Overexpression of ATF4 Inhibits Ferroptosis to Alleviate Anxiety Disorders by Activating the TGF-β Signaling Pathway

Wentao Wu,1,* Fei Wen,1,* Jiaxin Hu,1 Leijun Li2 1Department of Psychiatry, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou City, Guangdong Province, People’s Republic of China; 2Department of Psychiatry, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou City, Guangdong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Leijun Li, Email lljzssyxl@163.comBackground: Anxiety disorders seriously impair patients’ mental health and quality of life, with limited effectiveness of current treatments. Dysregulation of activating transcription factor 4 (ATF4) is involved in various mental diseases, but the research on its potential roles in alleviating anxiety disorders remains limited.Methods: ATF4 was screened out by bioinformatic analysis and its expression was verified in vivo. Mice were treated with 21 d of chronic restraint stress to establish the anxiety mice model. The anxiolytic effect of ATF4 was assessed by a battery of behavior tests and evaluation of hippocampal tissue damage after overexpressing ATF4. Ferroptosis-related indicators were detected by enzyme-linked immunosorbent assay and Western blotting. Then the transforming growth factor beta (TGF-β) signaling pathway was predicted as the downstream regulatory pathway of ATF4 by bioinformatic methods. Western blotting was conducted to detect the protein expression level of TGF-β 1, small mothers against decapentaplegic 3 (Smad3), and phospho-Smad3 (p-Smad3).Results: ATF4 was screened out as a ferroptosis-related anxiolytic gene after bioinformatics analysis and was down-regulated in the anxiety mice model. Mice with ATF4 overexpression spent more time in the open arms in the elevated plus-maze test, appeared more frequently in the central area in the open-field test, and decreased the immobility time in the forced swimming and tail suspension tests. Hippocampal tissue damage was alleviated, ferroptosis was suppressed, and the levels of TGF-β 1 and p-Smad3/Smad3 were increased by AFT4 overexpression.Conclusion: ATF4 overexpression can repress ferroptosis to improve anxiety disorders by activating the TGF-β signaling pathway.Keywords: anxiety disorders, ATF4, ferroptosis, hippocampus, TGF-β signaling pathway