Melatonin is a methoxyindole secreted mainly, but not exclusively by the pineal gland. Once formed melatonin is not stored within the pineal gland but diffuses out into the capillary blood 35 and CSF 31. Melatonin arrives early in the CSF of the third ventricle as compared to that of the lateral ventricles. Levels of melatonin released to the CSF were found to be 5 to 10 (up to 30) times higher than those simultaneously measured in the blood 31, whereas spinal CSF values did not much deviate from those in the serum. These findings indicate uptake of melatonin by the brain tissue, perhaps also metabolization to other compounds, such as substituted kynuramines, which are thought to display protective properties. Brain tissue may have higher melatonin levels than other tissues in the body 32.

It must be noted that the levels of a relatively lipophilic substance like melatonin reaching neurons under physiological or pharmacological conditions can differ considerably from circulating hormone concentrations. In early studies using high-pressure liquid chromatography 36 or radioimmunoassay 3738, hypothalamic melatonin concentrations were found to be about 50 times greater than in plasma. Two compartments of melatonin have been proposed to exist, which differentially affect physiological functions: in the plasma, melatonin would mainly act on peripheral organs, whereas, in the CSF, it might affect neurally mediated functions at a much higher concentration. Evidence interpreted as supporting this view has been presented in a study demonstrating that melatonin levels in the CSF of the third ventricle were 20-fold higher than nocturnal plasma concentrations 39. Therefore, it seems necessary to distinguish strictly between melatonin concentrations in the circulation and in tissues 4041.

While tissue melatonin sometimes shows only moderate circadian amplitudes 404142, circulating melatonin exhibits one of the most pronounced circadian rhythms known, at least prior to aging. The peak concentration occurs at night and is higher in younger age (18–54 yrs). With some exceptions 4344, a strong decline of melatonin during aging has been consistently reported by many investigators 45464748495051.

The age-associated decline in melatonin production and the flattening of the melatonin rhythm may be major contributing factors to the increased levels of oxidative stress and associated degenerative changes seen at old age. However, individuals of the same chronological age can exhibit considerable deviations in the degree of senescence-associated functional impairment. Some discrepancies between findings of different investigators can be attributed to the interindividual variation in melatonin levels of the same age group 8.

It is the physiological age of an individual rather than the chronological age that determines one's melatonin production. The varying extent of degenerative changes of cells and tissues may correspond to differences of melatonin production in the body 8.

Melatonin is involved in the control of various physiological functions such as coordination of other circadian rhythms including that of the central pacemaker, the suprachiasmatic nucleus (SCN) 52535455, sleep regulation 5657, immune function 5859, growth inhibition of malignant cells 60, blood pressure regulation 6162, retinal functions 636465, modulation of mood and behavior 666768, free radical scavenging and other antioxidant actions 4041697071. Many effects of melatonin, especially those concerning the circadian pacemaker system, are mediated by the G_i-protein (alternately G₀ or G_q) coupled membrane receptors MT₁ and MT₂ 727374. Additional binding sites exist. A previously assumed membrane receptor MT₃ was shown to represent an enzyme, quinone reductase 2 75, which may participate in antioxidative protection through elimination of prooxidant quinones 4076.

Other effects may be related to nuclear receptors of lower ligand sensitivity, RORα, which exists in at least four variants, and RZRβ 7778, but in these cases functional significance and target genes are less clear. Effects on the immune system have been partially attributed to these nuclear binding sites, but membrane receptors are obviously also involved 5559. To which extent the upregulation of antioxidant enzymes depends on nuclear receptors deserves clarification in detail. γ-Glutamylcysteine synthase, the rate-limiting enzyme of glutathione biosynthesis, is stimulated by melatonin at the transcriptional level; EMSA (electrophoretic mobility shift analyses) data have shown melatonin-dependent rises in DNA binding not only of AP-1, but also of RZRβ/RORα 79.

Further effects of melatonin do not require any of these membrane and nuclear receptors, since the indoleamine is able to directly bind to calmodulin 80, thereby inhibiting CaM-kinase II, and, moreover, to cause activation and Ca²⁺-dependent membrane translocation of protein kinase C 81, at concentrations in the nanomolar range, at a near-physiological level. Since these effects are related to the long-known cytoskeletal changes induced by melatonin, they may become of interest with regard to the abnormalities in cytoskeletal architecture and phosphorylation of cytoskeleton-associated proteins in AD. This aspect may be of higher relevance than obvious at first glance. Since cytoskeletal alterations, as observed in AD as well as tau hyperphosphorylation and related upregulations in the MAP kinase pathway, can be mimicked by the protein phosphatase inhibitor okadaic acid 82838485, the counteraction by melatonin of okadaic acid-induced AD-like lesions seems to indicate a common level of action 8687. Interestingly, okadaic acid also caused oxidative stress, which was again antagonized by melatonin 88, in an MT₁-receptor-independent fashion 89.

The relationship between melatonin and Ca²⁺ and, thus, with the activity state of the cell may be more profound than previously thought. The indoleamine was also found to bind, with a physiologically relevant K_d of about 1 nM, to calreticulin, a Ca²⁺-binding protein not only present in the endoplasmic reticulum, but also in the nucleus 90. These findings may turn out to be relevant with regard to the involvement of Ca²⁺ overload in overexcited neurons and cell death in neurodegenerative processes.

Concerning the antioxidative protection against amyloid-β, agonists of the MT₁ and MT₂ membrane receptors without antioxidant properties were not effective in neuroblastoma cells and primary hippocampal neurons, so that the neuroprotective and antiamyloidogenic properties of melatonin appeared to be independent of these receptors 91.

AD is an age-associated neurodegenerative disease that is characterized by a progressive loss of cognitive function, loss of memory, and other neurobehavioral manifestations. In spite of a large number of studies undertaken, the etiology of AD is largely unknown. Many mechanisms have been proposed, including genetic predispositions (e.g., expression levels and subforms of presenilins and ApoE), inflammatory processes associated with cytokine release, oxidative stress, and neurotoxicity by metal ions 9293949596979899. Pathological manifestations of AD include extracellular plaques of β-amyloid and intracellular neurofibrillary tangles composed of abnormally bundled cytoskeletal fibers. The deposition of amyloid plaques is thought to destabilize neurons by mechanisms which require further clarification. Tangles are associated with hyperphosphorylation of tau, a microtubule-associated protein, and of neurofilament H/M subunits, processes that lead to misfolding and accumulation of these proteins, along with a disruption of microtubules 9498100101102103. With regard to oxidative stress, prooxidant properties of the free amyloid-β molecule (Aβ) may be decisive, which is Fenton-reactive due to bound copper, and can, therefore, lead to hydroxyl radical-induced cell death. Additionally, Aβ initiates flavoenzyme-dependent rises in intracellular H₂O₂ and lipid peroxides, which also cause radical generation 27104105. Rises in Aβ protein have, in fact, been shown to induce oxidative stress 106. Moreover, an impairment of neurotrophin activity on associated tyrosine kinase receptors has been suggested to represent an important factor in AD pathology 107108109110.

