agonism has been generally considered undesirable, as S1PR2 signalling induces vascular permeability [141, 398] and a proinflammatory phenotype in microglia [189].

Additionally, S1PR2 expression is quite widespread with detection in the CNS [179, 189, 398], skeletal system [285, 399, 400], and immune system, amongst others [142, 401-404]. Thus, although current clinical S1P receptor agonists have a well-tolerated safety profile, the pursuit for

S1PR2 agonists as therapeutics for neurodegenerative diseases should be approached cautiously.

Nevertheless, S1PR2 agonists have also displayed neuroprotective properties in the context of chemotherapy-induced ototoxicity and neuropathy [286, 324], which - like most neurodegenerative conditions – is currently untreatable. Our data indicates that dual activation of

S1PR1 and S1PR2, rather than activation of either receptor in isolation, is crucial for an effective neuroprotective response. The current mechanism for clinical S1P receptor agonists is focused on immunosuppression due to downregulation of S1PR1, as opposed to the protective signalling properties of the native ligand S1P [210, 211, 268]. Future developments to S1P receptor agonists as therapeutic options for neurodegenerative diseases should consider the neuroprotective aspects of S1PR2 agonism.

In summary, the work from this chapter highlights the functional significance of LIF induction by

S1P, through an S1PR2-dependent signalling mechanism, in mediating neuroprotection against excitotoxic stimulus. Future studies should investigate whether this LIF-mediated mechanism affords protection in other neurotoxic insults present in AD such as Ab treatment or cerebral ischaemia.

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Chapter 6: Summary and Future Directions

Globally, society is facing a shift in demographics with population ageing. With this transition, countries will face major challenges to public health, especially in regard to diseases associated with ageing. AD, along with other neurodegenerative diseases for which the greatest risk factor is ageing, represents a major hurdle given the lack of effective disease modifying therapies. Current approved treatments aim to extend cholinergic signalling in the synaptic cleft to compensate the loss of cholinergic neurons and to antagonise excitotoxic cell death, however this does not significantly impact on disease progression [71, 72]. Recent developments in the field of S1P receptor agonists have identified potential neuroprotective properties associated with these compounds, independent of their immunosuppressive function [221, 250, 255, 263, 301]. These studies demonstrate that S1P agonists are brain-penetrant and target different resident CNS cells to exert protection from cytotoxic insults such as NMDA-mediated excitotoxic cell death and Ab- induced cytotoxicity [168, 188, 255, 264, 301]. However, development since the approval of

FTY720 have centred around improving the selective targeting of S1PR1 [245, 269]. Research into the effects of other S1P receptors in the context of neuroprotection is also significantly less compared to S1PR1.

This thesis describes the neuroprotective response in astrocytes following activation of S1P receptors, particularly noting the main driver of this neuroprotective response to be S1PR2. Results from this thesis demonstrate that astrocytes but not neurons respond to S1P by up-regulating the neurotrophic factors BDNF, PDGFB, HBEGF and LIF. The induction of neurotrophic mRNA is driven primarily by S1PR1 and S1PR2 in U251 cells, but involves co-ordinate activation of

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S1PR1, 2 and 3 in primary human astrocytes. G-protein analysis revealed a dual role for Gai and

Ga13, most likely coupling to S1PR1 and 2 respectively. Analysis of human hippocampal samples and in vitro studies identified a potential circuit involving levels of SphK2, S1P and LIF, as LIF expression was positively correlated with both SphK2 and S1P. This may be biologically important for basal neurotrophic factor expression in the CNS. This thesis also utilised, for the first time, a systems-wide approach to study the changes to the phosphoproteome following S1P receptor activation over time, and define the roles of S1PR1 and S1PR2 in this process. RhoA and MAPK signalling were highly affected by S1P. S1PR2 alone mediated RhoA signalling, while dual contributions of S1PR1 and S1PR2 mediated MAPK signalling. These signalling pathways resulted in activation of immediate early genes and target transcription factors downstream of

RhoA signalling. A comparison of neurotrophic gene induction found that FTY720-P was less effective than S1P, attributed to the lack of activation of S1PR2-associated pathways. This resulted in an inability for FTY720-P to protect neurons, co-cultured with glial cells, against a cytotoxic dose of NMDA, whereas S1P was protective. Finally, functional studies found LIF protein secretion was the primary neurotrophic factor mediating neuroprotection against NMDA excitotoxicity and this was dependent on cortical support cells, likely astrocytes.

6.1 Potential limitations and future directions

To investigate the effect of S1P in astrocytes, our in vitro experiments utilised the U251 cell line and primary human and murine embryonic astrocytes. Our use of embryonic cells may not accurately model what occurs in the aged brain with “older” astrocytes given that normal ageing favours the A1 astrocyte phenotype [405]. The A1 phenotype is thought to be neurotoxic as they no longer promote neuronal homeostasis and instead induce cell death in neurons and oligodendrocytes [55]. A recent study found that inhibiting this phenotypic change in astrocytes is

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Chapter 6 neuroprotective in the context of PD [406]. The use of primary human embryonic astrocytes as a model is the best option currently available to us, given primary human adult/aged astrocytes cannot be ethically sourced. An alternative approach would be differentiation of induced pluripotent stem cells into astrocytes. Future studies could also consider the use of aged murine astrocytes to investigate the effects of S1P. An RNA sequencing study investigating the effects of ageing in mice by the Barres lab showed drastic changes to S1P receptor expression in astrocytes with ageing, with S1PR1 mRNA more than tripling from 100 FPKM to greater than 400 FPKM in three different brain regions [405]. mRNA expression of S1PR2 and 3 remained at an average of

1 and 2 FPKM respectively. Murine astrocytes also show 10-fold higher mRNA expression of

S1PR1 than human astrocytes [163, 164]. How this may influence S1P signalling is yet to be determined but presents an important caveat when investigating the effects of S1P between species.

Our study into the effect of S1P on primary human embryonic neurons indicated a lack of induction for the neurotrophic factors of interest. However, we focused on four neurotrophic factors of interest, an approach that was justified given the functional relevance of LIF protein secretion established in Chapter 5 and prior reports of neuroprotection through BDNF up-regulation [221,

255] . Neurons express the same S1P receptors as astrocytes and previous literature has identified the induction of BDNF, the proinflammatory chemokine ligand 2 (CCL2) and transgelin in response to S1P [254, 255]. Studies into CCL2 reveal that it is a potent activator of microglia and promotes a proinflammatory phenotype [407-409]. In the context of AD, two independent studies noted that higher levels of CCL2 in CSF and plasma are associated with a faster rate of cognitive decline [410, 411]. Our focused qPCR approach may have resulted in us missing other important factors that are induced by S1P. Transcriptomic profiling or qPCR arrays in both cell types would

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Chapter 6 provide a system-wide view of target genes being affected by S1P and allow us to dissect the proinflammatory from the neurotrophic effect.

In the current study, our investigation into the effect of S1P on other glial cells was also limited.

