Conclusions
The reactivated mTORC1 could suppress the transcription levels of related mRNA, such as LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b. The research will expand the horizons that mTOR would negatively regulate autophagy at transcription and post-translation levels in neurons suffering ischemia-reperfusion injury.
Methods
The OGD/R or middle cerebral artery occlusion/reperfusion (MCAO/R)-treated neurons was used to simulate ischemia/reperfusion injury . Autophagy flux was monitored by means of microtubule-associated protein 1 light chain 3 (LC3) and p62. The reactivation of mTOR was determined by phosphorylation of ribosomal protein S6 kinase 1 (S6K1). Then the inhibitors of mTOR were used to confirm its existence form. Finally, the mRNA transcription levels were analyzed to observe the negative regulation of mTOR.
Results
The sequential phosphorylation of mTOR contributed to the neuronal autophagy flux blocking. mTOR was re-phosphorylated and existed as mTOR complex 1 (mTORC1), which was supported by phosphorylation of S6K1 at Thr 389 in neurons. In addition, the phosphorylation of S6K1 was decreased roughly by applying mTORC1 inhibitors, rapamycin and torin 1. However, the administration of mTORC1/2 inhibitor PP242 could recover the phosphorylation of S6K1, which suggested that mTORC2 was involved in the regulation of mTORC1 activity. In paralleling with reactivation of mTORC1, related mRNA transcription was repressed in neurons under ischemia-reperfusion exposure in vivo and in vitro. The mRNA expression levels of LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b were decreased in neurons after reperfusion, comparing with ischemia-treated neurons. Conclusions: The reactivated mTORC1 could suppress the transcription levels of related mRNA, such as LC3, Stx17, Vamp8, Snap29, Lamp2a, and Lamp2b. The research will expand the horizons that mTOR would negatively regulate autophagy at transcription and post-translation levels in neurons suffering ischemia-reperfusion injury.