Regulation Of Zinc On Protein Phosphatase2A And Its Role Inalzheimer’s Disease

Alzheimer’s Disease is one of the most common dementia which is characterized byextracellular senile plaques （SP） and intracellular neurofibrillary tangles （NTFs）. NTFs arecomposed of abnormal hyperphosphorylated microtubule-associated protein tau. Abnormalhyperphosphorylated tau loses the ability of promoting microtubules assembly and stabilityand forms paired helical filament （PHF）, inducing synapses degeneration and neuronloss.Thus, tau hyperphosphorylation plays an important role in the process of AD. As aphosphoprotein, tau phosphorylation is regulated by tau kinases such as GlycogenSynthesis Kinase3β （GSK-3β） and tau phosphatases such as Protein Phosphatase2A（PP2A）. Previous reports showed that PP2A is down-regulated in AD brain, but theupstream factor and underlying mechanism is still unknown.Zinc,one of the most important trace elements,plays an critical role in neuronaltransduction, anti-oxidation reaction and regulation of the activity of many enzymes andgrowth factors. Previous studies imply that the elavated extracellular zinc levels which isobserved in AD brain could induce tyrosine phosphorylation, and tyrosine phosphorylationat PP2A Y307site may inhibit PP2A potently, suggesting that zinc may take part in theregulation of PP2A activity and induces tau hyperphosphorylation. Till now, the regulationof zinc on PP2Aand the underlying mechanisms reamain unclarified.In the present study, we aimed to investigate the role of zinc in PP2A regulation and tauhyperphosphorylation,discussed the effect of zinc chelator CQ on tauopathy in tautransgenic mice. We also explored the underlying mechanisms of zinc regulation on PP2Aindirectly or directly. The main findings are as following: Part IZinc induces protein phosphatase2A inactivation and tauhyperphosphorylation through Src dependent PP2A （tyrosine307）phosphorylationThe activity of protein phosphatase2A （PP2A） is down-regulated and promotes thehyperphosphorylation of tau in the brains of Alzheimer’s Disease （AD）, but the mechanismfor PP2A inactivation has not been elucidated. We have reported that PP2Aphosphorylation at tyrosine307（Y307） is involved in PP2A inactivation. Here, we furtherstudied the upstream mechanisms for PP2A phosphorylation and inactivation. We foundthat zinc, a heavy metal ion that is widely distributed in the normal brain and accumulatedin the susceptible regions of AD brain, participates the inhibition of PP2A and tauhyperphosphorylation.【Aim】To explore the role zinc in PP2A regulation and tauopathy, and disclose theupstream factors.【Materials and Methods】The experiments were performed on the animal and cell levels.For the brain zinc injection, SD rats were deeply anesthetized and divided intoexperimental group for lateral ventricular injection with zinc sulfate （10mM,5μl） andcontrol group for injection with PBS （5μl）. The experimental group was further dividedinto two groups with or without treatment of zinc chelator CQ （Intraperitoneal injection,30mg/kg/48h）. One day or six days after the injection, the rats were sacrificed and thehippocampus was homogenized and then detect the phosphorylation site of tau PP2A andthe activity of PP2A. For the cell experiments, N2a cell were treated with100μM zincsulfate for3hours, with or without pre-incubation of zinc chelator CQ （10μM）, Srcinhibitor PP-2（10μM）, IGF-1R inhibitor AG1024（10μM）, EGFR inhibitor AG1478（10 μM）, or PP2Ac agonist DES （10nM） for30min, or transfected with PP2A Y307F orwtPP2A for24h, at the end of incubation, the cells were harvested for detecting thephosphorylation level of tau, PP2A and Src and the activity of PP2A. For brain zincchelating in hTau mice, animals were treated with CQ （30mg/kg/d by oral gavage）, orvehicle solution at9months of age for a total period of35days. Then the mice weresacrificed and the brains were isolated, half of the hippocampus was homogenized forfurther biochemistry detections of the phosphorylation level of tau, PP2A and Src and theactivity of PP2A, the other hemisphere was frozen and cryoprotected forimmunohistochemistry.【Results】We found that zinc could induce PP2A inhibition, phosphorylation of PP2A atY307and tau hyperphosphorylation both in rat brains and cultured N2a cells, while zincchelating prevented these changes completely. Up-regulation of PP2A chemically orgenetically attenuated zinc-induced tau hyperphosphorylation, whereas mutation of Y307to phenylalanine （F） abolished the zinc-induced tyrosine phosphorylation and inactivationof PP2A. Zinc could activate Src, while PP2, a specific Src family kinases （SFKs） inhibitor,attenuated zinc-induced PP2A phosphorylation and inactivation, indicating that zincinduces PP2A Y307phosphorylation and inactivation through Src activation. In human tautransgenic mice, zinc chelator rescued PP2Aactivity, prevented Src activation, and reducedhyperphosphorylated and insoluble tau levels.【Conclusion】 Zinc induces protein phosphatase2A inactivation and tauhyperphosphorylation through Src-dependent pathway, regulation of zinc homeostasis maybe a promising therapeutics for AD and the related tauopathies. PartⅡZinc inhibits Protein Phosphatase2A directly in vitroPrevious study indicated that zinc induces protein phosphatase2A （PP2A） inactivation andtau hyperphosphorylation through PP2A（tyrosine307） phosphorylation in cells and brains,but whether or not zinc ion has a direct inhibitory effect on PP2Ais not elucidated.【Aim】To explored the direct interaction and effect of zinc on PP2Ain vitro.【Materials and Methods】N2a cell lysates were incubated with100μM zinc sulfate or10nM okadaic acid （OA） for5,15,30min at30°C,then the tau phosphorylation level wasdetected by Western blotting; For the analysis of time and concentration dependent changeof PP2A activity in zinc-incubated cell lysates, N2a cell lysates were incubated with0,10,50,100μM zinc sulfate for30min, or with10μM zinc sulfate for0,10,30,60min at30°C. At the end of incubation, the lysate were collected for PP2A activity assay. For theanalysis of zinc interaction site, different fragments of PP2Ac (GST-PP2A_1-50、PP2AR51-270Rand PP2AR_271-309R were expressed and purified, and then incubated with or without100μMzinc. The PP2A activities were detected.For the zinc binding assay, IDA-agarose wasincubated with or without Zn²⁺PPfor30min and added with GST-PP2AcR（1-50）R,GST-PP2AcR_（51-270）Ror GST-PP2AcR（271-309）Rproteins separately at room temperature for30min. After the incubation, the proteins were washed with different buffers, the elutionswere collected for Western blotting by using antibody against GST.【Results】The results showed that zinc mimic the inhibitory effect of oadaic acid （OA） onPP2A and prevented tau dephosphorylation in N²a cell lysates. PP2A activity assay resultindicated that low concentration （10μM） of zinc inhibited PP2A directly. FurtherZn^2-PP-IDA-Agarose Affinity Binding Assay showed that zinc bound to and inhibitedPP2Ac_（51-270）but not PP2Ac_（1-50）Rand PP2Ac_{（271-309）}Rsegement directly. 【Conclusion】Zinc inhibits PP2Adirectly through binding to PP2Ac_（51-270）Rin vitro.