We investigated the effects of S1P on the immortalised microglial cell line BV-2, which did not show evidence for induction of the neurotrophic factors of interest. However, this does not exclude microglia as mediators of S1P-mediated neuroprotection. In future work, the neurotrophic and pro- inflammatory gene response to S1P should be investigated in primary microglia. We did not investigate the effect of S1P in oligodendrocytes. There are immortalised oligodendrocyte cell lines such as oli-neu and MO3.13 cells that were not utilised, and this should be considered in the future [412, 413]. Future attempts at investigating potential neuroprotective roles of microglia and oligodendrocytes could also consider differentiating induced pluripotent stem cells into these cell types [414-417]. As reviewed in Chapter 2, S1P receptor activation promotes myelination in OPCs and remyelination in vitro [191, 194]. Additionally, multiple reports demonstrate that microglial activation is reduced potentially through a mechanism dependent on S1PR1 activation [168, 253,

418]. In fact, microglia have been reported to secrete BDNF and glial-derived neurotrophic factor following FTY720 treatment, in addition to reduced secretion of pro-inflammatory cytokines such as TNFa [168]. Given the alterations to myelination in AD [419, 420], the fact that neuroinflammation plays a significant role in AD pathology [46, 47], and neurotrophic factors have well established cytoprotective properties [226, 239, 421], microglia and oligodendrocytes present a worthy target for future neuroprotection studies involving S1P and S1P agonists.

Of the neurotrophic factors induced by S1P, we did not measure protein secretion of HBEGF by

ELISA. HBEGF is a membrane bound protein that requires proteolytic cleavage by metalloproteases for secretion [313, 314]. Metalloproteases that cleave HBEGF include the

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ADAMs (a disintegrin and metalloproteinase) family of proteins, of which include ADAM9 and

17 [422-424]. Studies has noted the regulation of the ADAMs proteins, particularly ADAM17 activity to be regulated by ERK phosphorylation at Thr735 [425, 426]. However, in our phosphoproteomic data, we observed significant dephosphorylation of ADAM9, 17 and 19 at

S758, 791 and 818 respectively. How this affects their respective HBEGF-cleaving abilities is not yet known. HBEGF itself does not require ectodomain shedding to mediate its effects with studies showing cell-cell interactions sufficient for HBEGF activity [427-429]. Measuring the protein levels of HBEGF via traditional methods such as Western blotting may answer whether the induction of HBEGF translates to the protein level.

The results from this thesis utilised a systems-wide approach to investigate the phosphorylation signalling events activated by S1P, however there are signalling events this thesis is yet to elucidate. We were unable to establish whether FTY720-P activates signalling pathways distinct to S1P or is just at a lower magnitude (e.g. p-ERK, p-JUN, IEG induction). The phosphoproteomics results in Chapter 4 identified that major signalling events in response to S1P in U251 cells occur downstream of S1PR2, which is not activated by FTY720-P [208]. Given astrocytes express S1PR1-3 and FTY720-P is an agonist of four of the five S1P receptors, future phosphoproteomic studies comparing S1P and FTY720-P would potentially delineate differences in activated signalling pathways downstream of S1PR1 and 3. Additionally, we were unable to provide direct evidence that S1PR2 couples to Ga13. Previous studies have shown S1PR2 coupling to Ga13 but S1PR2 is also reported to couple to Gai and Gaq/11 [125, 143, 430-432]. The results from this thesis identified a major role for Ga13, since siRNA silencing resulted in significant suppression of neurotrophic gene induction. However, direct interactions were not shown.

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Assessing direct interactions through the use of co-immunoprecipitation, fluorescence or bioluminescence resonance energy transfer may provide evidence for S1PR2-Ga13 coupling.

S1P was demonstrated to be a potent neuroprotective molecule in the context of excitotoxicity.

This only occurred in the presence of glial cells, as direct treatment of neurons with S1P or

FTY720-P resulted in increased cell death. Although S1P protected neurons from excitotoxic cell death, this only represents one of the many mechanisms of cytotoxicity in AD, alongside neuroinflammation and Ab-mediated toxicity [13, 433, 434]. The choice of excitotoxic cell death for our studies was due to the inability to achieve consistent results using Ab as a cytotoxic molecule, potentially due to incorrect oligomer preparation. In the context of neuroinflammation, it is likely that FTY720-P, as reported by previous studies, would show greater efficacy protecting neurons given its well established properties in suppressing inflammation [168, 182, 253, 264].

Likewise, FTY720-P has previously been demonstrated to be neuroprotective against Ab [255,

301, 435]. S1P however, has been shown to promote inflammation in multiple cell types, including resident CNS cells such as microglia and astrocytes [145, 189, 190, 436-438]. It would be interesting to investigate whether S1P demonstrates neuroprotection in a neuroinflammatory context.

6.2 Targeting S1P receptors and sphingolipid metabolism for neurodegenerative disease therapy

Recent studies have demonstrated that S1P receptor agonists are neuroprotective and secrete neurotrophic factors in several neurodegenerative paradigms [188, 221, 250, 256, 266, 286, 323,

377, 378]. Repurposing these compounds may provide therapeutic benefit in neurodegenerative diseases like AD, especially FTY720 given its well-established safety profile and noted ability to

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The up-regulation of neurotrophic factors makes S1P receptor agonists a highly attractive therapeutic option not only for AD but for other neurodegenerative diseases as well. We and others have demonstrated the up-regulation of LIF in response to S1P and S1P agonists [256]. Aside from the effect on cholinergic function described in Chapter 5, LIF has previously been demonstrated to promote myelination and oligodendrocyte survival [392, 439-442]. This has ramifications not only for centrally occurring neurodegenerative diseases, but potentially peripheral demyelinating diseases as well. For example, individuals with HD, which is characterised by an accumulation of the mutant huntingtin (mHTT) protein, exhibit myelin breakdown and reduce white matter content

[443-445]. The mHTT protein itself has recently been shown to induce oligodendrocyte dysfunction and myelin defects in HD mice [446]. In the periphery, Schwann cells are the myelinating cell rather than oligodendrocytes. LIF has been demonstrated to not only promote the survival of Schwann cells, but also promote the expression of myelin basic protein, one of the markers for myelin expression [447, 448].

PD, characterised by the accumulation of Lewy bodies and loss of dopaminergic neurons [449], may also benefit from S1P receptor agonists. Aside from LIF, we and others have observed the up-regulation of HBEGF [256]. HBEGF has been demonstrated to promote the survival of midbrain dopaminergic neurons, which is an area of the brain heavily affected by PD pathology

[449-451]. Moreover, HBEGF has also been demonstrated to increase neurogenesis in the hippocampus and subventricular zone of aged mice [452], and reduce infarct size in a mouse model of cerebral ischaemia [453]. Consequently, the repurposing of S1P agonists that have demonstrated induction of neurotrophic factors may be beneficial for other neurodegenerative diseases.

