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Melatonin and brain inflammaging

Melatonin is known to possess several properties of value for healthy aging, as a direct and indirect antioxidant, protectant and modulator of mitochondrial function, antiexcitotoxic agent, enhancer of circadian amplitudes, immune modulator and neuroprotectant. It is levels tend to decrease in the course of senescence and are more strongly reduced in several neurodegenerative disorders, especially Alzheimer’s disease, and in diseases related to insulin resistance such as diabetes type 2. Although the role of melatonin in aging and age-related diseases has been repeatedly discussed, the newly emerged concept of inflammaging, that is, the contribution of low-grade inflammation to senescence progression has not yet been the focus of melatonin research. This review addresses the multiple protective actions of melatonin and its kynuramine metabolites that are relevant to the attenuation of inflammatory responses and progression of inflammaging in the brain, i.e. avoidance of excitotoxicity, reduction of free radical formation by support of mitochondrial electron flux, prevention of NADPH oxidase activation and suppression of inducible nitric oxide synthase, as well as downregulation of proinflammatory cytokines. The experimental evidence is primarily discussed on the basis of aging and senescence-accelerated animals, actions in the immune system, and the relationship between melatonin and sirtuins, having properties of aging suppressors. Sirtuins act either as accessory components or downstream factors of circadian oscillators, which are also under control by melatonin. Inflammaging is assumed to strongly contribute to neurodegeneration of the circadian master clock observed in advanced senescence and, even more, in Alzheimer’s disease, a change that affects countless physiological functions. 2015 Elsevier Ltd. All rights reserved.


Introduction
The term 'inflammaging' has been coined to denominate the contribution of inflammatory processes to the progression of aging (Franceschi et al., 2000;Boren and Gershwin, 2004;Capri et al., 2006;Salvioli et al., 2006;Cevenini et al., 2013). The importance of inflammation in senescence and its role in the development of ageassociated diseases is being increasingly perceived. Genetic predispositions such as an immune risk profile (IRP), which comprises an increased tendency toward inflammatory responses, may set limits to health and lifespan, whereas an ''inverted IRP'' found in centenarians may be the basis of successful aging (Strindhall et al., 2007;Candore et al., 2010). In fact, a wellfunctioning immune system is believed to be the strongest predictor of human longevity and healthy aging (Franceschi and Bonafè , 2003;Candore et al., 2006;DelaRosa et al., 2006;Ponnappan and Ponnappan, 2011). However, immunosenescence is not simply a process of deterioration, which proceeds more or less rapidly in the various individuals. Instead, the immune system is remodeled with aging in all subjects, a necessity that results from unavoidable changes in immune cell populations, which will be discussed in the following section. Apart from gradual functional losses, which are unavoidable despite the immune remodeling, immunosenescence has two additional undesired consequences, a higher incidence of autoimmune responses (Goronzy et al., 2013) and increased levels of inflammatory mediators . This latter observation is even made in centenarians. However, in these successfully aged subjects, the higher quantities of proinflammatory factors are associated with augmentations of anti-inflammatory cytokines and a protective genotype .
Inflammation is not only a matter of normal senescence, but is more strongly observed in a number of diseases, in particular, of the neurodegenerative type, as will be discussed in detail. With regard to the pathological aspects, brain inflammaging is a topic that is attracting increasing interest. As will be outlined in the respective section, the phenomenology of brain inflammation not caused by infections, but by degenerative processes may differ from acute or chronic inflammation elsewhere in the body and appear, in a sense, atypical. It may be of a slowly progressing, lingering type with moderate microglia activation that is sustained by the degenerative processes and oxidative stress resulting from release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by immune cells, astrocytes and neurons that is further enhanced by damage to mitochondria. Inflammaging, especially in neurodegenerative diseases, is multiply intertwined with other potentially deteriorating processes, among which mitochondrial dysfunction is of prime importance (De la Fuente and Miquel, 2009;Hardeland, 2013a). The connection between the immune system and oxidative stress has been extended by a recently discovered mechanism based on the senescence-associated secretory phenotype (SASP), which leads to a low-level but persistent inflammation, as will be outlined in the next section.
Melatonin is multiply interrelated to brain function, to the immune system and to the defense against damage by ROS and RNS. In its role as the hormone of the pineal gland, it is not only released to the circulation, from where it can enter the CNS, but also directly, via the pineal recess, to the third ventricle of the brain (Tricoire et al., 2002(Tricoire et al., , 2003a. It is additionally synthesized in various immune cells (Carrillo-Vico et al., 2013;Hardeland, 2013a). Melatonin receptors are found in many places within the CNS and also in the majority of the immune cells (summarized in: Hardeland et al., 2011). It does not only participate in the regulation of sleep and circadian rhythms, but exerts numerous additional actions in the CNS, notably as an antiexcitatory agent, a regulator of hormone secretion, of metabolic pathways and intracerebral blood flow. In the immune system including microglia, it modulates the release of various pro-and antiinflammatory cytokines as well as other immune stimulatory factors and can directly activate monocytes (Guerrero and Reiter, 2002;Carrillo-Vico et al., 2013;Hardeland, 2013a). It attenuates oxidative stress by various mechanisms that comprise avoidance of free radical-generating conditions, downregulation of strongly elevated inducible NO synthase (iNOS) and neuronal NO synthase  Hardeland et al. / Progress in Neurobiology xxx (2015) xxx-xxx (nNOS), upregulation of antioxidant enzymes and free radical scavenging Hardeland, 2005;Hardeland et al., 2011).
With regard to the eminent role of mitochondria in cell death, autophagy and free radical generation by electron dissipation from the electron transport chain (ETC), mitochondrial effects of melatonin are also of particular importance in the context of inflammation, which can lead to blockade of the ETC, enhanced electron overflow, breakdown of the mitochondrial membrane potential, and apoptosis. Protective actions of melatonin concerning maintenance of electron flux, decrease of electron leakage and structural integrity of the organelle have been extensively described and summarized Hardeland, 2013a). These effects have been observed under various conditions, from acute inflammation to normal and accelerated aging.
Without discussing at this point the gerontological significance of melatonin in detail, it should be mentioned that strong indications exist for its support of healthy aging (Poeggeler, 2005;Hardeland, 2013a). In laboratory animals, however, an extension of lifespan is, when demonstrable, only marginal. Several beneficial effects of melatonin have been observed in numerous models of neurodegenerative disorders, including Alzheimer's disease (AD), although a halting of disease progression is not or only poorly demonstrable in humans (Srinivasan et al., 2006;Cardinali et al., 2010).
A potentially important aspect of the relationship between melatonin and aging, especially inflammaging, concerns the changes of melatonin secretion that are frequently observed in the course of senescence. In many subjects, the nocturnal secretion of melatonin decreases with age, though with considerable interindividual variation. Reductions in melatonin levels are even more expressed in various diseases and disorders, most strongly in neurodegenerative diseases such as AD and other forms of dementia (Skene et al., 1990;Skene and Swaab, 2003;Hardeland, 2012Hardeland, , 2013a. In other words, melatonin, a regulatory molecule that can attenuate stimuli initiating inflammatory responses, that reduces damage to cells, mitochondria and DNA, and presumably prevents excessive conversion of cells to the DNA-damaged, senescent phenotype with SASP, undergoes a substantial decline in many aging individuals and, even more, in patients affected by neurodegeneration. The relationship between the decrease in melatonin and brain inflammaging merits future attention.

Inflammaging: new concepts and mechanisms
Earlier concepts of aging focused on various different mechanisms and hypotheses, such as assumed life-determining limits of energy expenditure, telomere attrition leading to reduced cell growth and progressive losses in number and proliferative potential of stem cells that cause deficits in tissue repair. All these processes do contribute to aging, but they should not be regarded as isolated, monocausal alterations that explain aging on an exclusive basis, but are rather interconnected and may become relevant in different stages of senescence, as recently discussed elsewhere (Hardeland, 2013a). The multiple nexus to other processes also apply to the frequently cited free radical theory of aging (Harman, 1956(Harman, , 2006Afanas'ev, 2010), including its variants focusing on mitochondrial dysfunction (Harman, 1972;Miquel et al., 1992;Gruber et al., 2008;Gomez-Cabrera et al., 2012). With regard to the immune system, attention was first focused on its functional losses. However, the perception that the immune system is not just decaying, but undergoing remodeling processes that include tendencies toward enhanced proinflammatory signaling have led to a new understanding of aging in conjunction with enhanced inflammation, as denominated by inflammaging. The importance of this view is supported by several findings, such as enhanced inflammation in neurodegenerative diseases, the effects of inflammatory signals and free radicals released by immune cells on neurons and astrocytes, and, more recently, the discovery of SASP with its consequences for sustained low-level inflammation.

