Involvement of the sphingosine 1-phosphate receptor 3 in neuro-inflammation

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Thesis

Reference

Involvement of the sphingosine 1-phosphate receptor 3 in neuro-inflammation

FISCHER, Iris

Abstract

The S1P3 receptor belongs to the family of G protein coupled receptors which were demonstrated to have regulatory functions in all systems of the body including the immune, cardiovascular and central nervous system. In this work, the role of S1P3 was characterized in neuro-inflammation in regard to its potential role in the neurodegenerative disease multiple sclerosis. It is shown that the SphK1/S1P3 axis is important in astrocyte signalling and probably involved in their activation in vitro. The work further shows that S1P3 is expressed on reactive astrocyte in MS lesions, but not in the mouse brain during neuro-inflammation. In an EAE study, an animal model used to investigate aspects of the human disease multiple sclerosis, it is demonstrated that S1P3 plays no major role in EAE. The therapeutic relevance of these findings remains open, as S1P3 seems to be able to mediate both deleterious and beneficial effects on astrocytes in MS.

FISCHER, Iris. Involvement of the sphingosine 1-phosphate receptor 3 in neuro-inflammation. Thèse de doctorat : Univ. Genève, 2011, no. Sc. 4338

URN : urn:nbn:ch:unige-172284

DOI : 10.13097/archive-ouverte/unige:17228

Available at:

http://archive-ouverte.unige.ch/unige:17228

Disclaimer: layout of this document may differ from the published version.

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Département de biochimie Professeur Howard Riezman

Département de Neurobiologie MERCK SERONO INTERNATIONAL S.A.

GENEVA RESEARCH CENTER Docteur Sandrine Pouly

Involvement of the Sphingosine 1-Phosphate Receptor 3 in Neuro-Inflammation

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimie

par

IRIS FISCHER

de

Walheim, Germany

Thèse N^o 4338

GENÈVE

Document Services Center, Merck Serono International

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Figures 32-37 and Figures 48 and 49 are part of a publication which was published in PloS ONE the 24^th of August 2011 under the following title: Sphingosine Kinase 1 and Sphingosine 1-Phosphate Receptor 3 are Functionally Upregulated on Astrocytes under Pro-Inflammatory Conditions

Iris Fischer¹, Chantal Alliod¹, Nicolas Martinier¹, Jia Newcombe², Corinne Brana¹, Sandrine Pouly¹

PLoS ONE 6(8): e23905. Doi:10.1371/journal.pone.0023905

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List of Figures

Figure 1. The main cellular components of the central nervous system ... 3

Figure 2. The cell associations at the Blood brain barrier ... 10

Figure 3. Immunopathogensis of multiple sclerosis ... 12

Figure 4. Sphingolipid metabolism ... 19

Figure 5. S1P receptor coupling ... 23

Figure 6. Proposed model of the mode of action of fingolimod in MS ... 26

Figure 7. Principle of the CellKey technology ... 50

Figure 8. Example of CDS response profiles of differentially coupled GPCRs ... 51

Figure 9. Location of the 5’ and 3’ flanking sequences of the SRA5069 construct on the mouse S1P3 gene. ... 65

Figure 10. Replacement of the entire coding region with LacZ lox-UB1-EM7- Neolox Cassette ... 65

Figure 11. RNA levels of S1P3 and GFAP in MOG-induced EAE ... 82

Figure 12. Differential mRNA expression levels of S1PRs in rat astrocytes and U251 astrocytomas ... 83

Figure 13. Differential mRNA expression levels of S1P1-3 in rat microglia ... 84

Figure 14. Upregulation of S1P₅ mRNA levels in differentiated oligodendrocytes ... 85

Figure 15. Differential MBP and S1P_1-5 expression in developing rat hippocampal slices ... 87

Figure 16. S1P receptors are differentially expressed on different brain tumours ... 89

Figure 17. Chemical structures of ligands of the S1P_1-5receptors ... 94

Figure 18. Dose and time dependent ERK-1/2 phosphorylation in primary rat astrocytes in response to stimulation with several S1P receptor agonists .... 96

Figure 19. Influence of S1P receptor antagonists on ERK-1/2 phosphorylation induced by agonists with different selectivity profiles. ... 98

Figure 20. S1P and pFTY720 induced ERK-1/2 phosphorylation is mainly mediated via Gi-proteins ... 99

Figure 21. ERK-1/2 phosphorylation in U251 indcued by different S1PR agonist ... 100

Figure 22. Influence of pre-treatment with different S1P receptor antagonist on S1P or pFTY20 induced ERK-1/2 phosphorylation in U251 cells. ... 101

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Figure 23. Dose response curves for different S1P receptor agonists in rat

astrocytes and measured by CellKey ... 102

Figure 24. All tested compounds induce a Gi protein response in astrocytes ... 104

Figure 25. Dose response curves for S1P, AUY954 and pFTY720 in the presence or absence of PTX or W146 ... 105

Figure 26. Effect of the S1P3 agonist compound 20 on ERK-1/2 phosphorylation and cell impedance ... 106

Figure 27. Dose response curve for different S1P receptor agonists in U251 astrocytoma measured by CellKey ... 108

Figure 28. Different G proteins are activated in U251 cells by S1P receptor modulators. ... 109

Figure 29. Dose response curves for S1P and pFTY720 in the presence or absence of PTX or W146 ... 110

Figure 30. Validation of tools ... 117

Figure 31. Dose-dependent LPS-mediated upregulation of S1P₃ mRNA in primary rat astrocytes ... 118

Figure 32. S1P3 and SphK1 mRNAs and protein are upregulated in rat primary astrocytes by LPS stimulation. ... 119

Figure 33. SphK1 is activated in response to LPS. ... 120

Figure 34. Increased S1P3-mediated ERK-1/2, but not Akt signalling by LPS. ... 121

Figure 35. S1P and LPS induce astrocyte migration in a scratch assay. ... 123

Figure 36. LPS-induced astrocyte migration is SphK1-dependent, but proliferation-independent. ... 124

Figure 37. S1P3 contributes to the S1P-induced CXCL1 release by primary astrocytes. ... 126

Figure 38. Influence of OGD/ReOx on S1PR and SphK1/2 mRNA expression levels in rat astrocytes ... 135

Figure 39. Influence of combined oxygen glucose deprivation on Hif1α, pERK and pAkt levels. ... 137