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Based on our findings, developing a small molecule agonist of both S1PR1 and S1PR2 may be beneficial for neuroprotection. GPCR signalling, however, is a complex process whereby ligands are able to bias downstream receptor signalling. GPCR signalling is dependent on ligand-receptor interaction, and different agonists have different efficacies in the activation of signalling pathways

[454, 455]. This biased signalling in GPCRs occurs as different ligands induce distinct receptor conformations that affect the receptor-G-protein complex [454, 456-459]. Although FTY720-P and other S1PR1 agonists bind in the orthosteric binding pocket [198, 269, 279, 460], current

S1PR2 agonists are allosteric activators [281]. However, these S1PR2 agonists demonstrate neuroprotective properties in the contexts of chemotherapy induced ototoxicity and neuropathy, through a similar mechanism of RhoA activation as established in this thesis [281, 286, 323, 324].

Structurally, the phenyl ring in FTY720 is crucial to its mechanism of action as an immunosuppressant, rather than as a natural agonist like S1P [199]. This structural difference between FTY720-P and S1P, and unique aspects of S1PR2 structure, contribute the lack of S1PR2 agonism by FTY720-P [461]. Therefore, future S1P receptor drug development projects wishing to develop a dual S1PR1/2 agonist should reconsider using FTY720 as the lead compound. One approach could be a combined administration of S1PR1 and S1PR2 agonists to achieve neuroprotection. However, as discussed in Chapter 5, there are drawbacks to S1PR2 agonisms including increased vascular permeability [141, 398] and promoting a pro-inflammatory phenotype in microglia [189].

Interestingly, two recent publications have noted that altering carbon chain length in S1P can affect its signalling at S1PR2 [462, 463]. One study found that C19 and C20 S1P both had greater potency but lower efficacy compared to S1P at S1PR2 [462]. Conversely, C20 S1P was found to act as a partial agonist and suppressed the up-regulation of prostaglandin-endoperoxide synthase 2 (COX-

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2) that was induced by C18 S1P [463]. Taken together, these results suggest how future compounds may be developed to induce neurotrophic factors while avoiding negative side-effects such as induction of pro-inflammatory proteins. Thus, future dual S1PR1 and 2 agonists that bind the orthosteric binding may have future therapeutic potential in neurodegenerative diseases.

While targeting S1P receptors presents a great therapeutic option, metabolic abnormalities in other sphingolipid enzymes have also been reported in AD. This presents other potential targets for novel small molecules. For example, activity of ceramide synthase 2, which is the enzyme that catalyses the formation of the long chain ceramides in myelin, has been found to be reduced prior to the deposition of tau in cortical regions of AD brains [396]. Conversely, the accumulation of ceramides, which is considered pro-apoptotic, has been reported in AD in multiple studies [108,

114, 464-466]. Decreased SphK2 activity and protein in AD has also been reported [109, 467]. In mouse models, loss of SphK2 activity resulted in reduced S1P, and surprisingly, appears to counteract Ab deposition [299, 467]. However, loss of SphK2 also resulted in demyelination and hippocampal volume loss in mice [299]. SphK2 inhibition, while presenting an opportunity to reduce Ab deposition, may therefore be detrimental to oligodendrocyte viability and overall neurological function. In summary, there are multiple points in the sphingolipid pathway that may be targeted to provide some therapeutic benefit in AD.

In summary, results arising from this thesis have described a neuroprotective role of dual S1P receptor signalling on astrocytes, specifically protecting neurons in an in vitro model of excitotoxic cell death. Astrocytes, but not neurons induce neurotrophic genes through cooperative signalling of multiple S1P receptors, with the main driver being S1PR2. The first report of phosphoproteomic analysis of S1P signalling in astrocytes identified how S1PR1 and S1PR2 activation by S1P synergises to activate signalling pathways that are both unique to each receptor and common to

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both receptors. These receptors likely couple to Gai and Ga13 to activate signalling pathways that converge on the induction of immediate early genes and other transcription factors in the vicinity of the transcription start site of neurotrophic genes. This marked induction of neurotrophic genes was significantly less with the clinical S1P receptor agonist Fingolimod, which does not activate

S1PR2. This reduced induction resulted in a lack of protection of neurons against NMDA-induced excitotoxic cell death compared to S1P. However, the protection mediated by S1P was absent when hippocampal neurons were cultured alone, indicating that glial cells, particularly astrocytes, may be mediating this response. Finally, this protective response by S1P was mediated through

LIF rather than BDNF, although both neurotrophic factors are secreted by S1P-stimulated astrocytes. Targeting S1PR1 and S1PR2 activation may provide therapeutic benefit when used in conjunction with other therapies for AD.

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Appendix

Table A1. Statistically significant phosphoproteins regulated by W146 only Gene Modified Protein names names residue q-value Ubiquitin-conjugating enzyme E2 H UBE2H S3 0.0408636 Protein FAM91A1 FAM91A1 S829 0.0231612 Actin-binding protein anillin ANLN S792 0.0168503 Apoptotic chromatin condensation inducer in the nucleus ACIN1 S240 0.00595574 Apoptotic chromatin condensation inducer in the nucleus ACIN1 S243 0.00595574 Nuclear factor 1 X-type;Nuclear factor 1 NFIX S265 0.0111975 Nuclear factor 1 X-type;Nuclear factor 1 NFIX S268 0.0111975 Protein LSM14 homolog A LSM14A S178 0.0462493 MKL/myocardin-like protein 2;Phosphatase and actin regulator MKL2 S211 0.043255 MKL/myocardin-like protein 2;Phosphatase and actin regulator MKL2 S225 0.0406777 Signal recognition particle subunit SRP72 SRP72 T624 0.030893 ETS domain-containing transcription factor ERF ERF S161 0.0442779 Sorting nexin-1 SNX1 S72 0.0286658

Table A.2 Statistically significant phosphoproteins regulated by JTE013 only Modified Protein names Gene names residue q-value UHRF1-binding protein 1-like UHRF1BP1L S989 0.044955 Alpha-endosulfine ENSA S2 0.035647 Afadin MLLT4 S1501 0.007122 Protein ITFG3 ITFG3 S21 0.001218 Rab5 GDP/GTP exchange factor RABGEF1 S302 0.009775 Ubiquitin-associated protein 2- like UBAP2L S416 0.034538 LIM domain only protein 7 LMO7 S704 0.01702 SH3 and PX domain- containing protein 2A SH3PXD2A S406 0.043535 Synembryn-A RIC8A S502 0.030241

