The Function And Mechanism Of The Signal Transduction Regulating The Anxiety Behavior Of Mice By G?i Mediated MANF(Mesoastrocyte Derived Neurotrophic Factor)

Purposes1 Analyze the molecular mechanism of Gai mediated MANF signal transduction;2 The correlation between the expression level and anxiety behavior of Gail/3 in the hippocampal area of the mouse was clarified.MethodsCultivation of wild type(WT)and Gail/3 double knockout mice(DKO)embryonic fibroblasts(MEFs),Gail single knockout MEFs cells,Gai3 single knockout MEFs cells,Gai2 single knockout MEFs,Gab1 knockout MEFs cells;The shRNA method was used to target and knock down Gail and/or Gai3 in MEFs;DKO MEFs cells were transferred to Gail and Gai3 expression plasmid to obtain Resurel and Resure3 MEFs;The above MEFs cells were treated with MANF respectively to detect the activation levels of the downstream joint protein Gabl and the MAPK/ERK signaling pathway(ERK)and AKT-mTORCl signaling pathway(AKT,S6,S6K,and GSK3?/3?);By using RNA interference technique,the mice were injected into the hippocampal area of ICR mice to detect the changes of anxiety behavior.ResultIn MEFs,G?i1/3 double knock(DKO)blocks the activation of the manf-induced AKT-mTORCl/MAPK/ERK signaling pathway;After the MANF stimulation,the Gail-KO,G?i3-KO and GaishRNA were slightly weakened after the stimulation of MANF,and the G?i2-KO after the treatment of MANF,the downstream signal did not change significantly;Resure1 and Resure3 MEFs,under MANF stimulation,the downstream signal activation has obvious recovery;Gab1 knockout MEFs,stimulated by MANF,showed significant reduction or even loss of activation level of downstream MAPK/ERK signaling pathway(ERK)and AKT-mTORCl signaling pathway(AKT Ser473,S6,S6K);In the hippocampus of the mice,the expression of anxiety was significantly increased after the injection of Gail/3shRNA lentivirus.The hippocampus of mice injected with slow virus parcel Gail/3 mutation plasmid(DN),anxiety behavior increase,but no significant cognitive impairment,and injection of sustained activation of Gail/3(CA),the mice showed no obvious anxiety behavior.Conclusion1.In vitro results suggest that Gail/3 May mediate the downstream signal transduction induced by MANF;2.Behavioral results indicate that the Gail/3 in the hippocampus may be involved in regulating the anxiety behavior of mice.