Chapter 1. Fingolimod attenuates ceramide induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes

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1 Chapter 1 Fingolimod attenuates ceramide induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes

2 CHAPTER SUMMARY

3 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS INTRODUCTION and S1P

4 CHAPTER METHODS Autopsy material HPLC and LC-ESI-MS/MS analyses g L L o 4

5 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS Case Age (years) Type of MS Sex Post-mortem delay (h:min) Disease Duration (years) 1 66 ND M 8 ND 48 SP F SP M SP F SP F PP M SP F PP M SP F SP F SP M PP F SP F SP M M 9 NA 75 F 6 NA 89 F 7 NA 56 M 9 NA 8 F 5 NA 89 F 6 NA Cause of death Sepsis Congestive heart failure Cardiac arrest - Euthanasia End stage MS Resp. failure - Euthanasia Urosepsis Pneumonia CVA Urinary tract infection Urosepsis with organ failure Pneumonia, metastasized kidney carcinoma Cachexia Cachexia Myocardial infarction Pneumonia Old age Table 1. Clinical data of MS patients and non-neurological controls. Immunohistochemistry 41

6 CHAPTER Primary antibody Proteolipid protein (clone plpc1) 1:5 Serotec Ltd, Oxford, UK Major histocompatibility complex class II 1:1 DAKO, Glostrup, Denmark ASM 1:1 Santa Cruz, California, USA Ceramide 1:1 Alexis, Lausen, Switzerland SMS 1: Santa Cruz, California, USA SGPP 1:1 Lifespan, Seattle, WA, USA SPHK 1:1 Abcam, Cambridge, UK ASAH1 1:45 Sigma-Aldrich, St. Louise, MO, USA CD68 1:1 DAKO, Glostrup, Denmark Glial fibrillary acidic protein (poly clonal) 1:3 DAKO, Glostrup, Denmark Glial fibrillary acidic protein (mono clonal) 1:1 DAKO, Glostrup, Denmark Table. Primary antibodies Astrocyte isolation, culture and treatment -6 Quantitative PCR

7 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS Primary antibody Major histocompatibility complex class II ASM Ceramide SMS SGPP SPHK ASAH1 CD68 Glial fibrillary acidic protein (poly clonal) Glial fibrillary acidic protein (mono clonal) Table 3. Primary and secondary antibodies Secondary antibody Alexa 594 goat anti-mouse Alexa 488 goat anti-rabbit Alexa 594 goat anti-mouse Alexa 488 donkey anti-goat Alexa 488 goat anti-rabbit Alexa 488 goat anti-rabbit Alexa 488 goat anti-rabbit Alexa 594 goat anti-mouse Alexa 488 goat anti-rabbit Alexa 594 goat anti-mouse Endothelial cell culture. Electrical Cell-substrate Impedance Sensing (ECIS) Monocyte migration 4 6

8 CHAPTER Statistical analysis a 15 RESULTS Increased ceramide expression by astrocytes in active MS lesions 6 nmol Cer/mg protein b ratio C16:/C4:-Cer c ratio C18:/C4:-Cer lesion lesion lesion Figure 1. Increased ceramide species expression in MS lesions. 44

9 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS a b c Active lesion d e f g 1 µm Figure. Enhanced ceramide expression in astrocytes in active MS lesions. Sphingomyelin OH NH O O P O- O N + Sphingomyelinase (ASM) O Sphingomyelin synthase (SMS) Ceramide OH NH OH Ceramidase (ASAH) Ceramide synthase O Sphingosine OH NH OH Sphingosine kinase (SPHK) Sphingosine phosphatase (SGPP) S1P OH NH O O P O- OH F de novo

10 CHAPTER a Active lesion c e Active lesion b d f expression involved in ceramide generation in active MS lesions. g h ceramide generation 46

11 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS a Active lesion c e Active lesion g i Active lesion k b d f h j l Few 47

12 CHAPTER a b Relative abundance (%) Relative Expression (%) C14: Cer 75 1 C16: Cer C18:1 Cer C18: Cer C: Cer C4:1 Cer C4: Cer c d Relative Expression (%) Ceramide (pmol/mg protein) TNF-α TNF-α Lesion Figure 6. Increased ASM levels in MS lesion derived astrocytes are attenuated by FTY7P. ** * FTY7P FTY7P astrocytes 48

13 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS FTY7P counteracts ceramide induced endothelial barrier alterations in vitro 9-3 a Resistance ( ) c Relative migration (%) e Relative migration (%) Time (min.) BSA Ctrl Cer 5 M Sphingomyelinase ** + DMSO TNF- Cer 5 M * TNF- FTY7P 7 8 b Resistance ( ) d Relative migration (%) C -Ceramide Time (min.) ** + DMSO Smase r (,16U/ml) Smase (,3U/m) Figure 7. Ceramide decreases transendothelial electrical resistance and increases monocyte migration. 49

14 CHAPTER DISCUSSION in vitro et al. et al.

15 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS. 6 in vitro and S1P 47

16 CHAPTER de novo ACKNOWLEDGEMENTS REFERENCES

17 FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS : :

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