With regard to the involvement of oxidative stress in AD, melatonin represents an interesting agent, since it displays multiple properties by which oxidative stress is antagonized 334041. In addition, other actions of melatonin exceed this aspect, but seem to have a beneficial potential in AD, too, as will be discussed in detail.

Accumulation of aggregated Aβ and tau hyperphosphorylation are highly common phenomena observed during aging of primates and other mammals 111. Though Aβ contributes directly or indirectly to neuronal degeneration, the potential of amyloid to cause AD depends on the individual's susceptibility to Aβ-mediated toxicity 28. In AD brains, oxidative end products are found to be significantly elevated. Metabolites of lipid peroxidation and oxidatively modified proteins and DNA are abundantly present in post mortem brain samples of AD patients 510112113. Inflammatory reactions associated with microglia and generation of nitric oxide (NO)-derived radicals contribute to cell stress and seem to be important especially in the degeneration of proximal neurons 114. Nevertheless, the inflammatory component in AD is clearly different from normal inflammation, since some classical hallmarks such as neutrophil infiltration and edema are usually absent, whereas other characteristics including acute-phase proteins and cytokines can be identified 17.

It seems important to distinguish between the extra- and intracellular sources of oxidants. Extracellular attack of neurons by oxidants may result either from inflammatory responses or from free radicals formed by Fenton-reactive Aβ molecules 27. Intracellular oxidative stress seems to be indirectly caused by Aβ, effects that may involve receptors or other surface molecules able to transduce oxidotoxicity 115116117118. Recently AD has been related to mitochondrial dysfunction 87119. This conclusion is based on several lines of evidence. First, cells depleted of mitochondrial DNA become insensitive to Aβ toxicity 120. Second, cybrid cells (cytoplasmic hybrid cells: mitochondrial DNA-depleted recipients of mitochondria from other sources) containing mitochondria from AD patients have shown enhanced vulnerability to Aβ 121. Third, cybrids with mitochondria from sporadic AD, which is associated with lower cytochrome c oxidase activity, due to a defective gene 122123124125126, exhibit various other signs of mitochondrial dysfunction, such as disturbed Ca²⁺ homeostasis 122 and Na⁺/Ca²⁺ exchange 127, enhanced formation of reactive oxygen species 122128129, lowered mitochondrial membrane potential 130 and, sometimes, abnormal morphology 125131.

These abnormalities may now be seen under aspects reminiscent of other mitochondrial diseases associated with pathological oxidant formation [cf. ref. 132], since similar changes are also present in PD, HD and Friedreich's ataxia, and sometimes already demonstrated in respective cybrid models 124128131. Collectively, all evidence convincingly demonstrates that the neural tissue of AD patients is subjected to increased oxidative stress. Therefore, its attenuation or prevention should be the goal of a strategic treatment of this neurodegenerative disease. However, a simplistic concept aiming to reduce oxidative damage and its consequences by applying classical radical scavengers is obviously insufficient. Vitamins E and C have been used for the treatment of AD patients with only limited success. Although several studies demonstrated a reduction in lipid peroxidation 133134, epidemiological data showed only minor or no clear-cut effects 135136137138. Moreover, these compounds remained relatively inefficient in preventing Aβ toxicity and fibrillogenesis 139140141.

In this regard, melatonin and other structurally related indolic compounds, such as indole-3-propionic acid, proved to be more potent 28140142143144. This may not only be a matter of radical-scavenging capacity, but also involve additional effects of these compounds and, perhaps, their metabolites. In particular, antifibrillogenic effects were observed in vitro 140144, but also in vivo in transgenic mouse models 145146. Moreover, protection from Aβ toxicity was observed, especially at the mitochondrial level 91143. For these reasons, melatonin appears as an antioxidant of superior potency, with additional effects relevant to intervention in AD.

As pointed out, antioxidative protection is not limited to radical scavenging and must be seen in a broader context involving many different mechanisms. Melatonin exerts several actions which collectively contribute to the prevention of oxidative damage assuring survival of cells even under adverse conditions. We shall, therefore, analyze in detail the different sources of oxidative stress and damage which allow for a broad spectrum of different counteractions by melatonin and a specific response to this antioxidant and adaptogenic agent. It should be kept in mind that coincidence does not indicate causality: Oxidative damage may sometimes be the consequence rather than the cause of the pathology observed in neurodegenerative disorders, with the mechanisms of protection exerted by melatonin often being quite complex and even interdependent. Neuroprotective effects induced by melatonin may act in concert to reduce oxidative stress and damage. Since it is not easily possible to distinguish between direct and indirect antioxidant actions mediated by melatonin, it is of utmost importance not to arrive at preliminary conclusions, which do not reflect the complexity of the multiple responses to this highly potent neuroprotective agent.

For several reasons, the central nervous system (CNS) exhibits a relatively high susceptibility to oxidative stress. As mentioned, one of these is a high oxygen consumption rate that inevitably accounts for increased generation of free radicals. Moreover, the brain is relatively rich in polyunsaturated fatty acids, a property which is not unfavorable per se, but which can become problematic under oxidative stress; especially docosahexaenoic acid is easily peroxidized, and this process has been discussed in relation to neurodegenerative diseases including AD 5147148149. Lipid peroxidation was found to initiate secondarily oxidative protein modifications, particularly in the AD brain 14. Numerous publications have demonstrated that lipid peroxidation can be suppressed by melatonin, and much of this work has been carried out in the CNS 3371150. Fewer data are available with direct relevance to AD. Melatonin did not only antagonize tau hyperphosphorylation induced by the PI3 kinase inhibitor wortmannin, but also a wortmannin-dependent stimulation of lipid peroxidation 151. Again, such findings shed light on the complexity of actions. While suppression of lipid peroxidation may be seen, at first glance, solely as an effect of an antioxidant, perhaps only by radical scavenging, the relationship to altered protein kinase activities reveals the involvement of additional actions in the metabolism.

Another aspect of vulnerability of the CNS is related to the availability of other, enzymatic and low molecular weight antioxidants. Antioxidant enzymes usually attain only moderate activities in the brain, but are in any case not that low as sometimes incorrectly stated (especially with regard to catalase). Among low molecular weight antioxidants, glutathione levels are comparable to those of other tissues, but ascorbate is usually by one order of magnitude higher than in the circulation. This basically protective scavenger turns into an extremely unfavorable and prooxidant agent in the presence of elevated iron concentrations, since the reductant is driving a Fenton reaction-based redox cycling. Iron levels are high in certain brain areas and, in addition, damage to the brain tissue, ischemia or neurotrauma can further mobilize iron so that radical-dependent destruction of biomolecules and cell death are strongly enhanced 152. Counteractions by melatonin against damage by Fenton reagents have been repeatedly demonstrated 153154155.

