Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene...Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene, was amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector. The recombinant plasmid pGL3-THprom was transfected into a dopammergic cell line MES23.5 or a α-Syn over-expressed MES23.5 （named MES23.5/hα-Syn^＋）. The promoter activity was detected by the Dual Luciferase Assay System. Results The luciferase activities in the MES23.5 cells transfected with pGl.,3-Basic, pGL3-THprom, and pGL3-Control vectors were 5.60±0.67, 26.80±4.11, and 32.90±4.75, respectively. On the other hand, the luciferase activity of pGL3-THprom in the MES23.5 （26.80±4.11） was significantly higher than that in the MES23.5/hα-Syn^＋（14.40±0.61） （P〈0.01）. Conclusion These results indicate that the -495 to ＋25 region in the TH gene possesses promoter activity for controlling the gene expression, and that α-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.展开更多
基金This work was supported by the Key Project of National Natural Science Foundation of China （No. 30430280）the National Natural Science Foundation of China （No. 30271437, No.30270482）the Natural Science Foundation of Beijing Municipality （No. 7022011 ）.
文摘Objective To approach the associated mechanism by which α-synuclein （α-Syn） might regulate the metabolism of dopamine. Methods A DNA fragment, located at --495 to ＋25 of the human tyrosine hydroxylase （TH） gene, was amplified by PCR and inserted into the pGL3-Basic luciferase reporter vector. The recombinant plasmid pGL3-THprom was transfected into a dopammergic cell line MES23.5 or a α-Syn over-expressed MES23.5 （named MES23.5/hα-Syn^＋）. The promoter activity was detected by the Dual Luciferase Assay System. Results The luciferase activities in the MES23.5 cells transfected with pGl.,3-Basic, pGL3-THprom, and pGL3-Control vectors were 5.60±0.67, 26.80±4.11, and 32.90±4.75, respectively. On the other hand, the luciferase activity of pGL3-THprom in the MES23.5 （26.80±4.11） was significantly higher than that in the MES23.5/hα-Syn^＋（14.40±0.61） （P〈0.01）. Conclusion These results indicate that the -495 to ＋25 region in the TH gene possesses promoter activity for controlling the gene expression, and that α-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.
