【作者】 王海红；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 第一部分Tau蛋白磷酸化对抗细胞凋亡及其机制的研究神经原纤维缠结(neurofibrillary tangles, NFTs)是阿尔茨海默病(Alzheimer’s disease, AD)和tau相关疾病患者脑中典型的病理学特征之一,NFTs的主要成分是异常过度磷酸化的微管相关蛋白tau。AD和tau相关疾病患者脑内大多数含有NFTs的神经元尽管长期持续处在促凋亡的环境中却不发生凋亡而是进行慢性的退行性变性死亡,目前对此现象的机制仍不清楚。本研究在两种细胞株鼠成神经瘤(mouse Neuroblastoma 2a, N2a)细胞和人胚肾(Human Embryonic Kidney 293, HEK293)细胞、大鼠和tau转基因小鼠中探讨神经细胞逃逸凋亡及其机制。结果显示:(1)细胞中过度表达微管相关蛋白tau显著抵抗凋亡诱导剂诱导的细胞凋亡,并且同时伴随有微管相关蛋白tau的过度磷酸化和糖原合酶激酶-3(glycogen synthase kinase 3, GSK-3)的酶活性升高;(2)在过度表达微管相关蛋白tau的细胞中过度表达GSK-3不仅完全抑制了GSK-3的促凋亡作用,并且使细胞抵抗凋亡诱导剂诱导的细胞凋亡;(3)细胞、大鼠和tau转基因小鼠中过度磷酸化的微管相关蛋白tau与caspase-3活性片段和(或)浓缩/碎裂的细胞核不存在共定位,去磷酸化的tau蛋白与caspase-3活性片段和(或)浓缩/碎裂的细胞核存在共定位;(4)与微管相关蛋白tau过度磷酸化相伴随的是β-catenin的磷酸化水平降低、β-catenin的水平升高和细胞核转位增加;(5)采用siRNA方法降低β-catenin的水平显著降低微管相关蛋白tau的抗凋亡作用;β-catenin的过度表达抗凋亡诱导剂诱导的细胞凋亡。综上所述,本实验独创地提出了微管相关蛋白tau过度磷酸化的抗凋亡作用:tau蛋白过度磷酸化抑制GSK-3β对β-catenin的磷酸化、升高β-catenin的水平和促进β-catenin的细胞核转位从而使细胞抵抗凋亡。本研究结果显示微管相关蛋白tau过度磷酸化使细胞逃逸灭绝性的凋亡性死亡,为揭示AD和tau相关疾病患者脑中神经退行性变性的本质提供了实验证据。第二部分在N2a细胞中过度表达dishevelled-1蛋白抑制渥曼青霉素诱导的细胞骨架蛋白的过度磷酸化神经原纤维缠结(neurofibrillary tangles, NFTs)是阿尔茨海默病(Alzheimer’s disease, AD)的特征性病理学特征之一,NFTs主要是由成对的螺旋丝(paired helical filaments, PHF)组成,而构成PHF的主要蛋白组分是过度磷酸化的微管相关蛋白tau。另外,异常过度磷酸化的神经细丝也是NFTs的组成成分。研究显示Wnt信号途径在AD的发病中起着重要的作用,并且过度表达dishevelled-1 (DVL-1)蛋白可以模拟Wnt信号。目前,DVL-1蛋白对神经细丝磷酸化的影响以及对神经细胞中微管相关蛋白tau磷酸化的影响尚未见报道。在本实验中,为了探讨DVL-1蛋白对阿尔茨海默病样骨架蛋白磷酸化的影响,首先,用渥曼青霉素处理鼠成神经瘤细胞2a (mouse Neuroblastoma 2a,N2a)建立细胞骨架蛋白异常过度磷酸化的细胞模型;然后,在N2a细胞中转染携带DVL-1基因的真核表达质粒过度表达DVL-1蛋白,用渥曼青霉素处理细胞后,采用免疫印迹和免疫荧光技术检测神经细丝和微管相关蛋白tau的磷酸化。结果显示:神经细丝在SMI31位点和tau蛋白在PHF-1位点的磷酸化在渥曼青霉素处理后1 h和3 h增强,6 h恢复至正常水平;神经细丝和微管相关蛋白tau磷酸化的高峰分别在渥曼青霉素处理后1 h和3 h;过度表达DVL-1蛋白显著抑制渥曼青霉素诱导的神经细丝在SMI31和SMI32位点和微管相关蛋白tau在PHF-1 (Ser-396/404)、M4 (Thr-231/Ser-235)和Tau-1 (Ser-198/199/202)位点的过度磷酸化。综上所述,我们的研究发现:在N2a细胞中过度表达DVL-1蛋白能够显著抑制渥曼青霉素诱导的神经细丝和微管相关蛋白tau的过度磷酸化。第三部分DVL-1 cDNA重组质粒在N2a细胞中的瞬时表达目的将鼠dishevelled-1 (DVL-1) cDNA重组质粒转染到培养的鼠成神经瘤细胞N2a (mouse Neuroblastoma 2a),建立瞬时表达系统,为研究Wnt信号途径在阿尔茨海默病(Alzheimer’s disease, AD)发病中的作用提供实验基础。方法用常规分子生物学方法扩增和纯化DVL-1 cDNA重组质粒,用紫外分光技术检测纯化质粒的纯度;用脂质体介导法将重组质粒转染入培养的鼠成神经瘤细胞N2a,用免疫印迹和免疫荧光方法从蛋白质水平检测转染和表达效果。结果质粒酶切电泳结果显示:鼠DVL-1 cDNA重组质粒PCS2+MT-MDVL1扩增和纯化成功,纯度达到要求;免疫印迹和免疫荧光结果显示鼠DVL-1 cDNA重组质粒在鼠成神经瘤细胞N2a中获得表达,基因转染和表达率为57.6%。结论成功将鼠DVL-1 cDNA重组质粒转染到培养的鼠成神经瘤细胞N2a中,并获得瞬时表达。更多还原

【Abstract】 Part 1 Phosphorylation of tau antagonizes apoptosis and the underlying mechanismsHyperphosphorylated tau is the major protein subunit of neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD) and related tauopathies. It is not understood, however, why the NFTs-containing neurons seen in the AD brains do not die of apoptosis but rather degeneration even though they are constantly awash in a proapoptotic environment. Here, we show that cells overexpressing tau exhibit marked resistance to apoptosis induced by various apoptotic stimuli, which also causes correlated tau hyperphosphorylation and glycogen synthase kinase-3 (GSK-3) activation. GSK-3 overexpression did not potentiate apoptotic stimulus-induced cell apoptosis in the presence of high levels of tau. The resistance of neuronal cells bearing hyperphosphorylated tau to apoptosis was also evident by the inverse staining pattern of PHF-1-positive tau and activated caspase-3 or fragmented nuclei in cells and the brains of rats or tau-transgenic mice. Tau hyperphosphorylation was accompanied by decreases inβ-catenin phosphorylation and increases in nuclear translocation ofβ-catenin. Reduced levels ofβ-catenin antagonized tau’s anti-apoptotic effect, while overexpressingβ-catenin