These effects are related to the remarkable efficacy of melatonin to scavenge various free-radicals, in particular, the extremely reactive hydroxyl radical 69156157158159. This property, which has been repeatedly reviewed 334041160161162, extends also to carbonate radicals (CO₃•^-) 163, reactive nitrogen species and to actions of metabolites of melatonin, such as cyclic 3-hydroxymelatonin, N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and N¹-acetyl-5-methoxykynuramine (AMK) 40164165166167168. Carbonate radicals, which have been shown to interact with both melatonin and AMK, abstract electrons (melatonyl cation radicals formed by CO₃•^- were demonstrated) or alternately, hydrogen atoms. •NO exhibits nitrosation reactions with melatonin, AFMK and AMK, whereas peroxynitrite-derived radicals, such as •NO₂ and •OH (from ONOOH) or •NO₂ and CO₃•^- (from ONOOCO₂ ^-) lead to nitration of these molecules.

Another poorly understood, but possibly important field is that of interactions with other antioxidants. In both chemical and cell-free systems, melatonin was shown to potentiate the effects of ascorbate, Trolox (a tocopherol analog), reduced glutathione, or NADH, in a non-additive and synergistic manner 158165169170. These findings indicate multiple interactions, via redox-based regeneration of antioxidants transiently consumed. Also in vivo, under conditions of long-lasting experimental oxidative stress, melatonin was shown to prevent decreases in ascorbate and α-tocopherol levels 171. It would be important to know whether this effect, to date only shown in the liver, may be demonstrable in the CNS, too.

Contrary to classical antioxidants, melatonin exerts several additional effects, which contribute either directly or indirectly to the decrease of free radicals, and some of these actions are particularly relevant to or specific for the brain. Antioxidant enzymes were repeatedly shown to be upregulated by melatonin. While activities or gene expression of enzymes like Cu,Zn- and Mn-superoxide dismutases and hemoperoxidase/catalase were stimulated by melatonin in a highly variable, tissue-specific fashion and usually only moderately in the CNS 4041, glutathione peroxidase was consistently and considerably upregulated in the brain 4041160172173. Glutathione reductase was usually found to rise after glutathione peroxidase, perhaps reflecting a secondary control by GSSG 40174175176177178. Additional stimulations of glucose-6-phosphate dehydrogenase 176 and γ-glutamylcysteine synthase 79161177 indirectly support the action of glutathione peroxidase by providing reducing equivalents (NADPH) for the action of glutathione reductase and by increasing the rate of glutathione synthesis, respectively. In addition, melatonin downregulates prooxidant enzymes such as lipoxygenases 161177 and NO synthases 404176161176177179180181182183184185. In this way, oxidative and nitrosative damage is attenuated, not only by avoiding peroxynitrite-derived radicals, but also by reducing NO-dependent neuronal excitation, and by antagonizing inflammatory reactions. The antiinflammatory potential of melatonin extends to downregulation of cyclooxygenase 2, an effect which may represent an action of the metabolite AMK 40186, a substance which is additionally a cyclooxygenase inhibitor much more potent than acetylsalicylic acid 187. Signaling mechanisms of AMK, in terms of receptors and interactions of transcription factors with the cyclooxygenase-2 promoter, have not been investigated to date.

Especially in the brain, melatonin contributes indirectly to the avoidance of radical formation, owing to several actions which are frequently overlooked, but which may be highly relevant in practice. First, melatonin is known to exert pronounced antiexcitatory and antiexcitotoxic effects, associated with inhibition of calcium influx and NO release and, consequently, prevention of the enhanced, excitation-dependent generation of free radicals 40. Melatonin was shown to possess strong anticonvulsant properties and to counteract efficiently the actions of various excitotoxins 188. When analyzed in detail, neuroprotection by melatonin against excitotoxins turned out to be a superposition of antiexcitatory and direct antioxidant effects, as shown for glutamate and its agonists, ibotenate, kainic acid, domoic acid, and, in particular, also for quinolinic acid (summarized by Hardeland 40).

Indirect antioxidative protection in terms of radical avoidance may be also assumed for the chronobiological role of melatonin as an endogenous regulator of rhythmic time structures. The importance of appropriate timing for maintaining low levels of oxidative damage has been overlooked for quite some time. However, it turned out that temporal perturbation as occurring in short-period or arrhythmic circadian clock mutants leads to enhanced oxidative damage 76. This action may be particularly important under the aspect of melatonin supplementation in the elderly, who exhibits a strongly reduced amplitude in the circadian melatonin rhythm, and in the AD patients in which the circadian system is disturbed. Finally, radical avoidance under the influence of melatonin is also a consequence of mitochondrial effects, as exerted by the indoleamine and by its metabolite AMK.

With regard to the mitochondrial aspect of AD and other neurodegenerative diseases – concerning radical generation, excitation-dependent calcium overload and its consequences for the mitochondrial membrane potential and for the permeability transition pore (mtPTP), involvement in apoptosis and sensitivity towards excitotoxins including Aβ- the actions of melatonin at the level of this important cellular compartment deserve particular attention.

The electron transport chain (ETC) represents a major source of reactive oxygen species (ROS) within the cell, due to electron leakage towards molecular oxygen 189. Complexes I and III of the ETC have been identified as the two principal sites of superoxide anion (O₂•^-) generation 190. While much of the O₂•^- is released from complex III to either side of the inner membrane 191, the iron-sulfur cluster N2 of complex I appears to be the main site of O₂•^- release to the matrix 190192193194. This seems to hold also for the brain, at least, under normal conditions. The fate of O₂•^- can be different. A certain proportion re-donates electrons to the ETC at cytochrome c 195196. Another fraction is converted to H₂O₂ and O₂ by the mitochondrial, manganese-containing subform of superoxide dismutase (MnSOD) 197. However, H₂O₂ produced by cytosolic Cu,Zn-SOD can likewise enter mitochondria owing to its high membrane permeability. A certain amount of H₂O₂ is eliminated intramitochondrially by interaction with cytochrome c 195196, while another fraction should be detoxified by peroxidases; though, the destruction of this oxidant and potential source of hydroxyl radicals is never complete. A third fraction of O₂•^- combines with NO, having a similar affinity to this oxygen radical as SODs, to give peroxynitrite, a source of hydroxyl and carbonate radicals as well as NO₂, in other words, an additional origin of destruction and nitration of proteins 198 or aromates 168198.

Mitochondria are not only a major site of ROS generation, but also the primary target of attack for ROS and reactive nitrogen species (RNS) 199. Damage to the mitochondrial respiratory chain can either cause breakdown of the proton potential, opening of the mtPTP and, thus, induce apoptosis or lead to further generation of free radicals maintaining a vicious cycle, which ultimately also ends up in cell death 189, either of the necrotic or apoptotic type 200.

