酒精介导的 Mcl-1 pre-mRNA 剪接错误参与神经毒性。
Alcohol-Mediated Missplicing of Mcl-1 Pre-mRNA is Involved in Neurotoxicity.
机构信息
Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, Pennsylvania.
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.
出版信息
Alcohol Clin Exp Res. 2017 Oct;41(10):1715-1724. doi: 10.1111/acer.13474. Epub 2017 Sep 1.
BACKGROUND
Heavy and chronic ethanol (EtOH) exposure can cause significant structural and functional damage to the adult brain. The most devastating consequence of EtOH exposure is the neurotoxicity associated with the depletion of neurons. Regulation of splice variants in the brain can modulate protein functions, which may ultimately affect behaviors associated with alcohol dependence and EtOH-mediated neurotoxicity. As alcohol consumption is associated with neurotoxicity, it is possible that altered splicing of survival and pro-survival factors during the development of alcoholism may contribute to the neurotoxicity.
METHODS
Primary human neurons and a neuroblastoma cell line were exposed to different concentrations of EtOH for various time periods. Cell viability and neuronal marker expression were analyzed by MTT assay and immunoblotting, respectively. Effect of EtOH exposure on splicing regulatory protein expression and alternative splicing of candidate genes was analyzed by a biochemical approach. Transcriptional activity of serine/arginine-rich splicing factor 1 (SRSF1) gene was determined by reporter gene analysis.
RESULTS
Our results suggest that EtOH exposure to neuronal cells at 25 mM and higher concentrations are detrimental. In addition, EtOH exposure caused a dramatic reduction in SRSF1 expression levels. Furthermore, EtOH exposure led to pre-mRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by down-regulating the expression levels of SRSF1. Moreover, ectopic expression of both SRSF1 and Mcl-1L isoform was able to recover EtOH-mediated neurotoxicity.
CONCLUSIONS
Our results suggest that EtOH exposure can lead to pre-mRNA missplicing of Mcl-1 in neuronal cells. Our results indicate that EtOH exposure of neurons leads to a decrease in the ratio of Mcl-1L/Mcl-1S by favoring pro-apoptotic Mcl-1S splicing over anti-apoptotic Mcl-1L isoform suggesting that Mcl-1S may play a crucial role in neurotoxicity associated with alcohol consumption.
背景
大量且长期的乙醇（EtOH）暴露会对成年人大脑造成严重的结构和功能损伤。EtOH 暴露最具破坏性的后果是与神经元耗竭相关的神经毒性。大脑中剪接变体的调节可以调节蛋白质功能，这可能最终会影响与酒精依赖和 EtOH 介导的神经毒性相关的行为。由于饮酒与神经毒性有关，因此在酒精中毒发展过程中，存活和促生存因子的剪接改变可能导致神经毒性。
方法
将原代人神经元和神经母细胞瘤细胞系暴露于不同浓度的 EtOH 中不同时间。通过 MTT 测定法和免疫印迹法分别分析细胞活力和神经元标志物表达。通过生化方法分析 EtOH 暴露对剪接调节蛋白表达和候选基因的可变剪接的影响。通过报告基因分析测定丝氨酸/精氨酸丰富剪接因子 1（SRSF1）基因的转录活性。
结果
我们的结果表明，神经元细胞暴露于 25mM 及更高浓度的 EtOH 是有害的。此外，EtOH 暴露导致 SRSF1 表达水平急剧下降。此外，EtOH 暴露通过下调 SRSF1 的表达水平导致抗凋亡的 Bcl-2 家族成员 Mcl-1 的前体 mRNA 错误剪接。此外，SRSF1 和 Mcl-1L 同工型的异位表达均能够恢复 EtOH 介导的神经毒性。
结论
我们的结果表明，EtOH 暴露可导致神经元细胞中 Mcl-1 的前体 mRNA 错误剪接。我们的结果表明，神经元细胞暴露于 EtOH 会导致 Mcl-1L/Mcl-1S 的比例降低，通过有利于促凋亡的 Mcl-1S 剪接而不是抗凋亡的 Mcl-1L 同工型，表明 Mcl-1S 在与酒精消耗相关的神经毒性中可能发挥关键作用。
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