Department of Physiology and Pharmacology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
Department of Neurology, Chang Gung Memorial Hospital, Keelung Branch, Keelung, Taiwan.
J Pineal Res. 2019 Jan;66(1):e12538. doi: 10.1111/jpi.12538. Epub 2018 Dec 2.
Cyclic 3-hydroxymelatonin (C3-OHM) and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) are two major cascade metabolites of melatonin. We previously showed melatonin provides multiple levels of mitochondria-targeted protection beyond as a mitochondrial antioxidant during ionomycin-induced mitochondrial Ca (mCa ) stress in RBA1 astrocytes. Using noninvasive laser scanning fluorescence coupled time-lapse digital imaging microscopy, this study investigated whether C3-OHM and AFMK also provide mitochondrial levels of protection during ionomycin-induced mCa stress in RBA1 astrocytes. Interestingly, precise temporal and spatial dynamic live mitochondrial images revealed that C3-OHM and AFMK prevented specifically mCa -mediated mitochondrial reactive oxygen species (mROS) formation and hence mROS-mediated depolarization of mitochondrial membrane potential (△Ψ ) and permanent lethal opening of the MPT (p-MPT). The antioxidative effects of AFMK, however, were less potent than that of C3-OHM. Whether C3-OHM and AFMK targeted directly the MPT was investigated under a condition of "oxidation free-Ca stress" using a classic antioxidant vitamin E to remove mCa -mediated mROS stress and the potential antioxidative effects of C3-OHM and AFMK. Intriguingly, two compounds still effectively postponed "oxidation free-Ca stress"-mediated depolarization of △Ψ and p-MPT. Measurements using a MPT pore-specific indicator Calcein further identified that C3-OHM and AFMK, rather than inhibiting, stabilized the MPT in its transient protective opening mode (t-MPT), a critical mechanism to reduce overloaded mROS and mCa . These multiple layers of mitochondrial protection provided by C3-OHM and AFMK thus crucially allow melatonin to extend its metabolic cascades of mitochondrial protection during mROS- and mCa -mediated MPT-associated apoptotic stresses and may provide therapeutic benefits against astrocyte-mediated neurodegeneration in the CNS.
循环 3-羟色胺（C3-OHM）和 N1-乙酰-N2-甲酰基-5-甲氧基犬尿氨酸（AFMK）是褪黑素的两种主要级联代谢物。我们之前的研究表明，褪黑素在离子霉素诱导的 RBA1 星形胶质细胞线粒体 Ca（mCa）应激期间不仅作为线粒体抗氧化剂，还提供了多个层次的线粒体靶向保护。本研究采用非侵入性激光扫描荧光偶联时间 lapse 数字成像显微镜技术，研究 C3-OHM 和 AFMK 是否也能在离子霉素诱导的 RBA1 星形胶质细胞 mCa 应激期间提供线粒体水平的保护。有趣的是，精确的时间和空间动态活线粒体图像显示，C3-OHM 和 AFMK 特异性地阻止了 mCa 介导的线粒体活性氧（mROS）形成，从而阻止了 mROS 介导的线粒体膜电位（△Ψ）去极化和线粒体通透性转换孔（MPT）的永久性开放（p-MPT）。然而，AFMK 的抗氧化作用不如 C3-OHM 强。为了研究 C3-OHM 和 AFMK 是否直接靶向 MPT，在使用经典抗氧化维生素 E 去除 mCa 介导的 mROS 应激的“无氧化钙应激”条件下，研究了它们的潜在抗氧化作用。有趣的是，两种化合物仍能有效地延缓“无氧化钙应激”介导的△Ψ和 p-MPT 去极化。使用 MPT 孔特异性指示剂 Calcein 的测量进一步表明，C3-OHM 和 AFMK 不是抑制而是稳定 MPT 处于其短暂保护性开放模式（t-MPT），这是减少过载 mROS 和 mCa 的关键机制。C3-OHM 和 AFMK 提供的这种多层次的线粒体保护，使褪黑素在 mROS 和 mCa 介导的 MPT 相关凋亡应激期间，能够扩展其线粒体保护的代谢级联，这可能为中枢神经系统中星形胶质细胞介导的神经退行性变提供治疗益处。
