Drug Discovery Today: Therapeutic Strategies

Editors-in-Chief Raymond Baker formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein GlaxoSmithKline, USA
DRUG DISCOVERY

Vol. 7, No. 34 2010

STRATEGIES

TODAY THERAPEUTIC

Aging

Aging control with resveratrol

` Palla ` s1, Felix Junyent1,2, Ester Verdaguer3, Carlos Beas-Zarate4, Merce Antoni Camins1,*
1 ` sia Facultat de Farma ` cia, Centros de Investigacio n Biome dica en Red Enfermedades Neurodegenerativas (CIBERNED), Unitat de Farmacologia i Farmacogno Institut de Biomedicina (IBUB), Universitat de Barcelona, Nucli Universitari de Pedralbes, 08028 Barcelona, Spain 2 ` ncies de la Salut, Centro de Investigacio n Biome dica en Red de Enfermedades Neurodegenerativas (CIBERNED), Unitat de Bioquimica, Facultat de Medicina i Cie Universitat Rovira i Virgili, C./ St. Llorenc , 21 43201 Reus, Tarragona, Spain 3 n Biome dica en Red Enfermedades Neurodegenerativas (CIBERNED), Facultat de Biologia, Universitat Departament de Biologia Cellular, Centros de Investigacio de Barcelona, Spain 4 n de Neurociencias, Centro de Investigacio n Biome dica de a Celular y Molecular, CUCBA, Universidad de Guadalajara, Divisio Departamento de Biolog Occidente, IMSS, Sierra Mojada 800, Col. Independencia, Guadalajara, Jalisco 44340, Mexico

The prevalence of age-related pathologies such as neurodegenerative disease and diabetes type II has increased with rise in average age of people. Finding antiaging molecules is necessary to prevent or delay the alterations taking place in age which are manifested as age-associated illnesses. Currently, dietary restriction (DR) is one of the most effective antiaging interventions. Thus, the biochemical pathways involved in DR lifespan increase are under intensive investigation. Sirtuin 1 and mTOR (mammalian target of rapamycin) are two well characterized antiaging pathways. Resveratrol constitutes a natural compound which mimics the effects of DR with potential application on age-associated diseases and thus as antiaging therapy. Introduction
It is evident that the increase in human life expectation has important socio-economic and health consequences in developed countries which are related to an increase in the load of diseases associated with aging. The age-associated diseases that accompany increased life expectancy include: cardiovascular diseases, stroke, neurodegenerative diseases, osteoporosis, diabetes and hypertension [1]. Thus, an antiaging pharmacological treatment should increase the quality
*Corresponding author.: A. Camins (camins@ub.edu) 1740-6773/$ 2011 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddstr.2011.01.001

Section editors: Vijay Kumar Kapoor G.H.G. Khalsa College of Pharmacy, Gurusar Sadhar, Ludhiana 141104, India Janhvi Dureja Swift School of Pharmacy, Rajpura, Punjab, India

of life via a decreased risk of such diseases associated with aging [1,2]. The search for drugs with an antiaging effect is complicated by the requirements of low toxicity and easy oral administration, because they must be suitable for long-term administration [1]. It is important to understand the biochemical mechanisms responsible for the aging process which will help to design drugs to inhibit this natural physiological process. The present review focuses on the role of resveratrol (RESV), a natural product. RESV has been shown to have benecial effects on health and longevity in experimental models and, together with new synthetic derivatives, it is being studied in humans as a means of treating age-related diseases [3].