Figure 40. OGD influences the responsiveness of astrocytes to agonistic stimulation of S1PRs. ... 138

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Figure 41. VEGF release induced by OGD in rat astrocytes in the presence or

absence of DMS. ... 139

Figure 42. Dose response for DMS-mediated inhibition of VEGF release and increase in LDH release in astrocytes exposed to OGD. ... 141

Figure 43. VEGF release in primary rat astrocytes exposed to OGD and reoxygenation ... 143

Figure 44. Illustration of rat organotypic slice cultures undergoing oxygen glucose deprivation ... 144

Figure 45. Influence of OGD/ReOx on S1PR and SphK1/2 expression levels in rat cerebellar slices ... 145

Figure 46. S1P₃ and SphK1 and pERK, pAkt protein expression in response to OGD ... 146

Figure 47. VEGF release in cerebellar slices in response to OGD... 147

Figure 48. S1P₃ receptor and SphK1 enzyme expressions are increased in a chronic active MS lesion ... 153

Figure 49. Expression of S1P₃ and SphK1 in reactive astrocytes and macrophages in MS lesions ... 154

Figure 50. X-Gal/S1P3 expression in the naive mouse brain ... 155

Figure 51. Comparison of GFAP and X-Gal staining ... 156

Figure 52. Cross section of a CNS capillary is shown in cartoon form ... 157

Figure 53. In naive mouse brain X-Gal colocalizes with NG2 a marker for pericytes ... 158

Figure 54. S1P3 expression and signalling in CNS-pericyte cultures of mouse ... 161

Figure 55. X-Gal expression in S1P3+/- animals injected with LPS ... 169

Figure 56. Developmental weight loss and disease score in response to LPS injection ... 170

Figure 57. Relative S1P receptor and SphK1, 2 mRNA expression levels in response to LPS ... 171

Figure 58. Elevated β-Galactosidase activity in response to LPS injection ... 172

Figure 59. LPS-induced neuroinflammation ... 173

Figure 60. Astrocyte activation in response to LPS injection ... 175

Figure 61. Microglia activation in response to LPS injection ... 176

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Figure 62. Comparison of EAE development in S1P3-/-

, S1P3+/-

and wt mice... 181

Figure 63. Upregulation of S1P3 and SphK1 mRNA level in MOG-induced EAE ... 182

Figure 64. Upregulation of CXCL1 and CXCL2 mRNA level in MOG-induced EAE ... 184

Figure 65. Enhanced betagalactosidase activity in MOG35-55-induced EAE... 185

Figure 66. Analysis of the T cell infiltration into the CNS in MOG35-55-induced EAE ... 186

Figure 67. Analysis of myeloid cells in the CNS in MOG35-55-induced EAE ... 187

Figure 68. Treg reduction in the CNS in S1P₃^+/-and S1P₃^-/-group ... 188

Figure 69. Composition of lymphocytes population in the spleen ... 189

Figure 70. Histological analysis spinal cord pathology in acute EAE ... 190

Figure 71. Comparison of EAE development in S1P₃^-/-, S1P₃^+/- and wt mice... 191

Figure 72. Histological analysis spinal cord pathology in chronic EAE ... 192

Figure 73. Correlation of X-Gal expression with inflammatory lesions in the white matter ... 194

Figure 74. Representative X-Gal expression in spinal cord and cerebellum in a S1P3-/- mouse ... 195

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List of Tables

Table 1. Score for LPS-induced septic shock symptoms ... 66

Table 2. Program for embedding of mouse CNS tissue in paraffin ... 69

Table 3. Scoring Index to evaluate spinal cord inflammation ... 72

Table 4. Clinical score criteria in EAE ... 76

Table 5. Compensation setup... 79

Table 6. The cocktail of antibodies for CNS ... 79

Table 7. Overview of properties and pharmacological characteristics of ligands for S1P_1-5 receptors ... 95

Table 8. Summary of in vivo LPS experiments ... 167

Table 9. Summary of performed EAE experiments ... 180

Table 10. Regulation of genes of the S1P receptor family in spinal cord probes^a ... 183

Table 11. Regulation of myelin genes in spinal cord probes ... 183

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Acknowledgments

First of all I want to thank my supervisor Sandrine Pouly who took care about me and who provided me this project after a difficult start in the institute. I further want to thank Sandrine for her strong scientific support and guidance of my project even when facing the various difficulties that came along with it. I am also very grateful for her continous personal support during the last four years.

I would like to thank Prof. Howard Riezman, my supervisor at the university, for his scientific advice and support and Astrid Alewijnse for accepting to review my thesis and to be a member of the jury of my thesis.

I am forever grateful to Chantal and Marie who helped and supported me so much during the whole time; from a technical point of view, by helping me with the work involving the different primary cultures I used, but moreover for their personal support during the last years. I further want to thank Chantal a lot for her experimental advice and for the accountability she took over for the authorizations of my LPS in vivo experiments.

Furthermore I want to thank my whole group of cellular neurobiology and all the persons of the neurobiology department for all their personal and professional support and the good time I shared with them during my thesis.

My sincere thank goes to Anthony Nichols who introduced me into the CellKey technology, for reviewing parts of my thesis and for all the great scientific discussions I had with him about my project and the big world of sphingolipid biology.

I am very grateful for the help of Nicolas Martinier and Simon Laustela in the work involving the histology part of my thesis and further for being so nice write-up colleagues. I also want to thank Tim Seabrook and Corinne Brana for their advice in the histological analysis.

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I am very grateful to the whole in vivo pharmacology group especially Paul Smith who made it possible that I could perform my EAE experiments in the company and who pushed me forward in writing the manuscript on the EAE studies. I am also very thankful to Fréderique Bernard for his scientific and experimental advice and for the responsibility he took over in leading and guiding the experiments. I also want to thank Alla Zozulya-Weidenfeller for her help in the immunophenotyping experiments, Sébastien Nock, Stefan Rudin, Patricia de Lys and Cheryl Baker for their great support during all the EAE studies.

I want to thank Catherine Salvant and the members of her group, Marie-Laure Curchoud, Helene Peixoto, Mireille Guerrier and Emmanuel Guedj for their help and experimental advice which they gave me in the work related to molecular biology.

I am also very gratedul to Jean-Francois Arrighi for his personal support and scientific guidance during my first year in the company.

