Received 2002-04-09 Accepted 2002-04-29

This work was supported by grants from National Natural Science Foundation of China (No.39825109) and National Key Project of Basic Science Research (No.G1999054007). *Corresponding author. Tel: +86-21-64041900-2774;

E-mail: fysun@shmu.edu.cn 生理学报, Aug. 2002, 54 (4): 287~293 Acta Physiologica Sinica

研究论文

岗田酸诱导大鼠脑神经细胞表达谷氨酸转运体EAAT1

魏建设, 张玲妹, 黄娅琳, 朱粹青, 孙凤艳*

复旦大学上海医学院医学神经生物学国家重点实验室, 上海 200032

摘 要: 为研究tau蛋白高度磷酸化与谷氨酸转运体功能之间的关系, 实验采用免疫组织化学、荧光双标记技术及大鼠额叶皮质定位注射的方法, 观察了蛋白磷酸酶抑制剂岗田酸(okadaic acid, OA)所致神经细胞退化对谷氨酸转运体亚型EAAT1表达的影响。结果如下: (1) 在OA注射中心区神经元早期出现胞体固缩、肿胀、核移位, 在注射3 d时细胞破碎, 发生坏死, 并有大量炎性细胞浸润等病理现象; 边周区细胞呈AT8(微管相关蛋白tau磷酸化指标)免疫阳性反应; (2) OA首先诱导神经细胞突起远端tau蛋白磷酸化, 并逐渐向胞体发展, 形成营养不良的神经细胞突起和神经纤维缠结样病理改变; (3) AT8免疫阳性反应脑区的神经细胞高表达谷氨酸转运体EAAT1, 在12 h阳性表达细胞数显著增多(P<0.01), 1 d时达峰值(P<0.001), 3 d时明显减少。在OA作用下EAAT1表达于星形胶质细胞和神经元。结果提示, OA致微管相关蛋白tau高度磷酸化时可诱导该区星形胶质细胞和神经元高表达谷氨酸转运体EAAT1。EAAT1高表达的病理生理意义有待进一步的阐明。

关键词: 岗田酸; tau蛋白; 磷酸化; 神经纤维缠结; 谷氨酸转运体EAAT1

学科分类号: Q421; R338; R741.02

Okadaic acid induces the expression of glutamate transporter EAAT1

in the neurons of rat brain

WEI Jian-She, ZHANG Ling-Mei, HUANG Ya-Lin, ZHU Cui-Qing, SUN Feng-Yan*

State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai 200032

Abstract: To study the relationship between tau hyperphosphorylation and the function of glutamate transporter okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5 µl volume, was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling, nuclear pyknosis, and dislocation at the early stage, and necrosis appeared 3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters with immuno-positive reation for AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the cell body at 1 d, which brought about dystrophic neurites and neurofibrillary tangle (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the mechanism and significance of the induction of glutamate transporter EAAT1 expression remain to be further elucidated.

Key words: okadaic acid; tau protein; phosphorylation; neurofibrillary tangle (NFT); excitatory amino acid transporter 1 (EAAT1)