Dietary restriction: an effective antiaging approach

Some theories of aging attempt to explain the complex mechanism of the aging process [4]. These include theories of changes in gene regulation, telomere loss theory and oxidative stress theory. All theories of aging focus on age-related physiological changes that are closely interrelated, such as: oxidative stress, mitochondrial alteration, membrane changes,
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apoptosis and genetic mechanisms; all of which contribute to the aging process [46]. Understanding the molecular processes that cause aging is important for preventing or postponing the onset of age-related diseases [6]. Most experimentation in the eld of aging research is performed on experimental model systems such as the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the fruit y Drosophila melanogaster [7]. Research in this area has demonstrated that different types of damage contribute to aging such as protein misfolding and aggregation, inammation, mitochondrial alteration and reactive oxygen species (ROS). One of the most widely accepted theories suggests that free radicals are responsible for the aging process; particularly, ROS damage biological macromolecules and are thus responsible for aging [4]. This theory is supported by much experimentation that demonstrates a close correlation between resistance to oxidative stress and lifespan in different organisms. However, this theory has recently been questioned. Experiments on various mouse models demonstrate that although the overexpression of several antioxidant enzymes makes mice more resistant to oxidative challenge, it does not increase their lifespan [8]. Thus, a deciency of superoxide dismutase reduced the lifespan in mice, whereas a deciency of other antioxidant enzymes had no effect. Likewise, not all antioxidant drugs prolong life in mice [8]. Interestingly, a well-characterized and effective nonpharmacological antiaging intervention is dietary restriction (DR), dened as a reduction in nutrient availability in the absence of malnutrition [9]. Thus, experimental studies have demonstrated that DR increases lifespan in a variety of different organisms in the laboratory, including yeast, nematodes, ies and rodents [9,10]. In a recent study, DR was also shown to reduce age-related deaths in a nonhuman primate: the rhesus monkey [11]. Importantly, DR not only increases lifespan in model organisms, but also increases the period of life spent in relatively good health and free of age-associated disease [911]. Many different age-associated diseases seem to be positively affected by DR, including: cancer, cardiovascular disease, neurodegenerative diseases and diabetes [10]. Although how DR inhibits the aging process is unknown, an analysis of gene expression in brain of aging mice under DR as compared to controls demonstrated that DR signicantly decreases the expression of a family of genes involved in inammation and stress response [12]. This could explain the benecial effects of DR on neurodegenerative diseases. Because DR could reduce age-associated diseases, the next step was to develop a pharmacological treatment that reproduces the effects of DR. In theory, a drug that alters the activity of genes and proteins in a manner similar to DR will act as a DR mimetic by slowing aging and retarding ageassociated diseases without requiring reduced food intake.
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RESV is a suitable candidate as an antiaging medicine, because of its low toxicity and easy oral administration [13].

Resveratrol acts as an antiaging drug through sirt1 activation

The development of specic antiaging treatments is a current challenge for preventive medicine. The rst antiaging medicines proposed were mainly compounds with antioxidant properties such as Ginkgo biloba extracts, carnosine, melatonin, RESV and avonoids [36]. RESV is of special interest after the discovery of the benecial effects of red wine on human health; specically protection against coronary heart disease [6]. However, the antioxidant properties of this natural compound are not exclusively responsible for its therapeutic properties. RESV has been reported to act as a sirtuin activator, and it has been suggested that this mechanism accounts for its antiaging effects (Fig. 1). Sir2 (silent information regulator 2) belongs to the family of histone deacetylases (HDACs) which are nicotinamide adenine dinucleotide (NAD+)-dependent enzymes [3,13]. NAD+ is necessary for the histone deacetylase activity. It has been demonstrated that Sir2 is one of the proteins involved in mediating the caloric restriction-dependent lifespan extension in invertebrates [14]. Sir2 is phylogenetically conserved from bacteria to humans; this is a very important point which suggests the importance of this gene in nature [15]. Silent information regulator two ortholog 1 (SIRT1) is the human ortholog of the yeast Sir2 protein [15,16]. Up to now, seven mammalian sirtuins, called SIRT17, have been characterized, which have very important physiological functions in the human body,

mTOR INHIBITION AMPK (PGC1-)

OH HO

NF-kB
OH

AUTOPHAGY

FOXO 3A

ANTIOXIDANT

SIRT 1 ACTIVATION
Drug Discovery Today: Therapeutic Strategies

Figure 1. Potential mechanisms involved in antiaging effects of resveratrol. Resveratrol through multiple mechanisms such as SIRT1 activation, autophagia activation, mTOR inhibition, inhibition of the inammatory response, inhibition of oxidative stress and inhibition or activation of other cellular components can participate actively in the antiaging process.

