Studies On GluK2-PSD-95Module-mediated Brain Injury After Ischemia Via Fas Signaling Pathway And The Relative Mechanisms

Background and purposeOne of the crucial mechanisms of ischemia-induced brain injury is that theexcessively released glutamate during reperfusion induces excitoxicity. Whenexcessively stimulated, GluK2, one subunit of glutamate receptors, recruits MLK3through the scaffold PSD-95and assemblies GluK2-PSD-95-MLK3module. Then,MLK3auto-activates via cross phosphorylation and activates its substrates throughMAPK cascade, resulting in activation of c-Jun and subsequent increased FasLexpression. This procedure has been named the nuclear pathway mediated by MAPKsignaling pathway. Since Fas is one of the important death receptors, the increase ofFasL expression after ischemia suggests that FasL-Fas pathway might act at theexecution phase. However, this postulation requires further investigations. Themechanisms of Fas-mediated ischemic brain injury also remain to be clarified.Fas-induced apoptosis pathway ubiquitously functions in various tissues and cells.When stimulated by its ligand FasL, the cytosolic DD of Fas interacts with FADD,which subsequently recruits caspase8and assemblies DISC. DISC formation facilitatesthe cluster and auto-activation of caspase8. Once activated, caspase8initiates caspasecascade or induces Cyt c release from mitochondria, which activate the excecutorcaspase, caspase3, and resulting in DNA cleavage and apoptosis. Multiple mechanismsregulate Fas apoptosis-inducing pathway. S-nitrosylation of Fas facilitates itstrans-localization to cell membrane, aggregation into CD95hiand internalization intoplasma. CD95hican form hiDISC with FADD and caspase8, which is the preferredmode that Fas mediate apoptosis.In this study, we investigated ischemic brain injury in two sections. We adoptedPDZ1domain inhibitor peptide and Fas shRNA to prove that GluK2-PSD-95modulemediates ischemic brain injury via Fas pathway. Then, we elucidated the mechanisms that GSNO protects neurons from ischemia injury to reflect the mechanisms that Fasmediate ischemic brain injury.Part Ⅰ GluK2-PSD-95mediates brain injury after ischemiavia Fas pathwayMethods1. Use TUNEL method to test the effect of PDZ1peptide on cell apoptosis in rathippocampal CA1region at5d of reperfusion.2. Use immumofluence method to test the effect of PDZ1peptide on Fas andFasL expression during reperfusion; use immunoblot and immunopricipitation to testthe effect of PDZ1peptide on DISC assembly and the activation of downstreammolecules during reperfusion.3. Use DAPI staining to examine the effect of Fas shRNA on apoptosis ofprimary hippocampal neurons after OGD.Results1. TUNEL staining showed that pretreatment of PDZ1peptide significantlyattenuated neuronal apoptosis in rat hippocampal CA1region.2. Pretreatment of PDZ1peptide attenuated the increase of FasL expression,DISC assembly and the activation of downstream molecules at6h of reperfusion,without effect on Fas expression.3. Pretreatment of Fas shRNA significantly reduced primary hippocampal neuronapoptosis after OGD.Part Ⅱ Neuroprotection of S-nitrosoglutathione againstischemia injury by down-regulating Fas S-nitrosylationand downstream signalingMethods1. Use cresyl viollet staining to examine the effect of GSNO pretreatment onneurons death in rat hippocampal CA1region after ischemia.2. Use immunoblot to examine the effect of GSNO pretreatment on activation of 3. Use Biotin-switch method to examine the S-nitrosylation of Fas duringreperfusion and the effect of GSNO pretreatment on Fas S-nitrosylation.4. Use Biotin-switch method to examine the effect of GSNO pretreatment on theS-nitrosylation of nNOS. Use NOS activity testing kit and NO testing kit to examine theeffect of GSNO pretreatment on NOS activity and NO creation.5. Use Biotin-switch method to examine the effect of7-NI pretreatment on theS-nitrosylation of Fas.6. Use immunoblot to test the expression of Fas and CD95hiat cell membrane andin plasma at vary reperfusion times and the effect of GSNO pretreatment on them.7. Use immunoprecipitation to examine the effect of GSNO pretreatment onDISC and hiDISC assembly.Results1. Pretreament of GSNO before ischemia significantly attenuated neurons deathin hippocampal CA1region.2. Pretreament of GSNO before ischemia significantly inhibited the activation ofapoptosis-associating proteins at6h of reperfusion.3. Fas S-nitrosylation was significantly enhanced during reperfusion and peakedat6h. Pretreament of GSNO before ischemia significantly inhibited Fas S-nitrosylationat6h of reperfusion. Although Fas S-nitrosylation was also increased in GSNO controlgroup, it was extremely lower than ischemia/reperfusion group.4. nNOS S-nitrosylation was significantly decreased at6h of reperfusion,however, its activity and NO creation was enhanced. Pretreament of GSNO beforeischemia significantly enhanced nNOS S-nitrosylation, but inhibited its activity and NOcreation.5. Pretreament of7-NI before ischemia significantly decreased FasS-nitrosylation at6h of reperfusion.6. Fas expression at cell membrane was significantly increased during reperfusion,and peaked at6h of reperfusion. Consistently, Fas expression in plasma decreased andreached the lowest level at6h. CD95hiexpression at cell membrane was alsosignificantly increased during reperfusion, and peaked at6h. However, CD95hiexpression in plasma showed the same increase. Pretreament of GSNO beforeischemia significantly blocked the variation of Fas and CD95hi. 7. Pretreament of GSNO before ischemia significantly blocked the assembly ofDISC and hiDISC.ConclusionsBased on the above results, we conclude as below:1. GluK2-PSD-95mediates ischemic brain injury via Fas pathway.2. GSNO inhibits nNOS activation and NO creation after brain ischemia throughS-nitrosylation on nNOS. GSNO-induced NO reduction during reperfusion causesdecrease of Fas S-nitrosylation, subsequent membrane translocation, aggregation andinternalization, which attenuates DISC assembly and activation of apoptosis proteins,thus protecting neurons from ischemic injury.