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)

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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.