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such as regulation of metabolism, inammation, prevention of cell death and lifespan control [3,1416]. SIRT1 is the most studied and best-characterized sirtuin because of its possible involvement in the aging process. For example, there is evidence that SIRT1 levels are reduced in patients with Alzheimers disease (AD) and that this is accompanied by an accumulation of tau protein, suggesting that SIRT1 could play a prominent role in AD pathology [17]. Studies in mice demonstrate the antiaging effects of a diet with a low dose of RESV (4.9 mg/kg/day). Such benecial effects of RESV are similar to those that DR has on gene expression [18]. Likewise, the same study of the effects of RESV on mice aging parameters conrms that RESV antioxidant effects are not responsible for the antiaging effects. In addition, studies of invertebrates show that RESV extends the lifespan of S. cerevisiae, C. elegans and D. melanogaster, but only if the gene that encodes Sir2 is present in these organisms [3,15,19]. RESV also increases the lifespan of the sh Nothobranchius furzeri (a very short-lived seasonal sh) by almost 60%, although the involvement of SIRT1 as a target for this process has not been demonstrated [2022]. Thus, from all these studies, it was hypothesized that Sir2 could be key to longevity [14,15,23,24]. An in vitro screening for activators of SIRT1 identied RESV as the most potent of 18 inducers of deacetylase activity [25]. Given these ndings, drugs that activate SIRT1 might also have antiaging effects and other benecial effects on metabolism. It is hoped that such compounds may thus be of benet in the treatment of diabetes and neurodegenerative diseases, as well as in the prevention of cardiovascular diseases [25]. So, current research is focused on understanding the mechanisms involved in the capacity of RESV to increase SIRT1 activity and on the intracellular pathways that are activated or regulated by SIRT1 [16,18]. No clinical trials to evaluate the effects of RESV on human lifespan have previously been carried out. However, the discovery of novel selective SIRT1 activators opened up an interesting new research area in the treatment of age-associated diseases. These newly synthesized compounds, among them SRT2183, SRT1460, SRT1720 and SRT501, are potent small-molecule activators of SIRT1 [23,25]. For example, SRT501 has been evaluated in patients with type 2 diabetes and phase II clinical trials indicate that this drug is well tolerated and safe for humans at oral doses of 1.25 or 2.5 g [23]. Moreover, a study with cancer patients has also been started.

How sirt1 extends lifespan

The most important antiaging gene that is upregulated by DR and RESV is SIRT1. Interestingly, mammalian SIRT1, and also Sir2, both are located primarily near the nucleus and physiological actions of SIRT1 are mediated in part of their ability to deacetylate nucleosomal histone proteins at specic residues for histone H4 lysine 16 [26,27]. It has been hypothe-