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Transmembrane and coiled- coil domain-containing protein 1 TMCO1 S60 0.005226 Catenin delta-1 CTNND1 S899 0.014657 Ribosomal L1 domain- containing protein 1 RSL1D1 S413 0.013618 Plasma membrane calcium- transporting ATPase 4 ATP2B4 S328 0.004094 Antigen KI-67 MKI67 S1937 0.001656 Antigen KI-67 MKI67 S2299 0.020605 Nuclear autoantigenic sperm protein NASP S497 0.026651 Nuclear autoantigenic sperm protein NASP S158 0.026651 Centromere protein F CENPF S3119 0.008015 Apoptosis-stimulating of p53 protein 2 TP53BP2 S480 0.006737 Scaffold attachment factor B2 SAFB2 S207 0.009236 Ataxin-2-like protein ATXN2L S306 0.009647 Proline-rich protein 11 PRR11 S307 0.006424 Protein LAP2 ERBB2IP S852 0.005187 Palmitoyltransferase ZDHHC5;Palmitoyltransferase ZDHHC5 S432 0.032008 Probable ATP-dependent RNA helicase YTHDC2 YTHDC2 S1221 0.019432 Dedicator of cytokinesis protein 5 DOCK5 S1781 0.032435 Protein ECT2 ECT2 S716 0.02354 Transmembrane protein 51 TMEM51 S182 0.004893 SH3 domain-binding protein 4 SH3BP4 S279 0.010839 Synergin gamma SYNRG S812 0.040678 Brain-specific angiogenesis inhibitor 1-associated protein 2 BAIAP2 S454 0.008895 CD2-associated protein CD2AP S458 0.002523 Nuclear mitotic apparatus protein 1 NUMA1 S1991 0.012041 Band 4.1-like protein 3;Band 4.1-like protein 3, N- terminally processed EPB41L3 S443 0.006325 Band 4.1-like protein 3;Band 4.1-like protein 3, N- terminally processed EPB41L3 S448 0.006325 CLIP-associating protein 2 CLASP2 S360 0.027229

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CLIP-associating protein 2 CLASP2 S364 0.027229 Lymphokine-activated killer T-cell-originated protein kinase PBK S32 0.015766 Pericentriolar material 1 protein PCM1 S428 0.040111 Pericentriolar material 1 protein PCM1 S431 0.040678 Microtubule-associated protein;Microtubule- associated protein 4 MAP4 S597 0.014184 Myb-binding protein 1A MYBBP1A S1308 0.030584 Myb-binding protein 1A MYBBP1A S1314 0.030584 Lamin-B2 LMNB2 S37 0.000657 Catenin delta-1 CTNND1 S920 0.0008 Antigen KI-67 MKI67 S538 0.001523 Antigen KI-67 MKI67 S1861 0.006424 Transcriptional coactivator YAP1 YAP1 S127 0.046925 Transcriptional coactivator YAP1 YAP1 S138 0.046925 Emerin EMD S49 0.01702 Arf-GAP domain and FG repeat-containing protein 1 AGFG1 S181 0.004542 Nuclear pore complex protein Nup98-Nup96;Nuclear pore complex protein Nup98;Nuclear pore complex protein Nup96 NUP98 S673 0.014657 Rho GTPase-activating protein 12 ARHGAP12 S213 0.044688 Rho GTPase-activating protein 12 ARHGAP12 S215 0.044688 Zinc finger CCCH domain- containing protein 13 ZC3H13 S1014 3.64E-05 Zinc finger CCCH domain- containing protein 13 ZC3H13 S1017 3.64E-05 Rho GTPase-activating protein 21 ARHGAP21 S1432 0.014757 Polymerase I and transcript release factor PTRF S300 0.040678 Rapamycin-insensitive companion of mTOR RICTOR S1174 0.005187 Rapamycin-insensitive companion of mTOR RICTOR S1177 0.005187

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Ras-specific guanine nucleotide-releasing factor RalGPS2 RALGPS2 S308 0.006661 Ras-specific guanine nucleotide-releasing factor RalGPS2 RALGPS2 S296 0.010151 Ankyrin repeat and LEM domain-containing protein 2 ANKLE2 S914 0.031503 Ankyrin repeat and LEM domain-containing protein 2 ANKLE2 S919 0.031503 Phosphatase and actin regulator 4 PHACTR4 S328 0.026183 General transcription factor 3C polypeptide 2 GTF3C2 S132 0.008415 Nucleolar and spindle- associated protein 1 NUSAP1 S352 0.012441 Palmitoyltransferase ZDHHC5;Palmitoyltransferase ZDHHC5 S296 0.000425 Palmitoyltransferase ZDHHC5;Palmitoyltransferase ZDHHC5 S299 0.000425 Nuclear mitotic apparatus protein 1 NUMA1 T2000 0.012041 Microtubule-associated protein;Microtubule- associated protein 4 MAP4 T602 0.02118 Lamin-B2 LMNB2 T34 0.001285 Catenin delta-1 CTNND1 T916 0.0008 Antigen KI-67 MKI67 T2325 0.026651 Antigen KI-67 MKI67 T2328 0.026651 Antigen KI-67 MKI67 T347 0.025817 Antigen KI-67 MKI67 T543 0.001523 Antigen KI-67 MKI67 T1869 0.006424 Arf-GAP domain and FG repeat-containing protein 1 AGFG1 T177 0.004542 Nuclear pore complex protein Nup98-Nup96;Nuclear pore complex protein Nup98;Nuclear pore complex protein Nup96 NUP98 T670 0.014657 Polymerase I and transcript release factor PTRF T302 0.040678 Phosphatase and actin regulator 4 PHACTR4 T342 0.026183 General transcription factor 3C polypeptide 2 GTF3C2 T137 0.020534

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RelA-associated inhibitor PPP1R13L T123 0.016131 Serine/arginine repetitive matrix protein 1 SRRM1 S424 0.030853 Serine/arginine repetitive matrix protein 1 SRRM1 S426 0.030853 Neuroblast differentiation- associated protein AHNAK AHNAK S5749 0.000425 Neuroblast differentiation- associated protein AHNAK AHNAK S5752 0.000425 Zinc finger protein 36, C3H1 type-like 2 ZFP36L2 S426 0.028956 Partitioning defective 3 homolog B;Partitioning defective 3 homolog PARD3B;PARD3 S924 0.008146 Myristoylated alanine-rich C- kinase substrate MARCKS S170 0.009854 BET1-like protein BET1L S37 0.009647 BET1 homolog BET1;DKFZp781C0425 S50 0.040222 Filamin A-interacting protein 1-like FILIP1L S791 0.003261 Protein LYRIC MTDH S298 0.001836 Protein NDRG1 NDRG1 S330 0.004826 Signal-induced proliferation- associated protein 1 SIPA1 S55 0.008895 FERM domain-containing protein 6 FRMD6 S542 0.016159 SLIT-ROBO Rho GTPase- activating protein 1 SRGAP1 S940 0.032828 Unconventional myosin-IXb MYO9B S1972 0.01019 Unconventional myosin-IXb MYO9B S1323 0.008415 Huntingtin-interacting protein 1 HIP1 S320 0.036791 Protein phosphatase 1 regulatory subunit 12A PPP1R12A S299 0.016931 Protein phosphatase 1 regulatory subunit 12A PPP1R12A S507 0.008895 Synemin SYNM S1107 0.028521 Protein phosphatase 1G PPM1G S527 0.018582 WD repeat-containing protein 62 WDR62 S1348 0.035816 Cyclin-dependent kinase 14 CDK14 S95 0.021238 Transcription factor AP-1 JUN S63 0.0008 Plastin-2 LCP1 S5 0.000749