Causes resulting from immunosenescence
Immunosenescence is one of the causes of chronic low-grade inflammation. The remodeling of the immune system is a necessity to which several processes contribute. One of these is progressive thymic involution, which leads to losses in the number of both CD4 + and CD8 + T lymphocytes as well as a disturbed balance between naïve, memory and effector T cells (Linton and Thoman, 2001;Franceschi and Bonafè , 2003;Pfister and Savino, 2008;Pinti et al., 2010;Goronzy et al., 2013). Typically an exhaustion of CD95 À , virgin T lymphocytes is observed. This concerns especially the subset of CD8 + cells, which are largely replaced by clonal expansion of CD28 À cells of lower proliferative activity (Sansoni et al., 2008). With regard to CD8 + T lymphocytes, type 1 cytokines are mainly elevated, such as interleukin-2 (IL-2), interferon-g (IFNg) and tumor necrosis factor-a (TNFa), and, to a smaller extent, type 2 cytokines, such as IL-4, IL-6, and IL-10. In balance, this contributes to a higher inflammatory state, however, with large interindividual differences. This divergence may be related to the degree of thymic involution and the maintenance of a population of ''recent thymic emigrants'' (Pinti et al., 2010). Age-dependent changes have been also reported for CD4 + T lymphocytes, mostly in the CD95 + CD28 + subset, i.e., activated/ memory cells, among which TNFa-positive cells are decreased, but IL-4-positive cells increased, reflecting, in these cells, a shift from type 1 to type 2 cytokines (Alberti et al., 2006).
Alterations in T cell subpopulations also affect the functionality and composition of B lymphocytes. A second cause is life-long exposure to numerous foreign antigens, which leads to changes in B cell subpopulations Colonna-Romano et al., 2010). B cell immunosenescence results in decreases of immunoglobulin M (IgM) and IgD production, whereas increased levels of IgG1 have been reported (Buffa et al., 2013). A presumably relevant regulatory dysfunctionality is apparent in strong losses of naïve IgD + B cells, which are replaced by exhausted memory IgD À B cells . Interestingly, in centenarian offspring, naïve IgD + CD27 À B cells were more abundant, whereas exhausted IgD À CD27 À B were mostly unchanged, findings that may indicate a favorable genetic predisposition . A recently described, TNF-producing CD19 + CD38 À CD24 À memory B cell subset was found to be lower in centenarian offspring, which would also indicate a lower inflammatory status (Buffa et al., 2013).
The alterations observed during immunosenescence do not concern only the adaptive, but also the innate immune system (Mocchegiani et al., 2007(Mocchegiani et al., , 2009Shaw et al., 2010;Gayoso et al., 2011), although the latter is usually less affected, which is especially apparent in very old individuals (Mocchegiani et al., 2009;Dewan et al., 2012). Centenarians were reported to have a relatively higher number of natural killer (NK) and natural killer T (NKT) cells, especially NKT cells bearing the gd-T cell receptor, in contrast to less successfully aging subjects. Moreover, these cells responded more strongly to activation in centenarians by releasing IFNg (Mocchegiani et al., 2007(Mocchegiani et al., , 2009, which would underline the importance of a robust innate immune system. However, this potential for a more efficient defense is partially compensated, at the inflammatory side, by a lower expression of the IL-6 receptor. This finding is therefore of particular importance as sustained elevated IL-6 release, which occurs under chronic inflammatory conditions, leads to high metallothionein expression and, thereby, to poor zinc availability because of its sequestration by this protein (Mocchegiani et al., 2007). Indeed, zinc is important to several aspects of the immune system, including function of the thymus.

Senescence-associated secretory phenotype (SASP)
SASP has turned out to be a continually active source of lowgrade inflammation and oxidative stress. Because it depends on DNA damage, which becomes more likely with increasing age and accumulates with time, its significance to aging tissues including the CNS steadily rises. Senescent cells carrying damaged DNA are usually mitotically arrested, a mechanism that keeps them alive and metabolically active, but prevents a neoplastic fate. However, these cells display a chronic DNA damage response (DDR) (Fumagalli and d'Adda di Fagagna, 2009). Apart from other effects concerning the cytoskeleton and formation of protein aggregates, the most important change in the behavior of these senescent, arrested cells concerns the release of proinflammatory cytokines, the hallmark of SASP (Coppé et al., 2008Young and Narita, 2009). These factors, which are notably secreted by nonimmune cells, attract and/or activate immune cells. On the one hand, they may lead to the elimination of a number of senescent cells, but, on the other hand, they cause low-grade inflammation. Since inflammation is a source of oxidative stress, the number of cells damaged by ROS and RNS is, on balance, steadily rising. In the extreme, ROS and RNS derived by the SASP mechanism can even induce cancer in previously nondamaged cells, in the worst case, in stem cells, which turn into telomerase-expressing cancer stem cells, which are usually not arrested (Davalos et al., 2010;Acosta et al., 2013). Thus, a mechanism that primarily prevents tumor formation, can secondarily become tumorigenic.
Interestingly, aging astrocytes express SASP characteristics (Salminen et al., 2011). Thus, low-level inflammation and, eventually, induction of cancer stem cells by SASP are also relevant to the aging brain.

Neuronal overexcitation and microglia activation
Neuronal overexcitation and inflammatory responses are intimately associated. These processes can be stimulated from either side. While glutamate excitotoxicity can lead to microglia activation (Campuzano et al., 2008;Arlicot et al., 2014;Lin et al., 2014;Nomaru et al., 2014), primary immune responses involving the microglia may, in turn, lead to excitotoxicity (Takeuchi et al., 2005;Brown, 2007;Brown and Neher, 2010). Astrocytes are frequently coactivated with microglial cells. They also contribute to both excitotoxicity and microglia activation, by mechanisms that involve impaired glutamate uptake, inflammatory signals, and/or oxidative/nitrosative stress (Tilleux and Hermans, 2007;Brown, 2007;Salminen et al., 2011;Morales et al., 2014). As will be discussed in Section 5, melatonin is capable of attenuating the detrimental changes at all these levels. A particular problem results from the fact that inflammatory responses can be initiated from different sides. Inflammasomes that may cause both release of proinflammatory cytokines such as IL-1b and IL-18 and apoptotic or pyroptotic cell death are found in neurons (NLRP1 and AIM2), astrocytes (NLRP2) and microglia (NLRP3) (de Rivero Vaccari et al., 2014). Histone H1 released from dying cells of whatever type acts as an additional pro-inflammatory signal and chemoattractant (Gilthorpe et al., 2013). Moreover, stimulation of proinflammatory processes in different cell types implies the possibility of positive feedback loops between neurons, astrocytes and microglia that expand the grade and area of inflammation and may be further aggravated by recruitment of other immune cells.
However, it seems important to remain aware of the conditionality of these immune-stimulatory processes and their activating interconnections. In particular, microglia is neither a basically inactive population of cells, as believed earlier, nor a uniformly responding entity. Even the ramified microglia has been shown to be continuously active in terms of movement and safeguarding the central nervous microenvironment (Blaylock, 2013). Activation of microglia can lead to different phenotypes, from neurodestructive and phagocytic cells to others that are primarily neuroprotective or growth promoting (Blaylock, 2013). To perceive these differences is important insofar as most of the information concerning the connections between overexcitation and microglial activity has been obtained in animal models of excitotoxicity, brain injury or immunological challenge, e.g., by endotoxemia. A crucial question concerns the applicability of these findings to the slow, lingering alterations that occur in the course of aging, with regard to both asymptomatic changes and ageassociated pathologies. According to current knowledge, the factors that initiate age-related deviations in neuronal activity and low-grade inflammation may frequently differ from the inducers applied in the experimental models, but the intercellular communication routes and the signaling pathways seem to be largely the same. At least, one hallmark of inflammaging, namely, SASP, has been demonstrated in astrocytes (Salminen et al., 2011) and, therefore, exists in an aging brain that is not compromised by more severe insults. When additional pro-inflammatory signals come into play in the course of neuropathological changes, the neuroinflammatory traits may be different from those observed after chemically induced excitotoxicity, brain injury or endotoxemia, but may still contribute to a considerable extent to the progressing anatomical and functional deterioration in the CNS.
The inflammatory component in neurodegenerative disorders was not obvious from the beginning. Meanwhile, inflammaging has been interpreted as a prodromal process to AD (Giunta et al., 2008). However, as discussed elsewhere (Srinivasan et al., 2006), differences exist between tissue acutely inflamed because of infection and the AD brain, in which some classical hallmarks are usually absent, such as neutrophil infiltration and edema, whereas other characteristics including microglia activation, release of proinflammatory cytokines (IL-1b, IL-6, IL-15, IL-18, TNFa), appearance of acute-phase proteins (e.g., C-reactive protein) and lymphocyte recruitment are clearly demonstrable. In addition, enhanced NO formation, which is largely caused by microglial upregulation of iNOS, elevated ROS generation by microglial NADPH oxidase (Brown and Neher, 2010) and impairments of mitochondrial electron flux, lead to critical levels of peroxynitrite formed by combination of NO and superoxide, and secondary radicals from the decay of peroxynitrite adducts, such as NO 2 , hydroxyl and carbonate radicals (Hardeland and Coto-Montes, 2010). The resulting nitroxidative stress and damage is reflected by widespread protein modifications induced by peroxynitrite or peroxynitrite-derived free radicals (Smith et al., 1997;Hensley et al., 1998). For further details concerning mitochondrial dysfunction, oxidative and nitrosative stress see Srinivasan et al. (2006) and Hardeland and Coto-Montes (2010). This includes the contribution of toxic mono-and oligomers of amyloid-b (Ab) peptides, Ab metal complexes that undergo redox cycling, and the depletion of copper and zinc by amyloid plaques, which impairs the formation of functionally active Cu,Zn-superoxide dismutase (summarized in: Hardeland, 2011a).
Inasmuch as early phases of neurodegenerative disorders are poorly distinguishable from normal inflammaging or other forms of low grade inflammation, these pathologies display important mechanisms of aggravation, in terms of vicious cycles. These comprise accumulation of toxic products, such as Ab peptides, oligomers and plaques in AD, microglial proliferation and phagocytosis after their activation, and progressive formation of pro-inflammatory signal molecules. Elevated levels of IL-6 and TNFa are demonstrable in the CNS as well as in the circulation, reflecting dysregulation of inflammatory pathways, and are meanwhile regarded as markers of frailty (Michaud et al., 2013). Increases in IL-15, a cytokine that stimulates activities and proliferation of cytotoxic T-cells, NK cells and B-cells, are associated in the CNS with microglial activation and are especially observed in AD and PD, but also under other neuroinflammatory conditions (Rentzos and Rombis, 2012). In AD, a specific deteriorating role may be attributed to IL-18, a cytokine that is not only a key mediator of inflammation involved in neurodegeneration (Bossù et al., 2010), but has been also shown to promote Ab formation (Sutinen et al., 2012). Interrupting the proinflammatory vicious cycles represents a main challenge for the combat against neurodegenerative diseases.