I furthermore want to thank my Thesis advisory committee, Sandrine Pouly, Howard Riezman, Christian Weidenfeller and Ursula Boschert for their critical feedback and scientific advice. Furthermore I am grateful to Amanda Proudfoot and the members of the Merck Serono student committee for giving me the opportunity to perform my thesis in the Institute.

I am very grateful to the head of TA NDD Ewen Sedman and the head of Global MS Jean Merrill as well as to Michaela Koehler, for the financial and especially for their personal support.

Last but not least my outmost thank goes to my family and friends. I am very grateful to my parents and my sisters, for their support, guidance and trust. I am very happy about the good friends that I have made in my suffering colleagues, Ulrike, Matthias, Sonja and Anne, who shared with me the good, bad and challenging moments during the thesis. Finally, I want to thank my better half Wolfgang, who stood by me all the time, trusting in me and who supported and encouraged me even when I was unbearable.

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Summary

Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system, where the myelin sheath, which insulates the nerve axons, is destroyed by myelin antigen specific autoreactive lymphocytes. MS is the most common neurodegenerative disease in young adults causing disability and heavily impacting the quality of patients life. Although a lot of progress was made in MS therapy over the last 20 years, until recently, there was no oral therapy available. Furthermore, so far there is no drug proven to directly promote regeneration of the damaged CNS.

However, at the end of 2010 a new drug, FTY720 (fingolimod), acting as a S1PR immunomodulator, was approved for the oral therapy of relapsing remitting MS.

FTY720 is a structural analog of sphingosine and is phosphorylated in vivo to the active form pFTY720 by the sphingosine kinase 2 (SphK2). After phosphorylation pFTY720 reduces immune cell infiltration into the CNS by down-modulating S1P₁ receptors on the surface of lymphocytes, thus preventing their exit out of the lymph nodes and subsequently their migration to the site of inflammation, the CNS.

The S1P receptor family (S1P1-5) of G protein coupled receptors was demonstrated to have regulatory functions in all systems of the body with important roles in the immune, cardiovascular and central nervous systems. These encompass functions such as induction of cell migration, proliferation, survival, angiogenesis, differentiation or apoptosis. The different functions are mediated by binding of their natural ligand, sphingosine 1-phosphate (S1P), but other small molecules like FTY720 can also activate S1P receptors.

In addition to its action as an immunosuppressive agent, FTY720 can cross the blood brain barrier (BBB) and may therefore exert direct effects in the CNS. S1P receptors are expressed in the CNS and receptor RNA expression levels were shown to be altered in the spinal cords of an experimental animal model (EAE) used to study MS.

Interestingly, the receptor S1P₃, which is associated with the mediation of pro- inflammatory processes, was found to be upregulated in EAE in parallel to GFAP (glial fibrillary acidic protein), which is upregulated on activated astrocytes.

Astrocytes are the main glial cell type in the CNS, become activated in MS and are thought to influence disease outcome in different ways. Astrocyte activation can be

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beneficial, by mediating trophic support to oligodendrocytes and neurons, but also detrimental by release of pro-inflammatory mediators that further activate and recruit inflammatory cells into the lesions, and by the formation of glial scars.

The correlated upregulation of S1P3 with GFAP over EAE disease progression indicated that S1P3 may play a role in the disease. In regard to the development of new S1PR modulators for MS, with a more selective activity profile than FTY720, we chose to investigate the role of S1P3 in neuro-inflammation with an emphasis on its potential role on astrocyte activation.

The aim of the study was to determine the tissue and cellular expression profile of S1P₃ in cells of the CNS, the signalling of S1P₃ in astrocytes in basal and under pro- inflammatory conditions, as well as the possible implication of the receptor in CNS- inflammation, by the use of two different in vivo models of neuro-inflammation in S1P₃ transgenic animals; LPS-induced neuro-inflammation, and EAE.

In the first part of the thesis, we aimed to define the differential mRNA expression profile of S1P3 receptor in relation to the other S1P receptors on cells of the CNS. We showed that S1P3 is abundantly expressed in primary cultures of rat astrocytes and on astrocytomas. We further showed that S1P3 mRNA is expressed at different extents in oligodendrocytes, microglia, hippocampal slices, and is upregulated in various brain tumours.

In a next step, we compared the basal downstream signalling of S1P receptors after agonistic stimulation in rat astrocytes and a human astrocytoma cell line (U251). ERK- 1/2 phosphorylation and G protein activation in response to receptor modulation was investigated by Western Blot and by the measurement of changes in cell impedance using the CellKey^TM technology. We showed that primary rat astrocytes respond to S1P and pFTY720 mainly via S1P₁ and S1P₃ activation, leading to ERK-phosphorylation in a Gi-dependent manner, whereas in the astrocytoma cell line ERK-1/2 phosphorylation also involves S1P₂ and probably G_q-protein activation.

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In the third part of the project we investigated the potential involvement of the SphK1/S1P3 axis in activated rat astrocytes in vitro. We showed that activation of primary rat astrocytes with the pro-inflammatory stimulus LPS, induced SphK1 activity, increased mRNA and protein expression of both SphK1 and S1P3, which resulted in increased activity of S1P3, as measured by elevated ERK-1/2 phosphorylation in response to stimulation with a selective S1P3 agonist. Moreover, we showed that LPS- induced astrocyte migration depends on SphK1 activity whereas S1P-induced migration was predominantly mediated by proliferation. In addition, we demonstrated the involvement of S1P₃ in the release of the potentially neuroprotective chemokine CXCL1, by the use of selective S1P₁ and S1P₃ agonists. Taken together, our data demonstrated that the SphK1/S1P₃ signalling axis appears to play a role in the establishment and maintenance of astrocyte activation.

The aim of the fourth part was to investigate the potential role of S1P₃ in the response of astrocytes and cerebellar slices to ischemic stress. We showed, by exposing the cells to a combination of oxygen and glucose deprivation (OGD) followed by reoxygenation (ReOx) that both S1P3 and SphK1 are upregulated at the RNA level during ReOx. We further showed that astrocyte signalling is increased during ReOx via S1P1 and S1P3 activation and that the release of proangiogenic VEGF is at least partially, dependent on SphK1 activation, probably through S1P/S1PR signalling.

