Cerebral ischemia–reperfusion (I/R) injury in ischemic penumbra is accountable for poor outcome of ischemic stroke patients receiving recanalization therapy. Compelling evidence previously demonstrated a dual role of autophagy in stroke. This study aimed to understand the traits of autophagy in the ischemic penumbra and the potential mechanism that switches the dual role of autophagy. We found that autophagy induction by rapamycin and lithium carbonate performed before ischemia reduced neurologic deficits and infarction, while autophagy induction after reperfusion had the opposite effect in the male murine middle cerebral artery occlusion/reperfusion (MCAO/R) model, both of which were eliminated in mice lacking autophagy (Atg7^flox/flox; Nestin-Cre). Autophagic flux determination showed that reperfusion led to a blockage of axonal autophagosome retrograde transport in neurons, which then led to autophagic flux damage. Then, we found that I/R induced changes in the protein levels of Sec22b and Ykt6 in neurons, two autophagosome transport-related factors, in which Sec22b significantly increased and Ykt6 significantly decreased. In the absence of exogenous autophagy induction, Sec22b knock-down and Ykt6 overexpression significantly alleviated autophagic flux damage, infarction, and neurologic deficits in neurons or murine exposed to cerebral I/R in an autophagy-dependent manner. Furthermore, Sec22b knock-down and Ykt6 overexpression switched the outcome of rapamycin posttreatment from deterioration to neuroprotection. Thus, Sec22b and Ykt6 play key roles in neuronal autophagic flux, and modest regulation of Sec22b and Ykt6 may help to reverse the failure of targeting autophagy induction to improve the prognosis of ischemic stroke.

SIGNIFICANCE STATEMENT The highly polarized architecture of neurons with neurites presents challenges for material transport, such as autophagosomes, which form at the neurite tip and need to be transported to the cell soma for degradation. Here, we demonstrate that Sec22b and Ykt6 act as autophagosome porters and play an important role in maintaining the integrity of neuronal autophagic flux. Ischemia–reperfusion (I/R)-induced excess Sec22b and loss of Ykt6 in neurons lead to axonal autophagosome retrograde trafficking failure, autophagic flux damage, and finally neuronal injury. Facilitated axonal autophagosome retrograde transport by Sec22b knock-down and Ykt6 overexpression may reduce I/R-induced neuron injury and extend the therapeutic window of pharmacological autophagy induction for neuroprotection.

缺血半影区的脑缺血再灌注 (I/R) 损伤是缺血性卒中患者接受再通治疗后预后不良的原因。令人信服的证据先前证明了自噬在中风中的双重作用。本研究旨在了解缺血半影区自噬的特征以及转换自噬双重作用的潜在机制。我们发现在缺血前通过雷帕霉素和碳酸锂进行的自噬诱导减少了神经功能缺损和梗死，而在雄性小鼠大脑中动脉闭塞/再灌注（MCAO/R）模型中，再灌注后的自噬诱导具有相反的效果，两者均被消除在缺乏自噬的小鼠中（Atg7 ^flox/flox; 巢-Cre)。自噬通量测定表明，再灌注导致神经元中轴突自噬体逆行转运受阻，进而导致自噬通量损伤。然后，我们发现 I/R 诱导神经元中 Sec22b 和 Ykt6 蛋白水平的变化，这两个自噬体转运相关因子，其中 Sec22b 显着增加，Ykt6 显着降低。在没有外源性自噬诱导的情况下，Sec22b 敲低和 Ykt6 过表达显着减轻了以自噬依赖性方式暴露于脑 I/R 的神经元或小鼠的自噬通量损伤、梗塞和神经功能缺损。此外，Sec22b 敲低和 Ykt6 过表达将雷帕霉素后处理的结果从恶化转变为神经保护。因此，Sec22b 和 Ykt6 在神经元自噬通量中起关键作用，

意义声明具有神经突的神经元的高度极化结构对物质运输提出了挑战，例如自噬体，自噬体在神经突尖端形成，需要运输到细胞体进行降解。在这里，我们证明 Sec22b 和 Ykt6 作为自噬体搬运工，在维持神经元自噬通量的完整性方面发挥着重要作用。缺血再灌注 (I/R) 诱导的神经元中过量的 Sec22b 和 Ykt6 的丢失导致轴突自噬体逆行运输失败、自噬通量损伤，最后导致神经元损伤。Sec22b 敲低和 Ykt6 过表达促进轴突自噬体逆行转运可减少 I/R 诱导的神经元损伤并延长药理学自噬诱导神经保护的治疗窗口。