Melatonin's age-dependent decline
The pleiotropically acting hormone melatonin which influences countless functions either directly or via circadian oscillators, has additional relevance to aging because of its roles as an immunomodulator (Carrillo-Vico et al., 2013), downregulator of nNOS and iNOS, antiexcitatory and partially anti-inflammatory agent , protector of mitochondrial electron flux (Acuñ a- Castroviejo et al., 2003Hardeland et al., 2009a;Hardeland, 2013a), and multiply acting antioxidant Hardeland, 2005;Tan et al., 2007). Deviations in the levels and/or circadian patterns of this molecule have to be expected to cause a plethora of physiological effects. On a statistical basis, the nocturnal maximum of melatonin decreases in the course of aging, although the interindividual variability is relatively large (Sack et al., 1986;Karasek and Reiter, 2002;Skene and Swaab, 2003). While several individuals maintain a fairly well pronounced rhythm with only moderate reductions of nocturnal values, others display nocturnal values close to those during daytime. For causes of reduced melatonin formation see Hardeland (2012). As soon as age-related reductions of melatonin become more profound, numerous changes are observed (Hardeland, 2012), among these considerable alterations in the immune system, as summarized earlier (Cardinali et al., 2008a). Decreased levels of melatonin are not only observed in pineal gland, blood and some other body fluids, but also in the cerebrospinal fluid (Brown et al., 1979;Liu et al., 1999). As long as a melatonin rhythm is still detectable in elderly individuals, the nocturnal peak of plasma melatonin is frequently phase-advanced relative to young subjects, indicating changes in the circadian oscillator system (Skene and Swaab, 2003). The interindividual variability of melatonin levels at advanced age raises the questions of whether maintenance of substantial melatonin rhythmicity can be taken as a marker of successful aging, and whether a more pronounced decrease is partially or more importantly causal to other physiological or even cognitive deteriorations.
In several neurodegenerative disorders, especially AD and other types of senile dementia, levels of melatonin are frequently more strongly decreased than in age-matched controls (Skene et al., 1990;Liu et al., 1999;Skene and Swaab, 2003;Srinivasan et al., 2005;Wu and Swaab, 2007;Hardeland, 2012). In quite a number of these patients, the melatonin rhythm is practically abolished. Decreased melatonin levels are also observed in various other disorders and diseases, but notably not PD (summarized in: Hardeland, 2012). These include neurological pathologies, painful conditions, and a number of metabolic disorders or diseases that seem, at first glance, unrelated to brain functions. This concerns especially diabetes type 2 (O'Brien et al., 1986;Peschke et al., 2007). Interestingly, the connection of this disease to melatonin is supported by other findings on altered melatonin signaling and concomitant age-associated impairments of cognitive functions, and the development of diabetes as a consequence of defective clock functions (Marcheva et al., 2010;Hardeland et al., 2012). These aspects have recently gained considerable actuality, as nexuses between insulin resistance, neuroinflammation and AD have become apparent (Clark and Vissel, 2013;Jiang et al., 2013;De Felice et al., 2014;de la Monte and Tong, 2014;Ferreira et al., 2014).

Two-sided role of melatonin in inflammation: proinflammatory vs. anti-inflammatory actions
In its role as an immune modulator, melatonin has been reviewed several times (Guerrero and Reiter, 2002;Carrillo-Vico et al., 2005Szczepanik, 2007;Cardinali et al., 2008a;Hardeland et al., 2011;Hardeland, 2013a). Its functions are diverse. As summarized in the reviews mentioned, it is produced by and acts on various types of leukocytes. Among these, T lymphocytes and NK cells may be of particular interest in the context of brain inflammaging. Although melatonin formation was demonstrated in monocytes, corresponding findings have not been published to date for microglia. In monocytes, it enhances ROS formation and cytotoxicity, representing one of the strongest direct prooxidant effects of melatonin reported (Morrey et al., 1994). Similar findings have been also observed in the promonocytic cell line U937 (Cristofanon et al., 2009;Radogna et al., 2009). Again, a direct demonstration of this effect is missing in microglia, although clarification of this possibility would be of considerable importance for the understanding of inflammatory dynamics in the brain. It may be that the threshold for this type of activation is higher in microglia than in other cells of the monocytic line and, therefore, not relevant to low-grade inflammation.
Numerous effects of melatonin known from melatonin in the peripheral parts of the immune system may well be relevant to immune functions in general and to inflammaging as well, but not necessarily to brain inflammation. This may especially concern the expansion of lymphocyte subtypes and changes in leukocyte abundance or activity in the various peripheral immune tissues including lymph nodes, gut, spleen and thymus (Carrillo-Vico et al., 2013;Cardinali et al., 2008a;Hardeland et al., 2011;Hardeland, 2013a). Nevertheless, the changes observed in the peripheral immune system during senescence may well influence health and indirectly affect the CNS.
If judged under the rubric of cytokine release, one arrives at the conclusion that melatonin can alternately exert pro-and antiinflammatory effects (Carrillo-Vico et al., 2013;Hardeland, 2013a). Among the immune-stimulatory and potentially or demonstrably pro-inflammatory cytokines, elevations of IL-1b, IL-2, IL-6, IL-12, TNFa, and IFNg can be mentioned (García-Mauriñ o et al., 1997, 1999Barjavel et al., 1998;Carrillo-Vico et al., 2005;Lardone et al., 2006). An additional classic finding describes the counteraction by melatonin of the inhibitory effect of prostaglandin E 2 (PGE 2 ) on IL-2 production (Carrillo-Vico et al., 2003). Moreover, a decrease in the anti-inflammatory IL-10 was reported (Kü hlwein and Irwin, 2001) as well as a reduction of IL-2-induced IL-10 secretion (Lissoni et al., 1996). However, these results seem to largely depend on cells, systems studied and especially conditions relevant to the grade of inflammation. In particular, downregulations of IL-1b, IL-6, IL-8, IL-12, and TNFa have been repeatedly documented under conditions of high oxidative stress, ischemia/reperfusion, brain trauma, hemorrhagic shock, and various forms of high-grade inflammation including sepsis (summarized in: Hardeland, 2013a). Two additional anti-inflammatory effects have to be taken into consideration. First cyclooxygenase-2 (COX-2) expression has been shown to be downregulated by melatonin in macrophages via nuclear factor-kB (NF-kB) (Mayo et al., 2005b;Deng et al., 2006;Korkmaz et al., 2012). The same signaling pathway seems to be involved in the suppression of iNOS in macrophages (Deng et al., 2006;Korkmaz et al., 2012), an effect observed in various organs and cells Escames et al., 2006aEscames et al., ,b, 2007, and notably also in microglia and astrocytes (Jimé nez-Ortega et al., 2009;Tapias et al., 2009).
The contrasting results on either pro-or anti-inflammatory actions seem to indicate that the former effects are observed under basal conditions, whereas the latters are typical for experimental high-grade inflammation. Under these aspects, melatonin has been regarded as an immune modulator with buffering properties, allowing immune stimulation in response to, e.g., infectious challenges, but mitigating severe damage in high-grade inflammation (Carrillo-Vico et al., 2013). Another interpretation has assumed that melatonin may only promote early phases of inflammation, but attenuates its continuation to avoid chronification of a disease (Radogna et al., 2010).
Therefore, the decisive question is how melatonin acts under conditions of low-grade inflammation, especially during inflammaging and specifically in the CNS, and under the consideration of SASP. This problem extends toward its utility in advanced neuroinflammation, e.g., in the different stages of AD. To date, a definitive answer based on evidence cannot be given. In particular, the effects of melatonin on SASP-dependent inflammation are practically unknown, because the proinflammatory cytokines are not mainly derived from immune cells, but from previously normal nonimmune cells that have been mitotically arrested after mutation and display a chronic DDR. However, there are several indications for a preferably anti-inflammatory role of melatonin in the course of aging. In old female rats, melatonin decreased the proinflammatory cytokines TNFa, IL-1b and, to a considerable extent, IL-6, whereas it strongly increased the anti-inflammatory IL-10 in the liver (Kireev et al., 2008). In the senescence-accelerated mouse strain SAMP8 (Chang et al., 2009) and in aged murine neurons (Tajes et al., 2009), melatonin upregulated sirtuin 1 (SIRT1). Recently, melatonin was shown to upregulate SIRT1 in the dentate gyrus of aged, ovariectomized rats, in conjunction with substantial decreases in proinflammatory cytokines (Kireev et al., 2014). It remains open to which extent these latter effects have been directly exterted by melatonin or indirectly by the sirtuin. SIRT1 is also known to have anti-inflammatory properties and its inactivation by posttranslational modification is believed to promote inflammaging (Hwang et al., 2013). Similar increases of SIRT1 were obtained in the pancreas of SAMP8 mice (Cuesta et al., 2013). Although SIRT1 and melatonin appear to be negatively coupled, when studied in tumor cells, this may not be a contradiction when analyzed on a chronobiological basis (Hardeland, 2013a). The observed upregulation of this sirtuin by melatonin in the context of aging might indicate an additional anti-inflammatory action of the methoxyindole in senescence. Notably, both melatonin and the SIRT1 ligand resveratrol were reported to protect cultured neurons from SAMP8 mice against frailty-inducing agents, including buthionine sulfoximine, which depletes reduced glutathione, and mitochondrial toxins (Cristò fol et al., 2012). The toxicological effects were more pronounced in SAMP8 than in the normally aging, otherwise isogenic SAMR1 mice. These authors also described upregulation of SIRT1 by both melatonin and resveratrol and counteractions by the SIRT1 inhibitor sirtinol. These findings are in line with directly studied anti-inflammatory effects of melatonin in SAMP8 mice obtained, however, in peripheral organs. In the liver, IL-1b and TNFa were downregulated and IL-10 upregulated by melatonin . Corresponding antiinflammatory effects were observed in the pancreas  and heart  of this strain. Collectively, these studies are in favor of the idea that melatonin behaves in a primarily anti-inflammatory way in the context of normal aging. What has been observed in the peripheral organs, may also apply to the brain, although the experimental basis for this concept is still limited. Whether the same may be the case under advanced neurodegenerative conditions, remains to be studied.

