Abstract：
Autophagy has been involved in protection of ischemia/reperfusion (I/R)-induced injury in many tissues including the brain. The upstream stimulatory factor 2 (Usf2) was proposed as a regulator in aging and degenerative brain diseases; however, the its role in autophagy during cerebral I/R injury remains unclear. Here, the middle cerebral artery occlusion (MCAO) operation was applied to establish an I/R mouse model. We showed that Usf2 was significantly upregulated in I/R-injured brain, accompanied by decreased levels of autophagy. Then, oxygen-glucose deprivation/recovery (OGD/R) treatment was used to establish a cellular I/R model in HT22 neurons, and lentiviral interference vector against Usf2 (LV-sh-Usf2) was used to infect the neurons. Our results showed that Usf2 was significantly upregulated in OGD/R-treated HT22 neurons that displayed an increased level in cell apoptosis and decreased levels in cell viability and autophagy, and interference of Usf2 largely rescued the effects of OGD/R on cell viability, apoptosis, and autophagy, suggesting an important role of Usf2 in neuron autophagy. In the mechanism exploration, we found that, as a transcription factor, Usf2 bound to the promoter of YTHDF1, a famous reader of N6-Methyladenosine (m6A), also induced by OGD/R, and promoted its transcription. Overexpression of YTHDF1 was able to reverse the improvement of Usf2 interference on viability and autophagy of HT22 neurons. Moreover, YTHDF1 suppressed autophagy to induce HT22 cell apoptosis through increasing m6A-mediated stability of Cdc25A, a newly identified autophagy inhibitor. Finally, we demonstrated that interference of Usf2 markedly improved autophagy and alleviated I/R-induced injury in MCAO mice.
摘要：
自噬已经参与保护包括脑在内的许多组织的缺血/再灌注(I/R)诱导的损伤。上游刺激因子2（Usf2）被提议作为衰老和退行性脑疾病的调节因子；然而，其在脑I/R损伤中自噬的作用尚不清楚.这里,采用大脑中动脉闭塞（MCAO）手术建立I/R小鼠模型。我们发现Usf2在I/R损伤的大脑中显著上调，伴随着自噬水平的降低。然后，氧-葡萄糖剥夺/恢复(OGD/R)处理用于建立HT22神经元的细胞I/R模型,使用针对Usf2的慢病毒干扰载体(LV-sh-Usf2)感染神经元。我们的结果表明，Usf2在OGD/R处理的HT22神经元中显著上调，细胞凋亡水平升高，细胞活力和自噬水平降低，Usf2的干扰在很大程度上挽救了OGD/R对细胞活力的影响，凋亡，和自噬,提示Usf2在神经元自噬中的重要作用。在机理探索中,我们发现,作为转录因子，Usf2与YTHDF1的启动子结合，YTHDF1是N6-甲基腺苷（m6A）的著名读者，也是由OGD/R诱导的，并促进了它的转录。YTHDF1的过表达能够逆转Usf2干扰对HT22神经元活力和自噬的改善。此外，YTHDF1通过增加m6A介导的Cdc25A稳定性抑制自噬诱导HT22细胞凋亡,一种新发现的自噬抑制剂。最后,我们证明，Usf2的干扰显著改善了MCAO小鼠的自噬，减轻了I/R诱导的损伤。