conferred resistance to apoptosis. These results reveal a novel anti-apoptotic function of tau hyperphosphorylation, which likely inhibits competitively phosphorylation ofβ-catenin by GSK-3βand hence facilitates the function ofβ-catenin. Our findings suggest that tau phosphorylation may lead the neurons to escape from an acute apoptotic death, implying the essence of neurodegeneration seen in the AD brains and related tauopathies. Part 2 Overexpression of dishevelled-1 attenuates wortmannin-induced hyperphosphorylation of cytoskeletal proteins in N2a cellNeurofibrillary tangles (NFTs) are one of the hallmark lesions of Alzheimer’s disease (AD) and NFTs are made up of paired helical filaments (PHF), whose main component is the abnormally hyperphosphorylated microtubule-associated protein tau. In addition to tau, abnormally hyperphosphorylated neurofilament is also the protein component of NFTs. Studies have shown that Wnt signaling pathways play important roles in AD and overexpression of dishevelled-1 (DVL-1) mimics the Wnt signal. There is no report about the role of DVL-1 in neurofilament phosphorylation, and the role of DVL-1 in tau phosphorylation in neuronal cells is also not known. To investigate the effect of DVL-1 on Alzheimer-like hyperphosphorylation of cytoskeletal proteins, we used wortmannin to produce a cell model with hyperphosphorylation of cytoskeletal proteins in mouse Neuroblastoma 2a (N2a) cell. Then, cultured N2a cells were transiently transfected with DVL-1 expression plasmid using LipofectamineTM 2000 and were treated with wortmannin. Western blot and immunofluorescence microscopy were used to measure the phosphorylation of neurofilament and tau. Level of phosphorylated neurofilament at SMI31 epitope and phosphorylated tau determined by PHF-1 was increased at 1 h and 3 h and back to normal at 6 h after wortmannin 1μM treatment. The highest level of phosphorylated neurofilament and phosphorylated tau was seen at 1 h and 3 h after wortmannin treatment, respectively. When DVL-1 protein was overexpressed, the hyperphosphorylation of neurofilament at SMI31 and SMI32 epitopes and tau at PHF-1 (Ser-396/404), M4 (Thr-231/Ser-235), and Tau-1 (Ser-198/199/202) epitopes was attenuated. Taken together, our findings suggests that overexpression of mouse DVL-1 protein inhibits wortmannin-induced hyperphosphorylation of neurofilament and tau in N2a cells.Part 3 Transient Expression of Mouse DVL-1 cDNA Recombinant Plasmid in Cultured Mouse Neuroblastoma 2a CellObjective To establish a transient expression system of mouse DVL-1 cDNA recombinant plasmid in cultured wild-type mouse Neuroblastoma 2a (N2a) cell for the further use in studying the role of Wnt signaling in Alzheimer’s disease (AD). Methods After being amplified and purified, the recombinant plasmid was transfected into cultured N2a cell by LipofectamineTM 2000. The transfection and expression were examined by Western blot and immunofluorescence microscopy. Results A successful amplification and purification of the recombinant plasmid PCS2+MT-MDVL1 was evaluated by Cla I digestion, and the successful transfection and expression of the fusion protein DVL-1-c-Myc in N2a cells was determined by Western blot and immunofluorescence microscopy using anti-c-Myc tag antibody. The transfection efficiency was 57.6% evaluated by immunofluorescence. Conclusion A transient expression system of the fusion protein DVL-1-c-Myc was established in the present study, which can serve as a tool in studying the role of Wnt signaling in AD.更多还原