Prevention of inflammation initiation by melatonin
With regard to neuroinflammation, it is important to distinguish between the causes of initiation and the selfsustaining, continually aggravating processes in disease progression. This distinction is also of relevance to the perspectives of medicinal intervention. The chances for a successful therapy are presumably much better in early than in advanced stages. Progressed inflammation has already led to numerous deteriorations and, in diseases with pathological accumulations of misfolded proteins and intracellular cytoskeletal tangles, to dysfunctions and dysregulations that perpetually re-stimulate inflammatory responses.
Beyond infectious causes, inflammation via primary microglia activation that is not initiated by an interplay with neurons or astrocytes seems to be the exception. However, this possibility cannot be generally ruled out with regard to SASP, since damage of DNA does not only occur upon oxidative stress, but also takes place as a stochastical process that hits here and there at one or other cell. Nevertheless, one of the most frequent causes of microglia activation and resulting inflammation is neuronal overexcitation, e.g., by overshooting glutamatergic stimulation, which may also be caused by insufficient glutamate reuptake by astrocytes. Another important cause of neuroinflammation is mitochondrial dysfunction, which may be associated with overexcitation, enhanced NO formation and Ca 2+ overload. It leads to enhanced electron dissipation and, thus, formation of ROS, peroxynitrite and additional peroxynitrite-derived free radicals Hardeland, 2009a,). Although apoptotic cell death does not lead to inflammation, and although mitochondrial dysfunction can be attenuated by mitophagy, the spreading of ROS and RNS may suffice to initiate microglia activation. Moreover, the mitochondrial changes are associated with imbalanced fusion/fission homeostasis and peripheral loss of these organelles causing functional impairments of neurotransmission (Hardeland, 2009b). Melatonin is known to antagonize both excitotoxicity and mitochondrial dysfunction Hardeland and Coto-Montes, 2010). Therefore, it may halt early stages of neuroinflammation.

Antiexcitatory actions
The antiexcitatory actions of melatonin comprise different mechanisms. They have not only been studied in the context of sedation, but also of anticonvulsive effects (Fariello et al., 1977;Golombek et al., 1992aGolombek et al., ,b, 1996Muñ oz-Hoyos et al., 1998;Molina-Carballo et al., 2007;Solmaz et al., 2009) and they may be also partially related to anxiolytic, antihyperalgesic and antinociceptive properties of the methoxyindole (Golombek et al., 1991a(Golombek et al., ,b, 1993Pang et al., 2001;Papp et al., 2006;Ulugol et al., 2006;Srinivasan et al., 2010). The antiexcitatory effects should not be confused with sleep-inducing and -promoting effects, because they are also observed in nocturnally active animals, in which high melatonin levels are associated with physical exercise and neural activity. Instead, the antiexcitatory function should rather be understood in terms of setting limits to overexcitation and, thus, to avoid all the negative consequences of excitotoxicity and possibly resulting in nitroxidative stress, mitochondrial impairments and microglia activation.
The anticonvulsant actions of melatonin seem to be, at least partially, mediated by the membrane receptors, MT 1 and/or MT 2 , because this property was also reported for ramelteon, a synthetic, MT 1 /MT 2 -selective melatonergic agonist (Fenoglio-Simeone et al., 2009). The antiexcitatory/antiexcitotoxic functions steered by melatonergic signaling may likewise largely depend on these receptors, although this has not always been demonstrated directly. Among these, the inhibition of nNOS (Leó n et al., 2000;Acuñ a-Castroviejo et al., 2005;Entrena et al., 2005;Jimé nez-Ortega et al., 2009) may be of particular importance. However, this effect seems to be only effective as soon as nNOS is strongly upregulated, since the normal circadian maximum of NO in the rat CNS almost coincides with that of melatonin (Clé ment et al., 2003;Hardeland et al., 2003). The value of this mechanism should be mainly seen in the prevention of overexcitation. Other antiexcitatory actions concern the modulation of GABA and glutamate receptors (Rosenstein and Cardinali, 1990;Molina-Carballo et al., 2007), in particular, decreases in cytosolic Ca 2+ via GABA c  or metabotropic glutamate mGlu 3 receptors , and GABAergic facilitation (Cardinali et al., 2008b). Using patch clamp techniques and measurements of cytosolic Ca 2+ , inhibitory effects on high voltage-activated calcium channels were described in dorsal root ganglion neurons (Ayar et al., 2001). Moreover, modulations of the opioid system (Golombek et al., 1991a;Ulugol et al., 2006;Srinivasan et al., 2010Srinivasan et al., , 2012, changes in K + currents (Liu et al., 2007;Corthell et al., 2014), and, in retinal ganglion cells, an MT 2 -dependent potentiation of glycine receptor-mediated inhibitory post-synaptic currents Zhao et al., 2010;Ji et al., 2011) have been reported. Although the glycine-related effects are site-specific and may not be applicable to other CNS regions, they underline the more general antiexcitatory role of melatonin.