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Y-box-binding protein 3;Nuclease-sensitive element- binding protein 1;Y-box- binding protein 2 YBX3;YBX1;YBX2 S102 0.003859 Chromobox protein homolog 5 CBX5 S14 0.032098 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S1339 0.012341 Nestin NES S746 0.002069 A-kinase anchor protein 12 AKAP12 S651 0.015836 Rap guanine nucleotide exchange factor 1 RAPGEF1 S311 0.02743 AP2-associated protein kinase 1 AAK1 S652 0.031216 MAP7 domain-containing protein 1 MAP7D1 S113 0.013617 Vacuolar protein sorting- associated protein 26B VPS26B S304 0.009647 Rho GTPase-activating protein 29 ARHGAP29 S1019 0.026759 Centrosomal protein of 55 kDa CEP55 S428 0.046925 Cytospin-B SPECC1 S912 0.000508 Cytospin-B SPECC1 S131 0.002398 Tensin-3 TNS3 S811 0.007045 Protein-methionine sulfoxide oxidase MICAL3 MICAL3 S1310 0.004214 Phosphatase and actin regulator 4 PHACTR4 S612 0.001789 Phosphatase and actin regulator 4 PHACTR4 S574 0.000783 Palladin PALLD S893 3.64E-05 Caskin-2 CASKIN2 S858 0.024825 TBC1 domain family member 5 TBC1D5 S522 0.012389 Dedicator of cytokinesis protein 7 DOCK7 S1392 0.007045 Fasciculation and elongation protein zeta-1 FEZ1 S298 0.016931 Endophilin-A2 SH3GL1 S288 0.017798 FUN14 domain-containing protein 2 FUNDC2 S151 0.023042 182 kDa tankyrase-1-binding protein TNKS1BP1 S1666 0.030893 Rab3 GTPase-activating protein non-catalytic subunit RAB3GAP2 S450 0.003267

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Ras GTPase-activating protein nGAP RASAL2 S213 0.02743 MKL/myocardin-like protein 2;Phosphatase and actin regulator MKL2 S66 0.005226 FH1/FH2 domain-containing protein 1 FHOD1 S367 0.045334 Rho guanine nucleotide exchange factor 7;Rho guanine nucleotide exchange factor 6 ARHGEF7;ARHGEF6 T526 0.028552 AP2-associated protein kinase 1 AAK1 T653 0.031216 B-cell CLL/lymphoma 9-like protein BCL9L T957 0.026651 Mitogen-activated protein kinase 1 MAPK1 Y187 0.007122 Extended synaptotagmin-2 ESYT2 S660 0.022444 Rho-associated protein kinase 2 ROCK2 S1137 0.002523 Promyelocytic leukaemia protein PML S482 0.01007 Na(+)/H(+) exchange regulatory cofactor NHE-RF1 SLC9A3R1 S294 0.01571 Fos-related antigen 2 FOSL2 S320 0.001218 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S1265 0.009854 Condensin complex subunit 1 NCAPD2 S1330 0.009363 PHD finger protein 6 PHF6 S138 0.025817 Cytoplasmic dynein 1 light intermediate chain 1 DYNC1LI1 T513 0.000143 Mitogen-activated protein kinase 8;Mitogen-activated protein kinase 10 MAPK8;MAPK10 T183 0.0008 Calmodulin-regulated spectrin-associated protein 2 CAMSAP2 T865 0.000311 RalA-binding protein 1 RALBP1 T27 0.009647 Golgi reassembly-stacking protein 2 GORASP2 T222 0.005187 Golgi reassembly-stacking protein 2 GORASP2 T225 0.005187

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Table A.3 Statistically significant phosphoproteins regulated by both W146 and JTE013 Gene Modified Protein names names residue q-value Myomegalin PDE4DIP S195 0.012303 Filamin A-interacting protein 1-like FILIP1L S1049 0.025551 Nuclear factor 1 X-type;Nuclear factor 1 NFIX S265 0.002917 Unconventional prefoldin RPB5 interactor 1 URI1 S442 0.034662 Nuclear pore complex protein Nup214 NUP214 S1023 0.006847 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S1852 0.017601 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S1265 0.005493 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S832 0.029245 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S1797 0.002186 Nestin NES S680 0.042632 Nuclear pore complex protein Nup153 NUP153 S516 0.001656 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-3 PLCB3 S537 0.001513 Neuroblast differentiation-associated protein AHNAK AHNAK S210 0.001218 A-kinase anchor protein 13 AKAP13 S2543 0.031216 GTPase-activating protein and VPS9 domain- containing protein 1 GAPVD1 S929 0.009854 Phosphofurin acidic cluster sorting protein 1 PACS1 S430 0.045708 EH domain-binding protein 1 EHBP1 S759 0.041654 E3 ubiquitin-protein ligase Praja-1 PJA1 S265 0.005187 Exocyst complex component 4 EXOC4 S32 0.005187 Actin-binding protein anillin ANLN S485 0.00016 Mitochondrial import receptor subunit TOM22 homolog TOMM22 S15 0.004317 Homer protein homolog 3 HOMER3 S159 0.006137 Protein phosphatase methylesterase 1 PPME1 S243 0.000508 Nuclear factor of activated T-cells, cytoplasmic 3 NFATC3 S265 0.001285 Nuclear factor of activated T-cells, cytoplasmic 3 NFATC3 S269 0.002554 Paxillin PXN S313 0.032091 Eukaryotic translation initiation factor 4E-binding protein 1 EIF4EBP1 S83 0.034538 Cytospin-B SPECC1 S131 0.01007 Niban-like protein 1 FAM129B S692 0.044955 Niban-like protein 1 FAM129B S696 0.044955 Mitogen-activated protein kinase 1 MAPK1 T185 0.000642 Mitogen-activated protein kinase 14 MAPK14 T180 0.026759

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Mitogen-activated protein kinase 1 MAPK1 Y187 0.000642 Mitogen-activated protein kinase 14 MAPK14 Y182 0.026759 Myosin light chain kinase, smooth muscle;Myosin light chain kinase, smooth muscle, deglutamylated form MYLK S1773 0.001037 Myosin light chain kinase, smooth muscle;Myosin light chain kinase, smooth muscle, deglutamylated form MYLK S1776 0.000167 Myosin light chain kinase, smooth muscle;Myosin light chain kinase, smooth muscle, deglutamylated form MYLK S1779 0.000167 Wiskott-Aldrich syndrome protein family member 2 WASF2 S293 0.02427 Wiskott-Aldrich syndrome protein family member 2 WASF2 S296 0.024206 Wiskott-Aldrich syndrome protein family member 2 WASF2 S308 0.02427 Nuclear factor 1;Nuclear factor 1 B-type NFIB S295 0.030452 Pumilio homolog 1 PUM1 S124 0.047988 Poly [ADP-ribose] polymerase 1 PARP1 T368 0.015766 Spectrin beta chain, non-erythrocytic 1 SPTBN1 S2340 0.002795 Spectrin beta chain, non-erythrocytic 1 SPTBN1 S2319 0.004113 Afadin MLLT4 S1262 0.035485 Signal recognition particle subunit SRP72 SRP72 S625 0.020567 General transcription factor 3C polypeptide 2 GTF3C2 S167 0.031315 Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 GBF1 S1311 0.027014 Dedicator of cytokinesis protein 7 DOCK7 S1392 0.009854 Spectrin beta chain, non-erythrocytic 1 SPTBN1 T2320 0.004113 COP9 signalosome complex subunit 1 GPS1 T479 0.024092 Vimentin VIM T3 0.048241 Vimentin VIM S26 0.010881 Reticulon-1;Reticulon RTN1 S327 0.031822

