Study On The Mechanisms Of Calcium-dependent Phosphorylation Of PKA-RⅡα And PKA-CREB Pathway In Alzhimer's Disease

PartⅠThe effect on PKA-RⅡαof mouse forebrain homogenate and PC12 cell with calcium incubation in vitroObjective To investigate the expression change of regulation subunit II a of cAMP-dependent protein kinase(PKA) with calcium incubation and different intervention.Methods C57BL/6 mouse forebrain homogenate were divided into 8 groups:Control group1:no intervention; Control 2:only incubation at 30℃for 10min; Incubation with CaCl2 (2.5mmol/L); Pretreatment with EDTA then incubation with CaCl2; Incubation with CaCl2 then adding EDTA; Incubation with CaCl2 then adding alkaline phophatase; Pretreatment with Ht31 then incubation with CaCl2; Pretreatment with Ht31P then incubation with CaCl2. PC12 cells lysate were divided into 8 groups,treat with same factor.. The expression of RⅡαwas detected by using western blot.Results Apparent molecular weight of RⅡαof mouse forebrain homogenate and PC 12 cells were 51kDa in control groupl and control group2:After incubation with CaCl2 apparent molecular weight of RⅡαwas 53kDa; Pretreatment with EDTA then incubation with CaCl2, apparent molecular weight of RⅡαwas 51kDa; Incubation with CaCl2 then adding EDTA, apparent molecular weight of RⅡαwas 53kDa; Incubation with CaCl2 then adding alkaline phophatase, apparent molecular weight of RⅡαwas 51kDa; Pretreatment with Ht31 or Ht31P, apparent molecular weight of RⅡαwas 53kDa.Conclusion The change of molecular weight of RⅡαmay be relevant to calcium,and it can be blocked by alkaline phophatase.it has nothing to do with binding activity of RⅡαand AKAP. Part II The effect on PKA-RⅡαof PC12 cell with calcium overloadObjective To investigate the expression change of regulation subunitⅡαof cAMP-dependent protein kinase(PKA) with calcium overload.Methods The calcium overload was induced by Aβ1-42 oligomer and KCl.PC12 cells were incubated with 5μmol/L or 10μmol/L Aβ1-42 oligomer at 37℃for 0min,1h,2h,4h,12h,and were incubated with 50mmol/L or 100mmol/L KCl at 37℃for 5min,10min,20min,30min,60min。Cell viability was determined by MTT assay.The expression of RⅡαwas detected by using western blot.Results Cell viability was decreased with Aβ1-42 oligomer and KCl incubation by a time-dependent manner. Western blot shown that after incubation with Aβ1-42 oligomer and KCl, apparent molecular weight of RⅡαwas increased at first.,which result was same as PartⅠ's. Finally, apparent molecular weight of RⅡαwas reversed at last time point.Conclusion Aβ1-42 oligomer and KC1 have some toxic effects on PC12 cell, and they can increase molecular weight of RⅡα.the change of molecular weight can be reversed later on. PartⅢThe effect on molecular weight change of PKA-RⅡαwith different kinases inhibitorObjective To study which kinase make the change of molecular weight of RⅡαby using different kinases inhibitor.Methods The cultured cells were divide into such groups:control group received the administration of DMEM medium. Aβ1-42 groups were treated with 5μmol/L Aβ1-42 oligomer for 2h, Aβ1-42 oligomer+H89(PKA inhibitor). Aβ1-42 oligomer+staurosporine(PKC inhibitor). Aβ1-42 oligomer+W-7(CaM antagonists) respectively. KCl groups were treated with 100mmol/L KCl for 20min, KCl+H89(PKA inhibitor). KCl+staurosporine(PKC inhibitor). KCI+W-7(CaM antagonists) respectively. The expression of RⅡαwas detected by using western blot.Results Molecular weight of RⅡαrised to 53kDa after PC 12 cells were incubated with 5μmol/L Aβ1-42 oligomer for 2h and 100mmol/L KCl for 20min.it was only blocked by W-7.Conclusion These data are consistent with phosphorylation of Rlla being mediated by CaMK but not by PKC or PKA. PartⅣThe effect on PKA activity with Aβ1-42 incubationObjective To study PKA activity with Aβ1-42 incubation.Methods The cultured cells were divide into such groups:control group received the administration of DMEM medium. Aβ1-42 group was treated with 5μmol/L Aβ1-42 oligomer for 2h, W-7 group was treated with W-7 for 1h, W-7+ Aβ1-42 group was pretreated with 10μmol/L W-7 1h before treatment with 5μmol/L Aβ1-42 oligomer. Aβ1-42+Ht31 group was treated with 50μmol/L Ht31 and 5μmol/L Aβ1-42 oligomer, Aβ1-42+Ht31P group was treated with 50umol/L Ht31P and 5μmol/L Aβ1-42 oligomer. The expression of phospho-PKA substrate was detected by using western blot.Results The level of phospho-PKA substrate in PC 12 cell was increased at first then decreased by treatment with 5μmol/L Aβ1-42 oligomer for Oh to 12h. W-7 had no effect on the expression of phospho-PKA substrate in PC 12 cell. The increased expression of phospho-PKA substrate result for Aβ1-42 can be reversed by pretreatment with W-7. Ht31 and Ht31P had no effect on the expression of phospho-PKA substrate.Conclusion Aβ1-42 oligomer can increase PKA activity. Phosphorylation of RⅡαmay be involved in the effect on PKA activity with W-7.The change of PKA activity has nothing to do with binding activity of PKA and AKAP. Part V The effect on pCREB with calcium overloadObjective To study the expression of pCREB with calcium overload.Methods The cultured cells were divide into such groups:control group, W-7 group, Aβ1-42 group, W-7+Aβ1-42 group, Aβ1-42+H89 group, Aβ1-42+staurosporine group; control group, W-7 group, KCl group, W-7+KCl group. KCl+H89 group, KCl+staurosporine group. The expression of pCREB was detected by using western blot.Results The level of pCREB in PC12 cell was increased by Aβ1-42 oligomer and KCl, W-7 had no effect on the expression of pCREB in PC12 cell. The increased expression of pCREB result for Aβ1-42 and KCl can be reversed by pretreatment with W-7. Staurosporine had no effect on the expression of pCREB.Conclusion Aβ1-42 oligomer and KCl can increase the expression of pCREB. Phosphorylation of RⅡαmay be involved in the effect on pCREB with W-7.The change of pCREB is mainly result for PKA and it has nothing to do with PKC.