The aim of the fifth part was to characterize S1P3 expression in vivo in post-mortem MS brain tissues, as well as to study basal S1P3 tissue expression in S1P3 transgenic animals, carrying a reporter gene LacZ instead of the endogenous S1P3 receptor. We showed S1P3 and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. We further identified X-Gal/S1P3 in the brain of naïve mice with a strong expression on vessels and associated cells, on interneurons and granular cells of the hippocampus as well as on cortical neurons, but not on astrocytes.

In preliminary experiments, we finally revealed strong experimental evidences for S1P₃ expression on pericytes, supported by the finding of X-Gal positive pericytes in cultures of S1P₃^+/- animals. A potential role of S1P₃ in stimulated pericytes in culture

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was further provided by increased ERK-1/2 activation upon stimulation with pFTY720 or a S1P3 agonist.

The aim of the sixth part of this study was to investigate whether S1P3 is indeed overexpressed on astrocytes in brains of mice injected with LPS, used to induce a neuro-inflammatory response, and furthermore if S1P3 receptor expression plays a role in septic shock symptoms or neuro-inflammation. We showed by X-Gal staining, that S1P3 expression was not induced on astrocytes in response to LPS, although demonstrated to be upregulated on the RNA level in the cerebellum. Whereas the inflammatory response, measured by the production of pro-inflammatory mediators, was similar in the wild-type and S1P₃ knockout animals, a hint towards a potential role of S1P₃ in neuroinflammation was however given by the observation that astrocyte and microglia activation in the cortex of LPS-injected animals was reduced compared to wild-type mice.

In the last part of the thesis, EAE was induced and disease progression compared in S1P3 wild-type, S1P3+/-

and S1P3-/-

mice, to investigate the role of S1P3 in this model.

We studied the role of S1P3 at the peak of the disease, by comparing acute inflammatory parameters, as well as in the chronic phase, when neurodegeneration occurs. We demonstrated that the receptor S1P3 plays no major role in EAE, as shown by the comparison of diseases onset and progression, immunophenotyping, gene expression analysis, proliferation and cytokine measurements, and by histopathological examination. Finally, we demonstrated that X-Gal/S1P3 expression is associated with inflammatory lesions in EAE, but it was, however, not possible to clearly identify the responsible cell type.

Taken together, this work showed that SphK1/S1P3 is important in astrocyte signalling and probably involved their activation in vitro. We further showed that S1P₃ is expressed on reactive astrocyte in MS lesions, but not in the mouse brain in neuroinflammation. In the EAE study, we finally demonstrated no major role of S1P₃ in the disease. We further showed that there is a discrepancy between the in vitro and the in vivo situation in rodents, showing the limits of in vitro models, but also maybe of

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genetically modified animals. The therapeutic relevance of our findings remains open, as S1P3 seems to be able to mediate both deleterious and beneficial effects on astrocytes in MS.

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Resumé

La sclérose en plaques (SEP) est une maladie neurodégénérative du système nerveux central (SNC), où la gaine de myéline, qui isole les axones, est détruite par des lymphocytes autoréactifs contre la myéline. La SEP est la maladie neurodégénérative la plus commune chez les jeunes adultes, provoquant des infirmités motrices et cognitives, et un lourd impact sur la qualité de vie des patients. Bien que de nombreux progrès aient été faits d’un point de vue thérapeutique dans les 20 dernières années, il n’y avait encore jusqu’à récemment aucun médicament oral. De plus, aucun traitement n’a jusque là montré de potentiel de régénérescence dans du SNC endommagé.

Néanmoins, à la fin 2010, un nouveau médicament, le FTY720 (fingolimod), qui agit comme un immunomodulateur des récepteurs S1PR, a été approuvé comme thérapie orale pour la SEP de type poussées-rémissions. FTY720 est un analogue structurel de la sphingosine, et est phosphorylé in vivo en forme active, le pFTY720, par la sphingosine kinase 2 (SPHK2). Après sa phosphorylation, le pFTY720 réduit l’infiltration lymphocytaire dans le SNC en diminuant l’expression des récepteurs S1P1

à la surface des lymphocytes, prévenant ainsi leur sortie des ganglions lymphatiques, et en conséquence, leur migration vers le site d’inflammation, le SNC :

Il a été démontré que la famille de récepteurs S1P (S1P1-5), des récepteurs couplés à la protéine G, a des fonctions régulatrices dans tous les systèmes du corps, avec des rôles importants dans les systèmes immunitaire, cardiovasculaire, et nerveux central.

Ceci comprend des fonctions telles que l’induction de la migration cellulaire, de la prolifération, de la survie, de l’angiogenèse, de la différentiation ou de l’apoptose. Les différentes fonctions sont contrôlées par la liaison de leur ligand naturel, la sphingosine 1-phosphate (S1P), mais des molécules chimiques telles que le FTY720 peuvent également activer les récepteurs S1P.

En plus des son activité comme agent immunosuppressif, le FTY720 traverse aisément la barrière hémato-encéphalique, et peut donc exercer des effets directs dans le SNC. Les récepteurs S1P y sont exprimés, et il a été montré que leurs niveaux d’expression (ARN messager) sont modifiés dans le modèle animal EAE, utilisé pour étudier la SEP. D’une façon intéressante, l’expression du récepteur S1P₃, qui est associé au contrôle de processus pro-inflammatoires, est augmenté dans la moëlle épinière d’un

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modèle EAE, en parallèle à GFAP (protéine acide gliale fibrillaire), qui est augmentée dans des astrocytes activés.

Les astrocytes représentent le type majeur de cellules gliales dans le SNC. Ils deviennt « réactifs » dans la SEP, et sont considérés comme pouvant influencer l’évolution de la maladie de différentes façons : l’activation astrocytaire peut être bénéfique, en contrôlant le support trophique des oligodendrocytes et des neurones, mais aussi nuisible, en relâchant des médiateurs pro-inflammatoires, qui vont à leur tour activer et recruter des cellules inflammatoires dans les lésions, ou en formant une cicatrice gliale.

Le fait que l’expression de S1P₃ soit augmentée en parallèle de GFAP au cours de l’évolution du modèle EAE indique que ce récepteur joue peut-être un rôle dans la maladie. En vue du développement de nouveaux modulateurs des récepteurs S1P ayant des profils plus sélectifs que le FTY720, nous avons choisi d’étudier la fonction de S1P3

dans la neuro-inflammation, avec une emphase particulière sur son rôle potentiel dans l’activation astrocytaire.

