Srikethan Mahavadi Multiple Sclerosis Autoimmune disease of the CNS Demyelination axonal degeneration and cell death More common in women than men Women are 23 times more likely to be diagnosed with ID: 932411
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Slide1
S1P effect on cIAP2 knockout mice microglia during EAE, an animal model of multiple sclerosis
Srikethan Mahavadi
Slide2Multiple Sclerosis
Autoimmune disease of the
CNS
Demyelination, axonal degeneration, and cell deathMore common in women than men Women are 2-3 times more likely to be diagnosed with MSLoss of motor skills, vision loss Experimental Autoimmune Encephalomyelitis (EAE) is the mouse experimental model of MS
Slide3Microglia
Glial
cells which function as immune cells of the CNS
Secrete cytokines such as TNF- and IL-1These are both signaling proteins involved in inflammation and regulation of immune cellsCytokines are small proteins that have an effect on other cells in this case immune cells
Slide4TNF Signaling Pathway
Slide5cIAP2
E3
ubiquitin ligase necessary to activate NF-kB pathway
When cIAP is bound, the cell is thought to be saved from apoptosisLippopolysaccharide (LPS) treated cIAP2-/- mice survived better than wildtype mice cIAP2-/- mice exhibited high inflammation and exaggerated symptoms of EAE
, TNF was highly expressed
Slide6Previous Experiments
Slide7Experimental Questions
Why is every cell but microglia dying
?
Is S1P being released, which is saving microglia?
Slide8Sphingosine-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
Slide9Experiment 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, S1PInflammatory cytokines added 30 min prior to S1P and incubated for 24 hoursCytotoxicity measured in 490 nm plate reader
Slide10LDH-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
Slide11LDH Cytotoxicity
Slide12Possible 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
Slide13Discussion
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?
Slide14References
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), 4853-4865. doi:10.1096/fj.15-275180What Is MS? (n.d.). Retrieved from https://www.nationalmssociety.org/What-is-MS 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), 1079-1106. doi:10.1111/j.1476-5381.2011.01302.xSilke, J., & Meier, P. (2013). Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation. Cold Spring Harbor Perspectives in Biology, 5(2). doi:10.1101/cshperspect.a008730Conte, 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), 699-708. doi:10.1128/mcb.26.2.699-708.2006Chen, G. (2002). TNF-R1 Signaling: A Beautiful Pathway. Science,296(5573), 1634-1635. doi:10.1126/science.1071924Cuvillier, 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), 800-803. doi:10.1038/381800a0Dojindo
Molecular Technologies (2012). LDH Cytotoxicity Assay