Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, 37604, USA.
Neurotox Res. 2014 Feb;25(2):193-207. doi: 10.1007/s12640-013-9421-4. Epub 2013 Aug 31.
Dopamine β-hydroxylase (DBH) and norepinephrine (NE) transporter (NET) are the noradrenergic phenotypes for their functional importance to noradrenergic neurons. It is known that in vivo N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) treatment induces degeneration of noradrenergic terminals by interacting with NET and depleting intracellular NE. However, DSP4's precise mechanism of action remains unclear. In this study various biochemical approaches were employed to test the hypothesis that DSP4 down-regulates the expression of DBH and NET, and to determine molecular mechanisms that may be involved. The results showed that treatment of SH-SY5Y neuroblastoma cells with DSP4 significantly decreased mRNA and protein levels of DBH and NET. DSP4-induced reduction of DBH mRNA and proteins, as well as NET proteins showed a time- and concentration-dependent manner. Flow cytometric analysis demonstrated that DSP4-treated cells were arrested predominantly in the S-phase, which was reversible. The arrest was confirmed by several DNA damage response markers (phosphorylation of H2AX and p53), suggesting that DSP4 causes replication stress which triggers cell cycle arrest via the S-phase checkpoints. Moreover, the comet assay verified that DSP4 induced single-strand DNA breaks. In summary, the present study demonstrated that DSP4 down-regulates the noradrenergic phenotypes, which may be mediated by its actions on DNA replication, leading to replication stress and cell cycle arrest. These action mechanisms of DSP4 may account for its degenerative consequence after systematic administration for animal models.
多巴胺β-羟化酶（DBH）和去甲肾上腺素（NE）转运体（NET）是去甲肾上腺素能神经元功能重要的去甲肾上腺素能表型。已知，体内 N-（2-氯乙基）-N-乙基-2-溴苯甲胺（DSP4）通过与 NET 相互作用和耗尽细胞内 NE 来诱导去甲肾上腺素能末梢变性。然而，DSP4 的确切作用机制仍不清楚。在这项研究中，采用了各种生化方法来检验 DSP4 下调 DBH 和 NET 表达的假设，并确定可能涉及的分子机制。结果表明，DSP4 处理 SH-SY5Y 神经母细胞瘤细胞显著降低了 DBH 和 NET 的 mRNA 和蛋白水平。DSP4 诱导的 DBH mRNA 和蛋白减少以及 NET 蛋白减少呈现时间和浓度依赖性。流式细胞术分析表明，DSP4 处理的细胞主要停滞在 S 期，这是可逆转的。通过几种 DNA 损伤反应标志物（H2AX 和 p53 的磷酸化）证实了细胞停滞，这表明 DSP4 引起复制应激，通过 S 期检查点触发细胞周期停滞。此外，彗星试验证实 DSP4 诱导了单链 DNA 断裂。总之，本研究表明，DSP4 下调了去甲肾上腺素能表型，这可能是通过其对 DNA 复制的作用介导的，导致复制应激和细胞周期停滞。DSP4 的这些作用机制可能解释了其在动物模型中系统给药后的退行性后果。
