The Role Of Parkin-dependent Mitophagy In Acidic Postconditioning Mediated Neuroprotection

Ischemic stroke is one of the leading causes of death worldwide. Currently, the only available treatment is thrombolysis with tissue plasminogen activator (tPA). Unfortunately, because of the narrow therapeutic window (<4.5 h) and the side effects, less than 5% of patients are treated with tPA. In recent years, many efforts have been made to find effective neuroprotective agents in the laboratory. However, these experimental treatments often have failed in large clinical trials. Therefore, it is imperative to search for effective and safe therapeutic approaches for ischemic brain injury.Ischemic postconditioning (IPC) is a novel therapeutic strategy to prevent detrimental effects of ischemic brain injury. It is defined as a series cycles of brief ischemia and reperfusion applied at the onset of reperfusion, which can activate the endogenous protective pathways. However, the narrow therapeutic time window and complicated operation limit the clinical application of IPC. Thus, it is necessary to develop a type of postconditioning which is safe and convenient.Acidosis is a key component in ischemia besides glucose depletion and hypoxia. The pH of brain tissues decreases from 7.0 to 6.6 during focal cerebral ischemia. It has been reported that transient acidic reperfusion applied at the onset of reperfusion mimics the protection of IPC against cardiac ischemia in isolated heart model. Our previous study found that acidic preconditioning protects against ischemia-induced injury. Due to the unpredictable occurrence of stroke, it is promising to study the action of acidosis treatment after reperfusion. Our study aims to investigate the effect of acidosis postconditioning (APC) on ischemic brain injury and the underlying mechanisms.Adult male C57BL/6J mice were subjected to 60 min middle cerebral arterial occlusion and acidosis treatment by inhaling 10%,20%, or 30% CO2 for 5 or 10 min at 5,50, or 100 min after reperfusion. For in vitro models, primary cultured cortical neurons and acute corticostriatal slices were subjected to oxygen-glucose deprivation and acidosis postconditioning by incubation in CO2-balanced medium (pH=6.8) after reperfusion. Our results showed that inhaling 10%,20%, but not 30%, CO2 for 5 min at 5 min after reperfusion induced prominent neuroprotection, as revealed by reduced infarct volume. The protection of APC, but not IPC, was still observed when applied 50 min after reperfusion, which suggests a wide time-window of APC. Attenuating brain acidosis by NaHCO3 significantly compromised the APC or IPC-induced neuroprotection. In addition, APC induced mitophagy shown by reduced Tomm20 and COX IV both in vivo and in vitro. The neuroprotection of APC was compromised by mitophagy inhibition achieved by 3-methyladenine, Mdivi-1 or ATG7 silencing treatment. Furthermore, we found that APC enhanced Parkin recruitment to mitochondria and parkin knockout abrogated the APC-conferred neuroprotection. Moreover, APC activated mitophagy extended reperfusion-induced neuroprotective time window, and up-regulation of mitophagy extended the therapeutic time window for APC.Taken together, these findings indicate that transient mild acidosis treatment at reperfusion protects against ischemic brain injury by inducing Parkin dependent mitophagy. With regard to its wide effective time window, APC can be a potential therapeutic strategy for ischemic brain injury, and Parkin dependent mitophagy may serve as a therapeutic target.