Effects of DDIT4 in Methamphetamine-Induced Autophagy and Apoptosis in Dopaminergic Neurons
Effects of DDIT4 in Methamphetamine-Induced Autophagy and Apoptosis in Dopaminergic Neurons
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DDIT4 对甲基苯丙胺诱导的多巴胺能神经元自噬和凋亡的影响
DOI：
10.1007/s12035-015-9637-9
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发表时间：
2017-04-01
影响因子：
5.1
通讯作者：
Wang, Huijun
中科院分区：
文献类型：
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作者：
Li, Bing;Chen, Rui;Wang, Huijun
甲基苯丙胺（冰毒）是一种非法的精神活性药物，可以对神经系统，特别是多巴胺能通路造成各种有害影响。我们假设DNA损伤诱导转录本4 （DNA damage-inducible transcript 4, DDIT4）参与了甲基甲氧麻黄素诱导的多巴胺能神经元自噬和凋亡。为了验证这一假设，我们检测了大鼠儿茶酚胺能PC12细胞和人多巴胺能SH-SY5Y细胞以及暴露于冰毒的大鼠海马、前额叶皮层和纹状体中DDIT4蛋白表达和自噬水平的变化。我们还通过荧光显微镜和电镜观察了沉默DDIT4表达对甲基甲醚诱导的多巴胺能神经元自噬的影响。采用流式细胞术和Western blot检测合成siRNA阻断DDIT4在PC12细胞和SH-SY5Y细胞中的表达后的凋亡和凋亡标志物（cleaved caspase-3和cleaved PARP）的表达，以及利用立体定位系统注射LV-shDDIT4慢病毒阻断大鼠纹状体中的表达。我们的研究结果表明，甲基苯甲胺暴露增加了大鼠PC12细胞（3 mM）和SH-SY5Y细胞（2 mM）以及海马、前额叶皮层和纹状体中DDIT4的表达，并伴有自噬和凋亡的增加。抑制DDIT4的表达可减少meth诱导的自噬和细胞凋亡。然而，在低浓度（1 μ M）的甲基安非他明下，没有观察到ddit4相关的效应。这些结果表明，DDIT4在高剂量甲基醚诱导的多巴胺能神经元自噬和凋亡中起重要作用，可能是高剂量甲基醚诱导的神经毒性治疗的潜在基因靶点。
Methamphetamine (METH) is an illicit psychoactive drug that can cause a variety of detrimental effects to the nervous system, especially dopaminergic pathways. We hypothesized that DNA damage-inducible transcript 4 (DDIT4) is involved in METH-induced dopaminergic neuronal autophagy and apoptosis. To test the hypothesis, we determined changes of DDIT4 protein expression and the level of autophagy in rat catecholaminergic PC12 cells and human dopaminergic SH-SY5Y cells, and in the hippocampus, prefrontal cortex, and striatum of Sprague Dawley rats exposed to METH. We also examined the effects of silencing DDIT4 expression on METH-induced dopaminergic neuronal autophagy using fluorescence microscopy and electron microscopy. Flow cytometry and Western blot were used to determine apoptosis and the expression of apoptotic markers (cleaved caspase-3 and cleaved PARP) after blocking DDIT4 expression in PC12 cells and SH-SY5Y cells with synthetic siRNA, as well as in the striatum of rats by injecting LV-shDDIT4 lentivirus using a stereotaxic positioning system. Our results showed that METH exposure increased DDIT4 expression that was accompanied with increased autophagy and apoptosis in PC12 cells (3 mM) and SH-SY5Y cells (2 mM), and in the hippocampus, prefrontal cortex, and striatum of rats. Inhibition of DDIT4 expression reduced METH-induced autophagy and apoptosis in vitro and in vivo. However, DDIT4-related effects were not observed at a low concentration of METH (1 mu M). These results suggest that DDIT4 plays an essential role in METH-induced dopaminergic neuronal autophagy and apoptosis at higher doses and may be a potential gene target for therapeutics in high-dose METH-induced neurotoxicity.