Findings of several investigators indicate that the neuroprotective role of melatonin in AD and PD is primarily due to mitochondrial effects. This is not only a matter of radical scavenging (see above) – which may support protection, but can be only of limited efficacy for reasons of stoichiometry – but also of additional actions exceeding the direct elimination of free radicals. Some of these are rather conventional, concerning protection of mitochondrial membranes and DNA from oxidative insults, stimulation of glutathione (GSH) synthesis and support of the reduction of oxidized glutathione (GSSG) [reviews: refs. 4041]. Some others may be also regarded as indirect antioxidant effects of melatonin, which are, however, based on the maintenance of mitochondrial electron flux, something that is notably observed even under adverse conditions 201202203204205206.

Melatonin's mitochondrial actions are taking place within the organelle. This statement is important since it strongly contrasts with many other antioxidants. Melatonin, disposing of a balanced amphiphilicity, crosses the cell membranes with ease and may be able to concentrate within subcellular compartments 207. Mitochondrial accumulation has been discussed 205, but this issue has not yet been finally settled. Its amphiphilicity may allow melatonin to act at or even within the membrane. Whether effects on the fluidity of the mitochondrial inner membrane 208 reflect such a property is uncertain, since these experiments were performed under oxidative stress, which leads to membrane rigidization. Moreover, [¹²⁵I]-iodomelatonin was shown to bind to mitochondrial membranes 209. It will be of future importance to study directly the entrance, penetration and presence of melatonin in mitochondrial inner membranes.

Effects of the indoleamine on electron flux seem to have, at least, two aspects. Melatonin administration increased the activities of mitochondrial respiratory complexes I and IV in a time dependent manner in brain and liver 204205210. However, these results were obtained in submitochondrial particles and, therefore, reflect activities of some more or less isolated proteins islets in the membrane, but not natural electron flux. What they do show is an improvement of electron transport capacity by melatonin. This is the more remarkable as these effects were also observed in aging and, especially, senescence-accelerated mice 211212213. Some studies of this type were also accompanied by determinations of ATP 204205210. With due caution, which is necessary because of the fact that a measured ATP concentration does not necessarily reflect ATP production rates, these results seem to indicate that also ATP formation is, in a sense, safeguarded by melatonin. If relevant, such effects should be also detectable at the level of the proton potential. In fact, processes perturbing the mitochondrial membrane potential such as calcium overload, either due to overexcitation, to protein misfolding or to damage by free radicals, are antagonized by melatonin. In cardiomyocytes, astrocytes and striatal neurons, melatonin prevented calcium overload 214215, counteracted the collapse of the mitochondrial membrane potential induced by H₂O₂ 214, doxorubicin 216 or oxygen/glucose deprivation 215, and also inhibited the opening of the mitochondrial permeability transition pore (mtPTP), thereby rescuing cells from apoptosis. In addition to the antioxidant actions, melatonin directly diminished mtPTP currents, with an IC₅₀ of 0.8 μM 215, a concentration that would require mitochondrial accumulation of melatonin, as discussed above.

Such findings require explanations exceeding the conventional antioxidant concept. In a recently proposed model 4076, single-electron exchange reactions of melatonin are assumed to be the basis of interactions with the ETC, at low, quasi-catalytic concentrations. Under this perspective, radical scavenging by melatonin is not the principal, decisive property, but rather an indicator for melatonin's capability of undergoing single-electron transfer reactions. A cycle of electron donation to the ETC, e.g. at cytochrome c, followed by electron acceptance at N2 of complex I by the resulting cation radical was proposed. This cycle may reduce electron leakage at N2, with the cation radical as a potent competitor of O₂. Such a cycle would enhance the net electron flux through ETC by diminishing electron leakage, thus safeguarding the proton potential and ATP synthesis 404176. In fact, reduction of electron leakage by melatonin was stated in neuroblastoma cells 217. Moreover, similar properties were assumed for the melatonin metabolite AMK 404176, which also easily undergoes single electron-transfer reactions and which is sufficiently amphiphilic, too 166167. Mitochondrial protection was, in fact, demonstrated also for AMK 203.

Indole antioxidants such as melatonin and their kynuric metabolites have multiple effects on oxygen and energy metabolism in improving, supporting and maintaining mitochondrial function and integrity 40. In this context, the similarity of melatonin, its metabolites and other indolic as well as kynuric antioxidants to natural or synthetic electron and proton carriers such as ubiquinones and nitrones is remarkable and they all may be considered to act primarily as mitochondria-targeted bioenergetic agents 4076217. By enabling, catalyzing and safeguarding single electron transfer reactions these mitochondrial antioxidants may enhance energy and oxygen metabolism efficacy and thereby act as very potent adaptogenic agents with profound neuroprotective activity 4076217. Much like melatonin, nitrone and quinone compounds can prevent the mitochondrial toxicity of Aβ and thereby increase cellular viability and survival 2891120217. Since neuronal energy metabolism is strongly affected by Aβ 2891120217, the preservation of mitochondrial activity may be one of the most important features shared by indole, nitrone and quinone antioxidant agents 2891120217. In aging and dementia, melatonin as well as any other mitochondrial metabolism modifier with a similar pharmacological profile may be able to restore brain energy supply, an activity that would distinguish these compounds from conventional antioxidant agents devoid of such neurotrophic effects. Catalytic antioxidants acting at the mitochondrial level would thereby allow for enhanced neuronal survival and synaptogenesis even under a severe amyloid burden 28407076217. Since mitochondria are a primary source and target of oxidative stress and damage, much of the neuroprotection seen after melatonin treatment in experimental models of AD, PD and HD may be somehow related to the specific effects of this indoleamine and its kynuramine metabolites in maintaining energy and oxygen metabolism of the organelles even under adverse conditions related to the neuropathology of these diseases.

Several actions of melatonin have been described which antagonize the deleterious effects of Aβ. These actions concern different molecular processes, but may be interrelated; however, the possible connections require further investigation. One might classify the effects of melatonin as (i) antioxidant, including influences on mitochondrial metabolism, (ii) antifibrillogenic and (iii) cytoskeletal, including the suppression of protein hyperphosphorylation. Some of these actions were demonstrated at elevated, pharmacological concentrations, but any judgment of the relevance of such findings has to consider the relatively high rates of melatonin secretion into the CSF, uptake into the brain tissue and, presumably also, the metabolization to other protective compounds, such as the kynuramines AFMK and AMK 4041, processes which are impaired during aging and in neurodegenerative diseases.

