Background Ischaemic stroke triggers neuronal mitophagy, while the involvement of mitophagy receptors in ischaemia/reperfusion (I/R) injury-induced neuronal mitophagy remain not fully elucidated. Here, we aimed to investigate the involvement of mitophagy receptor FUN14 domain-containing 1 (FUNDC1) and its modulation in neuronal mitophagy induced by I/R injury. Methods Wild-type and FUNDC1 knockout mice were generated to establish models of neuronal I/R injury, including transient middle cerebral artery occlusion (tMCAO) in vivo and oxygen glucose deprivation/reperfusion in vitro. Stroke outcomes of mice with two genotypes were assessed. Neuronal mitophagy was analysed both in vivo and in vitro. Activities of FUNDC1 and its regulator Src were evaluated. The impact of Src on FUNDC1-mediated mitophagy was assessed through administration of Src antagonist PP1. Results To our surprise, FUNDC1 knockout mice subjected to tMCAO showed stroke outcomes comparable to those of their wild-type littermates. Although neuronal mitophagy could be activated by I/R injury, FUNDC1 deletion did not disrupt neuronal mitophagy. Transient activation of FUNDC1, represented by dephosphorylation of Tyr18, was detected in the early stages (within 3 hours) of neuronal I/R injury; however, phosphorylated Tyr18 reappeared and even surpassed baseline levels in later stages (after 6 hours), accompanied by a decrease in FUNDC1-light chain 3 interactions. Spontaneous inactivation of FUNDC1 was associated with Src activation, represented by phosphorylation of Tyr416, which changed in parallel with the level of phosphorylated FUNDC1 (Tyr18) during neuronal I/R injury. Finally, FUNDC1-mediated mitophagy in neurons under I/R conditions can be rescued by pharmacological inhibition of Src. Conclusions FUNDC1 is inactivated by Src during the later stage (after 6 hours) of neuronal I/R injury, and rescue of FUNDC1-mediated mitophagy may serve as a potential therapeutic strategy for treating ischaemic stroke. Data are available upon reasonable request.

中文翻译：

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Src 抑制可挽救缺血性中风中 FUNDC1 介导的神经元线粒体自噬

背景 缺血性中风触发神经元线粒体自噬，而线粒体自噬受体在缺血/再灌注（I/R）损伤诱导的神经元线粒体自噬中的作用尚未完全阐明。在这里，我们的目的是研究线粒体自噬受体 FUN14 结构域 1 (FUNDC1) 的参与及其在 I/R 损伤诱导的神经元线粒体自噬中的调节作用。方法 制备野生型和FUNDC1基因敲除小鼠，建立神经元I/R损伤模型，包括体内短暂性大脑中动脉闭塞（tMCAO）和体外氧糖剥夺/再灌注。评估了具有两种基因型的小鼠的中风结果。在体内和体外分析神经元线粒体自噬。对 FUNDC1 及其调节器 Src 的活性进行了评估。通过施用 Src 拮抗剂 PP1 评估 Src 对 FUNDC1 介导的线粒体自噬的影响。结果令我们惊讶的是，接受 tMCAO 治疗的 FUNDC1 敲除小鼠显示出与野生型同窝小鼠相当的中风结果。尽管 I/R 损伤可以激活神经元线粒体自噬，但 FUNDC1 缺失并不会破坏神经元线粒体自噬。FUNDC1 的瞬时激活（以 Tyr18 去磷酸化为代表）在神经元 I/R 损伤的早期（3 小时内）被检测到；然而，磷酸化的 Tyr18 再次出现，甚至在后期（6 小时后）超过基线水平，同时 FUNDC1-轻链 3 相互作用减少。FUNDC1 的自发失活与 Src 激活相关，以 Tyr416 的磷酸化为代表，在神经元 I/R 损伤期间，其与磷酸化 FUNDC1 (Tyr18) 的水平平行变化。最后，I/R 条件下 FUNDC1 介导的神经元线粒体自噬可以通过 Src 的药理学抑制来挽救。结论 FUNDC1在神经元I/R损伤后期（6小时后）被Src失活，挽救FUNDC1介导的线粒体自噬可能成为治疗缺血性脑卒中的潜在治疗策略。数据可根据合理要求提供。