1. S1P effect on cIAP2 knockout mice microglia during EAE, an animal model of multiple sclerosis
Srikethan Mahavadi

2. Multiple Sclerosis Autoimmune disease of the CNS
Demyelination, axonal degeneration, and cell death
More common in women than men
Women are 2-3 times more likely to be diagnosed with MS
Loss of motor skills, vision loss
Experimental Autoimmune Encephalomyelitis (EAE) is the mouse experimental model of MS

3. Microglia Glial cells which function as immune cells of the CNS
Secrete cytokines such as TNF-  and IL-1
These are both signaling proteins involved in inflammation and regulation of immune cells
Cytokines are small proteins that have an effect on other cells in this case immune cells

4. TNF Signaling Pathway

5. cIAP2 E3 ubiquitin ligase necessary to activate NF-kB pathway
When cIAP is bound, the cell is thought to be saved from apoptosis
Lippopolysaccharide (LPS) treated cIAP2- /- mice survived better than wildtype mice
cIAP2-/- mice exhibited high inflammation and exaggerated symptoms of EAE , TNF was highly expressed

6. Previous Experiments

7. Experimental Questions
Why is every cell but microglia dying?
Is S1P being released, which is saving microglia?

8. Sphingosine-1-phosphate (S1P)
S1P is a lipid mediator involved in extracellular signaling
A study done by Cuviller et al. shows that S1P prevents the features of apoptosis.
It is believed that S1P is secreted by dead cells
S1P is present at elevated levels in the CNS during inflammation

9. Experiment Overview Cell Line: Microglia from mice
Control Group: cells treated with no inflammatory cytokines or S1P
Experimental Group: cells treated with TNF-,, TNF- and S1P, S1P
Inflammatory cytokines added 30 min prior to S1P and incubated for 24 hours
Cytotoxicity measured in 490 nm plate reader

10. LDH-Cytotoxicity
LDH is an enzyme found in all living cells which catalyzes the conversion of lactate to pyruvate as well as NAD+ to NADH.
An enzyme transfers NADH to NAD+ and reduce WST to formazan(a salt) which shows up orange

11. LDH Cytotoxicity

12. Possible Outcomes
Cytotoxicity levels go down as the S1P is increasing the survival of microglia
Cytotoxicity levels go up as the S1P is not changing the survival of microglia
Possible Further Experiments would include identifying if the S1P is working on the DISC complex or elsewhere or even doing the same experiment and inhibiting the R4 receptor
Limitations

13. Discussion
How exactly is S1P increasing the survival of microglia when inflammatory cytokines are released?
Can S1P be further used as drug therapy for patients with MS?

14. References
Yester, J. W., Bryan, L., Waters, M. R., Mierzenski, B., Biswas, D. D., Gupta, A. S., Kordula, T. (2015). Sphingosine-1-phosphate inhibits IL-1–induced expression of C-C motif ligand 5 via c-Fos–dependent suppression of IFN-β amplification loop. The FASEB Journal, 29(12), doi: /fj
What Is MS? (n.d.). Retrieved from
Constantinescu, C. S., Farooqi, N., Obrien, K., & Gran, B. (2011). Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). British Journal of Pharmacology, 164(4), doi: /j x
Silke, J., & Meier, P. (2013). Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation. Cold Spring Harbor Perspectives in Biology, 5(2). doi: /cshperspect.a008730
Conte, D., Holcik, M., Lefebvre, C. A., Lacasse, E., Picketts, D. J., Wright, K. E., & Korneluk, R. G. (2005). Inhibitor of Apoptosis Protein cIAP2 Is Essential for Lipopolysaccharide-Induced Macrophage Survival. Molecular and Cellular Biology, 26(2), doi: /mcb
Chen, G. (2002). TNF-R1 Signaling: A Beautiful Pathway. Science,296(5573), doi: /science
Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., & Spiegel, S. (1996). Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature, 381(6585), doi: /381800a0
Dojindo Molecular Technologies (2012). LDH Cytotoxicity Assay