DeTure Michael, Ko Li-Wen, Easson Colin, Yen Shu-Hui
Department of Neuroscience, Mayo Clinic Jacksonville, Florida, USA.
Am J Pathol. 2002 Nov;161(5):1711-22. doi: 10.1016/S0002-9440(10)64448-3.
Conditional expression systems for 4-repeat wild-type (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.
在人神经胶质瘤H4细胞中建立了4重复野生型（WT）tau或相应突变体V337M和R406W的条件表达系统，以研究tau突变对tau理化性质的影响，并建立一个细胞模型，用于形成与17号染色体连锁的额颞叶痴呆伴帕金森综合征（FTDP - 17）和阿尔茨海默病特征性的丝状tau。诱导后tau表达增加，在5至7天达到最高水平。WT tau在氨基酸T181、S202/T205、T231和S396/S404处被磷酸化。R406W突变降低了这些位点的tau磷酸化，V337M突变除了S396/S404位点增加外也降低了这些位点的tau磷酸化。核后细胞裂解物中的大多数tau在200,000×g离心后的上清液部分中回收。当诱导时间延长超过5天时，与WT相比，突变体转染细胞沉淀部分中的tau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。这种不溶性部分硫黄素S呈阳性，含有15至5纳米宽的具有tau免疫反应性的细丝。R406W细丝比WT或V337M转染细胞类似制剂中检测到的细丝更丰富。在光学显微镜水平上，大多数tau与微管在一起，或分散分布在细胞质中，但这些都没有硫黄素S阳性。结果表明，条件性tau转染细胞处于缠结前阶段，使其成为研究细胞内缠结积累和测试作为tau聚集抑制剂的潜在治疗药物的有吸引力的模型系统。
