Department of Biochemistry and Molecular Biology, School of Basic Medicine, Qingdao University, Qingdao 266000, China.
Department of Biotechnology, School of Basic Medicine, Qingdao University, Qingdao 266000, China.
Cells. 2022 Aug 30;11(17):2706. doi: 10.3390/cells11172706.
Neuronal oxidative stress caused by mitochondrial dysfunction plays a crucial role in the development of Parkinson's disease (PD). Growing evidence shows that autophagy confers neuroprotection in oxidative-stress-associated PD. This work aims to investigate the involvement of TMEM166, an endoplasmic-reticulum-localized autophagy-regulating protein, in the process of PD-associated oxidative stress through the classic cellular PD model of neuroblastoma SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP). Reactive oxygen species (ROS) production and mitochondrial membrane potential were checked to assess the oxidative stress induced by MPP and the cellular ATP generated was determined to evaluate mitochondrial function. The effect on autophagy induction was evaluated by analyzing p62 and LC3-II/I expression and by observing the LC3 puncta and the colocalization of LC3 with LAMP1/ LAMP2. The colocalization of mitochondria with LC3, the colocalization of Tom20 with LAMP1 and Tom20 expression were analyzed to evaluate mitophagy. We found that TMEM166 is up-regulated in transcript levels, but up-regulated first and then down-regulated by autophagic degradation in protein levels upon MPP-treatment. Overexpression of TMEM166 induces mitochondria fragmentation and dysfunction and exacerbates MPP-induced oxidative stress and cell viability reduction. Overexpression of TMEM166 is sufficient to induce autophagy and mitophagy and promotes autophagy and mitophagy under MPP treatment, while knockdown of TMEM166 inhibits basal autophagic degradation. In addition, overexpressed TMEM166 suppresses AMPK activation, while TMEM166 knockdown enhances AMPK activation. Pharmacological activation of AMPK alleviates the exacerbation of oxidative stress induced by TMEM166 overexpression and increases cell viability, while pharmacological inhibition mitophagy aggravates the oxidative stress induced by MPP treatment combined with TMEM166 overexpression. Finally, we find that overexpressed TMEM166 partially localizes to mitochondria and, simultaneously, the active AMPK in mitochondria is decreased. Collectively, these findings suggest that TMEM166 can translocate from ER to mitochondria and inhibit AMPK activation and, in response to mitochondrial oxidative stress, neuronal cells choose to up-regulate TMEM166 to promote autophagy/mitophagy; then, the enhancing autophagy/mitophagy degrades the TMEM166 to activate AMPK, by the two means to maintain cell survival. The continuous synthesis and degradation of TMEM166 in autophagy/mitochondria flux suggest that TMEM166 may act as an autophagy/mitochondria adaptor.
线粒体功能障碍引起的神经元氧化应激在帕金森病 (PD) 的发展中起着关键作用。越来越多的证据表明，自噬在与氧化应激相关的 PD 中提供神经保护。这项工作旨在通过经典的神经母细胞瘤 SH-SY5Y 细胞暴露于 1-甲基-4-苯基吡啶鎓 (MPP) 的细胞 PD 模型来研究内质网定位的自噬调节蛋白 TMEM166 在 PD 相关氧化应激过程中的作用。通过检测活性氧 (ROS) 产生和线粒体膜电位来评估 MPP 诱导的氧化应激，通过检测细胞产生的 ATP 来评估线粒体功能。通过分析 p62 和 LC3-II/I 的表达以及观察 LC3 斑点和 LC3 与 LAMP1/LAMP2 的共定位来评估自噬诱导的影响。通过分析 TMEM166 与 LC3 的共定位、Tom20 与 LAMP1 的共定位以及 Tom20 的表达来评估线粒体自噬。我们发现，TMEM166 的转录水平上调，但在蛋白水平上先上调然后被自噬降解下调。TMEM166 的过表达诱导线粒体碎片化和功能障碍，并加剧 MPP 诱导的氧化应激和细胞活力降低。TMEM166 的过表达足以诱导自噬和线粒体自噬，并促进 MPP 处理下的自噬和线粒体自噬，而 TMEM166 的敲低抑制基础自噬降解。此外，过表达的 TMEM166 抑制 AMPK 的激活，而 TMEM166 的敲低增强 AMPK 的激活。AMPK 的药理学激活减轻了 TMEM166 过表达引起的氧化应激加剧，并增加了细胞活力，而 AMPK 抑制剂抑制线粒体自噬加剧了 MPP 处理与 TMEM166 过表达联合引起的氧化应激。最后，我们发现过表达的 TMEM166 部分定位于线粒体，同时线粒体中活性 AMPK 减少。总的来说，这些发现表明 TMEM166 可以从内质网转位到线粒体，并抑制 AMPK 的激活，并且在应对线粒体氧化应激时，神经元细胞选择上调 TMEM166 以促进自噬/线粒体自噬；然后，增强的自噬/线粒体自噬降解 TMEM166 以激活 AMPK，通过这两种方式维持细胞存活。自噬/线粒体通量中 TMEM166 的持续合成和降解表明 TMEM166 可能作为自噬/线粒体衔接物发挥作用。