Table A.4 Statistically significant phosphoproteins regulated by neither W146 or JTE013 Modified Protein names Gene names residue q-value Smoothelin SMTN S277 0.046232 E3 ubiquitin-protein ligase HECTD1 HECTD1 S1533 0.008415 Nucleolar and coiled-body phosphoprotein 1 NOLC1 S397 0.007046 Nucleolar and coiled-body phosphoprotein 1 NOLC1 S643 0.026175 Protein FAM50A;Protein FAM50B FAM50A;FAM50B S165 0.025877 Protein DEK DEK S51 0.027267

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Metastasis-associated protein MTA1 MTA1 S576 0.044955 Cytoplasmic dynein 1 light intermediate chain 1 DYNC1LI1 S487 0.028873 Cadherin-11 CDH11 S788 0.041402 Catenin delta-1 CTNND1 S920 0.030121 Eukaryotic initiation factor 4A- III;Eukaryotic initiation factor 4A- III, N-terminally processed EIF4A3 S12 0.040985 Lamina-associated polypeptide 2, isoform alpha;Thymopoietin;Thymopentin TMPO S351 0.045247 Antigen KI-67 MKI67 S2466 0.047899 Antigen KI-67 MKI67 S1207 0.036791 Transcriptional coactivator YAP1 YAP1 S61 0.042181 Transcriptional coactivator YAP1 YAP1 S276 0.007122 Transcriptional coactivator YAP1 YAP1 S109 0.030771 Neuroblast differentiation- associated protein AHNAK AHNAK S511 0.027877 Dihydropyrimidinase-related protein 3 DPYSL3 S665 0.048795 Histone H3.1t;Histone HIST3H3;HIST1H3A;HIS H3.1;Histone H3.2 T2H3A S29 0.028552 Rho GTPase-activating protein 12 ARHGAP12 S215 0.021793 TBC1 domain family member 10B TBC1D10B S687 0.043255 Torsin-1A-interacting protein 1 TOR1AIP1 S305 0.020795 Proline/serine-rich coiled-coil protein 1 PSRC1 S122 0.035158 Histone H3 HIST2H3PS2 S29 0.028552 Protein ELYS AHCTF1 S1541 0.035158 Condensin complex subunit 3 NCAPG S390 0.027179 Nucleolar and spindle-associated protein 1 NUSAP1 S352 0.028552 182 kDa tankyrase-1-binding protein TNKS1BP1 S1652 0.032008 1 ligand 1 CD274 S283 0.031162 Hematological and neurological expressed 1 protein;Hematological and neurological expressed 1 protein, N-terminally processed HN1 S17 0.043954 Protein VPRBP VPRBP S255 0.009236 Eukaryotic translation initiation factor 4 gamma 2 EIF4G2 T508 0.031755 Importin subunit alpha-1 KPNA2 T9 0.044955

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Retinoblastoma-associated protein RB1 T841 0.036791 Antigen KI-67 MKI67 T1193 0.003097 Antigen KI-67 MKI67 T1801 0.043255 Fragile X mental retardation syndrome-related protein 2 FXR2 T411 0.029305 Dihydropyrimidinase-related protein 3 DPYSL3 T623 0.011198 Xenotropic and polytropic retrovirus receptor 1 XPR1 T690 0.00302 Ribonucleoprotein PTB-binding 1 RAVER1 S668 0.032405 Ribonucleoprotein PTB-binding 1 RAVER1 S680 0.032405 FERM, RhoGEF and pleckstrin domain-containing protein 1 FARP1 S889 0.046925 Treacle protein TCOF1 S1350 0.040111 Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein NOS1AP S27 0.014657 Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein NOS1AP S30 0.014657 DNA (cytosine-5)- methyltransferase 1 DNMT1 S152 0.021635 DNA (cytosine-5)- methyltransferase 1 DNMT1 S154 0.021635 Nuclear factor 1;Nuclear factor 1 B-type NFIB S295 0.025948 Zinc finger C3H1 domain- containing protein ZFC3H1 S949 0.044258 Catenin delta-1 CTNND1 S268 0.048919 Catenin delta-1 CTNND1 S269 0.048919 DNA topoisomerase 2-alpha TOP2A S1469 0.020567 DNA topoisomerase 2-alpha TOP2A S1471 0.007045 Antigen KI-67 MKI67 S1253 0.045247 Antigen KI-67 MKI67 S1983 0.009775 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S1252 0.00302 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S1256 0.002398 RNA-binding protein 25 RBM25 S677 0.016931 RNA-binding protein 25 RBM25 S683 0.016931 Neuronal membrane glycoprotein M6-a GPM6A S267 0.025009

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Iroquois-class homeodomain protein IRX-1 IRX1 S267 0.04914 Iroquois-class homeodomain protein IRX-1 IRX1 S280 0.04914 A-kinase anchor protein 12 AKAP12 S780 0.005469 A-kinase anchor protein 12 AKAP12 S783 0.005469 cAMP-specific 3',5'-cyclic phosphodiesterase 4B PDE4B S42 0.032514 Mediator of DNA damage checkpoint protein 1 MDC1 S1820 0.030192 DNA excision repair protein ERCC-6-like ERCC6L S980 0.017043 DNA excision repair protein ERCC-6-like ERCC6L S984 0.017043 MAP7 domain-containing protein 1 MAP7D1 S442 0.014894 MAP7 domain-containing protein 1 MAP7D1 S446 0.014894 Rho GTPase-activating protein 21 ARHGAP21 S1431 0.021532 Tensin-3 TNS3 S1149 0.007837 Tensin-3 TNS3 S1154 0.007837 Cell division cycle-associated protein 3 CDCA3 S199 0.046249 Cell division cycle-associated protein 3 CDCA3 S209 0.046249 La-related protein 6 LARP6 S447 0.039884 La-related protein 6 LARP6 S451 0.039884 Protein TANC1 TANC1 S63 0.016931 Protein TANC1 TANC1 S67 0.016931 Interferon regulatory factor 2- binding protein-like IRF2BPL S662 0.033371 Hematological and neurological expressed 1 protein;Hematological and neurological expressed 1 protein, N-terminally processed HN1 S87 0.002795 Hematological and neurological expressed 1 protein;Hematological and neurological expressed 1 protein, N-terminally processed HN1 S87 0.002795 Neurabin-1 PPP1R9A S190 0.033471 Spectrin beta chain, non- erythrocytic 1 SPTBN1 T2328 0.043954 Lymphokine-activated killer T- cell-originated protein kinase PBK T24 0.025817 Treacle protein TCOF1 T1358 0.040111 151

