The Effect Of Mutant Huntingtin Expression On CRE-dependent Transcription

Aim: To study the effect of mutant N-terminal Htt-552 and Htt-171 expression on CRE-dependent transcription and investigate the role of cAMP response element mediated transcription in Huntington's disease.Methods: PC12 cells were transfected with pGL4[luc2P/CRE/Hygro] reporter vector using Lipofectamine2000, the stable PC12 cells expressing this plasmid were selected with hygromycin. So the CRE regulated gene expression can be deteced by luciferase activity. Then, Huntington's disease cellular models of PC12-CRE were established by adenoviral expression system. The luciferase activity of PC12-CRE cells expressing Htt-552 and Htt-171 were detected by Luciferase Reporter Gene Assay Kit. The regulation of BDNF mRNA expression by Htt-552 and Htt-171 were detected by Real-time PCR.Results: The construction of stable PC12 cell line expressing cAMP response element regulating reporter gene was successful. Wild type and mutant Htt-552 and Htt-171 transfected by adenovirus successfully expressed in PC12-CRE cells. Htt-552 and Htt-171 distributed predominantly in the cytoplasm with relatively low levels in the nucleus. Little Htt aggregate formed in htt-552 expressing cells while lots of Htt aggregates exist in cytoplasm in htt-171. The luciferase activity detection indicated that mutant Htt-552 and Htt-171 expression havn't any effect in CRE-dependent transcription both in normal and stresses conditions (3-NP; KA; Dopamine; CuSO4; FeSO4.7H2O; NH4Cl). The expression of BDNF mRNA was up-regulated by wide type Htt-552 and Htt-171 and down-regulated by mutant Htt-552 and Htt-171.Conclusion: Mutant Htt-552 and Htt-171 expression havn't any effect in CRE-regulated gene transcription both in normal and some stresses conditions (3-NP; KA; Dopamine; CuSO4; FeSO4.7H2O; NH4Cl). The expression of BDNF mRNA was up-regulated by wide type Htt-552 and Htt-171 and down-regulated by mutant Htt-552 and Htt-171.