Le but de cette étude était de déterminer le profil d’expression tissulaire et cellulaire de S1P3 dans les cellules du SNC, la “signalisation” du récepteur dans des astrocytes non-activés ou activés, ainsi que son implication possible dans l’inflammation du SNC, en utilisant deux modèles in vivo de neuro-inflammation, avec des animaux transgéniques pour S1P3, soit le modèle de neuro-inflammation induite par le LPS, et le modèle EAE.

Dans la première partie de la thèse, nous avons eu pour objectif de définir le profil d’expression différentielle des ARN messagers du récepteur S1P3 relativement aux autres récepteurs S1P sur les cellules du SNC. Nous avons montré que S1P3 est exprimé de façon abondante dans des cultures primaires d’astrocytes de rat, et dans des astrocytomes. Nous avons de plus montré que l’ARN messager de S1P3 est exprimé à différents niveaux dans les oligodendrocytes, la microglie, des tranches d’hippocampe, et qu’il est augmenté dans différentes tumeurs cérébrales.

Dans l’étape suivante, nous avons comparé la signalisation en aval des récepteurs S1P après une stimulation avec des agonistes, dans des astrocytes de rat ou dans une lignée d’astrocytome humain (U251). La phosphorylation de ERK1/2 et l’activation de la protéine G en réponse à la modulation des récepteurs ont été étudiées par Western

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blot, et par la mesure du changement d’impédance cellulaire, en utilisant la technologie CellKey. Nous avons montré que les astrocytes primaires de rat répondent à S1P ou à pFTY720 principalement via l’activation de S1P1 et S1P3, conduisant à la phosphorylation de ERK1/2, de façon dépendante de Gi, alors que la phosphorylation de ERK1/2 dans la lignée d’astrocytome implique également S1P2, et probablement l’activation de la protéine Gq.

Dans la troisième partie du projet, nous avons étudié l’implication potentielle de l’axe SphK1/S1P3 dans des astrocytes activés in vitro. Nous avons montré que l’activation d’astrocytes primaires de rat avec le stimulus pro-inflammatoire LPS induit l’activité de SphK1, augmente les ARN messagers et les protéines de SphK1 et de S1P₃, ce qui résulte en une augmentation de l’activité du récepteur S1P3, tel que démontré par l’élévation de la phosphorylation de ERK1/2 en réponse à la stimulation par un agoniste sélectif de S1P₃. De plus, nous avons montré que la migration astrocytaire induite par le LPS dépend de l’activité de SphK1, alors que la migration induite par la S1P est essentiellement dépendante de la prolifération. De surcroît, nous avons démontré l’implication de S1P3 dans la production de la chemokine potentiellement neuroprotective CXCL1, en utilisant des agonistes sélectifs de S1P1 et S1P3. Ensemble, nos résultats démontrent que l’axe de signalisation SphK1/S1P3 semble jouer un rôle dans l’établissement et la maintenance de l’activation astrocytaire.

Le but de la quatrième partie était d’étudier le rôle potentiel de S1P3 dans la réponse d’astrocytes et de tranches de cervelet à un stress ischémique. Nous avons montré, en exposant les cultures à une combinaison de déprivation d’oxygène et de glucose (OGD), suivi par une période de réoxygénation (ReOx), que S1P3 et SphK1 sont augmentés au niveau de l’ARN messager pendant la période ReOx. Nous avons de plus montré que la signalisation astrocytaire est augmentée pendant la réoxygénation, via l’activation de S1P1 et de S1P3, et que la production du facteur pro-angiogénique VEGF est au moins partiellement dépendante de l’activation de SphK1, probablement à travers la signalisation S1P/S1PR.

Le but de la cinquième partie du projet était de caractériser l’expression de S1P3

dans des tissus cérébraux de SEP, ainsi que dans des souris transgéniques S1P₃, qui expriment le gène reporteur LacZ au lieu du récepteur endogène S1P₃. Nous avons montré dans des lésions SEP que S1P₃ et SphK1 sont exprimés par des astrocytes

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réactifs, et que SphK1 est également exprimé par des macrophages. De plus, nous avons montré une expression importante de X–Gal/S1P3 dans le cerveau de souris naïves sur les vaisseaux sanguins, ainsi que sur des interneurones, des cellules granulaires de l’hippocampe, et sur des neurones corticaux, mais pas sur les astrocytes.

Le but de la sixième partie de cette étude était d’étudier si le récepteur S1P3 est effectivement surexprimé par des astrocytes de souris injectées avec du LPS, qui est utilisé pour induire une réponse neuro-inflammatoire., et de plus, si l’expression de ce récepteur joue un rôle dans des symptômes de choc septique ou de neuro-inflammation.

Nous avons montré par coloration au X-Gal que l’expression de S1P₃ n’est pas induite dans les astrocytes par le LPS, malgré que l’ARN messager fût augmenté dans le cervelet. Alors que la réponse inflammatoire, telle que mesurée par la production de médiateurs pro-inflammatoires, est similaire dans les animaux « sauvage » et les animaux « knock-out », une petite tendance semble néanmoins montrer un rôle potentiel de S1P₃ dans la neuro-inflammation, car l’activation astrocytaire et microgliale dans le cortex de souris injectées au LPS était réduite dans les souris « knock-out » par rapport aux souris « sauvages ».

Dans la dernière partie de la thèse, le modèle EAE a été induit, et la progression de la maladie a été comparée dans des souris “sauvages”, S1P3+/-

ou S1P3-/-, afin d’étudier le rôle de S1P3 dans ce modèle. Nous avons examiné le rôle du récepteur au pic de la maladie, permettant la comparaison de paramètres inflammatoires aigüs, ainsi que dans la phase chronique, où il y a plus de neurodégénérescence. Nous avons montré que le récepteur S1P3 ne joue pas de rôle majeur dans EAE, en comparant l’apparition de la maladie et sa progression, les cellules immunitaires impliquées, l’expression de gènes, la prolifération, la production de cytokines et l’examen histopathologique. Finalement, nous avons montré que l’expression de X-Gal/S1P3 est associée aux lésions inflammatoires de EAE, mais il n’a néanmoins pas été possible d’identifier de façon claire le type cellulaire impliqué.

