Papasozomenos S C, Su Y
Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston 77030, USA.
J Neurochem. 1995 Jul;65(1):396-406. doi: 10.1046/j.1471-4159.1995.65010396.x.
We heat shocked 21- and 35-day-old fetal rat cerebral explants at 45 degrees C for 18 min and performed immunocytochemistry and immunoblot analysis of sodium dodecyl sulfate extracts using the monoclonal anti-tau antibodies Tau-1, Tau-5, Tau-46, and PHF-1 and the peroxidase-antiperoxidase technique or 125I-labeled protein A. Tau-1 and PHF-1 recognize nonphosphorylated and phosphorylated epitopes, respectively, and both Tau-5 and Tau-46 recognize phosphate-independent epitopes. tau immunoreactivity was confined to neurons and increased in heat-shocked perikarya but not axons. At 0 h after heat shocking, there was dephosphorylation of tau exemplified by (1) faster migration of tau isoforms with resultant loss or attenuation of the 60- and 52-kDa tau isoforms recognized by all four anti-tau antibodies and concomitant accentuation of the fastest moving 50-kDa tau isoform recognized by Tau-1, Tau-5, and Tau-46; and (2) significant increase in the nonphosphorylated Tau-1 epitope with resultant decreases in the ratio of total (phosphorylated plus nonphosphorylated) tau to nonphosphorylated tau and the difference of total tau minus nonphosphorylated tau. tau was phosphorylated back to the control level by 12 h and remained so at 24 and 48 h after heat shocking. Treatment of explants with cycloheximide, a protein synthesis inhibitor, did not prevent the heat shocking-induced dephosphorylation of tau. Treatment of explants with the inhibitors of protein phosphatases PP1 and PP2A, okadaic acid or calyculin A, produced hyperphosphorylated tau polypeptides, prevented the heat shocking-induced dephosphorylation of tau, and intensified the immunoreactivity of the neurofilament subunit H with the only antiphosphoneurofilament antibody that reacts with intraneuronal neurofibrillary tangles. In 35-day-old explants, in addition to the three 50-, 52-, and 60-kDa tau isoforms seen in 21-day-old explants, a 66-kDa tau polypeptide was also present.
我们将21日龄和35日龄的胎鼠脑外植体在45摄氏度下热休克18分钟，然后使用单克隆抗tau抗体Tau-1、Tau-5、Tau-46和PHF-1以及过氧化物酶-抗过氧化物酶技术或125I标记的蛋白A对十二烷基硫酸钠提取物进行免疫细胞化学和免疫印迹分析。Tau-1和PHF-1分别识别非磷酸化和磷酸化表位，而Tau-5和Tau-46均识别不依赖磷酸盐的表位。tau免疫反应性局限于神经元，且在热休克后的胞体中增加，但轴突中未增加。在热休克后0小时，tau发生去磷酸化，表现为：（1）tau异构体迁移加快，所有四种抗tau抗体识别的60 kDa和52 kDa tau异构体消失或减弱，同时Tau-1、Tau-5和Tau-46识别的迁移最快的50 kDa tau异构体增强；（2）非磷酸化Tau-1表位显著增加，导致总tau（磷酸化加非磷酸化）与非磷酸化tau的比值以及总tau减去非磷酸化tau的差值降低。热休克12小时后，tau磷酸化恢复到对照水平，在热休克后24小时和48小时仍保持该水平。用蛋白质合成抑制剂环己酰亚胺处理外植体，不能阻止热休克诱导的tau去磷酸化。用蛋白磷酸酶PP1和PP2A的抑制剂冈田酸或花萼海绵诱癌素A处理外植体，产生过度磷酸化的tau多肽，阻止热休克诱导的tau去磷酸化，并增强神经丝亚基H与唯一能与神经元内神经原纤维缠结反应的抗磷酸化神经丝抗体的免疫反应性。在35日龄的外植体中，除了在21日龄外植体中可见的三种50 kDa、52 kDa和60 kDa tau异构体外，还存在一种66 kDa的tau多肽。
