锌转运体ZnT3通过调节突触锌离子释放参与tau蛋白磷酸化的分子机制研究

Zinc transporter ZnT3 is a transmembrane protein located in the membrane of synaptic vesicles of excitatory zinc-ergic neurons in the brain. ZnT3 is responsible for zinc sequestration in synaptic vesicles. During synaptic activity, synaptic zinc is released to the synaptic cleft and enters the post-synaptic neurons through certain ion channels,such as voltage-gated calcium channels. Recent studies have shown that ZnT3 is highly expressed in the brains of Alzheimer's disease (AD). When ZnT3 gene is knocked out from the APP transgenic mouse brain, the mice showed both a reduced β-amyloid deposition and an ameliorated study/memory behavior. Interestingly, in vitro studies have shown that low micromolar zinc could accelerate the fibrillization of human tau via bridging of Cys-291 and Cys-322. Moreover, synaptic released zinc promotes tau hyperphosphorylation has been confirmed from a rencent study with rat hippocampal slices. Therefore, we raise the hypothesis that overexpression of ZnT3 enhances synaptic zinc release into the post synaptic neurons in AD brain. These zinc ions may promote tau phosphorylation and neurofibrillary tangles (NFTs) formation through certain protein kinases pathway, such as glycogen synthase kinase 3β (GSK-3β), calpain, cyclin-dependent kinase 5 (CDK5), extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNK), p38 mitogen-activated protein kinases (p38) and protein phosphatase 2A (PP2A). Therefore, in the present project, we will breed the ZnT3 knockout and human tau transgenic (tau/ZnT3-/-) mice to analyse whether knockout of ZnT3 gene could inhibit synaptic zinc release and tau phosphorylation in tau transgenic mouse brain. Furthermore, we will use the brain slices of tau transgenic and tau/ZnT3-/- mice combined with KCl and/or protein kinase inhibitor/agonists treatments, to analysis which protein kinase pathway is involved in synaptic released zinc-induced tau hyperphosphorylation. This study will be helpful to understand the involvement of brain zinc metabolism in the pathophysiology of Alzheimer disease.

锌转运体ZnT3是定位于兴奋性锌能神经元突触小泡的跨膜蛋白，参与锌离子在突触小泡内的聚集和释放。在阿尔茨海默病(AD)脑内ZnT3表达增加，敲除ZnT3基因的APP小鼠脑内β-淀粉样蛋白沉积明显减少且学习记忆能力得到改善。体外研究表明低浓度锌即可加剧tau蛋白聚集，大鼠脑片实验证实突触锌离子释放可促进tau蛋白过度磷酸化。因此，我们推测：ZnT3在AD脑内的高表达促进锌离子突触释放并进入突触后神经元，通过调节与tau磷酸化相关的蛋白激酶活性，促进tau蛋白磷酸化和神经原纤维缠结形成。本项目拟通过繁育ZnT3基因敲除且tau转基因的杂交小鼠，以及应用该杂交小鼠海马脑片培养给予突触兴奋性刺激等手段，分析突触锌离子释放与tau蛋白磷酸化及其树突异常定位的相关性；阐明ZnT3通过调节脑锌稳态参与tau蛋白磷酸化和NFTs形成的机理，为揭示脑锌代谢参与AD的发病机制提供充分的理论依据。

阿尔茨海默病（AD）的主要神经病理特征之一是变性神经元内出现由过度磷酸化的tau蛋白聚集形成的神经原纤维缠结，研究表明脑内金属离子代谢紊乱与此病理改变密切相关。体外实验证实低浓度锌即可加剧tau蛋白聚集，大鼠脑片实验证实突触锌离子释放可促进tau蛋白过度磷酸化。脑内突触锌离子的聚集和释放主要有锌转运体ZnT3来调控。本项目以ZnT3基因敲除小鼠与tau转基因小鼠的杂交小鼠（tau/ZnT3-/-）为模型，应用行为学、形态学、分子生物学等实验方法，探讨ZnT3调控突触锌离子释放参与tau蛋白磷酸化的相关机制。同时，项目组也探讨了同为2价金属离子的镁、钙离子代谢紊乱参与AD病理进展的相关机制。.本项目的研究表明，ZnT3基因敲除能明显抑制tau模型小鼠脑内突触锌离子释放，降低的锌离子进入突触后神经元，通过调节tau蛋白磷酸化相关蛋白激酶（p38，GSK-3β，ERK1/2，JNK/c-Jun）的活性，抑制tau蛋白不同位点（ser202、ser396、Ser400/Thr403/Ser404、thr181）过度磷酸化，说明突触锌离子释放可促进tau蛋白过度磷酸化。另外还发现突触锌离子释放可上调炎症因子TNF-α的表达，并诱导环氧合酶-2表达升高，高表达的环氧合酶-2及其下游代谢产物PGI2、PGF2α通过PI3-K/AKT、ERK1/2和JNK/c-Jun通路，刺激tau蛋白在Ser202和Ser400/Thr403/Ser404两个位点的磷酸化水平增加。综上所述，锌离子代谢紊乱可通过tau蛋白磷酸化参与AD的病理学发生与发展。 此外，镁离子可通过调节γ分泌酶活性，钙离子通过调节钙相关蛋白激酶calpain1和CAMKII活性参与AD的病理进展。以上研究结果为进一步揭示金属离子代谢紊乱参与AD发生发展的机理，以及确立将调节金属离子稳态作为AD等神经退行性疾病治疗的新策略提供充分的理论依据。.本项目已发表SCI收录期刊论文14篇，获得专利1项，培养了9名博硕士研究生，为项目组将来以金属离子为靶点的抑制致病蛋白沉积的化合物筛选与研发奠定基础。