Bcl-2 phosphorylation triggers autophagy switch and reduces mitochondrial damage in limb remote ischemic conditioned...

Autophagy, an important intracellular degradation pathway, has been reported to clear impaired mitochondria and reduce mitochondria-mediated injury in ischemic disease. Our study and other recent investigations have shown that AKT-dependent autophagy contributes to the neuroprotection afforded by limb remote ischemic conditioning (RIC) in experimental stroke. However, how AKT triggers RIC-based autophagy and whether RIC-afforded autophagy is beneficial for mitochondrial function after cerebral ischemia remains unclear. The disruption of the Bcl-2/Beclin1 complex has been reported to trigger autophagy formation in the condition of Bcl-2 phosphorylation at Ser70. We investigated whether Bcl-2 phosphorylation triggers RIC-based autophagy and thereby confers RIC-induced neuroprotection against mitochondrial injury, using a transient cerebral ischemic rat model. We demonstrated that rats undergoing RIC treatment 30 min after the onset of ischemia (I-30) and at reperfusion (R-0) significantly upregulated Bcl-2 phosphorylation. Immunoprecipitation revealed that RIC increased dissociation of the Bcl-2/Beclin1 complex, leading to a higher level of autophagy than in ischemia/reperfusion rats. Furthermore, AKT activation was shown to play a critical role in regulating Bcl-2-mediated autophagy, as an AKT inhibitor (LY294002, AKTi) administered 30 min prior to ischemia significantly suppressed Bcl-2 phosphorylation and Bcl-2/Beclin1 complex dissociation, thereby reducing autophagy in RIC rats. Blocking Bcl-2 phosphorylation-dependent autophagy with AKTi suppressed RIC-afforded protection on mitochondrial potential and mitochondrial-dependent cell death effector pathway. These findings indicate that Bcl-2 phosphorylation and thereby Bcl-2/Beclin1 complex disruption play a crucial role in triggering autophagy and reducing mitochondrial damage in RIC rats after cerebral ischemia and require the involvement of the AKT activation.