Attempts of using melatonin for antagonizing Aβ effects were based on the initial observation that the peptide induces oxidative stress, which leads to damage of mitochondrial DNA, formation of protein carbonyl, lipid peroxidation, changes in mitochondrial membrane structure, changes in respiration and breakdown of the mitochondrial membrane potential, induction of antioxidant enzymes and heat-shock proteins 3106218219220221222223224. Notably, many of these findings were mitochondria-related. The pioneering work of Pappolla's research group first published compelling evidence for potent neuroprotection against the toxicity of Aβ in AD 218225. In fact, application of melatonin prevented the death of neuroblastoma cells exposed to Aβ peptide 91142225226. Similar results were obtained in astroglioma cells 227, findings of potential interest with regard to astrocyte-neuron interactions 228. The indoleamine significantly reduced several features of apoptosis, like cellular shrinkage or formation of membrane blubs 225. Additionally, lipid peroxidation in the cultured neuroblastoma cells was diminished, a finding first interpreted in terms of scavenging of free radicals generated by Aβ. However, and in accordance with our present point of view, the situation appears more complicated, since lipid peroxidation can also be a secondary consequence of mitochondrial dysfunction, and a support of mitochondrial integrity and electron flux should diminish the secondary formation of free radicals. It should also be noted that protection from Aβ-induced oxidative stress was achieved by the melatonin metabolite AFMK, too 164. The relatively high concentrations required in this case may be seen on the background of the redox properties of AFMK, which preferentially undergoes two-electron transfer reactions and, therefore, is a less potent radical scavenger than its product AMK; this type of experiments should be repeated with AMK, which correspondingly disposes of a higher protective potential in mitochondria [cf. ref. 40].

The second type of melatonin's antiamyloid actions concerns fibrillogenesis. Melatonin was shown by different techniques to inhibit the formation of amyloid fibrils, more efficiently than other, classical antioxidants 28140144229. Such effects were seen with both Aβ_1–40 and Aβ_1–42 peptides 229. A structural analog of melatonin, indole-3-propionic acid, sharing the property of a good radical scavenger 230, had a similar or even higher antifibrillogenic activity 142144. Despite the similarities in redox chemistry, the effects on protein structure cannot be easily attributed to radical scavenging and are by far not understood. Notwithstanding, inhibition of amyloid plaque deposition by melatonin was also observed in vivo, using a transgenic mouse model 145146. Therefore, the antifibrillogenic actions are not just in vitro effects, although relatively high, pharmacological doses were required in the transgenics. However, despite the obvious histologically and behaviorally evident protection in these independent studies, antiamyloidogenic effects were not seen when the treatment was started in old transgenic mice, after 14 months of life 231. In other words, after the disease has reached a certain severity, a substance like melatonin is no longer capable of efficiently antagonizing amyloid deposition and amyloid-dependent damage. However, nothing else should have been expected, after numerous amyloid plaques have been formed and neuronal damage has progressed. Consequently, one should see the value of melatonin mainly in its preventive potential rather than pinning unrealistic hopes on curative effects in later stages of disease. This does, however, not exclude symptomatic alleviations even in the progressed disease, concerning sleep, sedation, sundowning etc. (see below).

In the last years, the influence of lipoproteins on fibrillogenesis has received particular attention. Lipoproteins were found to interact with soluble Aβ, and levels of the free peptide may be crucial for parenchymal deposition 232. However, the respective composition of lipoproteins including their content in cholesterol and apolipoprotein subtypes can modulate fibrillogenesis. Melatonin was shown to reverse the particularly profibrillogenic activity of apolipoprotein E4 and to antagonize the neurotoxic combinations of Aβ and apoE4 or apoE3 140. ApoE4, which aggravates Aβ effects, is also produced by astrocytes. A mutual potentiation between Aβ protein and apoE4 may, thus, be regarded as particular kind of astrocyte-neuron interactions in AD 228.

The third aspect, suppression of protein hyperphosphorylation and cytoskeletal disorganization, has largely been studied in experimental systems aiming to mimic by pharmacological means the changes which are typical of AD. Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, not only induced cell death in two lines of neuroblastoma cells, but also mitochondrial dysfunction 828687 and other characteristics of AD cytoskeletal changes (see above). Addition of melatonin prevented the okadaic acid-induced decline in cell viability and mitochondrial metabolic activity, attenuated lipid peroxidation and protected cytoskeletal integrity 8687. Similar data were obtained in neuroblastoma N2a cells, using calyculin A, another inhibitor of the same protein phosphatases. This study revealed an activation of GSK-3 (glycogen synthase kinase 3), a redox-controlled enzyme involved in various regulatory mechanisms of the cell 233. Melatonin decreased not only oxidative stress and tau hyperphosphorylation, but also reversed GSK-3 activation, thereby showing that melatonin's actions exceeded its antioxidant effects, and also interfered with the phosphorylation system, especially stress kinases 233. Tyrosine kinase (trk) receptors, representing other, particularly important elements of the phosphorylation system, and neurotrophins were also shown to be affected by oxidotoxins, including Aβ. In neuroblastoma cells, melatonin was capable of normalizing trk and neurotrophin expression 109. In other experiments, tau hyperphosphorylation was induced by wortmannin 234 and isoproterenol 235; again, melatonin was found to attenuate this process.

Several studies show that melatonin levels are lower in AD patients compared to age-matched control subjects 236237238239240241. Decreased CSF melatonin levels observed in AD patients reflect a decrease in pineal melatonin production rather than a diluting effect of CSF. CSF melatonin levels decrease even in preclinical stages when the patients do not manifest any cognitive impairment (at Braak stages I-II), suggesting thereby that the reduction in CSF melatonin may be an early marker for the first stages of AD 242243. The reduction in nocturnal melatonin levels with the abolition of diurnal melatonin rhythmicity may be the consequence of dysfunction of noradrenergic regulation and depletion of the melatonin precursor 5-HT by increased MAO-A activity, as already seen in the earliest preclinical AD stages 242. Alternately, changes in the pathways of light transmission, from physical properties of the dioptric apparatus to a defective retino-hypothalamic tract or SCN-pineal connections have been discussed as possible reasons of declines in melatonin amplitude and corresponding changes in the circadian system 244. One should, however, be aware that light is inhibitory to the pineal 3552, so that dysfunction in the transmission of light signals would not easily explain a decrease in melatonin. In any case, the changes in melatonin secretion could contribute to some frequent symptoms like sleep disruption, nightly restlessness and sundowning seen in AD patients 245. Other reasons may be sought in an altered metabolism of AD patients, e.g., in relation to known genetic predispositions. The presence of apolipoprotein E-ε4/4, which is associated with enhanced Aβ toxicity and more rapid disease progression, also leads to considerably stronger declines in melatonin in the respective AD subpopulation than in patients with other apoE subtypes 238. From this point of view, the relative melatonin deficiency may appear as a consequence rather than one of the causes of AD, although the loss in melatonin may aggravate the disease. Decreased nocturnal melatonin levels were also shown to correlate with the severity of mental impairment of demented patients 246.

