Chapter 1 Fingolimod attenuates ceramide induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes 1 1 1 1
CHAPTER SUMMARY
FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS INTRODUCTION 1.. 6-1. 1 1 1 and S1P 18 1 19
CHAPTER METHODS Autopsy material HPLC and LC-ESI-MS/MS analyses g L L o 4
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 6 1 3 69 SP M 1 34 4 66 SP F 6 3 5 48 SP F 5 5 6 64 PP M 7 34 7 5 SP F 8 8 41 PP M 7 14 9 53 SP F 11 7 1 7 SP F 7 4 11 56 SP M 8 7 1 55 PP F 17 19 13 7 SP F 6 7 14 47 SP M 7 7 79 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
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 18 1 4
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
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 1 5 4 3 1 3 1 lesion lesion lesion Figure 1. Increased ceramide species expression in MS lesions. 44
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
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
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
CHAPTER a b Relative abundance (%) Relative Expression (%) 1 1 8 6 4 1 15 1 5 1 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) 14 1 1 8 4 TNF-α - + - + 1 1 8 TNF-α - + - + Lesion Figure 6. Increased ASM levels in MS lesion derived astrocytes are attenuated by FTY7P. ** * FTY7P FTY7P astrocytes 48
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 (%) 15 14 13 1 11 1 9 8 14 1 1 8 175 15 15 1 75 5 5 1 3 4 5 6 Time (min.) BSA Ctrl Cer 5 M Sphingomyelinase ** + DMSO TNF- Cer 5 M * TNF- FTY7P 7 8 b Resistance ( ) d Relative migration (%) 17 16 15 14 13 1 175 15 15 1 75 5 5 C -Ceramide 1 3 4 5 6 7 8 Time (min.) ** + DMSO Smase r (,16U/ml) Smase (,3U/m) Figure 7. Ceramide decreases transendothelial electrical resistance and increases monocyte migration. 49
CHAPTER DISCUSSION in vitro et al. et al.
FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS. 6 in vitro 4-4 1 and S1P 47
CHAPTER de novo ACKNOWLEDGEMENTS REFERENCES 1.. 4. 6. 7. 8. 9. 1. 11. 1. 14. 16. 17. 18. 19.
FINGOLIMOD ATTENUATES CERAMIDE PRODUCTION IN MS LESIONS. 1.. 4. 6. 7. 8. 9. 1949. 8:7879-7887. 77:196-1969. 4. 41. 4. 44. 46. 47. 48. 49.