The Effect Of Regulating The Phosphorylation Level Of CREB On APP And Secretase ? And Its Significance In The Prevention And Treatment Of AD

Objective:In Alzheimer's disease(AD),the A? precursor protein(APP)plays an important role in the pathogenesis of AD,and the increase of APP is an inevitable stage in which amyloid ?(A?)increases and then precipitates.Phosphorylation of cAMP-response element binding protein(CREB)is down-regulated in AD patients and transgenic mice overexpressing APP.However,whether up-regulated phosphorylation of CREB can reverse the expression of APP and affect the expression of PS1 and PEN2 secretase remains unclear.The main purpose of this study is to test the changes of AD-associated proteins like Preselin 1(PS1)and Preselin enhencer 2(PEN2)in Gaq-overexpression and APP-overexpression SH-SY5Y neuron-like cells respectively by activating and inhibiting CREB,and to analyze the feasibility to prevent and treat AD by regulating CREB.Methods:(1)Three groups of neuron-like cell line SH-SY5Y were cultured as Gaq-SY5Y(over-expression of G protein a subunit q),Vector-SY5Y(transfection with empty vector)and APP-SY5Y(over-expression of APP).Expression levels of APP,PS1 and PEN2 were measured by Western Blot.(2)According to the recommended concentration of purchased reagents,Forskolin cyclic phosphoric acid adenosine(cAMP)agonist 35 ?mol and H89 2 HCL protein kinase A(PKA)receptor 30 ?mol were applied to the above three groups of cells at 37? for one hour.And each group of cells was treated by activator of CREB,inhibitor of CREB and PBS as control respectively.Western Blot assay was used to determine the protein levels of APP,PS1 and PEN2.(3)The morphology of cells was observed and compared after administering activator and inhibitor of CREB respectively.Results:(1)Western Blot results showed that the level of phosphorylated CREB(p-CREB)in Gaq-SY5Y cells was significantly higher than that in the Vector-SY5Y and APP-SY5Y cells,while the p-CREB level in APP-SY5Y cells was lower than that in Vector-SY5Y cells.(2)The expression levels of PEN2 and PS1-CTF(C-terminal of PS 1)were significantly increased in Gaq-SY5Y cells compared with the other 2 group of cells.(3)Western Blot experiments showed that:the expression levels of PEN2 and PS1-CTF in the CREB-activated group were higher than those in the control group and the CREB-inhibited group.The levels of PEN2 and PS1-CTF in the CREB-inhibited group were the lowest among the three groups,whereas level of APP was the highest.(4)Activation of CREB improved proliferation and attenuated aging state of both Gaq-SY5Y and APP-SY5Y cells,inhibition of CREB slowed proliferation and deteriorate aging state of both Gaq-SY5Y and APP-SY5Y cells.Conclusion:(1)Activation of CREB can up-regulate the expression levels of components of y secretase complex PEN2 and PS1-CTF and decrease the APP level,and improve proliferation and attenuate aging state of neuron like cells.There is the possibility to design strategy based on CREB for prevention and treatment of AD.(2)Over-expression of Gaq can enhance the phosphorylation level of CREB,the underlying mechanism deserves further exploring.