Dans l’ensemble, nos résultats montrent que l’axe SphK1/S1P₃ est important dans la signalisation astrocytaire, et est probablement impliqué dans l’activation des astrocytes in vitro. De plus, nous avons montré que S1P₃ est exprimé sur des astrocytes réactifs dans des lésions SEP, mais pas dans un modèle de neuroinflammation chez la souris.

Dans nos études EAE, nous avons finalement démontré un rôle mineur de S1P₃ dans la

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maladie. Nous avons de plus mis en évidence une divergence entre les modèles in vitro et in vivo chez le rongeur, démontrant les limites des modèles in vitro, mais peut-être également des animaux génétiquement modifiés. La relevance thérapeutique des résultats présentés reste ouverte, car dans la SEP, S1P3 semble avoir des rôles à la fois délétères, mais également bénéfiques sur les astrocytes.

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Abbreviations

Abbreviation Full name

% Percent

[γ-³²P]ATP Adenosine-5'-triphosphate-γ -³²P

°C Degree Celsius (centigrade) a

ABC ATP-binding cassette

AC Adenyl cyclase

ATP Adenosine triphosphate

b

BBB Blood brain barrier

BCA Bicinchoninic acid

BcL-2 B-cell lymphoma 2

BM Bone marrow

BSA Bovine serum albumine

c

C/D Collagenase/Dispase

C1P Ceramide 1-phosphate

Ca²⁺ Calcium

cDNA Complementary deoxyribonucleic acid CDS Cellular dielectric spectroscopy

CERK Ceramide kinase

CERT Ceramide transfer protein

CFA Complete Freund`s Adjuvant

CHO Chinese hamster ovary

CINC-1 Cytokine induced neutrophil chemoattractant-1

CLS2 Collagenase-2

cm Centimetre

CNS Central nervous system

CNTF Ciliary neurotrophic factor

CO2 Carbon dioxide

CPRG Chlorophenol red-β-D-galactopyranoside

CTL Cytotoxic T lymphocyte

CXCL CXC chemokine ligand

d

DA Dark agouti

DAB 3,3´-diaminodbenzidine

DC Dendritic cell

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DIV Days in vitro

DMEM Dulbecco’s modified eagle medium

DMS Dimethylsphingosine

DMSO Dimethyl sulfoxide

DTT Dithiothreitol

e

EC Endothelial cell

EAE Experimental autoimmune encephalomyelitis EDTA Ethylenediamine-tetraacetic acid

ELISA Enzyme-linked immunosorbent assay eNOS enodthelial nitric oxide synthase

ER Enodplasmatic reticulum

ERK Extracellular signal-regulated kinase

EtOH Ethyl alcohol

f

FACS Fluorescence Activated Cell Sorting

FBS Fetal bovine serum

FGF Fibroblast growth factor g

g Gram

g Gravital force

GA Glatiramer acetate

GABA Gamma aminobutyric acid

GAPDH Glyceraldehyde 3-phosphate dehydrogenase GDNF Glial cell line-derived neurotrophic factor

GlcCer Glucosylceramide

GPCR G protein-coupled receptor

GRO Growth regulated oncogene

h

h Hours

H₂O Water

HA Hyaluronan

HBSS Hank's Balanced Salt Solution

HCl hydrochloric acid

HE Heterozygous

HEPES 4-(2-hydroxyethyl)-piperazine-1-ethane-sulfonic acid HGEC Human gingival epithelial cell

Hif-1α Hypoxia inducible factor-1α

HKG House keeping gene

HLA Human leukocyte antigen

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HO Homozygous

Hz Hertz

i

IBA-1 Ionized calcium binding adapter molecule 1 ICAM-1 Intercellular adhesion molecule 1

Iec Extracellular current

IFNβ Interferon β

IFNγ Interferon γ

Ig Immunoglobulin

IGEPAL

IGF-1 Insulin-like growth factor 1

IL Interleukin

IL-6 Interleukin-6

Itc Transcellular current

j

JNK c-Jun NH2-terminal kinase k

KCl Potassium chloride

kg Kilogramm

KO Knockout

l

l Litre

LDH Lactate dehydrogenase

LFA-1 Lymphocyte function-associated antigen 1 LIF leukemia inhibitory factor

LN Lymph node

LPA1 Lysophosphatidic acid receptor 1

LPS Lipopolysaccharide

m

M Molar

MAdCAM-1 Mucosal addressin cell adhesion molecule 1 MAG Myelin-associated glycoprotein

MAPK Mitogen-activated protein kinase

MBP Myelin basic protein

MEKi Mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor

mg Milligram

MHC Major histocompatibility complex

min Minutes

ml Millilitre

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mM Millimolar

MOG Myelin Oligodendrocyte Glycoprotein

MRI Magnetic resonance imaging

mRNA Messenger RNA

MS Multiple sclerosis

MW Molecular weight

MZ Marginal zone

n

Na₃VO₄ Sodium orthovanadate

NaCl Sodium chloride

NADPH Nicotinamide adenine dinucleotide phosphate

NaF Sodium fluoride

ng Nanogram

NGS Normal goat serum

NK Natural killer

nM Nanomolar

NO nitric oxicde

NT-3 Neurotrophin-3

o

OFA Oncins France Strain A

OGD Oxygen glucose deprivation

ON Over night

OPC Oligodendrocyte progenitor cell p

PAR1 Proteinase-activated receptor 1 PBS Phosphate-buffered saline PCR Polymerase chain reaction PDGF Platelet derived growth factor

