【作者】 熊艳；

【导师】 刘蓉； 周新文；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 阿尔茨海默病（Alzheimer’s Disease, AD）是最常见的老年性痴呆症，其主要病理学改变是脑内神经元纤维缠结（NFTs）和老年斑（SP）的形成。NFTs的主要成分是过度磷酸化的微管相关蛋白tau,异常磷酸化的tau丧失其促微管组装和稳定微管的功能,并聚集成为成对螺旋丝（PHF），最终导致NFT的形成、突触的退变和神经元的丢失,在AD的发生、发展中起关键作用。而tau的磷酸化主要受相关蛋白激酶（protein kinases, PK）和蛋白磷酸酯酶（protein phosphatases,PP）的调节。大量的研究事实表明，蛋白磷酸酯酶2A（PP2A）的活性在阿尔茨海默病人脑中下降并参与AD样tau蛋白异常磷酸化的形成，但导致PP2A活性下降的上游因素和机制尚未阐明。锌（Zn）离子，是脑内含量较为丰富的微量元素之一，参与神经传导，抗氧化反应，并调节多种酶和生长因子的活性。大量研究结果表明，胞外锌离子水平增高可导致多种蛋白质发生酪氨酸磷酸化修饰。前期研究结果显示PP2A催化亚单位酪氨酸307位点（Y307）磷酸化后可明显抑制PP2A活性，而在AD易感脑区锌离子水平增高，提示锌离子可能参与了AD脑内PP2A活性的下调而促进tau蛋白过度磷酸化。且到目前为止Zn对PP2A的调节及机制尚无报道。我们在本研究中重点探讨了锌离子对PP2A的调节及其在tau过度磷酸化中的作用，并且从分子水平解释了Zn对PP2A的直接和间接抑制作用机制。主要结果如下：第一部分锌离子通过激活Src途径抑制PP2A活性和导致tau蛋白过度磷酸化蛋白磷酸酯酶2A（PP2A）的活性在阿尔茨海默病（AD）人脑中下降并参与AD样tau蛋白异常磷酸化的形成，但导致PP2A活性下降的上游因素和机制尚未阐明。有文献报道显示，PP2A催化亚基的酪氨酸307位点（Y307）磷酸化修饰是导致PP2A失活的直接原因，但何种机制促使PP2A发生失活性修饰还不清楚。大量研究结果表明，胞外锌离子水平增高可导致多种蛋白质发生酪氨酸磷酸化修饰，而在AD易感脑区锌离子水平增高，提示锌离子可能参与了AD脑内PP2A活性的下调而促进tau蛋白过度磷酸化。【目的】阐明锌离子是否通过调节PP2A活性促进tau蛋白异常过度磷酸化及调节PP2A活性的机制。【材料和方法】实验在整体和细胞水平进行。整体水平采用雄性SD大鼠，侧脑室定位注射硫酸锌，分组如下：生理盐水对照组、30mM硫酸锌给药组，30mM硫酸锌给药+锌离子特异性螯合剂CQ腹腔注射组。在侧脑室注射6天后，麻醉取脑分离海马组织匀浆，用免疫印迹检测tau磷酸化水平的变化，以及PP2Ac Y307磷酸化水平的变化,检测PP2A的活性；细胞水平采用小鼠成神经瘤N2a细胞系，用100μM硫酸锌处理3小时后提取蛋白，同样方法检测PP2Ac Y307磷酸化及tau磷酸化水平。同时在细胞水平给予PP2或DES预孵育30min或转染野生型PP2A和突变性PP2A，在用用100μM硫酸锌处理3小时后提取蛋白，同样方法检测PP2Ac磷酸化及tau磷酸化水平。在转基因动物水平，我们采用转人tau的转基因小鼠，锌离子螯合剂CQ灌胃35天，检测PP2Ac磷酸化及tau磷酸化水平及可溶性tau和不溶性tau的变化情况。