CAFFEINE AS A PHARMACOLOGICAL TOOL Caffeine is one of the most consumed psychoactive drugs and acts mostly by blocking adenosine

A1 and A2A receptors (A1R and A2AR respectively). These receptors have a crucial neuromodulatory role and regulate both synaptic transmission and plasticity either by directly modulating synaptic responses or by interfering with other receptors. Adenosine receptors are Gprotein coupled receptors that regulate, in opposite directions, the second messenger cAMP, while A1R are inhibitory Gi/o coupled thus decreasing cAMP levels,A2AR are excitatoryGs coupled, increasing cAMP levels. The activation of these receptors is also able to modulate Ca2+ channels and the phospholipase C pathway. Through these actions and by modulating both the release and the uptake of different neurotransmitters, the balance between adenosine A1 and A2A receptors activation, allows the ne tuning of synaptic transmission and plasticity in the hippocampus. In pathological situations, the balance in the adenosine neuromodulatory system is compromised and thus by blocking adenosine receptors, caffeine emerges as a potential drug to modulate brain function and cognition (Fig. 1). The widespread consumption of caffeine underlies the importance of studying the consequences of its chronic intake to the agingassociated cognitive decline, particularly in pathological conditions. Caffeine consumption increases alertness and improves cognition in physiological conditions. Chronic caffeine intake for one month (20mg/kg/day/L orally; described to reach9Min plasma and6M in the cerebrospinal uid which is equivalent in humans to approximately 23 cups of coffee/day is able to potentiate synaptic plasticity and spatial working memory in young adult rats, having also positive effects in the exploratory behavior in the elevated plus maze test (Fig. 2). However, only recently the relationship between its consumption and the pathophysiology of many neurodegenerative diseases has been rmly established. Caffeine consumption has benecial effects not only in aging, reducing the associated cognitive decline in healthy subjects, but also in AD patients. Different epidemiological reports highlight the inverse correlation between caffeine consumption and the incidence or severity of AD. These observations are supported by several animal studies. Caffeine is protective against Ab toxicity in different models of AD, possibly by decreasing Ab levels in plasma and brain. This translates into improvements of memory function in the animals pretreated with caffeine, when comparing to matched controls. The less severe age-associated decline in memory is also reduced by caffeine intake in animal models. In light of the reports on the effects of caffeine in AD subjects and in animal models, the rationale for using caffeine as a therapeutic strategy for AD is growing stronger. However, caffeine itself has some drawbacks. One of the major aspects is the non-selectivity of caffeine towards adenosine receptors thatmay lead to side effects, particularly at the cardiovascular level. Another one is the possibility of effects that are independent of adenosine receptors blockade. At higher doses, caffeine inhibits phosphodiesterases, increases intracellular calcium, and affects directly GABAA receptors (reviewed by). Despite this, the side effects of caffeine consumption

