The Effects Of Changed Expression Of α7 Neural Nicotinic Receptors On Synaptic Proteins In SH-SY5Y

Objective: To investigate the effects of changed expression of ?7neural nicotinic acetylcholine receptors(nAChR) on synaptic proteins and the synaptic effects induced by Aβ in SH-SY5 Y cells, to understand the neuroprotective role of ?7 nAChR in the pathogenesis of Alzheimer’s disease(Alzheimer diseases,AD). Methods:(1) Constrcting recombinant plasmid(pcDNA3.1-?7 nAChR) by ligating ?7 nAChR gene, got by reverse transcription PCR(RT-PCR), and pcDNA3.1by T4 DNA ligase.(2) Selecting the cells with ?7 nAChR stabling high expression and silencing through the stable transfection method.(3) The m RNA and protein level of ?7 nAChR, vesicle associated protein(synaptophysin), presynaptic membrane proteins(SNAP-25) and postsynaptic membrane proteins(PSD-95) detected by real-time PCR and western blotting, respectively.(4) The cells with ?7 nAChR silencing, enhancing and with empty plasmids and SH-SY5 Y cells were treated with the final concentration of 1 μmol/L of beta amyloid protein(Aβ1-42), the mRNA and protein level of synaptophysin, SNAP-25, PSD-95 were determined by Real- time PCR and method and Western Blot, respectively. Results:(1) We got the cell clone strains with stable transfection of pcDNA3.1-?7 nAChR recombinant plasmid by screened with G418 in culture medium, and as compared with controls, the expression levels of α7 nAChR mRNA and protein in such cells were increased by 533% and110%, respectively; Also we got the cell clone strains with stable transfection of α7nAChR shRNA recombinant plasmid by screened with puromycin in culture medium,and as compared with controls, the expression levels of α7 nAChR mRNA and protein in such cells were decreased by the inhibitory efficiency with 95% and 80%,respectively.(2) The mRNA and protein level of PSD- 95, SNAP- 25, SYP were increased obviously in cells with α7 nAChR up-regulated, while the PSD- 95, SNAP- 25, SYP mRNA and protein levels were reduced significantly in cells with α7nAChR silencing.(3) When SH-SY5 Y cells and cells with empty plasmid were exposed to Aβ1-42, the mRNA and protein expression levels of PSD-95, SNAP-25,SYP were decreased significantly(P<0.01); when treated the α7 nAChR enhancing cells with Aβ, the mRNA and protein expression level of PSD- 95, SNAP- 25, SYP were increased compared to SH-SY5 Y cells treated with Aβ, while compared to Aβtreated cells, the levels were reduced more obvious in α7 nAChR silencing cells exposed to Aβ. Conclusion: Enhancing the level of α7 nAChR in SH-SY5 Y cells increase the level of synaptic proteins, and inhibiting α7 nAChR expression reduce the synaptic proteins in cells. Meanwhile, increasing α7 nAChR level can resist the synapse damage induced by Aβ, and inhibiting the level of α7 nAChR enhance the toxic effects of Aβ on the synapse. The results indicated that α7 nAChR is closely related to the synaptic function in cells, can resist the synapse damage induced by Aβ,suggesting that the receptor might play an important neuroprotective role in connection with the pathogenesis of AD.