人全长Tau蛋白过度表达对线粒体分裂融合动态及细胞退变的影响

发布时间：2018-01-29 11:16

本文关键词： tau 线粒体分裂融合退行性变阿尔茨海默病　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：Tau蛋白既是组装微管和维持微管稳定性的重要骨架蛋白，也是AD脑内神经元纤维缠结的重要组分。最近研究发现AD患者或AD样动物大脑神经元有显著的线粒体形态分布异常和功能障碍。曾有研究报道被半胱天冬酶酶切后的tau在神经元可引起线粒体碎裂；AD病人神经元突触线粒体内富含N端截断tau蛋白且与突触病变和细胞器损伤相关。但是，AD脑内也存在全长tau蛋白，而tau蛋白是否影响线粒体动态，且其在细胞及神经退变中是否起作用未有研究报道。目的：探讨人全长tau（htau）蛋白对细胞的线粒体分裂融合动态的影响及其在神经元退变中的作用。本研究在HEK293细胞、大鼠原代海马神经元、htau转基因动物水平进行了研究，旨在阐明过度表达tau蛋白对线粒体分裂、融合动态，线粒体功能，及其对细胞突起退变的影响及其机制。方法：首先采用转染标记线粒体的质粒进行激光共聚焦成像技术结合线粒体形态分布定量分析，在HEK293细胞检测了稳定或瞬时htau过度表达对线粒体形状、分布的影响；采用ZEISS510激光共聚焦实时动态成像观测htau过度表达对细胞内线粒体分裂、融合动态，及细胞突起变化。使用免疫印迹、荧光定量PCR检测了细胞及动物过表达htau对线粒体分裂融合蛋白及mRNA水平的影响。给予放线菌酮抑制mRNA合成来证实tau蛋白是否通过影响Mfns的泛素化降解引起融合蛋白水平升高。进一步通过免疫印迹实验检测了tau过度表达对Mfns的泛素化修饰的影响。酶活性测定检测了tau过度表达时蛋白酶小体活性及Complex I活性的变化。测定了tau过度表达对细胞内ATP水平的影响。采用JC-1染色分析细胞内线粒体膜电位。分离线粒体检测了细胞及动物tau过度表达对线粒体PINK1水平的影响。采用碱或胰酶处理纯化的线粒体来检测tau蛋白是否分布在线粒体。结果：在本实验中我们观察到以下结果：（1） htau稳定或瞬时过度表达引起大量HEK293细胞和大鼠原代海马神经元出现线粒体形态和分布障碍，表现为线粒体延长并聚集在细胞核周围，细胞突起部位分布的线粒体数目显著减少；(2)稳定或过度表达htau蛋白引起HEK293细胞线粒体融合速度显著加快；（3）htau过度表达引起HEK293细胞及小鼠海马线粒体融合蛋白水平Mfn1、Mfn2和OPA1水平显著升高;(4)下调融合蛋白Mfn1和Mfn2可部分逆转稳定表达htau的HEK293细胞的线粒体形态和分布障碍;(5)荧光定量PCR发现Mfn2mRNA水平升高；蛋白酶小体活性下降；抑制mRNA合成后， Mfn2水平高于对照组；（6）证明鼠tau蛋白可作为一个完整的膜蛋白定位于线粒体内膜，htau蛋白定位于线粒体膜间隙或靠近内膜处；（7）发现htau蛋白过度表达升高线粒体基础膜电位，PINK1水平显著下降；（8）发现在HEK293细胞htau过度表达早期不影响线粒体Complex I活性及ATP水平，晚期使二者下降，，htau转基因动物内Complex I活性降低；（9）htau稳定过度表达或瞬时表达48h引起HEK293细胞出现退行性变化，突起变短变细，或出现肿胀、断裂等变化；（10）发现htau过度表达48h引起原代海马神经元的线粒体聚集在核周围或短树突内，突起内线粒体密度显著降低，突起数目显著减少，轴突长度显著缩短，突起出现串珠样断裂等退行性变化。结论：htau过度表达早期引起线粒体融合加速、聚集在细胞核周围，晚期引起线粒体呼吸酶Comlex I活性降低和ATP生成减少，使细胞出现退行性改变。htau分布在线粒体升高线粒体基础膜电位，抑制PINK1转位于线粒体，使融合蛋白Mfns泛素化降解障碍，从而导致线粒体动态改变的机制。
[Abstract]:Tau protein is an important skeleton protein to assemble the microtubules and maintain the stability of the microtubules . It is also an important component of the entanglement of the neurons in AD . Recent studies have found that the brain neurons of AD patients or AD - like animals have significant abnormal mitochondrial morphology and dysfunction . There is a study that the tau protein is rich in N - terminal truncated tau protein in the synaptic mitochondria of AD patients and is associated with synaptic lesions and organelle damage . However , there is also a full - length tau protein in AD brain , and whether tau protein affects the dynamics of mitochondria and whether it plays a role in the cell and neurodegeneration . Objective : To investigate the effect of human full - length tau ( htau ) protein on mitochondrial DNA fragmentation and its role in neuronal degeneration . Methods : The effect of the overexpression of htau on the mitochondrial membrane protein and mRNA level of the mitochondria was detected by using the plasmid of the transfection marker mitochondria . The effect of the overexpression of htau on the mitochondrial membrane protein and mRNA level in the cells was investigated by using the fluorescence quantitative polymerase chain reaction ( PCR ) . The effects of the overexpression of htau on the mitochondrial membrane protein and mRNA level in the cells were investigated by using the method of Western blot . Results : In this experiment , we observed the following results : ( 1 ) the stable or transient overexpression of htau resulted in a significant decrease in mitochondrial morphology and distribution in the neurons of the hippocampus and in the rat primary hippocampal neurons . Conclusion : In the early stage of htau overexpression , mitochondrial fusion was accelerated , and the activity of mitochondrial respiratory enzymes Comlex I was decreased and ATP formation was decreased in the late stage .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

【共引文献】