are described to fade away after 34 days and chronic consumption of low to moderate doses of caffeine appears to have no side effects. The mechanism of action of caffeine is still relatively unknown, however the blockade of A2AR seems to be the major mechanism through which caffeine acts. Caffeine effects are mimicked whenever A2AR selective antagonists are used [26, 27]. Both the pharmacological blockade or gene disruption ofA2ARconfers neuroprotection against Ab toxicity. Moreover, administration of Ab to A2AR KO mice does not induce the traditionally observed decits. This reinforces the importance of A2ARinA toxicity and thus drives the therapeutic research towards A2AR antagonists. ADENOSINE A2A RECEPTORS AS TARGETS FOR INTERVENTION A2AR are excitatory receptors, whose effects are exacerbated in situations of compromised brain homeostasis due to the increase in adenosine levels. In such situations, blockade of A2AR has been shown to be neuroprotective. The high abundance of A2AR in striatal areas, their ability to tightly regulate dopaminergic transmission, and the low levels of these receptors in other brain regions, render A2AR particularly interesting targets in Parkinsons disease (PD) [57]. The administration of A2AR antagonists attenuates the motor impairments in different animal models of PD and recent clinical trials are probing A2AR antagonists as an alternative to traditional therapeutic approaches. The evidence that A2AR have an important role in extra-striatal regions through the regulation of hippocampal excitability is not consensual. Under physiological conditions, there is now evidence that exogenous activation of A2AR with selective agonists facilitates the release of acetylcholine and enhances glutamatergic synaptic transmission in the hippocampus. However, their activation by endogenous adenosine does not contribute to the basal synaptic transmission unless brain homeostasis is compromised. Remarkably, in the hippocampus of aged animals the effects of adenosine A2AR on synaptic transmission are more pronounced, most likely due to the increased expression of these receptors in glutamatergic neurons. This is particularly relevant since cognitive decits, and particularlyAD, are tightly associated with the aging process. Thus, despite the low levels of A2AR in the hippocampus, they become relevant in noxious brain conditions. Notably, this is observed in animal models of AD [26], following Ab exposure [52] and, more importantly, it was also conrmed in the AD brain. Blockade of the A2AR seems effective against Ab toxicity. The neuronal cell death and memory impairments induced by exposure to Ab are prevented by both A2AR antagonists and in A2AR KO mice. These benecial effects mediated by A2AR blockade in the hippocampus are

not exclusive against Ab toxicity. A2AR antagonism also prevents acute stress induced effects, convulsive episodes and even age associated memory impairments. Nevertheless, the therapeutic use of A2AR antagonists raises an important question,which is the result of administering A2AR antagonists in situations of underlying chronic caffeine intake. In order to address this question additional studies are required. Genetic polymorphisms in the gene encoding A2AR, ADORA2A, are also of great importance. These poly- morphisms are being widely studied and can inuence not only caffeine intake and effects but eventually also the progression of neurodegenerative diseases such as Huntingtons disease [74, 75], as well as the incidence of anxiety and panic disorders. These genetic variations account for two major issues concerning A2AR directed therapy: they can either impact on the outcome of possible therapies, or discriminate a specic population that can be more or less prone to be treated by these compounds. Overall, the major drawback for the use of A2AR antagonists in therapeutics is the lack of clinical studies assessing its effects on AD or on cognitive impairment in humans. The only indirect evidence is epidemiological and solely regarding caffeine consumption. Akey point to understanding the mechanism responsible for the changes observed in the function of the A2AR receptor and the effectiveness of caffeine in AD is the epigenetic modulation of the A2AR gene. CAFFEINE AS A POTENTIAL EPIGENETIC MODULATOR IN AD Currently, there are multiple studies suggesting that epigenetics is involved in (patho) physiological processes. The epigenome comprises the heritable, but potentially reversible, changes in gene expression that occur in the absence of changes to the DNA sequence itself. These changes are brought about by DNA methylation and modications of chromatin, such as acetylation, methylation, phosphorylation, or ubiquitylation of histones (Fig. 3). The epigenotype shows far greater plasticity than the genotype, since it varies among tissues and throughout life, whereas the DNA sequence remains essentially the same. The loss of responsiveness to stress that occurs with aging could be dependent of epigenetic modulation and explain, at least in part, the age dependency of complex disorders such as heart disease, diabetes, and neurodegenerative ones. The common disease genetic and epigenetic (CDGE) model speculates that age-related susceptibility to disease is due to the incorporation of epigenetic with genetic variation. Aging promotes a generalized decrease in DNA methylation, in association with an increase in hypermethylation at specic promoters [82]. Consequently, since AD is highly affected by the aging process, it is plausible to think that epigenetic modulation may also have a role onADpathophysiology. Indeed, increasing evidence supports a role for epigenetic modications in AD. Some cytosines in the promoter region of the AbPP gene are frequently methylated in patients 70 years old and are signicantly demethylated in patients >70 years old [83]. In another study, in a set of monozygotic twins discordant for AD, global levels of DNA methylation were reduced in the temporal neocortex of

