Lysosome vacuolation disrupts the completion of autophagy during norephedrine exposure in SH-SY5Y human neuroblastoma cells
Lysosome vacuolation disrupts the completion of autophagy during norephedrine exposure in SH-SY5Y human neuroblastoma cells
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DOI：
10.1016/j.brainres.2012.10.056
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发表时间：
2013-01-15
期刊：
影响因子：
2.9
通讯作者：
Uemura, Koichi
中科院分区：
文献类型：
--
作者：
Funakoshi, Takeshi;Aki, Toshihiko;Uemura, Koichi
在我们目前的研究中，我们研究了SH-SY 5 Y人神经母细胞瘤细胞中去甲麻黄碱引起神经细胞损伤的机制。发现去甲麻黄碱诱导细胞质空泡化和由此导致的细胞活力丧失。在用去甲麻黄碱处理的细胞中，自噬标记物LC 3也转化为其LC 3-II活化形式，表明诱导自噬。在用RFP-LC 3和GFP-LAMP 1转染的细胞中，观察到LC 3表达的点状模式和LAMP 1与形成的空泡的共定位，突出了空泡的溶酶体性质及其与自噬体的关联。使用tfLC 3（mRFP-GFP-LC 3）的自噬通量测定表明在早期时间点（类似于3小时）通过去甲麻黄碱刺激形成自噬体和自溶酶体。然而，在稍后的时间点（类似于6 h），还观察到自溶酶体/溶酶体扩张和空泡pH值中和。因此，这些结果表明去甲麻黄碱在早期时间点诱导自噬，并在较晚时间点诱导细胞死亡伴溶酶体功能障碍和自噬破坏。(c)2012爱思唯尔有限公司版权所有。
In our current study, we examined the mechanism underlying neuronal cell injuries caused by norephedrine in SH-SY5Y human neuroblastoma cells. Norephedrine was found to induce cytoplasmic vacuolation and a resultant loss of cell viability In the cells treated with norephedrine also, an autophagic marker LC3 was converted to its LC3-II activated form, suggesting the induction of autophagy. In cells transfected with RFP-LC3 and GFP-LAMP1, a punctate patterning of LC3 expression and colocalization of LAMP1 with the formed vacuoles were observed, highlighting the lysosomal nature of the vacuoles and their association with autophagosomes. An autophagic flux assay using tfLC3 (mRFP-GFP-LC3) indicated the formation of autophagosomes and autolysosomes by norephedrine stimulation at an early timepoint (similar to 3 h). However, at a later timepoint (similar to 6 h), both the dilation of autolysosomes/lysosomes and the neutralization of the vacuolar pH were also observed. These results thus indicate that norephedrine induces autophagy at an early timepoint and cell death with lysosomal dysfunction and autophagy disruption at a later timepoint. (c) 2012 Elsevier B.V. All rights reserved.