Despite the multifactorial etiology, the pronounced decline in nocturnal melatonin synthesis is common to AD patients. Therefore, the circadian system is impaired, and the circadian sleep-wake cycle is more strongly disturbed than in age-matched non-demented control subjects. The sleep-wake disturbances become more marked with progression of the disease. With the progressing neurodegeneration, the neuronal basis of the circadian system can be increasingly affected. Sleep-wake disturbances of elderly AD patients finally result from changes at different levels, such as reductions in the strength of environmental synchronizers or their perception, a lack of mental and physical activity, age- or disease-induced losses of functionality of the circadian clock. Cross-sectional studies have shown that sleep disturbances are associated with increased memory and cognitive impairment in AD patients 247.

AD patients with disturbed sleep-wake rhythms did not only exhibit reduced amounts of melatonin secreted, but also a higher degree of irregularities in the melatonin pattern, such as variations in phasing of the peak 239. Therefore, the melatonin rhythm has not only lost signal strength in clock resetting, but also reliability as an internal synchronizing time cue. Loss or damage of neurons in the hypothalamic SCN and other parts of the circadian timing system may account for the circadian rhythm abnormalities seen in demented patients 240248249, especially as the number of neurons in the SCN of AD patients is reduced 248250251.

Clinical findings strongly argue in favor of disruption of the circadian timing system in AD, since numerous overt rhythms are disturbed, including body temperature and concentrations of other hormones such as glucocorticoids 252253. Circadian alterations, which are detectable at an advanced stage of AD, also concern phase relationships, such as the phase difference between the rest-activity and core body temperature cycles, the last one being significantly delayed 248249. Another criterion for a weakened circadian system may be seen in the possibility of improving rhythmicity in AD patients by well-timed light treatment 254. In practical terms, this may be important as AD patients were found to be less exposed to environmental light than their age-matched controls 255, so that dysfunction of the SCN may be aggravated by low strength of the synchronizing signal light 256. In other words, the AD patient is gradually deprived of the photic input and even more of the non-photic, darkness-related internal signal melatonin.

A chronobiological phenomenon in AD observed in conjunction with disturbances of the sleep-wake cycle is "sundowning ", symptoms appearing in the late afternoon or early evening, which include reduced ability to maintain attention to external stimuli, disorganized thinking and speech, a variety of motor disturbances including agitation, wandering and repetitious physical behaviours and perceptual and emotional disturbances 254257. A chronobiological approach with bright light, restricted time in bed and diurnal activity represents a therapeutic alternative for the management of sleep-wake disorders in AD patients 256. Indeed bright light exposure in selected circadian phases markedly alleviated sundowning symptoms, such as wandering, agitation and delirium and improved sleep wave patterns in AD patients 258259260.

As outlined, melatonin acts at different levels relevant to the development and manifestation of AD. The antioxidant, mitochondrial and antiamyloidogenic effects may be seen as a possibility of interfering with the onset of the disease, although a balanced judgment requires due caution. While there can be no doubt that melatonin antagonizes Aβ toxicity and fibrillogenesis in vitro, at pharmacological levels also in vivo (see above), the beginning of treatment will be decisive [cf. ref. 231]. One cannot expect a profound inhibition of disease progression once a patient is already in an advanced demented state, notwithstanding a very few case reports with anecdotal evidence of slight mental improvements [cf. refs. 28261]. Whether melatonin exerts a preventive effect, is a hope, but can be judged only after extensive epidemiologic studies. The possibility exists that melatonin is particularly useful in a subpopulation which is more susceptible to oxidative stress for reasons of genetic predispositions, such as defects in mitochondrial genes, apolipoprotein variants etc., and an epidemiologic evaluation will have to consider this complexity.

At least, melatonin has several obvious advantages over other comparable compounds, in particular, most other antioxidants. Because of its balanced amphiphilicity, it crosses the blood-brain barrier and enters any cellular compartment, including mitochondria 2840262.

The question whether melatonin has a causal value in preventing or treating AD, affecting disease initiation or progression of the neuropathology and the driving mechanisms, remains to be answered in future studies. Double-blind multicenter studies are urgently needed to further explore and investigate the potential and usefulness of melatonin as an antidementia drug. Its apparent usefulness in symptomatic treatment, concerning sleep, sundowning etc., even in a progressed state, further underlines the need for such decisive studies.

Melatonin as a sleep-promoting agent has been tried in a small non-homogenous group of elderly patients with primary insomnia (3 mg p.o. for 21 days) associated with dementia or depression. Seven out of ten dementia patients having sleep disorders treated with melatonin (3 mg p.o. at bed time) showed a significant decrease in sundowning and reduced variability of sleep onset time 263. In another study, administration of 6 mg of melatonin to 10 individuals with mild cognitive impairment improved sleep, mood, and memory 264. Similar observations were made by other groups, too. Seven AD patients who exhibited irregular sleep-wake cycles, treated with 6 mg for 4 weeks, showed a significantly reduced percentage of nighttime activity compared to a placebo group 49. The efficacy of 3 mg melatonin/day at bedtime in improving the sleep and alleviating sundowning was shown in 11 elderly AD patients 265 and in 7 patients of another study 266. Long-term administration of melatonin in the dose of 6–9 mg to 14 AD patients with sleep disorders and sundowning agitation for a period of 2–3 years improved sleep quality 267. Sundowning, diagnosed clinically in all patients examined was no longer detectable in 12 patients. Another study on 45 AD patients with sleep disturbances, in which 6 mg of melatonin was given daily for 4 months, confirmed sleep improvement and suppression of sundowning 268. Along with these ameliorations, which can already be seen as an important improvement, also with regard to the efforts of a caregiver, the evolution of cognitive alterations in melatonin receiving patients seemed to be halted in several individuals, as compared to AD patients not receiving melatonin.

The major findings were confirmed in a double-blind study, with regard to sleep-wake rhythmicity, cognitive and non-cognitive functions 269. In a larger multicenter, randomized, placebo-controlled clinical trial, two dose formulations of oral melatonin were applied: 157 subjects with AD and nighttime sleep disturbance were randomly assigned to 1 of 3 treatment groups: (i) placebo, (ii) 2.5 mg slow-release melatonin, or (iii) 10 mg melatonin given daily for 2 months 270. In this study, a statistical problem became apparent, since melatonin facilitated sleep in a certain number of individuals, but collectively the increase in nocturnal total sleep time and decreased wake after sleep onset, as determined on an actigraphic basis, were only apparent as trends in the melatonin-treated groups. On subjective measures, however, caregiver ratings of sleep quality showed significant improvement in the 2.5 mg sustained-release melatonin group relative to placebo 270. Large interindividual differences between patients suffering from a neurodegenerative disease are not uncommon. It should be also taken into account that melatonin, though having some sedating and sleep latency-reducing properties, does not primarily act as a sleeping pill, but mainly as a chronobiotic. Since the circadian oscillator system is obviously affected in AD patients showing severe sleep disturbances, the efficacy of melatonin should be expected to also depend on disease progression.

