Analysis of the effect with the, expanded CTG repeat for cell toxicity, especially tauopathy in the central nervous system in patietnt of myotonic dystrophy type 1 (DM 1)

携带mRNA的扩展CUG三联体似乎与强直性肌营养不良1型(DM1)有关。为了研究DM-1的发病机制，特别是它在中枢神经系统中的相互作用，我们用PC-12神经细胞系建立了DM-1细胞培养模型，并筛选出改善DM-1功能的类黄酮类化合物。将扩增的250个CTG重复序列亚克隆到荧光素酶基因的3‘-非翻译区，得到稳定的转化子PC-12。为了寻找改变扩展的CTG重复序列的毒性效应的药物，筛选了235个生物黄酮类化合物。经神经生长因子诱导分化后，顺式效应和细胞毒作用增强。此外，在该DM1模型细胞中还证实了tau基因选择性剪接模式的改变。抗caspase-3抗体免疫印迹结果显示，细胞死亡是由细胞凋亡引起的。筛选分析证实，黄酮类、异黄酮类、黄酮类和脱氢表雄酮-S均可抑制CTG重复序列的细胞毒性和顺式效应，而单黄酮类、双异黄酮类和黄曲霉素能强烈抑制CTG重复序列的顺式效应。综上所述，我们发现这一表达CUG重复序列基因的神经细胞系在体外通过报告基因和细胞分化后的神经元死亡表现出顺式作用。此外，一些黄酮类化合物和脱氢表雄酮-S同时抑制顺式作用和细胞毒性，表明它们的化学结构对这两种毒性作用都有改善作用。这一系统使评估扩大的毒性效应变得容易。CTG重复，因此应该有助于筛选其他DM1治疗的有效性。

Expanded CUG triplet repeats carrying mRNA seem to be responsible for myotonic dystrophy type 1 (DM1). To study the pathogenesis of DM 1, especially of its tauopathy in CNS, we constructed a DM 1 cell culture model using a PC 12 neuronal cell line and screened flavonoids that ameliorate this mRNA gain of function. The expanded 250 CTG repeat was subcloned into the 3'-untranslated region of the luciferase gene yielding a stable transformant of PC 12. The cis-effect of expanded repeat for this cell was evaluated with luciferase. To find agents that alter the toxic effect of expanded CTG repeat, 235 bioflavonoids were screened. An increased cis-effect and cytotoxicity were found when this cell was treated with nerve growth factor to induce differentiation. Furthermore, modification of alternative splicing pattern of tau gene was also confirmed in this DM1 model cell. Western blotting with anti-caspase-3 antibody suggested that cell death was caused by apoptosis. Screening analysis confirmed that a flavone, an isoflavone, a flavanone and DHEA-S prevent both the cytotoxicity and cis-effect of expanded CTG repeat and that a flavanone, two isoflavones, and xanthylatin strongly inhibit the cis-effect of CTG repeats. In conclusion, we found that this neuronal cell line, which expresses the CUG repeat-bearing mRNA, showed cis-effects through the reporter gene and neuronal death after cell differentiation in vitro. Moreover, some flavonoids and DHEA-S inhibit both the cis-effect and cytotoxicity, indicate that their chemical structures work to ameliorate both these toxic effects. This system makes it easy to evaluate the toxic effects of expanded.CTG repeats and therefore should be useful for screening other DM1 treatments for their efficacies.