【结果】将锌离子直接注射入整体动物侧脑室，6天后海马PP2Ac Y307磷酸化水平明显增高，伴随PP2A活性下降，tau蛋白在多个丝氨酸/苏氨酸位点如Ser214、Thr205、Ser396、Ser404发生过度磷酸化；给动物同时腹腔注射锌离子螯合剂CQ,则可部分逆转PP2Ac Y307磷酸化水平和PP2A活性的抑制。用硫酸锌处理小鼠成神经瘤N2a细胞，同样导致PP2Ac发生酪氨酸磷酸化修饰和活性下降，tau蛋白在多个位点发生过度磷酸化,伴随Src失活性磷酸化位点酪氨酸529（Tyr529）磷酸化水平降低，Src激活。给予锌同时给予PP2A的激动剂或过表达野生型PP2A可逆转锌导致的PP2Ac活性下降以及tau蛋白的过度磷酸化。给予Src家族的特异性抑制剂PP2同样可部分逆转PP2Ac Y307磷酸化和PP2A活性的抑制以及tau蛋白的磷酸化。转基因小鼠的给予锌离子螯合剂CQ灌胃后，Src活性抑制，PP2Ac Y307磷酸化水平降低，PP2A活性抑制和tau蛋白磷酸化被逆转，伴随海马内不可溶性tau水平明显下降，【结论】锌离子可通过激活Src而对PP2A进行酪氨酸磷酸化修饰下调PP2A活性,从而导致tau蛋白发生过度磷酸化，促进AD样病变的形成。干预tau转基因小鼠脑内锌离子水平可上调PP2A活性并降低tau蛋白的磷酸化水平。因此干预锌离子水平有可能成为在体上调PP2A活性的新策略。第二部分锌离子体外直接抑制PP2A活性及机制研究我们已经在第一部分阐明锌离子在整体动物脑和细胞内可通过激活Src而使PP2AY307磷酸化失活和tau蛋白过度磷酸化。那么在体外锌离子对PP2A是否有直接调节作用？其内在机制是什么到目前为止尚无报道。【目的】阐明锌离子直接调节PP2A活性及其内在机制。【材料和方法】收集培养的N2a细胞并裂解，在不加入ATP的情况下直接将细胞裂解液和100μM硫酸锌或10nM冈田酸（OA）孵育0，5，15，30min，用免疫印迹检测tau磷酸化水平的变化。同样的方法用不同浓度（0，10，50，100μM）硫酸锌与细胞裂解液共孵育不同时间（0，5，15，30，60min），检测PP2A活性。构建不同片段的PP2Ac原核表达质粒并纯化，将纯化的GST-PP2A_1-50、PP2A_51-270和PP2A_271-309分别和100μM硫酸锌孵育，检测PP2A活性。最后我们用亲和层析柱（IDA）预先孵育硫酸锌或不孵育硫酸锌，分别让纯化的GST-PP2A_1-50、GST-PP2A_51-270和GST-PP2A_271-309自然流过IDA柱，然后用EDTA洗脱，收集不同组分的液体用免疫印迹检测GST的变化以确定不同片段PP2Ac与Zn的结合情况。【结果】将硫酸锌和细胞裂解液共孵育可延缓tau蛋白发生去磷酸化的进程，共孵育15min时已和强PP2A抑制剂冈田酸具有同等的抑制tau蛋白去磷酸化的效果，同时随着时间和硫酸锌浓度的变化，PP2A的活性被抑制，在10μM，30min时到达最低水平，抑制率下降30%。而且给予硫酸锌10μM时，PP2A的抑制效应有时间依赖性。说明在体外锌离子直接抑制PP2A活性。为了进一步确定锌离子对PP2A的直接抑制作用，将截断的PP2A与锌离子直接孵育发现锌离子可以直接抑制GST-PP2A_51-270片段的活性。通过IDA实验发现，锌离子仅和GST-PP2A_51-270有直接结合作用。【结论】在体外，锌离子通过与PP2A_51-270结合发挥直接抑制PP2A活性的作用，这为阐明PP2A活性调节的全新途径提供了重要线索，并为AD的防治提供新靶点。更多还原

【Abstract】 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.更多还原