The mechanisms that account for these therapeutic effects of melatonin in AD patients remain to be elucidated. Since the symptomatic actions become relatively rapidly apparent, they should be of mainly chronobiological nature. Melatonin treatment has been shown to promote mainly non-REM sleep in the elderly 56 and is found beneficial in AD by supporting restorative phases of sleep. Whether this includes in AD additional mechanisms known from non-demented elderly humans or animals, such as augmented secretion of GH 271 and neurotrophins 272, remains to be analyzed. The chronobiological aspect is underlined by a study on golden hamsters, in which melatonin was able to protect against the circadian changes produced by Aβ_25–35 microinjection into the SCN 273. From this point of view, changes in melatonin receptor density in AD – increases in arterial MT₁274 and decreases in hippocampal MT₂ 275 – may be less important than a remaining responsiveness of the SCN, perhaps in conjunction with the sedating effects of melatonin based on downregulation of neuronal NO synthase and actions on the GABAergic system 276. Regardless of the mechanistic details, all pertinent data unanimously direct to a sleep-promoting effect of melatonin in AD patients, as generally in elderly insomniacs [review: ref. 277].

Parkinsonism, the other major neurodegenerative disease, is caused by a progressive loss of dopaminergic neurons in the substantia nigra. We do not refer to this disease with the intention of extensively reviewing here all its facets, but rather to outline some parallels with and differences to AD concerning the actions and applicability of melatonin. Oxidative stress was shown to play a major role in PD, too 278279. In post-mortem samples of the substantia nigra from PD patients, lipid peroxidation and oxidative modification of proteins and DNA were increased 26280281, whereas GSH was decreased 282. A particular problem of vulnerability of the substantia nigra neurons is resulting from iron incrustations in the dopamine-derived melanins. The high levels of iron 280 are redox-active and generate hydroxyl radicals by the Fenton reaction. This situation appears to be aggravated by an enhanced rate of H₂O₂ formation, to which dopamine oxidation by MAO contributes 278.

Animal models of PD frequently use 6-hydroxydopamine (6-OHDA) to destroy the nigrostriatal pathway, or the neuronal oxidotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Behavioral and motor deficits in rats or monkeys, such as akinesia, rigidity and tremor, reminiscent of those seen in PD patients 283, are commonly used to study the efficacy of therapeutic agents used in this disease.

MPTP administered to rats is mainly taken up by astrocytes and is metabolized into the 1-methyl-4-phenylpyridinium ion (MPP⁺). This cation is selectively taken up by dopaminergic neurons. Its actions are predominantly based on redox cycling involving redox-active enzymes 284 and, in particular, mitochondrial effects especially at complex I, thereby causing increased generation of free radicals, depletion of NAD and ATP and apoptosis 285286. In the 6-OHDA model, the neurotoxin acts selectively on nigrostriatal neurons because it is substrate of the reuptake transporter, and induces cell death by increased generation of free radicals due to autoxidation. In this context, one of the limits of l-DOPA medication may be noticed, namely, the iron-mediated hydroxylation of dopamine to 6-OHDA in the substantia nigra of PD patients 287.

Among the studies undertaken in animal models, some of them support the possible beneficial effects of melatonin in arresting the neurodegenerative changes, while others report adverse effects of melatonin in exacerbating motor deficits. In an MPTP model, melatonin counteracted the induced lipid peroxidation in striatum, hippocampal, and midbrain regions 288. In a study using 6-OHDA, melatonin inhibited lipid peroxidation in cultured PC12 cells 289, effects that were associated with rises in antioxidant enzymes. Prevention of MPTP-induced dopaminergic cell death by melatonin was demonstrated by determining tyrosine hydroxylase levels and the number of normal DA cells 290.

The major MPP⁺ effect, inhibition of complex I, leads to enhanced electron leakage, decrease of mitochondrial electron flux and ATP deficiency. Rises in complex I activity, as observed with melatonin 201202203205, may antagonize this action and contribute to protection. Apart from such effects seen in submitochondrial particles, direct interactions of melatonin at N2 of complex I have been discussed 404176. In unilaterally 6-OHDA-injected, hemi-parkinsonian rats, protective effects by melatonin were also attributed to normalizations of complex I activity 291.

One should be aware that electron leakage at complex I causes secondary oxidative stress, by combination of the superoxide anions hereby formed with NO, to give peroxynitrite and radicals deriving from it (see above); in other words, a connection between mitochondrial dysfunction and the excitational vulnerability of the neuron becomes evident. In this regard, suppression of NO formation and scavenging of reactive nitrogen species by melatonin and its metabolite AMK [review: ref. 40] should additionally support cell survival, along with other protective effects, such as upregulation of the antioxidant enzymes Cu,ZnSOD, MnSOD, GPx, which has been demonstrated in cultured dopaminergic cells, too 289.

While complex I inhibition is a plausible cause of neurodegeneration in the toxicological animal models, it would be of particular importance to know whether mitochondrial dysfunction is relevant in the PD patient. In fact, decreases in complex I activity were reported for mitochondria from platelets and in the substantia nigra of parkinsonian individuals 292293294. However, recent investigations did not reveal any differences in complex I, II/III and IV activities in mitochondria from platelets 295, so that a genetically based dysfunction in electron transport is not evident. However, this does not entirely rule out striatal mitochondrial dysfunction in advanced stages of PD, because of an impairment by iron-mediated oxidative stress.

The pleiotropy of melatonin's antioxidant and otherwise protective effects is, on the one hand, a hindrance for relating cell survival to a particular, single mechanism in a given experimental situation, but, on the other hand, may give an impression of the powerful concerted actions of this indoleamine. Protection by melatonin was demonstrated in a variety of experimental PD models. [reviews: refs. 8933]. If studied in detail, the phenomenology of protection is complex, and melatonin may have acted on multiple targets, even though they may be partially interrelated. MPTP-induced stress was antagonized by melatonin at the levels of mitochondrial radical accumulation, mitochondrial DNA damage as well as breakdown of the proton potential 296. As already outlined in the context of AD, cytoskeletal abnormalities are associated and, to a certain degree, caused by oxidative stress, but represent an own type of phenomenology, with additional regulatory mechanisms and additional sites of possible intervention. Lewy bodies, which are considered cytopathologic markers of parkinsonism, comprise abnormal arrangements of tubulin and microtubule-associated proteins, MAP1 and MAP2. Melatonin effectively promotes cytoskeletal rearrangements and was, thus, assumed to have a potential therapeutic value in the treatment of parkinsonism, and, perhaps, generally in dementias with Lewy bodies 297.

