The Effects Of Changed Expression Of а3 Nicotinic Receptors On Synaptic Related Proteins In SH-SY5Y Cells

Objective: To investigate the effects of changed expression of ? 3nicotinic acetylcholine receptors(nAChR)on synaptic related proteins and calcium signaling pathway related proteins,to understand the neuroprotective role of ? 3nAChR in the pathogenesis of Alzheimer’s disease(Alzheimer diseases,AD).Methods:(1)Constracting recombinant plasmid(?3 nAChR-pcDNA3.1)by ligating?3 nAChR gene,got by reverse transcription PCR(RT-PCR),and pcDNA3.1 by T4 DNA ligase.(2)Obtaining the cells with ?3 nAChR high expression and silencing through the transient transfection method.(3)The mRNA and protein level of vesicle associated protein(synaptophysin,SYP),postsynaptic membrane proteins(PSD-95),adaptor protein 180(AP180),dynamin protein 1(DYN1),Calmodulin(CaM),Calmodulin-binding protein kinase II(CaMKII)cAMP responsive element-binding protein(CREB)and phosphorylated CREB(pCREB)detected by real-time PCR and western blotting,respectively.Results:(1)We successfully got recombinant plasmid(? 3 nAChR-pcDNA3.1)with increasing ? 3 nAChR.(2)After transfecting recombinant plasmid(?3 nAChR-pcDNA3.1)into SH-SY5 Y cells,and as compared with controls,the expression levels of α3 nAChR mRNA and protein in such cells were increased by 418% and 116%,respectively;After transfecting α3 nAChR shRNA Plasmid into SH-SY5 Y cells,and as compared with controls,the expression levels of α3 nAChR mRNA and protein in such cells were decreased by the inhibitory efficiency 76% and 67%,respectively.(3)The mRNA and protein level of SYP,PSD-95,DYN1,AP180,CaM,CaMKII,CREB,pCREB were increased obviously in cells with α3 nAChR up-regulated,while the SYP,PSD-95,AP180,DYN1,CaM,CaMKII,CREB,pCREB m RNA and protein levels were reduced significantly in cells with α3 nAChR silencing.Conclusion: Enhancing the level of α3 nAChR in SH-SY5 Y cells increase the level of synaptic related proteins SYP,PSD-95,AP180,DYN1,CaM,CaMKII,CREB,pCREB;and inhibiting α3 nAChR expression reduce the synaptic related proteins SYP,PSD-95,AP180,DYN1,CaM,CaMKII,CREB,pCREB in cells.The results indicated that synaptic function is closely related to α3nAChR in cells,suggesting that the α3 nAChR might play an neuroprotective role in connection with the pathogenesis of AD.