Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan.
Department of Biomedical Research Laboratory, Gifu Pharmaceutical University, Gifu, Japan.
Free Radic Biol Med. 2020 May 20;152:235-247. doi: 10.1016/j.freeradbiomed.2020.03.012. Epub 2020 Mar 23.
Daily phagocytosis of shed photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) is required to sustain the visual function. Recent reports revealed that POS phagocytosis is progressed with LC3-associated manner. Patients with age-related macular degeneration (AMD) had impaired autophagic degradation in the RPE. Nrf2 is a key antioxidant transcriptional regulator that ameliorates oxidative stress which is another contributor to AMD pathogenesis. Nrf2 activation also induces the autophagy receptor protein, p62. However, the role of the Nrf2-p62 pathway in LC3-associated phagocytosis of POS is poorly understood. Here, we investigated the relationships between Nrf2 activation and POS phagocytosis progression. A triterpenoid Nrf2 activator, RS9, facilitated POS uptake into phagolysosomes in RPE cells. RS9 also induced the expression of the autophagy-related proteins, LC3-II and p62, as well as phase-2 antioxidant enzymes. The effect of RS9 on POS phagocytosis was abolished by autophagy inhibition. Unexpectedly, p62 knockdown did not inhibit the effect of RS9 on POS phagocytosis, although, RS9-mediated LC3-II induction by RS9 was inhibited in p62 knockdown RPE cells. We also found that RS9 activated the AMPKα-mTOR signaling pathway earlier than p62 induction. Knockdown of AMPKα, but not α, inhibited the RS9-mediated activation of LC3-associated phagocytosis and RS9-mediated induction of LC3-II. Furthermore, intravitreal treatment of RS9 to adult mice decreased the size of POS phagolysosomes after light exposure. Collectively, these results showed that RS9-mediated activation of POS phagocytosis was mainly ascribed to the enhancement of autophagy via AMPKα activation. Our findings reveal novel effects of Nrf2 and AMPK α activation that contribute to the maintenance of the RPE function via LC3-associated POS phagocytosis.
每天视网膜色素上皮（RPE）吞噬脱落的光感受器外节（POS）是维持视觉功能所必需的。最近的报告显示，POS 的吞噬作用是通过 LC3 相关的方式进行的。年龄相关性黄斑变性（AMD）患者的 RPE 自噬降解受损。Nrf2 是一种关键的抗氧化转录调节因子，可改善氧化应激，这也是 AMD 发病机制的另一个促成因素。Nrf2 的激活还诱导自噬受体蛋白 p62。然而，Nrf2-p62 途径在 POS 的 LC3 相关吞噬作用中的作用知之甚少。在这里，我们研究了 Nrf2 激活与 POS 吞噬作用进展之间的关系。一种三萜类 Nrf2 激活剂 RS9，促进 RPE 细胞中 POS 的摄取进入吞噬溶酶体。RS9 还诱导自噬相关蛋白 LC3-II 和 p62 的表达，以及相 2 抗氧化酶。RS9 对 POS 吞噬作用的影响被自噬抑制所消除。出乎意料的是，尽管 RS9 介导的 LC3-II 诱导被 p62 敲低 RPE 细胞中的抑制，但 p62 敲低并不抑制 RS9 对 POS 吞噬作用的影响。我们还发现，RS9 激活 AMPKα-mTOR 信号通路早于 p62 诱导。AMPKα 的敲低，但不是α，抑制了 RS9 介导的 LC3 相关吞噬作用的激活和 RS9 介导的 LC3-II 的诱导。此外，RS9 对成年小鼠玻璃体腔给药可减少光照后 POS 吞噬溶酶体的大小。总之，这些结果表明，RS9 介导的 POS 吞噬作用的激活主要归因于通过 AMPKα 激活增强自噬。我们的研究结果揭示了 Nrf2 和 AMPKα 激活的新作用，这些作用通过 LC3 相关的 POS 吞噬作用有助于维持 RPE 功能。