Pen Penicillin

PFA Paraformaldehyde

PGE₂ Prostaglandin E₂

pH Negative log of Hydrogen ion concentration

PKC Protein kinase C

PLC Phospholipase C

PNS Peripheral nervous system

PPMS Primary progressive MS

PTX Pertussis toxin

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Abbreviation Full name r

Ras RAt sarcoma

ReOx Reoxygenation

RNA Ribonucleic acid

ROCK Rho-associated protein kinase

rpm Revolutions per minute

RRMS Relapsing-remitting MS

RT Room temperature

s

S1P Sphingosine 1-phosphate

S1PR Sphingosine 1-phosphate receptor

SDS Sodium dodecyl sulfate

SEM Standard error of the mean

SKI 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole

SMA Smooth muscle actin

SMC Smooth muscle cell

SphK Sphingosine kinase

SPL S1P lyase

SPMS Secondary progressive MS

SPT Serine palmitoyltransferase

Strep Streptomycin

SVZ subventricular zone

t

T3 Thyroxine-3

TBS Tris-buffered saline

TCM Central memory T cell

TEM Effector memory T cell

TGFα Transforming grwoth factor α

TH T helper cell

TLC Thin layer chromatography

TLR4 Toll-like receptor 4 TNFα Tumor necorsis factor α

TRAIL TNF-related apoptosis-inducing ligand Tris Tris(hydroxymethyl)-aminomethane Tween20 Polyoxyethylenesorbitan monolaurate v

v/v/v

VCAM-1 Vascular cell adhesion molecule 1 VEGF Vascular endothelial growth factor VLA-4 Very late antigen 4

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VSMC vascular smooth muscle cell w

WT Wildtype

μ Micro

μCi Micro Curie

μm Micrometer

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1 Introduction

1.1 Preface

The human brain is the most complex organ of the human body and controls every movement, thought, sensation and emotion that comprises the human experience. The brain collects, converts, and transforms all the information it receives from the body and the environment, making it possible for us to experience and reflect our environment.

The research on brain function is growing rapidly and in the last few decades the over 100 years lasting dogma of the unchangeable adult brain turned out to be wrong.

Today we know that even in the adult brain there is neurogenesis and furthermore the glial cells with an emphasis on astroglia, which were originally seen as the glue of the central nervous system (CNS), come into the front as important players in regulating neuronal functions and even in discussion as substrate of memory and consciousness (Ming & Song 2005; Paton & Nottebohm 1984; Reynolds & Weiss 1992; Kukekov et al. 1999; Eriksson et al. 1998).

With the complexity of the brain, the difficulty to study the brain comes along, and reveals how little we still know about this complex organ. This becomes especially evident when malfunction or injury of the brain leads to disorders like depression, Alzheimer’s or Parkinson’s diseases. All of these diseases have their own pathomechanism and complexity but have in common their enormous social and economic impacts.

Multiple sclerosis (MS) is another devastating inflammatory driven neurodegenerative disease, where autoreactive lymphocytes infiltrate the CNS and mediate an inflammatory response that results in multifocal demyelination, axonal injury, neuronal loss and brain atrophy (Hauser & Oksenberg 2006; Lucchinetti et al.

2000; Trapp & Nave 2008). MS affects mainly young adults between their 20ties to 40ties and women are at least twice as affected as men (Orton et al. 2006; Debouverie et al. 2007). Demyelination manifests in clinical symptoms like paralysis, sensory disturbances, lack of coordination, fatigue or visual disturbances and many other symptoms strongly impacting the quality of patients life (Smith & McDonald 1999).

The etiology of MS, especially the initial event remains unknown and it is unlikely that

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one single causative factor is responsible, but that MS instead develops in genetically susceptible subjects with a critical environmental contribution (Ascherio & Munger 2007a; Ascherio & Munger 2007b). Furthermore, there is increasing evidence that epigenetic influences also play a role (Burrell et al. 2011).

For a better understanding of the pathomechanism in MS and why the further development of more accurate therapies is needed but complex, the different cell types of the CNS and their main physiological functions will be presented in the following sections.

1.2 CNS cells – an overview

Neurons and glia compose the two cell types of all complex nervous systems and both of these cell types are involved in pathological processes in MS (Figure 1).

Although it is estimated, that there are > 100 billion neurons within the human brain, this number is outnumbered by glial cells by ten times and the evolution of increasing complexity in the nervous system is accompanied by a steady rise in glial cell number.

In the CNS, glial cells are represented by three types of cells of neural (i.e. ectodermal) origin, often referred to as ´macroglial` or ´neuroglial` cells. These include the astrocytes, oligodendrocytes and the ependymal cells. In contrast to neuroglia, microglia are of non-neuronal (mesodermal) origin and originate from macrophages that invade the brain during early development and settle throughout the CNS (Verkhratsky A, and Butt A, (2007); Glial Neurobiology, Chichester, England).

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Figure 1. The main cellular components of the central nervous system (Le et al. 2005)

1.2.1 Neurons and oligodendrocytes

The neurons take over a specialised role in the CNS, as they are electrical excitable cells, which can receive chemical information and convert it into electrical impulses by generation of plasmalemmal ´all-or-non` action potentials, which are then transmitted throughout the axon to its terminals, which build synapses with other dendrites and cell bodies allowing the propagation of information throughout the CNS. The morphology of neurons is variable, some with many intricately branched dendrites and others, even though a small minority, with no dendrites at all. The dendritic and soma membrane represents the main region through which the neuron receives its synaptic input. Some neurons receive many thousands of such inputs. Dendrites form synapses with axon terminals from other neurons. The more complex the branching of the dendritic arbour

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the more inputs the neuron receives. The input from the synapses is then collected and the action potential is generated at the axon hillock and propagated through the nerve axon to a synapse with another nerve cell. Neurons can interact with their environment through neuronal membrane proteins like ion-channels, receptors and cell adhesion molecules.

Most of the neurons in the mammalian CNS are myelinated allowing the fast transmission of electrical signals along the axons. The myelin sheath is produced by two types of specialized glial cells, the oligodendrocytes in the CNS and the Schwann cells in the peripheral nervous system (PNS). In contrast to Schwann cells, which perform many of the diverse functions performed by astrocytes in the CNS, the main function of oligodendrocytes is the production of myelin sheaths. Oligodendrocytes originate during CNS development from a pool of progenitors within the ventricular and subventricular zone (SVZ) (LeVine & Goldman 1988; Hardy 1997; Menn et al. 2006).

Once the immature and highly proliferative progenitors reach their destination in the CNS, they begin to maturate and differentiate by increasing the complexity and number of their processes and by upregulating myelin protein expression. The compact myelin internodes are formed by close enwrapping of the axons with their glial plasma membrane, followed by the extrusion of cytoplasm and the compaction of the stacked bilayers (Yu et al. 1994; Butt & Ransom 1993; Armstrong 1998). One oligodendrocyte myelinates multiple axons (average 10) within approximately 10-30 μm of the cell body, but does not produce consecutive myelin sheaths along the same axon. This membrane structure allows membrane depolarisation of the axon only at the nodes of Ranvier, where the myelin sheath is interrupted, and hence results in rapid, saltatory nerve conduction (Verkhratsky A, and Butt A, (2007); Glial Neurobiology, Chichester, England). In CNS demyelinating diseases, axo-glial interactions, axolemmal organization, electrical conduction and connectivity are all disrupted. Consequently, chronically demyelinated axons become vulnerable, with axon loss being a major cause of long lasting disability.

