Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes
Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes
复制标题
雷帕霉素可防止培养的星形胶质细胞中突变型亨廷顿蛋白抑制的 GLT-1 表达
DOI：
10.1038/aps.2011.162
复制
发表时间：
2012-03-01
影响因子：
8.2
通讯作者：
Lin, Fang
中科院分区：
文献类型：
--
作者：
Chen, Lei-lei;Wu, Jun-chao;Lin, Fang
目的：研究雷帕霉素对表达突变型亨廷顿蛋白（Htt-552）N端552位残基的大鼠星形胶质细胞摄取谷氨酸的影响。用携带亨廷顿蛋白N端552个氨基酸残基的科登基因的腺病毒感染星形胶质细胞，建立了亨廷顿病的星形胶质细胞模型。采用蛋白质印迹法检测星形胶质细胞中谷氨酸转运蛋白GLT-1和GLAST、自噬标志物微管相关蛋白1A/1B轻链3（LC 3）和自噬底物p62的蛋白水平。采用Real-time PCR检测星形胶质细胞中GLT-1和GLAST的mRNA表达水平。结果：突变型Htt-552的表达可显著降低星形胶质细胞GLT-1的mRNA和蛋白水平，但对GLAST的mRNA和蛋白水平无明显影响。此外，Htt-552显著降低星形胶质细胞对3 H]谷氨酸的摄取。自噬抑制剂3-MA（10 mmol/L）可显著增加突变型Htt-552在星形胶质细胞中的蓄积，降低GLT-1的表达和3 H]谷氨酸摄取。自噬刺激剂雷帕霉素（0.2mg/mL）可显著降低突变型Htt-552在星形胶质细胞中的蓄积，逆转GLT-1表达和3 H]谷氨酸摄取的变化。结论：自噬刺激剂雷帕霉素可逆转突变型Htt-552对星形胶质细胞GLT-1表达和3 H]谷氨酸摄取的抑制。
Aim:To investigate the effects of rapamycin on glutamate uptake in cultured rat astrocytes expressing N-terminal 552 residues of mutant huntingtin (Htt-552).Methods:Methods: Primary astrocyte cultures were prepared from the cortex of postnatal rat pups. An astrocytes model of Huntington's disease was established using the astrocytes infected with adenovirus carrying coden gene of N-terminal 552 residues of Huntingtin. The protein levels of glutamate transporters GLT-1 and GLAST, the autophagic marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and the autophagy substrate p62 in the astrocytes were examined using Western blotting. The mRNA expression levels of GLT-1 and GLAST in the astrocytes were determined using Real-time PCR. 3 H] glutamate uptake by the astrocytes was measured with liquid scintillation counting.Results:The expression of mutant Htt-552 in the astrocytes significantly decreased both the mRNA and protein levels of GLT-1 but not those of GLAST. Furthermore, Htt-552 significantly reduced 3 H] glutamate uptake by the astrocytes. Treatment with the autophagy inhibitor 3-MA (10 mmol/L) significantly increased the accumulation of mutant Htt-552, and reduced the expression of GLT-1 and 3 H] glutamate uptake in the astrocytes. Treatment with the autophagy stimulator rapamycin (0.2 mg/mL) significantly reduced the accumulation of mutant Htt-552, and reversed the changes in GLT-1 expression and 3 H] glutamate uptake in the astrocytes.Conclusion:Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.