Melatonin is an ancient antioxidant. After its initial development in bacteria, it has been retained throughout evolution such that it may be or may have been present in every species that have existed. Even though it has been maintained throughout evolution during the diversification of species, melatonin's chemical structure has never changed; thus, the melatonin present in currently living humans is identical to that present in cyanobacteria that have existed on Earth for billions of years. Melatonin in the systemic circulation of mammals quickly disappears from the blood presumably due to its uptake by cells, particularly when they are under high oxidative stress conditions. The measurement of the subcellular distribution of melatonin has shown that the concentration of this indole in the mitochondria greatly exceeds that in the blood. Melatonin presumably enters mitochondria through oligopeptide transporters, PEPT1, and PEPT2. Thus, melatonin is specifically targeted to the mitochondria where it seems to function as an apex antioxidant. In addition to being taken up from the circulation, melatonin may be produced in the mitochondria as well. During evolution, mitochondria likely originated when melatonin-forming bacteria were engulfed as food by ancestral prokaryotes. Over time, engulfed bacteria evolved into mitochondria; this is known as the endosymbiotic theory of the origin of mitochondria. When they did so, the mitochondria retained the ability to synthesize melatonin. Thus, melatonin is not only taken up by mitochondria but these organelles, in addition to many other functions, also probably produce melatonin as well. Melatonin's high concentrations and multiple actions as an antioxidant provide potent antioxidant protection to these organelles which are exposed to abundant free radicals.

Oxidative stress-induced mitochondrial dysfunction has been shown to play a crucial role in the pathogenesis of a wide range of diseases. Protecting mitochondrial function, therefore, is vital for cells to survive during these disease processes. In this study, we demonstrate that melatonin, a chief secretory product of the pineal gland, readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptotic events and death in rat brain astrocytes (RBA-1). The early protection provided by melatonin in mitochondria of intact living cells was investigated by the application of time-lapse conventional, confocal, and multiphoton fluorescent imaging microscopy coupled with noninvasive mitochondria-targeted fluorescent probes. In particular, we observed that melatonin effectively prevented exogenously applied H2O2-induced mitochondrial swelling in rat brain astrocytes at an early time point (within 10 min) and subsequently reduced apoptotic cell death (150 min later). Other early apoptotic events such as plasma membrane exposure of phosphatidyl serine and the positive YOPRO-1 staining of the early apoptotic nucleus were also prevented by melatonin. A mechanistic study at the mitochondrial level related to the early protection provided by melatonin revealed that the indole molecule significantly reduced mitochondrial reactive oxygen species (ROS) formation induced by H2O2 stress. Melatonin also prevented mitochondrial ROS generation caused by other organic hydroperoxides including tert-butyl hydroperoxide and cumene hydroperoxide. This antioxidative effect of melatonin is more potent than that of vitamin E. Via its ability to reduce mitochondrial ROS generation, melatonin prevented H2O2-induced mitochondrial calcium overload, mitochondrial membrane potential depolarization, and the opening of the mitochondrial permeability transition (MPT) pore. As a result, melatonin blocked MPT-dependent cytochrome c release, the downstream activation of caspase 3, the condensation and karyorrhexis of the nucleus and apoptotic fragmentation of nuclear DNA. Thus, the powerful mitochondrial protection provided by melatonin reinforces its therapeutic potential to combat a variety of oxidative stress-induced mitochondrial dysfunctions as well as mitochondria-mediated apoptosis in various diseases.

Defected mitochondrial respiratory chain (RC), in addition to causing a severe ATP deficiency, often augments reactive oxygen species (ROS) generation in mitochondria (mROS) which enhances pathological conditions and diseases. Previously, we demonstrated a potent endogenously RC defect-augmented mROS associated dose-dependently with a commonly seen large-scale deletion of 4977 base pairs of mitochondrial DNA (mtDNA), i.e. the common deletion (CD). As current treatments for CD-associated diseases are rather supplementary and ineffective, we investigated whether melatonin, a potential mitochondrial protector, provides beneficial protection for CD-augmented mitochondrial oxidative stress and apoptosis particularly upon the induction of a secondary oxidative stress. Detailed mechanistic investigations were performed by using laser scanning dual fluorescence imaging microscopy to provide precise spatial and temporal resolution of mitochondrial events at single cell level. We demonstrate, for the first time, that melatonin significantly prevents CD-augmented mROS formation under basal conditions as well as at early time-points upon secondary oxidative stress induced by H2O2 exposure. Thus, melatonin prevents mROS-mediated depolarization of mitochondrial membrane potential (DeltaPsim) and subsequent opening of the mitochondrial permeability transition pore (MPTP) and cytochrome c release. Moreover, melatonin prevents depletion of cardiolipin which appears to be crucial for postponing later MPTP opening, disruption of the mitochondrial membrane and apoptosis. Finally, the protection provided by melatonin is superior to those caused by the suppression of mitochondrial Ca2+ regulators including the mitochondrial Na+-Ca2) exchanger, the MPTP, and the mitochondrial Ca2+ uniporter and by antioxidants including vitamin E and mitochondria-targeted coenzyme Q, MitoQ. As RC defect-augmented endogenous mitochondrial oxidative stress is centrally involved in a variety of pathological conditions and diseases, melatonin thus may serve as a therapeutic drug to benefit many clinical conditions that involve malfunction of the mitochondria.

Melatonin exhibits extraordinary diversity in terms of its functions and distribution. When discovered, it was thought to be uniquely of pineal gland origin. Subsequently, melatonin synthesis was identified in a variety of organs and recently it was shown to be produced in the mitochondria. Since mitochondria exist in every cell, with a few exceptions, it means that every vertebrate, invertebrate, and plant cell produces melatonin. The mitochondrial synthesis of melatonin is not photoperiod-dependent, but it may be inducible under conditions of stress. Mitochondria-produced melatonin is not released into the systemic circulation, but rather is used primarily in its cell of origin. Melatonin’s functions in the mitochondria are highly diverse, not unlike those of sirtuin 3 (SIRT3). SIRT3 is an NAD+-dependent deacetylase which regulates, among many functions, the redox state of the mitochondria. Recent data proves that melatonin and SIRT3 post-translationally collaborate in regulating free radical generation and removal from mitochondria. Since melatonin and SIRT3 have cohabitated in the mitochondria for many eons, we predict that these molecules interact in many other ways to control mitochondrial physiology. It is predicted that these mutual functions will be intensely investigated in the next decade and importantly, we assume that the findings will have significant applications for preventing/delaying some age-related diseases and aging itself.