Appendix

Histone-lysine N-methyltransferase NSD2 WHSC1 T110 0.028552 Histone-lysine N-methyltransferase NSD2 WHSC1 T115 0.028552 Lamina-associated polypeptide 2, isoform alpha;Thymopoietin;Thymopentin; Lamina-associated polypeptide 2, isoforms beta/gamma;Thymopoietin;Thymo pentin TMPO T160 0.021532 Lamina-associated polypeptide 2, isoforms beta/gamma;Thymopoietin;Thymo pentin TMPO T211 0.033789 Lamina-associated polypeptide 2, isoforms beta/gamma;Thymopoietin;Thymo pentin TMPO T355 0.024092 Antigen KI-67 MKI67 T1991 0.009775 Centromere protein F CENPF T224 0.044955 Mediator of DNA damage checkpoint protein 1 MDC1 T1403 0.037998 Mediator of DNA damage checkpoint protein 1 MDC1 T1608 0.009854 Codanin-1 CDAN1 T71 0.047989 Codanin-1 CDAN1 T75 0.047989 Zyxin ZYX S267 0.044955 Zyxin ZYX S259 0.044955 Catenin delta-1 CTNND1 S346 0.044955 Catenin delta-1 CTNND1 S349 0.044955 Catenin delta-1 CTNND1 S352 0.044955 Antigen KI-67 MKI67 S2344 0.032499 Coiled-coil domain-containing protein 86 CCDC86 S58 0.034823 Coiled-coil domain-containing protein 86 CCDC86 S66 0.034823 Coiled-coil domain-containing protein 86 CCDC86 S69 0.034823 Zyxin ZYX T270 0.044955 Antigen KI-67 MKI67 T2352 0.032499 Coiled-coil domain-containing protein 86 CCDC86 T65 0.034823 B-cell CLL/lymphoma 9 protein BCL9 S104 0.021793

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C-Jun-amino-terminal kinase- interacting protein 4 SPAG9 S258 0.000425 Uncharacterized protein FLJ45252 AAK1 S456 0.049065 Mediator of RNA polymerase II transcription subunit 24 MED24 S862 0.036678 DLG1 S636 0.046187 Matrin-3 MATR3 S188 0.024978 Rho guanine nucleotide exchange factor 7;Rho guanine nucleotide exchange factor 6 ARHGEF7;ARHGEF6 S525 0.026759 Retrotransposon-derived protein PEG10 PEG10 S391 0.005956 Retrotransposon-derived protein PEG10 PEG10 S392 0.005956 Nuclear-interacting partner of ALK ZC3HC1 S344 0.020567 Translocon-associated protein subunit gamma SSR3 S105 0.032499 Amyloid beta A4 precursor protein-binding family B member 2 APBB2 S123 0.006325 Uncharacterized protein C1orf198 C1orf198 S289 0.013043 Apoptotic chromatin condensation inducer in the nucleus ACIN1 S710 0.030893 Pericentriolar material 1 protein PCM1 S93 0.029689 Alpha-crystallin B chain CRYAB S59 0.028666 Bystin BYSL S98 0.013144 Microtubule-actin cross-linking factor 1, isoforms 1/2/3/5 MACF1 S1371 0.048919 Phosphatase and actin regulator;Phosphatase and actin regulator 2 PHACTR2 S510 0.030893 RNA-binding protein 7 RBM7 S137 0.046187 Nuclear factor 1 X-type;Nuclear factor 1 NFIX S280 0.003859 DNA (cytosine-5)- methyltransferase 1 DNMT1 S143 0.000783 Unconventional myosin-IXb MYO9B S1405 0.019938 Syntaxin-7 STX7 S129 0.017601 Mitogen-activated protein kinase MAP3K7;DKFZp586F042 kinase kinase 7 0 S389 0.028666 Zinc finger C3H1 domain- containing protein ZFC3H1 S352 0.034538 Germinal-center associated nuclear protein MCM3AP S527 0.026884 Lysine-specific demethylase PHF2 PHF2 S1056 0.000786

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Histone deacetylase complex subunit SAP30 SAP30 S131 0.046925 Protein-methionine sulfoxide oxidase MICAL2 MICAL2 S515 0.036791 Ubiquitin conjugation factor E4 B UBE4B S31 0.017875 CD2 antigen cytoplasmic tail- binding protein 2 CD2BP2 S194 0.00302 Glucocorticoid receptor NR3C1 S134 0.025817 Heat shock protein beta-1 HSPB1 S15 0.028666 Transcription factor AP- 1;Transcription factor jun-D JUN;JUND S73 0.01571 Solute carrier family 2, facilitated glucose transporter member 1 SLC2A1 S490 0.010085 Zinc finger protein 22 ZNF22 S49 0.007122 Integrin alpha-2 ITGA2 S1181 0.001813 Dual specificity mitogen-activated protein kinase kinase 2 MAP2K2 S23 0.001205 Alpha-taxilin TXLNA S515 0.043853 Nuclear pore complex protein Nup98-Nup96;Nuclear pore complex protein Nup98;Nuclear pore complex protein Nup96 NUP98 S623 0.026651 Nuclear cap-binding protein subunit 1 NCBP1 S22 0.000425 Neuroblast differentiation- associated protein AHNAK AHNAK S5110 0.014783 Rho GTPase-activating protein 12 ARHGAP12 S213 0.013202 Transmembrane protein 132A TMEM132A S529 0.034538 DNA-directed RNA polymerase I subunit RPA43 TWISTNB S316 0.002151 Rho GTPase-activating protein 29 ARHGAP29 S949 0.023042 Rho GTPase-activating protein 29 ARHGAP29 S913 0.04573 Zinc finger protein 318 ZNF318 S527 0.019432 SPECC1L;SPECC1L- Cytospin-A ADORA2A S384 0.016931 Enhancer of mRNA-decapping protein 4 EDC4 S879 0.03345 Kinesin light chain 3 KLC3 S502 0.032182 Polyadenylate-binding protein 2 PABPN1 S95 0.023002 B-cell CLL/lymphoma 9-like protein BCL9L S116 0.044955 B-cell CLL/lymphoma 9-like protein BCL9L S118 0.001724 Protein AHNAK2 AHNAK2 S5175 0.024092

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EH domain-binding protein 1 EHBP1 S1058 0.006424 Fibroblast substrate 2 FRS2 S211 0.003772 Protein ELYS AHCTF1 S1944 0.043001 Golgi-specific brefeldin A- resistance guanine nucleotide exchange factor 1 GBF1 S1776 0.012341 Golgi-specific brefeldin A- resistance guanine nucleotide exchange factor 1 GBF1 S314 0.000143 Symplekin SYMPK S510 0.008888 Rho guanine nucleotide exchange factor 2 ARHGEF2 S174 0.035647 Rho guanine nucleotide exchange factor 2 ARHGEF2 S645 0.024092 Rho guanine nucleotide exchange factor 2 ARHGEF2 S886 0.003174 PRKC apoptosis WT1 regulator protein PAWR S259 0.045334 S211 0.030853 Pseudopodium-enriched atypical kinase 1 PEAK1 S572 0.044955 Kinase D-interacting substrate of 220 kDa KIDINS220 S1681 0.019938 TSC22 domain family protein 4 TSC22D4 S279 0.032828 Epsin-1 EPN1 S435 0.040111 Epidermal growth factor receptor;Receptor protein- EGFR T693 0.034555 KH domain-containing, RNA- binding, signal transduction- associated protein 1 KHDRBS1 T61 0.002398 Nuclear cap-binding protein subunit 1 NCBP1 T21 0.007003 Transcription intermediary factor 1-beta TRIM28 T541 0.000523 Nuclear fragile X mental retardation-interacting protein 2 NUFIP2 T220 0.009854 Golgi reassembly-stacking protein 2 GORASP2 T222 0.012824 Transforming acidic coiled-coil- containing protein 3 TACC3 T590 0.028552 Mitogen-activated protein kinase 3;Mitogen-activated protein kinase MAPK3 Y204 0.024092 Paxillin PXN Y118 0.002132