Mitochondrial protection
Mitochondrial function, integrity and intracellular distribution are central to various aspects of healthy aging, such as attenuation of aging-associated oxidative damage, free radical-induced microglia activation, neuronal overexcitation, losses of connectivity because of peripheral mitochondrial depletion and cell death. Melatonin interferes in multiple ways with these processes of deterioration. Thus, its mitochondrial actions that are relevant to aging and age-related diseases will have to be discussed in this section.
Mitochondrial dysfunction is both cause and consequence of excessive free radical formation and inflammatory responses. Therefore, any reduction of free radicals by melatonin, via diminished production of ROS and RNS, upregulation of antioxidant enzymes, avoidance of Ca 2+ overload, improvements of mitochondrial electron flux, radical scavenging or anti-inflammatory effects, contributes to the preservation of mitochondrial integrity and function. Antiapoptotic actions of melatonin are also associated with its modulation of the balance between pro-and antiapoptotic mitochondrial proteins of the intrinsic pathway of apoptosis, an influence on cardiolipin peroxidation, and prevention of a long-lasting breakdown of the mitochondrial membrane potential (DC mt ). For details regarding especially the progression of aging see Hardeland (2013a).
The protection of mitochondria is also important because of the high vulnerability of these organelles by oxidative and nitrosative damage. However, this is frequently misinterpreted in terms of its causes. The main reason is not an insufficient protection of mitochondrial DNA (mtDNA), which is, in fact, not naked, but rather relatively densely covered by mtTFA, the mitochondrial transcription factor A, which is structurally related to high mobility group (HMG) chromatin proteins and also serves functions in nucleoid structure, damage sensing and mitochondrial replication (Kang and Hamasaki, 2005). Other proteins bound to mtDNA are antioxidant enzymes that constitute integral components of the mitochondrial nucleoid and convey on-site protection (Kienhö fer et al., 2009). Moreover, investigations in mtDNA mutator mice revealed that the production of free radicals was not demonstrably increased in these animals during aging, relative to controls, although mitochondrial mutations had accumulated (Thompson, 2006).
Protection of mitochondria by melatonin has been described under various conditions. Two findings of potential relevance to changes that occur during aging are mtDNA copy numbers and attenuation of mitophagy, although they were not obtained in aged animals. Kainic acid-induced neurotoxicity in the mouse hippocampus, associated with mitophagy and a-synuclein aggregation, was antagonized by melatonin, which normalized the cellular content of mtDNA and cytochrome c (Chang et al., 2012). In a mouse model of opiate addiction, the progressive reduction of hippocampal mtDNA was reversed by melatonin, along with improvements of neuronal structure and functions (Feng et al., 2013). An important question is that of whether numerous findings obtained in models of sepsis, endotoxemia and other types of highgrade inflammation are really relevant to basic processes of aging. Damage and ETC blockade by the different ROS and RNS as well as their intramitochondrial sites of action and consequences to electron overflow have been extensively discussed elsewhere (Hardeland, 2009a(Hardeland, ,b, 2013aHardeland et al., 2009b;Hardeland and Coto-Montes, 2010). Because of its crucial importance, the role of cardiolipin in the breakdown of mitochondrial function should be emphasized. This compound is peroxidized earlier and more strongly than other mitochondrial lipids, which is explained by its interaction with cytochrome c, because the resulting proteolipid complex gains the function of a peroxidase Ott et al., 2007;Bayir et al., 2007;Kagan et al., 2009a,b). The peroxidation of cardiolipin was shown to precede that of other mitochondrial lipids, as demonstrated by a lipidomic approach (Samhan-Arias et al., 2011;Kagan, 2013). Moreover, damaged mitochondrial membranes externalize, by virtue of phospholipid scramblase-3, cardiolipin to the outer membrane, which represents an elimination signal for mitophagy . Cardiolipin peroxidation has been reported to be prevented by melatonin (Petrosillo et al., 2006(Petrosillo et al., , 2008Luchetti et al., 2007), but it remains uncertain whether this effect is based on a direct inhibition of the cytochrome c/cardiolipin peroxidase rather than radical scavenging (cf. Hardeland, 2013a). As discussed there, an alternative explanation might be deduced from the known upregulation of mitochondrial glutathione peroxidase (GPx), because overexpression of the respective subform, GPx4, strongly antagonizes cardiolipin peroxidation and cytochrome c release, whereas peroxidation was increased in heterozygous knockouts (Liang et al., 2009).
Formation of the primary free radical, the superoxide anion (O 2 À ), by mitochondria is a key step for processes leading to intraand extramitochondrial damage, which includes dismutation to O 2 and H 2 O 2 , a source of hydroxyl radicals ( OH), or combination with NO to peroxynitrite (ONOO À ), which decomposes after protonation to NO 2 and OH or, as a CO 2 adduct (ONOOCO 2 À ) to NO 2 and the carbonate radical (CO 3 À ), as summarized elsewhere (Hardeland and Coto-Montes, 2010; Hardeland, 2011b). Electron leakage from the ETC can occur either by overflow at Complexes I and III, especially because of bottlenecks of electron flux (Hardeland, 2009a;Hardeland et al., 2009a), or by permeability transition. The opening of the mitochondrial permeability transition pore (mtPTP) was originally regarded as a fatal event that ends up in apoptosis or, at least, mitophagy. However, mtPTP opening has been found to already occur under relatively basal conditions and was described as ''superoxide flashes'' . This process can be stimulated by oxidative stress . Meanwhile, the duration of the opening has turned out to be decisive. Usually a transient DC mt breakdown of short duration does not lead to apoptosis, but may be rather favorable, because it also represents a means for releasing unfavorable amounts of Ca 2+ from the mitochondrial matrix, whereas long-lasting Ca 2+ overload typically initiates cell death. Interestingly, melatonin inhibited prolonged permeability transition in astrocytes, whereas it still allowed transient mtPTP opening (Jou, 2011). This is in good agreement with melatonin's counteraction of apoptosis induction by the Ca 2+ ionophore, ionomycin (Jou et al., 2004). Moreover, melatonin was reported to directly inhibit mtPTP opening, at an IC 50 of 0.8 mM (Andrabi et al., 2004), a concentration that would require mitochondrial accumulation of melatonin. This has, in fact, already been observed Paradies et al., 2010;Venegas et al., 2012), at physiological levels of the circulating hormone in vivo (Messner et al., 1998). The fate of superoxide anions released to the intermembrane space has been reported to be influenced by melatonin, which activates Cu,Zn-superoxide dismutase present in this intermembrane compartment (Iñ arrea et al., 2011). However, this mechanism should not be relevant under conditions of superoxide flashes, because O 2 À also activates this enzyme and is present in sufficiently high quantities after a permeability transition (Hardeland, 2013a). Mitochondria are protected by melatonin in manifold ways. Instead of actions observed in models of high-grade inflammation, such as downregulation of iNOS, enhanced expression of respirasomal components and antioxidant enzymes which are reviewed elsewhere (Acuñ a- Castroviejo et al., 2003Hardeland, 2005Hardeland, , 2009aHardeland et al., 2009aHardeland et al., , 2011, the following considerations should be the focus of aging-related findings. A senescence-associated decay of mitochondrial functions is generally observed, a process which is particularly aggravated in neurodegenerative disorders, in which inflammation contributes considerably to the deterioration of neuronal function. In brain mitochondria of aged rats, melatonin prevented cardiolipin peroxidation and dysfunction resulting there from (Petrosillo et al., 2008). Moreover, it upregulated, in a comparable setting, superoxide dismutase (Ö ztü rk et al., 2012). In the same study, a decrease of GPx levels was reported that may be interpreted as a normalization, because a senescence-associated increase of oxidative stress leads to a rise in GPx. However, this finding contrasts with results obtained in brain mitochondria from the senescence-accelerated mouse strain SAMP8, in which GPx decreased by age and was upregulated by melatonin (Carretero et al., 2009). Similar results in other organs of SAMP8 mice are summarized elsewhere (Hardeland, 2013a). Most of the protection experiments concerning SAMP8 mitochondria have been carried out in peripheral organs, but data from brain regions do exist. Melatonin was shown to prevent the age-related rigidization of mitochondrial membranes , and elevated DC mt . In the hippocampus of SAMP8 mice, the numerical number and surface volume of mitochondria was increased by melatonin . Collectively, data from brain (Carretero et al., 2009) and other organs of SAMP8 mice (summary: Hardeland et al., 2013) show numerous additional improvements by melatonin, such as increased activities of Complexes I, III, and IV, elevations of respiratory control index, efficiency in ATP synthesis, ATP content and/or ATP/ADP ratio, ATP/ O ratio, higher state 3 respiration and lower state 4 respiration, reduced electron leakage, increased activities of GPx and glutathione reductase, augmentations of reduced glutathione (GSH) or the GSH/oxidized glutathione (GSSG) ratio, and attenuated lipid peroxidation. In conclusion, the numerous positive effects of melatonin at the mitochondrial level indicate that the initiation of inflammatory processes via dysfunction of these organelles and resulting overproduction of free radicals is strongly antagonized by melatonin.

Anti-inflammatory actions of melatonin in the CNS
The question of whether and, if so, to what extent melatonin may reduce inflammatory responses in the CNS is of crucial importance. The answer has to give credit to the fact that melatonin can exert both pro-and anti-inflammatory effects, as outlined in a previous section. Without considering the specific situation in aging, the general experience has been that melatonin rather promotes low-grade but strongly antagonizes high-grade inflammation, as described by the ''buffering hypothesis'' (Carrillo-Vico et al., 2013). During normal aging and in the earlier stages of neurodegenerative disorders, the immunologically relevant changes have to be classified as low-grade inflammation and may also be regarded as, to a certain extent, atypical. Nevertheless, the evidence from studies in aging animals mostly speak for a predominantly anti-inflammatory, antioxidative and mitochondrial-protective role of melatonin (Petrosillo et al., 2008;Cheng et al., 2008;Tajes et al., 2009;Carretero et al., 2009;Ö ztü rk et al., 2012). Unfortunately, most of the direct evidence for antiinflammatory actions that are based on reductions of proinflammatory cytokines has been obtained in peripheral organs Forman et al., 2010Forman et al., , 2011. However, a more general effect of melatonin that concerns various aspects of senescence including inflammaging may be central importance, in peripheral organs as well as in the brain. Signaling via NF-kB has been shown to drive the majority of genes related to SASP and to be multiply involved in processes of aging, also in the CNS (Adler et al., 2007;Freund et al., 2011;Franceschi and Campisi, 2014). Melatonin has been shown to be a potent suppressor of NF-kB expression (Deng et al., 2006;Korkmaz et al., 2012).
Moreover, conclusions on a mostly anti-inflammatory role of melatonin in aging are supported by its antagonism to insulin resistance (Cuesta et al., 2013), which is in good agreement with counteractions against metabolic syndrome, prediabetic states and diabetes type 2 (Nishida et al., 2002;Nishida, 2005;Peschke et al., 2007;Peschke, 2008;Robeva et al., 2008;Korkmaz et al., 2009;Shieh et al., 2009;Peschke and Mü hlbauer, 2010;Hardeland et al., 2012). These new insights concerning a relationship between insulin resistance and low-grade neuroinflammation, which consequences to the development of AD (Clark and Vissel, 2013;Jiang et al., 2013;De Felice et al., 2014;de la Monte and Tong, 2014;Ferreira et al., 2014) indicates a substantial role of melatonin as an anti-inflammatory agent in the context of aging. An overview of melatonin's actions that antagonize brain inflammaging is presented in Fig. 1.

