Protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin's cascade metabolites C3-OHM and AFMK in RBA1 astrocytes

Abstract

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 Ca²⁺ (mCa²⁺ ) 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 mCa²⁺ stress in RBA1 astrocytes. Interestingly, precise temporal and spatial dynamic live mitochondrial images revealed that C3-OHM and AFMK prevented specifically mCa²⁺ -mediated mitochondrial reactive oxygen species (mROS) formation and hence mROS-mediated depolarization of mitochondrial membrane potential (△Ψ_m ) 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-Ca²⁺ stress" using a classic antioxidant vitamin E to remove mCa²⁺ -mediated mROS stress and the potential antioxidative effects of C3-OHM and AFMK. Intriguingly, two compounds still effectively postponed "oxidation free-Ca²⁺ stress"-mediated depolarization of △Ψ_m 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 mCa²⁺ . 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 mCa²⁺ -mediated MPT-associated apoptotic stresses and may provide therapeutic benefits against astrocyte-mediated neurodegeneration in the CNS.

Keywords: AFMK; C3-OHM; mCa2+; mROS; melatonin; mitochondria-targeted antioxidant; mitochondrial permeability transition.