Recently, a possible melatonin-sensitive link between mitochondria, hyperphosphorylation and neuronal apoptosis became apparent, with general implications for mental deficits. In a study conducted in cerebellar granular neurons, melatonin did not only antagonize MPP⁺-induced cell death, but also activation of Cdk5 and cleavage of p35 to the hyperactivator p25 298. This protein kinase which has received its name for reasons of homology, but is unrelated to the cell cycle, seems to play an important role in neuronal function and plasticity. Dysregulation of Cdk5 and, in particular, rises in p25 have not only been found to occur in parkinsonism, but also in other neurodegenerative disorders including AD 299300301. Moreover, inflammatory processes in the brain were shown to be associated with p25-dependent upregulation of Cdk5, along with tau hyperphosphorylation 302. These findings are not only relevant in terms of neurodegeneration, but also with regard to cognitive processes in general. Cdk5 was shown to be required for associative learning, and its transient activation by p25 facilitates hippocampal long-term potentiation, in conjunction with increases in the density of synapses and dendritic spines 303304305. However, this desirable effect on neuronal plasticity is turned into the opposite as soon as p25 formation and, thus, Cdk5 activation takes place for an extended period of time: a prolongued production of p25 led to synaptic and neuronal loss, impaired long-term potentiation and, consequenly, cognitive deficits 305306. Whether or not melatonin used at pharmacological concentrations in the MPP⁺ study 298 influences p25 and Cdk5 activity indirectly via mitochondrial actions and/or directly by receptor-mediated signal transduction pathways, remains to be elucidated.

While all experiments on MTPT- or 6-OHDA-induced oxidative stress unanimously report protection by melatonin 288289290307, the value of the pineal hormone may be judged entirely differently under systemic aspects. In rats treated with 6-OHDA or MPTP, pinealectomy or suppression of melatonin synthesis by bright light caused a remission of symptoms 308. The view that melatonin may be unfavorable in the case of parkinsonism, was further supported by respective experiments using the (putative) melatonin receptor antagonists ML-23 and S-20928, which, again, improved motor functions and, in the case of ML-23, prevented 6-OHDA-induced mortality 309310.

These findings show that antioxidative protection and even potentially beneficial mitochondrial effects do not suffice for judging the value of a drug under systemic aspects. The multiplicity of melatonin's actions, including the receptor-mediated ones, has to be a matter of responsible caution.

Melatonin secretion patterns have been studied in patients suffering from PD. A phase advance of the nocturnal melatonin maximum was noted in L-DOPA-treated but not in untreated patients, as compared to control subjects 311312313. Under medication with L-DOPA, daytime melatonin was additionally increased 313, a finding discussed in terms of an adaptive mechanism in response to the neurodegenerative process and possibly reflecting a neuroprotective property of melatonin 313.

In rats, fluctuations in serum melatonin levels were also related to variations in motor function and attributed to the interaction of monoamines with melatonin in the striatal complex 314. Melatonin's inhibitory effect on motor activity has been suggested as one of the possible causes for the wearing-off episodes seen during drug treatment of parkinsonism. Electrical stimulation of internal globus pallidus inhibited an increase in daytime melatonin in PD patients as compared to healthy subjects 315. Deep-brain stimulation of the internal globus pallidus had been shown to improve motor symptoms and complications in patients with Parkinson's disease 316.

Studies undertaken in elderly insomniacs have convincingly demonstrated that melatonin can increase sleep efficiency and decrease nighttime activity 317318. Administration of melatonin in 5 mg/day for 1 week reduced the nocturnal wake time for about 20 minutes in eight patients with PD 319. In a recent double-blind, placebo-controlled study on 40 subjects conducted over 10 weeks, Dowling et al. 320 noted that administration of a higher dose of melatonin, 50 mg/per day, increased actigraphically scored total nighttime sleep in PD patients, when compared with 5 mg or placebo-treated patients. Subjective reports of overall sleep disturbance improved significantly with 5 mg of melatonin compared to 50 mg or placebo 320. This study may indicate that very high doses of melatonin can be tolerated in PD patients over a 10-week period as in healthy older adults. Nevertheless, the caveat from the melatonin-antagonist studies (see above) remains and should be taken serious.

Among neurodegenerative disorders, HD is the most clearly mitochondria-related disease. Primary cause is a mutation in the huntingtin gene, leading to an extended polyQ repeat, which causes protein misfolding and secondary effects hereof. Although huntingtin misfolding has multiple consequences, including some concerning iron metabolism 321, mitochondrial dysfunction is particularly fatal, in an excitation-dependent way. Under high calcium load, mitochondria carrying huntingtin with an extended polyQ domain are no longer able to cope with calcium uptake; as a result, complex II/III activity is impaired 322323, the proton potential breaks down, and mtPTP-dependent cytochrome c release induces apoptosis 324325. Ca²⁺ dependence explains the relationship to NMDA receptor-mediated excitation, and the selective vulnerability of frequently excited neurons carrying this receptor 326327. For these reasons, excitotoxicity by quinolinic acid, which also acts via the NMDA receptor, has been used as a model of HD 328329330331. Additionally, quinolinic acid has strong prooxidant properties when complexed with iron 332, a finding that is, however, uncertain with regard to its in vivo relevance. An alternate experimental model, using 3-nitropropionic acid as a blocker at complex II 329333334, acts primarily at the mitochondrial level, but is, in our experience, sometimes affected by the problem of ATP deficiency as the primary cause of cell death. One should clearly see the differences between the two models. Quinolinic acid acts upstream of the mutated protein, and most of the oxidative stress measured at low dosage of the drug sufficient for causing excitotoxicity may be regarded as side or secondary effects in the compromised cell. 3-Nitropropionic acid aims to mimic the mitochondrial blockade caused by misfolded huntingtin under calcium overload. In this case, oxidative stress can result from multiple sources and may include enhanced electron leakage.

Melatonin was shown to prevent quinolinic acid-induced lipid peroxidation in rat brain homogenates 335 and cell death in the rat hippocampus 336. Since some effects of quinolinic acid differ from those of NMDA or glutamate and are obviously not mediated by its receptor, the question arose as to whether melatonin might protect mainly by antagonizing the NMDA receptor-dependent actions of the neurotoxin. Both quinolinic acid and NMDA induced lipid peroxidation in the rat hippocampus, but only damage by quinolinic acid was inhibited by melatonin; moreover, the action of melatonin was not inhibited by the MT₁/MT₂ blocker luzindole 337. Therefore, one should conclude that, at least, the induction of lipid peroxidation is not mediated via the NMDA receptor, nor the antagonizing effect of melatonin via its membrane receptors. Extensive lipid peroxidation after administration of quinolinic acid was not only seen in hippocampal, but also striatal and globus pallidum regions, again antagonized by melatonin, which additionally attenuated neurobehavioral signs associated with the neurotoxin 338. In brain tissue culture, melatonin antagonized the prooxidant effects of high doses of quinolinic acid, which strongly exceed the concentrations required for excitotoxicity 339. Lipid peroxidation induced by 3-nitropropionic acid in synaptosomes of rat striatal and cortical regions were attenuated by melatonin 340. Collectively, these results demonstrate the antioxidant capacity of melatonin, but the relevance for HD may greatly depend on the validity of the animal models for fully describing the situation in the disease. Melatonin's undoubtedly existing antiexcitotoxic properties are not clearly apparent in studies focusing on lipid peroxidation.