Actions in experimental models
In the brain, anti-inflammatory actions of melatonin have been described in several animal and cell culture models. Some studies shall be mentioned in advance, in which melatonin's antiinflammatory actions were investigated in high-grade inflammation induced by infection, transient focal ischemia, or endotoxemia, although such models may not be translated to normal aging and only with caution to advanced stages of neurodegenerative diseases. In acute Klebsiella pneumoniae meningitis, melatonin strongly inhibited, in the rat hippocampus, microglia activation (criterion: isolectin-B4 histochemistry), the microglial rise in cytosolic Ca 2+ , and reduced, in both hippocampus and serum, the levels of TNFa, IL-1b and IL-6 (Wu et al., 2011). The reduction of inflammation was associated with lower numbers of apoptotic and higher counts of cytochrome oxidase-positive neurons, indicating a support of mitochondrial function. In a median nerve injury model, TNFa, IL-1b and IL-6 were decreased by melatonin in the rat cuneate nucleus (Chiang et al., 2013). In the dentate gyrus of ovariectomized rats, which exhibit higher levels of proinflammatory cytokines than age-matched control animals, melatonin also decreased TNFa, IL-1b and IL-6 and, additionally, upregulated SIRT1 mRNA expression (Kireev et al., 2014). Substantial attenuations of microglia activation were also observed after hypoxiaischemia (Balduini et al., 2012) or ischemia-reperfusion, along with reduced neutrophil and macrophage/monocyte infiltration (Lee et al., 2007). Melatonin antagonized several lipopolysaccharide (LPS)-induced immunological changes, such as expression of COX-2, microsomal PGE synthase-1, and iNOS in the rat astrocytoma cell line C6 (Niranjan et al., 2012), rises in TNFa and IL-1b in several rat brain regions (Tyagi et al., 2010), and expression of three chemokines (CCL2, CCL5, CCL9) in the murine microglial cell line BV2 (Min et al., 2012). Another finding of possibly fundamental importance for the protective role of melatonin in neuroinflammation has been obtained in LPS-treated rats (Pinato et al., 2013). In the cerebellum, which has previously discussed as one of the extrapineal CNS sites of melatonin formation , these authors showed that LPS induced aralkylamine N-acetyltransferase (AANAT) and melatonin formation in this brain region, but not in cortex and hippocampus, although circulating melatonin in the blood plasma was decreased by this treatment. The induction of cerebellar melatonin formation was not prevented by pinealectomy. The physiological melatonin levels formed in response to LPS were sufficient to protect cerebellum but not cortex and hippocampus against neuronal loss, and the effect was abolished by the melatonergic antagonist luzindole (Pinato et al., 2013).
Several publications have studied anti-inflammatory actions of melatonin in microglial cell lines. In BV-2 cells, Yan et al. (2013) investigated the increase of oxidative stress by fluoride, showing rises in NADPH oxidase, iNOS activity, the release of TNFa and IL-1b as well as downstream effects on c-Jun N-terminal kinase (JNK) phosphorylation, which were antagonized by melatonin. In HAPI cells, melatonin inhibited the amphetamine-induced rise in iNOS (Tocharus et al., 2008) and the methamphetamine-induced overexpression of TNFa, IL-1b and IL-6 (Tocharus et al., 2010). Similar upregulations of iNOS and TNFa by methamphetamine and their suppression by melatonin were also observed in SH-SY5Y neuroblastoma cells (Permpoonputtana and Govitrapong, 2013).
With regard to the newly emerged nexus between insulin resistance and neuroinflammation, as mentioned in Section 3, a Fig. 1. Overview of the multiple actions of melatonin that antagonize brain inflammaging.
R. Hardeland et al. / Progress in Neurobiology xxx (2015) xxx-xxx 9 study on experimental diabetic neuropathy is of particular interest. In the sciatic nerve, melatonin suppressed a number of inflammatory responses, including rises in NF-kB expression and inhibitor of NF-kB-a (IkB-a) phosphorylation, the decrease of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), increased expression of iNOS and COX-2, as well as elevated levels of TNFa and IL-6 (Negi et al., 2011). Although some of melatonin's actions may have been related to its antioxidative effects, others are clearly associated with a counteraction of inflammation. This distinction may be only of interest from a mechanistic point of view, because antioxidant and anti-inflammatory actions are in any case intertwined in systems that can respond by inflammation.

Actions in neurodegenerative diseases
Effects of melatonin in neurodegenerative diseases have been repeatedly reviewed in their multiple aspects (Mayo et al., 2005a;Srinivasan et al., 2005Srinivasan et al., , 2006Srinivasan et al., , 2011Cardinali et al., 2010;Pappolla et al., 2000;Esposito and Cuzzocrea, 2010;Rosales-Corral et al., 2012;Lin et al., 2013). Several of the beneficial effects described are poorly or only indirectly linked to inflammation. This may be by support of the circadian system, which is deteriorating by age and even more under conditions of neurodegeneration, and in which melatonin can improve sleep and behavioral symptoms such as sundowning. Other indirect effects concern the support of mitochondrial function and intraneuronal distribution, along with reductions in ROS formation and mitophagy, prevention of tau hyperphosphorylation, antioxidant and antiamyloidogenic actions. Although this functional spectrum is, at certain points, either connected to inflammation or even may, under conditions of improvement by melatonin, substantially reduce neuroinflammation, the whole body of evidence would exceed the scope of this article and shall not be repeated here in full. Instead, the focus shall be laid on described reductions of inflammatory signals. In this context, it should also be recalled that melatonin is more strongly reduced in most neurodegenerative disorders, with the exception of PD, relative to age-matched control subjects (Hardeland, 2012), as discussed is Section 3. Therefore, neurodegeneration is frequently associated with a relative melatonin deficiency that has to lead to specific functional losses which may be relieved by melatonin supplementation. It should be also noted that the relationship between melatonin and SIRT1, as it appears to exist in aging nontumor cells (Chang et al., 2009;Tajes et al., 2009;Cristò fol et al., 2012;Cuesta et al., 2013;Kireev et al., 2014), may extend to the also observed age-associated decreases in SIRT1 and its cosubstrate, NAD + , changes that have been implicated in neurodegenerative disorders, too (Imai and Guarente, 2014;Herskovits and Guarente, 2014;Rehan et al., 2014). These reductions in SIRT1, NAD + , and the rate-limiting enzyme of NAD + formation, nicotinamide phosphoribosyltransferase (NAMPT), are additionally associated with circadian dysfunction and mitochondrial impairments, changes that may be corrected by melatonin.
An important, both direct and indirect inflammatory signal with particular importance to AD are Ab oligomers, with a contribution of amyloid plaques. Microglia activation is induced by Ab peptides/oligomers  and can be easily achieved by Ab 1-42 injection (McLarnon, 2014). In both astrocytes and neurons, Ab 1-40 and Ab 1-42 oligomers stimulate NADPH oxidase, thereby increasing ROS production and causing secondary effects of oxidative stress (Narayan et al., 2014). Moreover, astrocytes respond to Ab 25-35 by upregulating Ab 1-42 formation and secretion (Dal Prà et al., 2014). In addition to the peptides, amyloid plaques are also capable of activating microglia and cause release of proinflammatory cytokines (Rodriguez et al., 2014). Not only are micro-and astroglia activated by Ab oligo-and polymers, but also neurons were shown to be stimulated to express inflammatory molecules, including TNFa, IL-1b, COX-2, and the T-cell and monocyte attractant chemokine CX3CL1 (Hanzel et al., 2014). For this reason, any reduction of Ab formation should also attenuate neuroinflammation and oxidative stress. While several antifibrillogenic effects by melatonin are known (reviewed in: Srinivasan et al., 2006;Rosales-Corral et al., 2012), only indirect evidence is available concerning the formation of Ab. In the pheochromocytoma cell line, melatonin caused a decrease in the expression of b-amyloid precursor protein (APP) mRNA (Song and Lahiri, 1997;Lahiri, 1999). To what extent these findings can be translated to and reproduced in the brain, is of considerable importance.
Several publications have described beneficial effects of melatonin in AD models of single, double or triple transgenic mice (Matsubara et al., 2003;Feng et al., 2006;Olcese et al., 2009;Dragicevic et al., 2011;García-Mesa et al., 2012;Peng et al., 2013). However, the relationship to brain inflammaging largely remains a matter of indirect interpretations. Perhaps, findings of reduced Ab aggregation and oxidative stress markers in melatonin-treated transgenic animals (Olcese et al., 2009) may be indicative of an anti-inflammatory action. This conclusion may be in line with similar results obtained after injection of Ab 1-42 , Ab 1-40 or Ab 25-35 peptides (Masilamoni et al., 2008;Gunasingh et al., 2008;Jun et al., 2013), although some of these findings have to be critically seen with regard to dosage.
A specific aspect of microglia activation by Ab 1-42 consists of NADPH oxidase (Nox) activation. Melatonin was shown to inhibit this process by a previously unknown antioxidant effect. It reduced the translocation of the Nox subunit p47 phox to the plasma membrane by preventing its phosphorylation via the phosphatidylinositol 3-kinase (PI3K)/Akt cascade and, thereby its association with the subunits gp91 phox and p67 phox that are essential parts of the active Nox complex (Zhou et al., 2008). As a result, the formation of superoxide and secondary free radicals generated is considerably reduced and, thus, the oxidative stress component of inflammation diminished.
Effects of melatonin concerning the reduction of proinflammatory cytokines formed in response to Ab 1-40 were investigated in an organotypic mouse brain slice culture (Clapp-Lilly et al., 2001). Melatonin inhibited the Ab 1-40 -induced rises of both IL-1b and IL-6. Interestingly, no melatonin effect was observed in the absence of the Ab peptide, but instead unexpectedly caused a rise in IL-6 when given at the high concentration of 500 mM. The latter finding indicates another type of proinflammatory action by melatonin when administered in excess, a possibility that has not been sufficiently considered in a number of studies. Similar repressions of interleukin secretion were observed in a more recent study in organotypic slices from rat hippocampus, in which Ab 25-35 was applied (Bender Hoppe et al., 2010). This peptide, which also stimulates Ab 1-42 secretion (Dal Prà et al., 2014), caused microglial and astrocytic activation. The resulting enhanced release of TNFa and IL-6 was blocked by melatonin, along with other beneficial effects such as reduction of glycogen synthase kinase-3b (GSK-3b) activation and tau hyperphosphorylation. Attenuation of both GSK-3b activation and tau hyperphosphorylation by melatonin has been repeatedly observed, in different experimental settings, and seems to also comprise effects beyond the prevention of cytokine release (Deng et al., 2005;Li et al., 2005;Yin et al., 2006;Gutierrez-Cuesta et al., 2007;Peng et al., 2013). GSK-3b is known to act as an accessory component of cellular circadian oscillators (Hirota et al., 2008;Sahar et al., 2010;Kozikowski et al., 2011;Kinoshita et al., 2012). With regard to the effects of melatonin on central and peripheral circadian clocks Hardeland, 2013b), the pineal hormone can be assumed to influence GSK-3b activity additionally via the oscillators. In SAMP8 mice, melatonin also inhibited another tau kinase, cyclindependent kinase 5 (cdk5), and reduced the cleavage of its activator p35 to its hyperactivator p25 (Gutierrez-Cuesta et al., 2007). The suppression of p25 formation has been observed in experimental models of both Alzheimer's (Srinivasan et al., 2006) and Parkinson's disease (Alvira et al., 2006).
The suppression of TNFa secretion by melatonin may be additionally important to the production of Ab peptides. Combinations of TNFa and IFNg were shown to inhibit the release of soluble APP and to enhance the formation of Ab 1-40 and Ab 1-42 in neurons and astrocytes (Blasko et al., 1999(Blasko et al., , 2000. With regard to the proinflammatory cytokines, the time course is of particular interest. Although some changes were already present at 6 or 12 h, the increases in TNFa and IL-6 were extremely higher after 48 rather than 24 h of Ab 25-35 exposure (Bender Hoppe et al., 2010), a finding that may be interpreted in terms of a vicious cycle of the neuroinflammatory process. Nevertheless, the suppression of cytokine secretion by melatonin was still quite effective after 48 h, which may indicate melatonin's potential for breaking the positive feedback loop of the vicious cycle.

