| Ischemic brain injury is caused by the sudden shortage of blood supply to the brain. The rapid decrease of glucose and oxygen level leads to tissue injury and subsequently neurological dysfunction. Tens of millions of people suffer from cerebral ischemia worldwide, whereas unfortunately, very limited clinical approaches are available in the current stage. The pathophysiological mechanisms of cerebral ischemia are extraordinarly complicate, a variety of cellular events, including autophagy, can be trigerred by ischemia. Autophagy is an intracellular degradation process by delivering dysfunctional proteins or organelles to lysosomes. Autophagy is critical for cell homeostasis and thus helps cells to overcome stresses and has been proved to be involved in a variety of human diseases. Emerging evidences indicated that autophagy may play critical roles in ischemic brain injury. However, debates remain over the contributions of autophagy in ischemic brains. There are data showed that inhibition of autophagy reduce neurodegeneration after focal cerebral ischemia. In contrast, there are also evidences indicating the protective roles of autophagy in several neurological disorders. Therefore, the roles of autophagy in cerebral ischemia need further clarification. Selevtive degradation of dysfunctional or excessive mitochondira by autophagy is termed mitophagy, which may reduce cell apopotosis by clearing damaged mitochondira resulted from brain ischemia, and thus promote cell survival. Mitophagy is close related to several neurodegenerative diseases, yet, whether it was involved in the process of brain ischemia remains to be investigated, and further understanding the regulatory mechanisms of mitophagy in ischemic brains will prove opportunities to develop novel strategies agaist cerebral ischemia.In this investigation, we aimed to clarify the role of autophagy in cerebral ischemia, and focus on the existence and contributions of mitophagy to ischemic brains and further explore the regulatory mechanisms of mitophagy in this scenario.Part1Autophagy protects against transient ischemic brain injury by clearing damaged mitochondriaIn the present investigation, we found that autophagy was activated in the reperfusion phase after ischemia, as revealed in mice with middle cerebral artery occlusion. Interestingly, in contrast to that in permanent middle cerebral artery occlusion (pMCAO), inhibition of autophagy by 3-methyladenine in the reperfusion phase reinforced, rather than reduced, the brain injury induced by ischemia-reperfusion (I-R). These results indicated the different roles of autophagy during brain ischemia and subsequent reperfusion. By using electron microscopy, we found mitochondria were engulfed by vacuolar structures in the cortex from mice subjected to transient middle cerebral artery occlusion (tMCAO), besides, the mitochondrial membrane proteins TOMM20 and COX 411 were decreased after ischemia reperfusion. Further,3-MA treatment significantly attenuated the mtDNA reduction in the tMCAO but not in the pMCAO model, together suggesting that mitochondira were degraded by autophagic flow after ischemia reperfusion. Inhibition of autophagy with 3-methyladenine enhanced the I-R-induced release of cytochrome c. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. Parkin translocated to mitochondria during reperfusion and Parkin knockdown aggravated ischemia-induced neuronal cell death. Overall, these results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis.The aforementioned studies indicated that mitophagy protected against ischemia-reperfusion induced brain injury, which raises an open question about whether ischemic brains can be rescued by mitophagy. The endoplasmic reticulum (ER) stress is activated by ischemia and subsequently triggers autophagy by a variety of signaling pathways. Therefore, we next aimed to test the hypothesis that ER stress-reinforced mitophagy is beneficial for neuronal survival after ischemia-reperfusion.Part 2Mild ER stress protects against transient ischemic brain injury by selectively reinforcing mitophagy.Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress, which subsequently induces autophagy. To test the hypothesis that ER stress-reinforced mitophagy protects against ischemic brain injury, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons,4-PBA, the ER stress releasor, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. TM and TG reduced OGD-Rep.-induced cell apoptosis in a concentration-dependent manner, and selectively activated mitophagy without reinforcing autophagic flux. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Parkin silencing. The neuroprotection was also diminished in Parkin+/- mice. These data clarified that mitophagy activation is involved in the neuroprotection. Knocking down the ER stress sensor EIF2S1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. Moreover, EIF2S1 and downstream ATF4 silencing reduced Parkin expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.The molecular mechanisms underlying mitophagy biogenesis and regulation are far from fully understood. The present study highlighted that Parkin plays a key role in ischemia-induced mitophagy. Interestingly, however, the Parkin protein level was rapidly decreased after ischemia when substantial mitophagy remained observable, implying that the existence of Parkin-independent mitophagy pathways in cerebral ischemia reperfusion. Nix locates in mitochondrial out membrane and is responsible for the erythrocytes maturation by clearing mitochondria as a mitophagy receptor. We next investigate whether Nix is involved in mitophagy regulations in the context of cerebral ischemia reperfusion.Part 3Nix-mediated mitophagy protected against ischemic brain injury by aParkin-independent mannerTo address the involvement of Nix in transient cerebral ischemia-induced mitophagy, Nix+/+, Nix+/- and Nix-/- mice were subjected to transient MCAO. It showed that mitophagy was impaired by Nix deletion, as revealed by reversed TOMM20 and COX4I1 protein levers after 6 h of reperfusion. Further injection of GFP-Nix expression adeno-associated viruses (AAVs) rescued both mitophagy dysfunction and the brain infract enlargement with transient MCAO in Nix-/- mice, suggesting Nix is required for mitophagy in brain ischemia-reperfusion and protected against ischemic brain injury. To investigate whether Parkin was required in Nix-mediated mitophagy, we injected GFP_Nix AAVs to the Parkin-/- mice brain, and found that Nix could also compensate for Parkin loss-induced mitophagy dysfunction and exaggerate brain infract. In addition, Nix and Parkin double knockout mice (Nix-/- & Parkin-/-, DKO) displayed synergetic mitophagy impair and brain injury vs Nix and Parkin gene deletion alone, further suggested that Parkin is dispensable in Nix-mediated mitophagy. Taken together, these results indicated that Nix may be a potential target for protecting ischemic brains. |
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