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Ubiquitin carboxyl-terminal hydrolase 36;Ubiquitin carboxyl- terminal hydrolase USP36 S610 0.011528 DENN domain-containing protein 1A DENND1A S523 0.012179 ELM2 and SANT domain- containing protein 1 ELMSAN1 S661 0.035158 Syntaxin-12 STX12 S139 0.009647 Syntaxin-12 STX12 S142 0.009647 Serine/threonine-protein kinase D2;Serine/threonine-protein kinase PRKD2 S198 0.029305 Cdc42 effector protein 3 CDC42EP3 S89 0.007045 Cdc42 effector protein 3 CDC42EP3 S100 0.007045 Putative Polycomb group protein ASXL2 ASXL2 S834 0.000508 Cytoplasmic dynein 1 light intermediate chain 1 DYNC1LI1 S516 0.005339 ARF GTPase-activating protein GIT1 GIT1 S592 0.02021 ARF GTPase-activating protein GIT1 GIT1 S596 0.02021 Histone-lysine N-methyltransferase 2D KMT2D S3199 0.031315 Protein phosphatase 1 regulatory subunit 12A PPP1R12A S409 0.046884 Mediator of RNA polymerase II transcription subunit 14 MED14 S977 0.001724 Mediator of RNA polymerase II transcription subunit 14 MED14 S986 0.001724 Lysine-specific demethylase PHF2 PHF2 S899 0.041742 Zinc finger CCCH domain- containing protein 11A ZC3H11A S758 0.012179 Zinc finger CCCH domain- containing protein 11A ZC3H11A S761 0.012179 RAC-alpha serine/threonine- protein kinase AKT1 S473 0.04622 RAC-alpha serine/threonine- protein kinase AKT1 S477 0.04622 ETS translocation variant 3 ETV3 S245 0.031706 ETS translocation variant 3 ETV3 S250 0.031706 Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S992 0.029245

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Microtubule-associated protein 1B;MAP1B heavy chain;MAP1 light chain LC1 MAP1B S995 0.029245 Ribosomal protein S6 kinase alpha- 3 RPS6KA3 S369 0.000929 DNA topoisomerase 2-beta TOP2B S1550 0.005863 DNA topoisomerase 2-beta TOP2B S1552 0.005863 A-kinase anchor protein 12 AKAP12 S283 0.046701 A-kinase anchor protein 12 AKAP12 S286 0.046701 Disks large homolog 1 DLG1 S687 0.014273 Acetyl-CoA carboxylase 1;Biotin carboxylase ACACA S23 0.028324 Myosin light chain kinase, smooth muscle;Myosin light chain kinase, smooth muscle, deglutamylated form MYLK S1773 0.001836 La-related protein 7 LARP7 S298 0.000425 La-related protein 7 LARP7 S299 0.002186 La-related protein 7 LARP7 S300 0.000171 Microtubule-associated protein 1S;MAP1S heavy chain;MAP1S light chain MAP1S S741 0.009647 DENND1B S653 0.000143 B-cell CLL/lymphoma 9-like protein BCL9L S959 0.036976 B-cell CLL/lymphoma 9-like protein BCL9L S947 0.046701 Nuclear receptor coactivator 7 NCOA7 S208 0.023002 Palladin PALLD S766 0.006661 Protein FAM122A FAM122A S270 0.009775 BUD13 homolog BUD13 S175 0.000749 Dedicator of cytokinesis protein 5 DOCK5 S1803 0.010839 Protein TANC2 TANC2 S1824 0.009775 Protein TANC2 TANC2 S1827 0.009775 Neuronal-specific septin-3 Sep-03 S15 0.044955 Serine/arginine repetitive matrix protein 2 SRRM2 S536 0.000775 MORC family CW-type zinc finger protein 2 MORC2 S739 0.000749 MORC family CW-type zinc finger protein 2 MORC2 S743 0.000749 Dachshund homolog 1 DACH1 T580 0.026331 ELM2 and SANT domain- containing protein 1 ELMSAN1 T655 0.035158

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Mitogen-activated protein kinase 3;Mitogen-activated protein kinase MAPK3 T202 0.001037 Cyclic AMP-dependent transcription factor ATF-2 ATF2 T69 0.012824 Cyclic AMP-dependent transcription factor ATF-2 ATF2 T71 0.012824 Histone-lysine N-methyltransferase 2D KMT2D T3197 0.031315 Protein phosphatase 1 regulatory subunit 12A PPP1R12A T406 0.030893 Ribosomal protein S6 kinase alpha- 3 RPS6KA3 T365 0.000929 Calmodulin-regulated spectrin- associated protein 2 CAMSAP2 T864 0.009854 Disks large homolog 1 DLG1 T683 0.018458 B-cell CLL/lymphoma 9-like protein BCL9L T957 0.004214 Neuronal-specific septin-3 Sep-03 T10 0.005956 Mitogen-activated protein kinase 3;Mitogen-activated protein kinase MAPK3 Y204 0.035412 Protein SCAF11 SCAF11 S878 0.037998 Protein SCAF11 SCAF11 S880 0.037998 Protein SCAF11 SCAF11 S882 0.037998 Nuclear receptor corepressor 2 NCOR2 S2057 0.043255 Cytoplasmic dynein 1 light intermediate chain 1 DYNC1LI1 S510 0.007122 Protein phosphatase 1 regulatory subunit 12A PPP1R12A S402 0.005162 Protein phosphatase 1 regulatory subunit 12A PPP1R12A S409 0.011815 Ribosomal protein S6 kinase alpha- 3 RPS6KA3 S375 0.016132 Sequestosome-1 SQSTM1 S355 0.037998 Sequestosome-1 SQSTM1 S361 0.012441 Sequestosome-1 SQSTM1 S366 0.010663 STE20-like serine/threonine- protein kinase SLK S347 0.023002 Dedicator of cytokinesis protein 5 DOCK5 S1781 0.030136 Dedicator of cytokinesis protein 5 DOCK5 S1789 0.009647 DENN domain-containing protein 4C DENND4C S1608 0.008146 MAP/microtubule affinity- regulating kinase 3 MARK3 T536 0.000642

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Cytoplasmic dynein 1 light intermediate chain 1 DYNC1LI1 T513 0.007122 Protein phosphatase 1 regulatory subunit 12A PPP1R12A T406 0.004359 Dedicator of cytokinesis protein 5 DOCK5 T1794 0.007045

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