sized that a potential cause of aging is a failure to control chromatin structure. Although the vast majority of lifespan studies have been performed on yeast, chromatin structure is a target for Sir2 in the regulation of aging through histone acetylation and gene expression [7,15,27]. Thus SIRT1 and Sir2 control yeast lifespan through histone targets located near telomeres. Accordingly, in mammals, a mechanism involved in the antiaging effects of RESV could be mediated by changes in chromatin structure via SIRT1 activation (Fig. 1). In addition to chromatin, once SIRT1 is activated, it mediates intracellular responses through the regulation of nonhistone cellular substrates [28]. More than ten substrates of SIRT1 have been described to date. Among them, SIRT1 specically acts by deacetylating transcription factors, such as the tumor suppressor p53, the FOXO (also called FKHR) family of transcription factors FOXO1, FOXO3 and FOXO4, and the transcription factors NF-kB and Ku70 [13,16,19,28]. Therefore, SIRT1 is involved in the regulation of cell signals from all these transcription factors and will affect their ability to trigger apoptosis or induction in the expression of their target genes involved in cell cycle arrest, senescence or apoptosis. Moreover, SIRT1 also deacetylates nuclear receptor peroxisome-proliferator activated receptor-g (PPARg) and its transcriptional co-activator PPARc coactivator-a (PGC1a), which regulates a wide range of metabolic activities in muscle, adipose tissues, heart and liver [29,30]. Furthermore, PGC1-a is linked to the aging process, because it is activated by several intracellular signals and inuences longevity through its interaction with the mitochondria [31,32]. Early studies on C. elegans demonstrate that FOXO transcription factors (or the invertebrate ortholog DAF-16) interact with several pathways that regulate cellular lifespan and increase longevity [15,28]. This extension of lifespan is reversed when FOXO ortholog DAF-16 is mutated. Although the mechanism involved in the antiaging properties of FOXO is unknown, nuclear FOXO interacts with DNA and regulates the transcription of specic target genes. Furthermore, FOXO transcription factors can regulate the antiaging PI3KAkt signaling pathway, which responds to the insulin/IGF-I pathway and several other growth factors [31]. In addition, apart from regulating longevity, members of the FOXO family of transcription factors are also involved in the regulation of other cellular processes such as metabolism. For instance, FOXO1 modulates glucose tolerance in adipose tissue, liver and pancreas [15]. The importance of FOXO transcription factor in the mammalian aging process was recently revealed in reports of three independent human studies. This suggests that there is an association between an increase in longevity and the FOXO3A gene [3335]. Those studies were performed on an ethnic Japanese population in Hawaii, an Italian population from a region southeast of Naples with notably high longevity, and in a population of German centenarians. The
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results from those studies suggest an association between this gene and attaining exceptional old age. Experimental data also conrm that RESV is involved in FOXO3a regulation via SIRT1, because it induces the localization of FOXO3A at the nucleus, promoting FOXO-dependent gene expression [28]. Interestingly, studies of cultures of human dermal broblast FOXO3A protein deleting result in senescence induction and ROS increase [23]. Loss of FOXO3A in aged rats decreases the antioxidant properties of manganese-superoxide dismutase and favors cell injury due to aging [29]. Accordingly, RESV induction of FOXO transcription factors could also be involved in the antiaging effects of this compound [3]. As discussed above, aged mice showed a signicant increase in the brain inammatory and stress response [12]. It is well known that NF-kB is the master regulator of the immune/ inammatory response. Inammatory response is involved in the aging process, as we discuss below. Therefore, SIRT1, through the regulation of NF-kB, could inhibit the expression of genes involved in inammation which is directly related with the NF-kB signaling pathway [23]. Furthermore, the inhibition of cytokines by SIRT1 could have benecial effects on neurodegenerative diseases and cardiovascular diseases. The regulation of NF-kB by SIRT1 could constitute another pathway involved in the antiaging effects of RESV.

Other pathways known to regulate the aging process

In addition to SIRT1 activation, several other proteins are now known to inuence longevity, energy use and the response to DR. Such documented pathways, which are also involved in antiaging, include the receptors for insulin, for another hormone called IGF-1, and for a protein that warrants increasing interest and is known as TOR (target of rapamycin) [31,3638]. The role of insulin/insulin-like growth factor I (IGFI) signaling in lifespan extension is well established in invertebrates. The rst experiments were also performed on C. elegans and Drosophila. In addition, the reduction of insulin/IGF-I signaling also extends the lifespan of mice. The antiaging properties of this pathway may mainly be due to FOXO factor activation. The most important targets in this IGFI signaling pathway are FOXO factors among them FOXO3a which, as we discussed above, extends the lifespan of humans [31,3335]. Another interesting point is the recent demonstration that RESV inhibits the mammalian target of rapamycin (mTOR) pathway [37]. The mTOR pathway is currently an area of research in antiaging drug development, because mTOR inhibition is also involved in lifespan increase after DR [37,39,40]. Previous papers report that mTOR inhibition extends the lifespan of invertebrates (worms, yeast and fruit ies). Rapamycin (also known as sirolimus) is a macrolide antibiotic that inhibits mTOR activity and is used clinically as an immunosuppressive drug [39,40]. Recently, it was demon54
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strated that oral administration of rapamycin to mice at 600 days of age signicantly increased their lifespan [39]. These ndings are in agreement with another study performed on mice concerning the deletion of S6 protein kinase 1 (S6K1), a component of mTOR and also the IGF1 signaling pathway, which was found to increase lifespan [41]. In addition, in the same study, a strong correlation between gene expression by DR and S6K1 deletion in the liver was demonstrated. RESV also affects the PI3K/mTOR/S6K pathway suggesting that SIRT1 and mTOR could be members of the same network aging process control [41]. This is probably because mTOR is a pro-aging and SIRT1 is an antiaging pathway. Likewise, AMP-activated protein kinase (AMPK) has emerged as another potentially important age-related target of RESV [38]. This enzyme is involved in the regulation of insulin sensitivity and mitochondrial biogenesis, maintaining the energy balance of the cell by modulating ATP production. Experimental data demonstrate that RESV activates AMPK which increases lifespan in invertebrates. AMPK is involved in the effects on lifespan in the worm C. elegans after DR [38]. Moreover, AMPK activates the enzyme SIRT1, providing a possible mechanism for indirect activation of SIRT1 by RESV. In addition, RESV did not have such an effect on mice decient in the catalytic subunit of AMPK (alpha1 or alpha2). Furthermore, an increase in AMPK activity was demonstrated in mice lacking the ribosomal S6 protein [41]. With reference to this, the drug metformin, an AMPK activator which is used clinically in the treatment of diabetes type II, has been reported to increase longevity [41]. Therefore, AMPK activation could constitute a key master convergent point/regulator in all antiaging pathways. Another interesting hypothesis is that aging is associated with autophagy inhibition [42]. Autophagy could be described as a mechanism of cell protection mediated by the degradation of waste products during the aging process. It has been suggested that aging inhibition of autophagy could be responsible for the apparition of age-related diseases such as neurodegenerative diseases which are characterized by accumulation of anomalous proteins (b-amyloid or asynuclein) in the brain. Moreover, suppression of autophagy in neural cells is also responsible for the appearance of neurodegenerative disease in mice. Recent data indicate that the activation of SIRT1 by RESV triggers autophagy in nematode cells [41]. Moreover, Sirtuin-1 activators do not have such effects on autophagy-decient C. elegans. In addition, FOXO3A increases the expression of proteins associated with autophagy such as LC3. Accordingly, RESV could modulate autophagy and aging process through FOXO3A.