Formation of methoxylated kynuramines in the brain and the contribution of inflammation
One of the melatonin catabolic pathways is initiated by pyrrole ring cleavage, to yield N 1 -acetyl-N 2 -formyl-5-methoxykynuramine (AFMK), which is deformylated to N 1 -acetyl-5-methoxykynuramine (AMK). AFMK formation is possible by a remarkably large number of mechanisms, including enzymatic reactions (Hardeland et al., 2004, e.g., by indoleamine 2,3dioxygenase and myeloperoxidase, as well as various pseudoenzymatic and nonenzymatic reactions, including oxidation by free radicals and other oxidants. Deformylation of AFMK to AMK is also possible by enzymatic catalysis, involving arylamine formamidases or hemoperoxidases, and nonenzymatic reactions (Hardeland, 2010). Both AFMK and AMK had been discovered after injection of melatonin into the cisterna magna of rats (Hirata et al., 1974). Since the other melatonin metabolite known at this time, 6hydroxymelatonin, was not detected in that study, AFMK and AMK were concluded to represent major brain metabolites of melatonin. Meanwhile, it is known that 6-hydroxymelatonin is also formed in the brain, but, surprisingly, sulfated to 6-sulfatoxymelatonin (for details and discussion see: Hardeland, 2010). Meanwhile, some doubts had arisen as to whether these high amounts of AFMK and AMK detected in the original publication are generally present in the brain, although the finding is reliable. A possibility may consist in the activation of melatonin-catabolizing enzymes or generation of free radicals in the presence of very high concentrations of the methoxyindole (Hardeland, 2010). However, little is known about the effects of melatonin in unchallenged microglia, whereas the counteraction of proinflammatory treatments and signals has been repeatedly studied. Although macrophages, peripheral monocytes, cultured monocyte-derived cell lines and also postnatal microglia were reported to be activated by melatonin, resting microglia was believed to not respond (Shafer et al., 2001). Melatonin was found to be efficiently oxidized to AFMK by activated macrophages (Silva et al., 2004), but corresponding studies on microglia have not yet been conducted.
A relationship between AFMK and inflammation became evident when high AFMK concentrations were detected in the CSF of patients with viral meningitis (Silva et al., 2005). Interestingly, a negative correlation emerged between AFMK levels and the concentrations of the pro-inflammatory cytokines IL-1b and IL-8. Individuals with 10-50 nM AFMK had considerably higher levels of these interleukins than patients with more than 50 nM AFMK. It seems important to also take notice of the remarkably high amounts of AFMK detected in these subjects, since 50 nM is by orders of magnitude higher than nocturnal plasma concentrations of melatonin, which are maximally in the range of 1 nM in young individuals and frequently much less in middle-aged or elderly persons. This seems to indicate an accumulation of AFMK over time. If AFMK is formed as a consequence of inflammation, including oxidative stress resulting thereof, the negative correlation with pro-inflammatory cytokines might indicate an AFMK-related or -mediated dampening of the inflammatory responses. This assumption however, requires direct demonstration.
Nevertheless, it might be worth following this possibility in the future. AFMK is also capable of scavenging free radicals Burkhardt et al., 2001), although the antioxidant properties of its product AMK exceed those of AFMK (Ressmeyer et al., 2003;Hardeland et al., 2004). Moreover, several reports have demonstrated protection by AFMK against oxidotoxicity Onuki et al., 2005;Manda et al., 2007), excitotoxicity Onuki et al., 2005), mitochondrial dysfunction (Dragicevic et al., 2011), and prevention of radiation-induced inhibition of neurogenesis and memory impairment (Manda et al., 2008). The mitochondrial effects were notably observed in APP-expressing neuroblastoma cells. Mitochondrial protection was also shown in other systems with AMK (Acuñ a- Castroviejo et al., 2003;Tapias et al., 2009). AMK is also of particular interest as an antiinflammatory agent. It was reported to be a more potent COX inhibitor than acetylsalicylic acid (Kelly et al., 1984) and to downregulate COX-2 expression in macrophages (Mayo et al., 2005). Moreover, it downregulates iNOS (Tapias et al., 2009), in addition to antiexcitatory actions as a potent inhibitor of nNOS (Entrena et al., 2005;Leó n et al., 2006). It also scavenges NO very efficiently, thereby forming a stable product, 3-acetamidomethyl-6-methoxycinnolinone (AMMC), that does not re-donate NO like other nitrosated compounds including 1-nitrosomelatonin (Guenther et al., 2005;Hardeland et al., 2007). To what extent the potentially anti-inflammatory, antiexcitatory and mitochondrial protective actions of AMK are of relevance to the brain under conditions of low-grade inflammation and in neurodegenerative diseases remains to be clarified.