the individual with AD, which is consistent with the hypothesis that epigenetics may mediate the differences in risk for certain diseases despite the genetic similarities [84]. More recent studies focused on epigenetic analysis of specic gene loci linked to AD. PSEN1 and APOE, which are involved in Ab processing, and methylenetetrahydrofolate reductase (MTHFR), DNA methyltransferase-1 (DNMT1), which are responsible for methylation homeostasis show a signicant interindividual epigenetic variability,whichmay contribute to late-onset AD predisposition. Several dietary factors, such as ethanol, are also recognized as modulators of both the genome and the epigenome and, consequently, can modify the risk to develop complex diseases. In cardiovascular diseases (CVD), epigenetics is now being widely implicated, since a number of CVD risk factors, such as nutrition, smoking, pollution, stress, and the circadian rhythm, have been associated with modication of epigenetic markers. Hypomethylation of genomicDNAis present in human atherosclerotic lesions and methylation changes also occur at the promoter level of several genes involved in the pathogenesis of atherosclerosis, such as extracellular superoxide dismutase, estrogen receptor-alpha, endothelial nitric oxide synthase and 15-lipoxygenase. Nutritional alterations have been linked to epigenetic modulation of some AD-related genes and seem to have a role on AD pathology. There are also evidences for epigenetic modulation of genes involved in the pathways activated by some dietary factors, both in aging and pathology, suggesting its involvement in AD. While folate levels in CSF and in blood are signicantly lower in individuals with AD, those of homocysteine (Hcy) are elevated, indicating a strong association with atrophy of the cerebral cortex. These alterations suggest that the conversion of Hcy to methionine is altered in this disorder, as it is the production of S-adenosyl methyonine (SAM), the universal methyl donor [94, 95]. Indeed SAM levels, as well as the activity of methionine-S-adenosyltransferase, are decreased in the CSF and brains of individuals with AD compared with age-matched controls. In addition, hyperhomocysteinemia can increase S-adenosylhomocysteine (SAH), a potentmethyltransferase inhibitor. Elevated SAH in the AD brain inhibits methyltransferases and is related to markers of disease progression and cognitive impairment. In neuroblastoma cell lines, SAM/Hcy cycle alterations modify the DNA methylation status with the consequent deregulation of PS1, BACE, and Ab production. Some links between epigenetic mechanisms and ADORA2A regulation have recently arisen. As stated above, the modulatory effects of A2AR are modied whenever brain homeostasis is compromised and particularly in the aged hippocampus. Since aging is tightly linked to epigenetic modulation, the observed increase in these receptors during aging could be a result of hypomethylation or chromatin remodeling. Indeed, it was recently demonstrated that DNAmethylation plays a role in ADORA2A transcription and, subsequently, in constitutive A2AR cell surface levels. Increased YY1 levels and DNA methylation percentage in the

untranslated region of ADORA2A, were also found in the cerebellum with respect to the putamen of human brains, showing an inverse relationship with A2AR levels in the two cerebral regions. These studies showing that DNA methylation plays a role in ADORA2A transcription, may explain the overexpression of A2A receptors in aging and pathological conditions described earlier. Nevertheless, additional data specically evaluating if aging is accompanied by hypomethylation of the ADORA2A gene promoter is lacking. Since the neuroprotective role of caffeine attributed to the blockade of A2A receptors, one question that needs to be addressed is the hypothesis that caffeine regulates epigenetic changes in the ADORA2A gene. Caffeine, acting either directly or through A2A receptors, may increase the activity of the DNA methylating enzymes. This would lead to an increase in DNA methylation of the ADORA2Apromoter, resulting in decreased transcription and consequent synthesis of the receptor (Fig. 4). For therapeutic or even preventive purposes, these links should be further explored