and this suppression of FoxO1 on TH promoter was more pronounced in constitutive active FoxO1 (ADA FoxO1; Fig. 7e). In contrast, the promoter activity of TH gene was significantly increased after suppression of endogenous FoxO1 by overexpression of dominant-negative form of FoxO1 (DN FoxO1) or by FoxO1 knockdown with small interfering RNAs (siRNAs; Fig. 7e and Supplementary Fig. 6e and Supplementary Fig. 7). The effects of FoxO1s overexpression were not seen with the luciferase construct that does not contain FoxO1-binding site (Fig. 7e) indicating the specific binding of FoxO1 on the TH promoter. In addition, endogenous Th expression40 was markedly inhibited by the expression of WT and ADA FoxO1 (Fig. 7f and Supplementary Fig. 6f and Supplementary Fig. 7). Conversely, FoxO1 knockdown promoted endogenous Th expression (Fig. 7g and Supplementary Fig. 6g and Supplementary Fig. 7). Altogether, these results indicate that FoxO1 directly regulates TH expression both in vitro and in vivo and therefore might affect CA synthesis in DA neurons (Supplementary Fig. 6h).
