Effects And The Underlying Mechanisms Of SiRNA I₂^PP2A On The Learning And Memory In Tg2576 Mice

Background: Alzheimer's disease (AD) is the most common neurodegenerativedisorder, characterized pathologically with formation of neurofibrillary tangles andsenile plaques. Hyperphosphorylated tau is the major protein component of thetangles, which parallel with the duration and severity of AD, suggesting the crucialrole of tau hyperphosphorylation in AD. The massive research indicated that theprotein kinase activeness upward and/or the phosphate enzyme activity decline is thedirect cause for the AD-like tau hyperphosphorylation. Among them, glycogensynthase kinase-3β(GSK-3β) and protein phosphatase PP2A are the most implicated.I₂^PP2A, an endogenously produced inhibitory protein of PP2A, selectively up-regulatedin the areas of the AD brain affected with neurofibrillary pathology, and translocatedI₂^PP2A co-localize both with PP2A and the early- to middle-stage neurofibrillarychanges of abnormally hyperphosphorylated tau. This suggests that reducing I₂^PP2Amay attenuate or prevent tau hyperphosphorylation through activating PP2A and mayhave potential therapeutic value in the treatment of AD. However, the role of I₂^PP2A inAD is still uncertain.Objective: to investigate the influence of siRNA I₂^PP2A on the learning andmemory in mice and the underlying mechanisms in AD models (HEK293/tau cell andTg2576 APP mice).Methods: construction of siRNA plasmids targeting I₂^PP2A ( pSUPER (pSUP),pSUPER-siCon (pSUP-siC) , pSUPER-siI₂^PP2A (pSUP-siI₂^PP2A) and Lentiviral-siRNAtargeting I₂^PP2A (Lenti-NC, Lenti-siI₂^PP2A). Tg2576 mice were injected twomicroliters of Lenti-NC-GFP or Lenti-siI₂^PP2A-GFP into the left hippocampus or rightcortex with a stoelting stereotaxic instrument and HEK/293tau cells were transfactedwith pSUPER (pSUP), pSUPER-siCon (pSUP-siC) and pSUPER-siI₂^PP2A (pSUP-siI₂^PP2A) with Lipofectamine^TM 2000. Then employed Morris water maze,Step-down avoidance test, Western blotting, Real-time RT-PCR,Immunohistochemistry, Immunofluorescence, Protein phosphatase or kinases activityassay and Immunoprecipitation to measure the alterations of the learning and memoryin mice and tau hyperphosphorylation and the underlying mechanisms.Results: (1) siI₂^PP2A decreases the expression of I₂^PP2A in HEK293/tau cells andTg2576 APP mice; (2) siI₂^PP2A reduces Alzheimer-like alterations in tauhyperphosphorylation and behavioral abnormalities in transgenic mice; (3)knockdown of I₂^PP2A not only activates PP2A but also inhibits GSK-3β; (4)Knockdown of I₂^PP2A inhibits GSK-3βby increasing the phosphorylation level ofGSK-3βat Ser9 through activating PKA but not Akt; (5) I₂^PP2A preserves PP2A bydecreasing the inhibitory binding level of the inhibitor to PP2A catalytic subunit(PP2A_C).Conclusion: knockdown of I₂^PP2A not only activates PP2A but also inhibitsGSK-3β, the two major factors involved in AD-like tau hyperphosphorylation andbehavioral abnormalities. I₂^PP2A may serve as a promising target for AD therapy.