Inflammaging and the decay of the circadian pacemaker
A particular aspect of brain inflammaging concerns the impaired function of the circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), which is observed during normal aging and, even more, in neurodegenerative disorders. This change is frequently underrated, but may be of high relevance, because it systemically affects a host of other functions in the body. Several alterations can be involved in SCN insufficiency, from reduction of blue light perception to losses in signal transmission to the SCN (review: Hardeland et al., 2012), but a major factor is that of SCN degeneration. Because of the crucial role of the SCN in the control of mammalian pineal gland, dysfunction of the hypothalamic master clock strongly contributes to aging-or disease-related reductions of nocturnal melatonin secretion and changes in melatonin's secretory patterns and phasing (review: Hardeland, 2012). Although SCN deterioration is more strongly pronounced in AD and other forms of dementia, it seems that the relevance of this phenomenon is already high in normal aging (Skene et al., 1990;Skene and Swaab, 2003;Wu and Swaab, 2007;Hardeland, 2012Hardeland, , 2013a. In part, this may be related to changes in melatonin, but additional impairments of other rhythmic functions likely arise when a master clock decays. It has also been shown that the replacement of the poorly functional SCN of a senescent hamster by transplantation of a juvenile SCN not only restored the previously decomposed circadian rhythmicity, but caused a rejuvenation in terms of physical appearance and also extended the lifespan of the recipient (Hurd and Ralph, 1998). These findings impressively show how important the SCN and a well-operating circadian system are for preventing aging-related impairments.
The specific mechanisms of inflammaging in the SCN are poorly understood, compared to other brain regions. One may, of course, assume that the known processes of microglia and astrocyte activation contribute to the deterioration of the SCN. Nevertheless, an additional, chronobiological mechanism has emerged during the last years. The aging suppressor SIRT1 has been shown to act as an accessory component of cellular cicadian oscillators. It interacts transiently with the core oscillator complex BMAL1:CLOCK, is part of an amplifying positive feedback loop that involves NAMPT and NAD + and further influences the expression of the core oscillator gene Period2 (Per2) (Nakahata et al., 2008Grimaldi et al., 2009;Ramsey et al., 2009;Wijnen, 2009;Imai, 2010). More recently, it was shown that another sirtuin, SIRT3, can also participate in the NAD + cycle, thereby driving the rhythmicity of mitochondrial activity (Peek et al., 2013). Moreover, SIRT1 was shown to decrease with aging in the SCN of wild-type mice, with consequences to BMAL1 and CLOCK levels and circadian output functions (Chang and Guarente, 2013). Overexpression of SIRT1 prevented the aging-dependent circadian changes, whereas SIRT1 deletion in young mice led to decreased Bmal1 and Per2 expression and temporal patterns reminiscent of those in senescent animals (Chang and Guarente, 2013). Therefore, sirtuins such as SIRT1 and SIRT3 are obviously required for the maintenance of an appropriately operating circadian system at different levels including the coordinative function of the SCN and the crucial role of mitochondria in cellular metabolic adaptation. A major question that arises from these recent findings is that of the cause of the aging-related decline in S1RT1. Another parameter that decreases similarly during senescence is the melatonin level. Therefore, it is of utmost importance to confirm or reject results on upregulation of S1RT1 by melatonin in the context of aging, as discussed in Section 4, whereas suppression was repeatedly observed in tumor cells. In addition to its chronobiological role, SIRT1 has antiinflammatory properties and inflammaging is believed to be promoted by its absence (Hwang et al., 2013). If a positive relationship between melatonin and SIRT1 in aging nontumor cells is confirmed, either of these molecules may contribute to an attenuation of inflammation and, thus, inflammaging in the SCN. The next question of whether melatonin or SIRT1 tends to decline first, is to date entirely open. A SIRT1-depleted malfunctioning SCN would poorly stimulate the pineal gland, but also a reduction in melatonin secretion may impair SIRT1 expression in the SCN. It seems possible that both of these possibilities, i.e., primary decrease in either melatonin secretion or in SIRT1 expression, may lead to a vicious cycle that not only impairs the circadian pacemaker but ultimately other brain regions and organs, too, thus reducing the resistance to oxidative insults and aging-associated inflammatory responses.

Conclusion
Low-grade inflammation is a hallmark of senescence and contributes to brain inflammaging. Interindividual variability in the grade and progression of the underlying processes is high and seems to depend on a spectrum ranging from an IRP to an ''inverted IRP'', but may be overlaid by lifestyle and the occurrence of other diseases (Hardeland, 2013a). Brain inflammaging is considerably enhanced in neurodegenerative diseases, most impressively in AD. Interestingly, the degree of inflammation seems to be negatively correlated with the level of melatonin. This relationship also exists in diabetes type 2, which has now gained relevance with regard to the possible role of insulin resistance in the development and, perhaps, progression of AD (Clark and Vissel, 2013;Jiang et al., 2013;De Felice et al., 2014;de la Monte and Tong, 2014;Ferreira et al., 2014). However, with regard to the decline of melatonin, the distinction between cause and consequence is by far unclear to date. In several animal models including senescence-accelerated mice, melatonin has been reported to be protective at various levels, including mitochondrial protection and reduction of proinflammatory cytokines. The latter effect has also been obtained in models using Ab peptides, as outlined in Section 6.2. The primarily anti-inflammatory actions of melatonin, as reported, are surprising insofar as melatonin can behave conditionally either in a pro-or antiinflammatory way, with a mainly proinflammatory profile in lowgrade inflammation (Carrillo-Vico et al., 2013). The reasons for why primarily anti-inflammatory actions have been described in the aging brain deserves clarification. Several reasons may be discussed, but require experimental support. One possibility may be related to dosage. In the aging or senescence-accelerated animals, melatonin was usually administered via the drinking water, frequently at doses of 1 mg/kg/day. Although the time course of external melatonin in the blood was usually not followed, the effective dose may have been in favor of antiinflammatory actions, whereas lower endogenous levels may tend to enhance proinflammatory responses. However, marked acute inflammatory responses were observed, in experimental granulomatosis, also at an elevated dosis of 4 mg/kg (de la Rocha et al., 2004). Nevertheless, a treatment option may result from the anti-inflammatory actions observed in other experiments. Another possibility may be based on the assumption by Radogna et al. (2010), who discussed sequential actions of melatonin under low-grade inflammatory conditions. According to their concept, melatonin would behave in proinflammatory way under acute low-grade inflammation, but as an anti-inflammatory agent after chronification. Interestingly, melatonin also decreased proinflammatory cytokines in another type of low-grade inflammation induced by high-fat diet. In rats receiving 750 mg melatonin/day via the drinking water, levels of IL-1b, IL-6, TNFa, IFNg and also Creactive protein were decreased, whereas the previously reduced anti-inflammatory cytokines IL-4 and IL-10 were increased (Cano Barquilla et al., 2014). A limitation of inflammatory responses by physiological levels of melatonin may be also assumed because of elevated proinflammatory cytokine expression in aortic endothelial cells of pinealectomized rats , findings that would require confirmation in brain tissue and exclusion of other influences such as changes in the circadian system.
One of the problematic properties of chronic neuroinflammation has to be seen in its potential for aggravation by vicious cycles. Again, this seems to be particularly serious in AD, in which the released Ab peptides further stimulate neuroinflammation Hanzel et al., 2014;Rodriguez et al., 2014) and ROS production (Narayan et al., 2014), while inflammatory signals and cells may further enhance Ab levels and deposits (Eikelenboom et al., 2006;von Bernhardi, 2007;Choo et al., 2013). Whether a system containing positive feedback loops that progressively aggravate the disease condition can be halted at all after a certain stage has been reached may be doubted. The generally poor therapeutic outcome in the treatment of moderate to severe AD is indicative of this problem. Nevertheless, the stage of disease and start of intervention may turn out to be decisive. This has also become evident in an AD model using transgenic mice. When the animals were treated with melatonin relatively early in their life, this was partially successful in retarding symptoms and death (Matsubara et al., 2003), whereas no relevant improvements where stated when the treatment started later in life (Quinn et al., 2005). In any case the presence of a pathogenic transgene may not allow a higher curative efficacy, and what applies to a transgenic mice may not be equally valid for a diseased human. Nevertheless, melatonin may be nothing more than an agent for palliative improvements if treatment starts too late. If there is any chance for delaying symptoms in humans, given an early onset of therapy, remains to be shown. However, what may be critically seen in the case of advanced neurodegenerative disorders, may turn out to be of value during normal aging. However, this would require extensive clinical studies, in which subjects would have to be treated over long periods, from a timepoint on when the first symptoms of agedependent circadian dysfunctions become apparent, such as sleep disturbances or nocturia. It may also be difficult to control the treatment of persons who not yet perceive the changes as impairments of health over a long period of time.
With regard to the mechanistic aspects of melatonin effects in neuroinflammation and, more specifically, in brain inflammaging, a number of gaps have to be closed. If insulin resistance turns out to be as important in neuroinflammation as in diabetes type 2 and metabolic syndrome, improvements of insulin sensitivity by melatonin, as known from the latter area and discussed in Section 6, should be directly studied in the brain. Moreover, the spectrum of cytokines under control by melatonin should be expanded under conditions of aging and chronic neuroinflammation. First, it should be noted that microglia activation is not exclusively deleterious, but can also contribute to the clearance of senile plaques (Schlachetzki and Hü ll, 2009;Choo et al., 2013). As done in other studies on AD, anti-inflammatory cytokines should be studied as well. Moreover, one of the key regulators of neuroinflammation, IL-18, should be included in further investigations on immune modulation by melatonin in the CNS. Finally, another field with crucial relevance to inflammaging, namely, the roles of DDR and SASP deserve specific attention in future research on the aging CNS. This concerns both the in-depth analysis of the contribution of these newly emerged pathways to brain inflammaging and the elucidation of melatonin's role in this specific area, in which this direct and indirect antioxidant, neuroprotective and often antiinflammatory agent may be beneficial.