Neuroprotective Effect And Mechanism Of Cold Shock Protein RBM3 Against Rotenone-induced Apoptosis In PD Cell Model

BackgroundRotenone(ROT)is known to be an insecticide,leading to the denaturalization and loss of dopaminergic neurons.Studies showed that the cytotoxicity of ROT on central nervous system could be one important risk factor causing Parkinson's disease(PD).Cold shock protein RBM3,a highly conservative RNA binding motif protein,may regulate the protein translation by binding 60S subunit of ribosome,mi RNA,and mRNAs.RBM3can be strongly up-regulated by various mild stimuli such as hypothermia especially.Here,we utilizedthe PD cell model based on ROT-induced apoptosisin human neuroblastoma SH-SY5Y cells,to investigate the effects and the underlying molecular mechanism of mild hypothermia and RBM3 on neuroprotection.Meanwhile,the effect of RBM3overexpression on global protein synthesis in SH-SY5Y cells will be examined.Objectives1.To explore the effect of mild hypothermia on ROT-induced apoptosis in PD cell model SH-SY5Y cells;2.To clarify the molecular mechanism of RBM3 on ROT-induced apoptosis;3.To study RBM3 expressions after ROT treatment.Methods1.Apoptosis caused by ROT on SH-SY5Y cells.Take the cell model endangered by ROT and MTT assay to test the toxicity caused by various concentrations of ROT on SH-SY5Y cells after hours.2.Effects of mild hypothermia(32~oC)on apoptosis induced by ROT in SH-SY5Y cells.Incubation of SH-SY5Y cells in 32~oC for 24 h makes highly expressions of cold shock protein RBM3,followed by adding ROT to cells.Using MTT assay to detect cellviability,and use TUNEL staining assay to detect the level of DNA fragments in apoptosis.Then detect the expressions of apoptosis-related proteins cleaved caspase3,cleaved PARP,Bcl2,and Bax with immunoblotting(Western blotting),and of the autophagic markerBeclin-1.As highly RBM3 expressions induced by 32~oC in HEK293 cells,we chose MTT assay and Western blotting to determine the cell viability and the cleaved PARP expressions,respectively.3.Effects of over-expressions of RBM3 on apoptosis induced by ROT in SH-SY5Y cells.With transient transfection of the recombinant plasmidp XJ40-myc-RBM3intoSH-SY5Y cells,we added ROT insults to cause apoptosis.Using MTT assay to detect cellviability,and use TUNEL staining assay to detect the level of DNA fragments in apoptosis;then detect the expressions of apoptosis-related proteins cleaved caspase3,cleaved PARP,Bcl2,and Bax with Western blotting,and of the autophagic markerBeclin-1.As exogenous overexpression of RBM3 in HEK293 cells,we chose MTT assay and Western blotting to determine the cell viability and apoptosis,respectively.4.Effects of over-expressions of RBM3 on signaling pathway.Exogenous overexpression of RBM3 SHSY5Y cells were treated with ROT.In order to clarify the signaling pathway activated byROT,we tested the phosphorylation levels of p38,JNK,ERK,NF-?B,AMPK,and GSK-3?.5.Detecting the protection effects and the mechanism of RBM3 with siRNA.Silencing gene expression of RBM3 cells were treated with ROT.Then,we tested the level of cleaved caspase3 and cleaved PARP,and the phosphorylation levels of p38,JNK,and ERK by Western blotting.6.Effects of MAPK inhibitor on apoptosis induced by ROT.Employ MTT,Western blotting and DAPI staining to detect the anti-apoptosis effect of MAPK inhibitor.7.Effects of RBM3 on global protein synthesis(GPS).With SUnSET tech,we observed the GPS in SH-SY5Y cells treatment by ROT.Using Western blotting to figure out the effects of RBM3 and MAPK inhibitor on p-eEF2,respectively.Results1.ROTinduced cytotoxicity in SH-SY5Y cells in a concentration-and time-dependent manner.2.Mild hypothermia showed a significant protective effect on apoptosis induced by ROT.3.The overexpression of RBM3resembled the neuroprotective effect of mild hypothermia against ROT insult.4.Gene silencingofRBM3 abrogated the neuroprotective effect of mild hypothermia.5.RBM3 significantly repressed the activation of MAPK signaling pathway induced by ROT.6.MAPK inhibitor rescued SH-SY5Y cells from ROT-induced apoptosis.7.RBM3 might promot the level of GPS by inhibiting JNK and ERK signaling pathway in SH-SY5Y cells.ConclusionsRBM3 mediates the neuroprotective effect of mild hypothermia againstapoptosis induced by ROT in SH-SY5Y cells,by inhibiting JNK,p38,and ERK MAPKs.RBM3might promote GPS by interefering with JNK and ERK signaling pathways.To conclude,RBM3 overexpression or induction presents a potential strategy for the therapy of neurodegeneration diseases such as PD.