tau assembly in inducible transfectants expressing wild-type or FTDP-17 tau
tau assembly in inducible transfectants expressing wild-type or FTDP-17 tau
复制标题
DOI：
10.1016/s0002-9440(10)64448-3
复制
发表时间：
2002-11-01
影响因子：
6
通讯作者：
Yen, SH
中科院分区：
文献类型：
--
作者：
DeTure, M;Ko, LW;Yen, SH
在人神经胶质瘤H4细胞中建立了针对4重复野生型（WT）tau蛋白或相应突变体V337M和R406W的条件性表达系统，以研究tau突变对tau蛋白理化性质的影响，并为伴有帕金森综合征的额颞叶痴呆（与17号染色体相关，FTDP - 17）和阿尔茨海默病所特有的丝状tau形成建立细胞模型。诱导后，tau表达增加，在5到7天达到最高水平。野生型tau在氨基酸T181、S202/T205、T231和S396/S404位点被磷酸化。R406W突变降低了这些位点中每个位点的tau磷酸化水平，V337M突变也是如此，但S396/S404位点除外，该位点的磷酸化水平升高。在200,000×g离心后，细胞核后细胞裂解物中的大多数tau蛋白在上清液组分中被回收。当诱导时间延长超过5天时，与野生型相比，突变体转染细胞中沉淀组分中的tau蛋白量增加更多。这种颗粒状tau可以用盐、Triton X - 100或 Sarkosyl部分提取。在转染细胞中，到第7天，R406W具有最高比例的Sarkosyl不溶性tau。这种不溶性组分硫黄素S阳性，并含有15 - 5nm宽的具有tau免疫反应性的细丝。R406W细丝比在野生型或V337M转染细胞的类似制剂中检测到的细丝更丰富。在光学显微镜水平下，大多数tau与微管一起被发现，或弥散分布在细胞质中，但这些都没有呈现硫黄素S阳性。结果表明，条件性tau转染细胞处于缠结前阶段，这使它们成为研究细胞内缠结积累以及测试潜在治疗药物作为tau聚集抑制剂的有吸引力的模型系统。
Conditional expression systems for 4-repeat wildtype (WT) tau or the corresponding mutants V337M and R406W were established in human neuroglioma H4 cells to study the effect of tau mutations on the physicochemical properties of tau, and to develop a cellular model for the formation of filamentous tau characteristic of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and Alzheimer's disease. Upon induction tau expression increased, reaching maximal levels at 5 to 7 days. WT tau was phosphorylated at amino acids T181, S202/T205, T231, and S396/S404. The R406W mutation decreased tau phosphorylation at each of these sites as did the V337M mutation except for S396/S404 sites that increased. Most tau in postnuclear cell lysates was recovered in the supernatant fraction after centrifugation at 200,000 X g. The amount of tau in the pellet fraction increased more in mutant transfectants compared to WT when the induction was extended beyond 5 days. This particulate tau could be partially extracted with salt, Triton X-100, or sarkosyl. Of the transfectants, R406W had the highest proportion of sarkosyl-insoluble tau by day 7. This insoluble fraction was thioflavin S-positive and contained 15- to 5-nm-wide filaments with tau immunoreactivities. The R406W filaments were more abundant than those detected in similar preparations from WT or V337M transfectants. At the light microscopy level, most tau was found with microtubules, or diffusely distributed in the cytoplasm, but none of this appeared thioflavin S-positive. The results suggest that conditional tau transfectants are in a pretangle stage making them an attractive model system for studying intracellular tangle accumulation and for testing potential therapeutic agents as inhibitors for tau aggregation.