Ke Zun-Ji, Wang Xin, Fan Zhiqin, Luo Jia
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Alcohol Clin Exp Res. 2009 Jun;33(6):1097-103. doi: 10.1111/j.1530-0277.2009.00931.x. Epub 2009 Mar 23.
Heavy alcohol consumption causes cerebellar degeneration, and the underlying mechanism is unclear. Chronic alcoholism is usually associated with thiamine deficiency (TD) which is known to induce selective neurodegeneration in the brain. However, the role of TD in alcohol-induced cerebellar degeneration remains to be elucidated. The double-stranded RNA-activated protein kinase (PKR) is a potent antiviral protein. Viral infection or binding to dsRNA causes PKR autophosphorylation and subsequent phosphorylation of the alpha-subunit of eukaryotic translation factor-2alpha, leading to inhibition of translation or apoptosis. PKR can also be activated by cellular stresses.
In this study, we used an in vitro model, cultured cerebellar granule neurons (CGNs), to investigate the interaction between TD and ethanol and evaluate the contribution of their interaction to neuronal loss. TD was induced by treatment with amprolium in association with ethanol. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay. PKR expression/phosphorylation and subcellular distribution was analyzed with immunoblotting and immunocytochemistry.
Thiamine deficiency caused death of CGNs but ethanol did not. However, TD plus ethanol induced a much greater cell loss than TD alone. TD-induced PKR phosphorylation and ethanol exposure significantly promoted TD-induced PKR phosphorylation as well as its nuclear translocation. A selective PKR inhibitor not only protected CGNs against TD toxicity, but also abolished ethanol potentiation of TD-induced loss of CGNs.
Ethanol promoted TD-induced PKR activation and neuronal death. PKR may be a convergent protein that mediates the interaction between TD and ethanol.
大量饮酒会导致小脑变性，但其潜在机制尚不清楚。慢性酒精中毒通常与硫胺素缺乏（TD）有关，已知TD会在大脑中诱发选择性神经变性。然而，TD在酒精诱导的小脑变性中的作用仍有待阐明。双链RNA激活蛋白激酶（PKR）是一种有效的抗病毒蛋白。病毒感染或与双链RNA结合会导致PKR自磷酸化，随后真核翻译起始因子2α的α亚基发生磷酸化，从而抑制翻译或诱导凋亡。PKR也可被细胞应激激活。
在本研究中，我们使用体外模型，即培养的小脑颗粒神经元（CGNs），来研究TD与乙醇之间的相互作用，并评估它们的相互作用对神经元损失的影响。通过用氨丙啉联合乙醇处理来诱导TD。采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐法测定细胞活力。用免疫印迹和免疫细胞化学分析PKR的表达/磷酸化及亚细胞分布。
硫胺素缺乏导致CGNs死亡，但乙醇不会。然而，TD加乙醇诱导的细胞损失比单独的TD要大得多。TD诱导的PKR磷酸化以及乙醇暴露显著促进了TD诱导的PKR磷酸化及其核转位。一种选择性PKR抑制剂不仅保护CGNs免受TD毒性的影响，还消除了乙醇对TD诱导的CGNs损失的增强作用。
乙醇促进了TD诱导的PKR激活和神经元死亡。PKR可能是介导TD与乙醇相互作用的汇聚蛋白。
