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)

扩展 CTG 重复序列对强直性肌营养不良 1 型 (DM 1) 患者细胞毒性尤其是中枢神经系统 tau 蛋白病变的影响分析

• 批准号: 14570608
• 负责人: FURUYA Hirokazu
• 金额: $ 2.05万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570608/
• 关键词: myotonic dystrophy; triplet repeat disease; CTG repeat; PC12 neuronal culture cell line; tauopathy; cytotoxicity; bio-flavonoid; flavonoid

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.

携带mRNA的扩展CUG三联体重复序列似乎是导致1型肌强直性营养不良（DM1）的原因。为了研究DM - 1的发病机制，特别是其在中枢神经系统中的病变，我们利用pc12神经元细胞系构建了DM - 1细胞培养模型，并筛选了改善该mRNA功能增加的黄酮类化合物。扩增的250 CTG重复序列被亚克隆到荧光素酶基因的3'-非翻译区，产生了pc12的稳定转化。用荧光素酶评价扩增重复序列对该细胞的顺式效应。为了寻找能够改变扩增CTG重复序列毒性作用的药物，筛选了235种生物类黄酮。神经生长因子诱导细胞分化后，顺式效应增强，细胞毒性增强。此外，在DM1模型细胞中也证实了tau基因的选择性剪接模式的修饰。抗caspase-3抗体Western blotting提示细胞凋亡引起细胞死亡。筛选分析证实，a黄酮、a异黄酮、a黄酮和DHEA-S均能抑制扩增CTG重复序列的细胞毒性和顺式效应，a黄酮、2异黄酮和黄嘌呤均能强烈抑制CTG重复序列的顺式效应。综上所述，我们发现这一表达CUG重复基因mRNA的神经细胞系在体外分化后通过报告基因表现出顺式效应和神经元死亡。此外，一些类黄酮和DHEA-S同时抑制顺式效应和细胞毒性，表明它们的化学结构可以改善这两种毒性作用。该系统便于评价膨化剂的毒性作用。CTG重复，因此应该有助于筛选其他DM1治疗的有效性。

项目成果

Furuya H, Yasuda M, Terasawa K et al.: "A novel mutation (L250V) in the presemlin 1 gene in a Japanese familial Alzheimer's disease with myoclonus and generalized convulsion."J Neurol Sci.. 209. 75-77 (2003)

Furuya H、Yasuda M、Terasawa K 等人：“日本家族性阿尔茨海默病伴肌阵挛和全身惊厥的 presemlin 1 基因中的一种新突变 (L250V)。”J Neurol Sci.. 209. 75-77 (2003)

Furuya H.: "Pathogenesis and trial for treatment of Myotonic Dystrophy"Neurological Therapeutics. (in press).

Furuya H.：“强直性肌营养不良的发病机制和治疗试验”神经治疗学。

Kikuchi H, Yamada T, Furuya H et al.: "Involvement of cathepsin B in the motor neuron degeneration of amyotrophic lateral sclerosis."Acta Neuropathol. 105. 462-468 (2003)

Kikuchi H、Yamada T、Furuya H 等人：“组织蛋白酶 B 参与肌萎缩侧索硬化症的运动神经元变性。”《神经病理学报》。

Kikuchi H, Yamada T, Furuya H et al.: "Involvement of cathepsin B in the motor neuron degeneration of amyotrophic lateral sclerosis"Acta Neuropathol. (in press). (2003)

Kikuchi H、Yamada T、Furuya H 等：“组织蛋白酶 B 参与肌萎缩侧索硬化症运动神经元变性”Acta Neuropathol。

Furuya H, Yasuda M, Terasawa K et al.: "A novel mutation (L250V) in the presenilin 1 gene in a Japanese familial Alzheimer's disease with myoclonus and generalized convulsion"J Neurol Sci. (in press). (2003)

Furuya H、Yasuda M、Terasawa K 等人：“日本家族性阿尔茨海默病伴肌阵挛和全身性惊厥的早老素 1 基因中的新突变 (L250V)”J Neurol Sci。