Astrocytes and neurons regulate oligodendrocyte proliferation, differentiation survival and can furthermore promote or inhibit myelination by the release of several factors like platelet derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2),

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insulin-like growth factor 1 (IGF-1), neurotrophin-3 (NT-3) or leukaemia inhibitory factor (LIF) (Simons & Trajkovic 2006).

Oligodendrocytes in turn play a critical role in the maintenance of axonal structures and survival and they likely exert a neuroprotective function as they provide trophic support for the neuronal cell bodies as they were reported to synthesize factors such as ciliary neurotrophic factor (CNTF), IGF-1 and glial cell line-derived neurotrophic factor (GDNF). Oligodendrocytes are thought to be the primary target cells in MS, although there is accumulating evidence that neurons are also subject to extensive attack with occurrence of acute axonal damage already from the beginning of the disease (Kuhlmann et al. 2002).

In addition to myelin-forming oligodendrocytes, there are also many non- myelinating satellite oligodendrocytes and adult oligodendrocyte progenitor cells (OPCs) in the adult CNS (Ludwin 1979; Dawson et al. 2000), of which the anatomy and ultrastructre is known but their function remains elusive. The NG2 glia is now generally accepted as a distinct class of glia, that has the antigenic phenotype of OPCs and the morphological phenotype of astrocytes, with functional properties of both (Kettenmann and Ransom (Editors), Neuroglia 2^nd edition; 2005).

In culture, OPC differentiation can be regulated by different trophic factors, growth factors and morphogens. PDGF-AA is a mitogen and survival factor for OPCS.

Fibroblast growth factor 2 (FGF2) is a mitogen for OPCs and inhibits their differentiation into mature oligodendrocytes (OLs). Insulin-like growth factor I (IGF-1) is a survival factor for oligodendrocytes and with thyroid hormone promotes differentiation. cAMP (adenosine 3`:5` cyclic monophosphate) and retinoic acid regulate the differentiation of OPCs into more mature stages.

1.2.2 Microglia

Microglia are CNS resident cells with a myeloid origin appearing in the brain during early embryonic development. Microglia are so called immunocompetent cells forming the brain immune system. They comprise about 10 percent of all glia cells in the brain and appear in the mature CNS as resting, activated or phagocytic cells. In the healthy CNS, microglia exist in a resting state with a small soma and numerous very thin and

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highly branched processes. As they are equipped with certain receptors and immune recognition sites, they take part in the homeostatic activity of the normal CNS. This function is associated with a high motility of their ramified processes and the constant phagocytic clearance of cell debris.

Microglia can turn from their resting to an activated state in response to various kinds of pathological conditions of the brain, like viral (Polazzi et al. 1999) or bacterial (Liu et al. 2000a) infections, CNS injury and diseases (Teeling & Perry 2009). Some of theses activated microglia can turn into phagocytes. The activation of microglia has the function to provide an active brain defence system contributing to neuronal survival through cellular maintenance and innate immunity. However, an over activation can have detrimental neurotoxic effects by the release of a diverse set of cytotoxic substances like pro-inflammatory cytokines (TNFα: tumor necrosis factor α, PGE2:

prostaglandin E₂, IFNγ: Interferon γ), leading to astrocyte activation, which in a pro- inflammatory feedback loop also release pro-inflammatory factors. The pro- inflammatory factors, together with the release of oxidative stress factors from microglia (NO: nitric oxicde, H₂O₂: hydrogen peroxide, O₂^- and ONOO^-/ONOOH) exert direct toxic effects on both, neurons and oligodendrocytes (Block & Hong 2005).

1.2.3 Astrocytes

The typical astrocytes are stellate shaped cells and are the most abundant and diverse glial cells in the mammalian CNS. Based on their morphological and anatomical localization astrocytes are divided into two main subtypes, the protoplasmatic astrocyte of the grey matter, which exhibit a morphology of several stem branches, and the fibrous astrocytes of the white matter, which exhibit a morphology of many long fiber- like processes. Astrocytes extend their processes to neuronal synapses, nodes of Ranvier, and to the blood brain barrier (BBB), and they are interconnected via gap junctions, and connect to oligodendrocytes via heterotrophic gap junctions (Rash et al.

2001; Venance et al. 1995).

Another group of astroglial cells are the bipolar radial glia, which are the first cells to develop from neural progenitors and disappear from many brain regions after maturation and transform into stellate astrocytes. Other smaller populations of

Involvement of the sphingosine 1-phosphate receptor 3 in neuro-inflammation

Thesis

Reference

Involvement of the sphingosine 1-phosphate receptor 3 in neuro-inflammation

Involvement of the Sphingosine 1-Phosphate Receptor 3 in Neuro-Inflammation

THÈSE

IRIS FISCHER

Table of Contents

Table of Contents ... I List of Figures ... VII List of Tables ...XI Acknowledgments ... XII Summary ... XIV Resumée ... XIX Abbreviations ... XXIV

1 Introduction ... 1

2 Material and Methods ... 34

3 Differential expression of S1P receptors in cells of the CNS ... 81

4 Characterization of S1P receptor signalling in primary rat astrocytes and human astrocytomas ... 92

5 S1P

and SphK1 are functionally upregulated under pro- inflammatory conditions in astrocytes ... 114

6 Combined oxygen glucose deprivation in primary rat astrocytes and cerebellar slices ... 132

7 S1P

tissue expression in MS-lesions and mouse brain ... 151

8 LPS-induced neuroinflammation in S1P

wild-type vs. knockout mice... 166

9 EAE in S1P

transgenic animals ... 179

10 Final Conclusions and Perspectives ... 200

11 Reference List ... 207

List of Figures

List of Tables

Acknowledgments

Summary

Resumé

Abbreviations

1 Introduction

1.1 Preface

1.2 CNS cells – an overview

1.2.1 Neurons and oligodendrocytes

1.2.2 Microglia

1.2.3 Astrocytes