Role Of PUMA In Microglia Activation In Response To Methamphetamine

Objective:The present study was to investigate the role and detailed molecular mechanisms of PUMA in methamphetamine-mediated microglia activation in order to provide insights into the development of potential therapeutic target for neuroinflammatory effects induced by methamphetamine.Methods:The expression of PUMA and the different phenotypes of activated microglia (M1, M2a and M2c) were examined by Western blot in BV-2 cells and primary microglia cells; Activation of cell signaling pathways was also detected using Western blot; Carboxy-H2DCFDA was used to examine the formation of ROS; ChIP assay was performed to estimate the interaction between STAT3 and PUMA promoter; PUMA KO mice were used to further validate the role of PUMA in methamphetamine-mediated microglia activation using Western blot and immunostaining.Results:1) Exposure of BV-2 cells and primary microglia cells to methamphetamine increased the expression of PUMA in a time-dependent manner; 2) Engagement of Sigma-1R was critical for methamphetamine-induced PUMA expression in BV-2 cells; 3) ROS generation was involved in the increased expression of PUMA induced by methamphetamine; 4) Activation of MAPKs and PI3K/Akt pathways with consequent translocation of transcription nuclear factor STAT3 into nucleus contribute to the increased expression of PUMA induced by methamphetamine; 5) Exposure of BV-2 cells and primary microglia cells to methamphetamine resulted in a significant up-regulation of inflammatory (M1) phenotype marker (iNOS) with concomitant decreased expression of anti-inflammatory (M2) marker (Arginase), but methamphetamine failed to affect the expression of M2c phenotype marker-SOCS3; 6) Transfection of BV-2 cells with siRNA PUMA significantly inhibited methamphetamine-mediated microglia activation, while PUMA overexpression enhanced the activation of microglia; 7) In vivo relevance of this study was further confirmed in PUMA KO genetic mice, methamphetamine administration induced the activation of microglia in WT mice, but not in PUMA KO mice.Conclusions:PUMA was involved in methamphetamine-mediated microglia activation via its cognate receptor Sigma-1R as well as the formation of ROS, followed by the activation of MAPKs and PI3K/Akt pathways and the transcription factor STAT3 into nucleus, leading to the increased expression of PUMA.