Conclusion
The molecular and physiological processes of aging and longevity appear to have been conserved through a long period of evolution. Because there is no disease that can be termed

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Acknowledgements
OXIDATIVE STRESS

SIRT1
MITOCHONDRIA DAMAGE

mTOR
DNA DAMAGE

This study was supported by grants SAF-2009-13093 from the n y Ciencia, PI080400 from the Instituto Ministerio de Educacio de Salud Carlos III (Fondos FEDER) 2009/SGR00853 from the La Generalitat de Catalunya and (063230) from the Fundacio de TV3. 610RT0405 from Programa Iberoamericano de Marato Ciencia y Tecnologia para el Desarrollo (CYTED). We thank the Language Assessment Service of the University of Barcelona for revising the manuscript.

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AGING SWITCH
3
AUTOPHAGIA INHIBITION

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Drug Discovery Today: Therapeutic Strategies

Figure 2. The hypothetical molecular process involved in causing the aging switch. Irrespective of the primary stress signal (mitochondria damage, autophagia inhibition, DNA damage and oxidative stress), a complex response would be obtained by the activation of molecular proteins (SIRT1, mTOR and AMPK). Note that aging switch is expected to contain several positive o negative feedback loops that determine its activation.

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aging, the development of a unique antiaging drug does not seem viable. Thus, for a given intervention to be acknowledged as antiaging, delayed onset of age-related changes and pathologies must be demonstrated. RESV could constitute a pharmacological approximation of the reversal of the natural process of aging and age-associated diseases. Moreover, it represents an attempt to move antiaging interventions from the laboratory to the clinic. RESV is reported to increase lifespan and slow the progression of age-related diseases in multiple model systems, and RESV jointly with SIRT1 activators are under study for clinical applications against age-related diseases. Evidence suggests that the antioxidant properties of RESV are not the main mechanism involved in its antiaging properties. However, although it remains unknown whether RESV and SIRT1 activators show antiaging effects in humans, we should have some indication of this in the near future. In addition, RESV has several advantages because, to be effective in the prevention or delay of the aging process, a drug should act on more than one aged pathway. Thus it has been demonstrated that RESV affects mTOR (inhibition), SIRT1 (activation), AMPK (increase), autophagy (increase), antioxidant and anti-inammatory effects, and also affects intracellular transcription factors (Fig. 2). For all these reasons, the natural compound RESV is a potential candidate for an antiaging treatment and also the prevention of aging switch.

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