S1P effect on cIAP2 knockout mice microglia during EAE, an animal model of multiple sclerosis Srikethan Mahavadi
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
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
TNF Signaling Pathway
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
Previous Experiments
Experimental Questions Why is every cell but microglia dying? Is S1P being released, which is saving microglia?
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
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
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
LDH Cytotoxicity
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
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?
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