An Introduction to Anti-Aging Research
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Sirtuin is an essential factor that delays cellular senescence and extends the organismal lifespan through the regulation of diverse cellular processes. Suppression of cellular senescence by Sirtuin is mainly mediated through delaying the age-related telomere attrition, sustaining genome integrity and promotion of DNA damage repair. In addition, Sirtuin modulates the organismal lifespan by interacting with several lifespan regulating signaling pathways including insulin/IGF-1 signaling pathway, AMP-activated protein kinase, and forkhead box O. Although still controversial, it is suggested that the prolongevity effect of Sirtuin is dependent with the level of and with the tissue expression of Sirtuin. Since Sirtuin is also believed to mediate the prolongevity effect of calorie restriction, activators of Sirtuin have attracted the attention of researchers to develop therapeutics for age-related diseases. Resveratrol, a phytochemical rich in the skin of red grapes and wine, has been actively investigated to activate Sirtuin activity with consequent beneficial effects on aging. This article reviews the evidences and controversies regarding the roles of Sirtuin on cellular senescence and lifespan extension, and summarizes the activators of Sirtuin including Sirtuin-activating compounds and compounds that increase the cellular level of nicotinamide dinucleotide. The Sirtuin family is nicotinamide dinucleotide (NAD+)-dependent deacylases having remarkable properties in preventing diseases and reversing some aspects of ageing. Sirtuins are known to regulate diverse cellular processes including DNA repair, fat differentiation, glucose output, insulin sensitivity, fatty acid oxidation, neurogenesis, inflammation, and aging. Research interests increased after a report showed that extra copies of SIR2, a member of Sirtuin in budding yeast Saccharomyces cerevisiae, extended the lifespan by 30% by preventing the formation of extrachromosomal DNA circles. The main activity of Sirtuins is deacetylation); recent studies have indicated other enzymatic activities, including O-ADP-ribosylation, demalonylation, desuccinylation, and deproprionylation. Thus, a recent proposal has renamed Sirtuin as deacylase, and not deacetylase. Unlike budding yeast, multicellular organisms have more than one Sirtuin in their genome. Caenorhabditis elegans has four Sirtuins (sir-2.1, sir-2.2, sir-2.3, and sir-2.4), where sir2.1 is the most similar to the S. cerevisiae SIR2. In an experiment using sir-2.1 fused with mCherry fluorescence marker, sir-2.1 was shown to be expressed in the nerve cells of the head, hypodermis, muscle and intestinal cells in C. elegans. The expression of sir-2.1::mCherry was found to be partially nuclear-localized when excess food is available, and was localized in the nuclei of intestines and muscles under nutrient deprived conditions. Drosophila melanogaster has five Sirtuins (dSirt1, dSirt2, dSirt4, dSirt6, and dSirt7), of which Sirt1 (better known as dSir2) is most similar to S. cerevisiae SIR2, and high levels are found in the nuclei and/or cytoplasm of neurons and fat bodies. Although recently reported that fly dSirt4 contains a mitochondrial targeting sequence, the dSirt4 knockout fly appeared healthy, and the mitochondria respiratory function was not disrupted. In mammals, there are seven Sirtuins (SIRT1-SIRT7) having different profiles of enzymatic activity and subcellular ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 2 compartmentation. SIRT1, having the highest sequence homology to yeast SIR2, is predominately found in the nucleus but also shuttles between the cytoplasm and nucleus. SIRT2 is generally found in the cytoplasm, but binds to the chromatin during mitosis. SIRT3 resides in the mitochondria and is translocated to the nucleus in response to stress (such as DNA damage). SIRT4 and SIRT5 are localized in the mitochondria and SIRT6 and SIRT7 are mostly localized in the heterochromatic regions and nucleoli, respectively. The main activity of SIRT4 and SIRT6 are ADP-ribosylation, whereas SIRT5 exerts demalonylation and desuccinylation activities. The subcellular localization and enzymatic activities of Sirtuins are summarized in ANTIAGING EFFECTS OF SIRTUINS Cellular senescence is a physiological phenotype aimed at permanent cell cycle arrest, and is morphologically identified as flattening, increased size of nucleus and nucleoli, and the appearance of vacuoles in the cytoplasm. In addition, several biomarkers developed for cellular senescence are targeted towards the senescence-associated β-galactosidase (SA-β-gal), telomere attrition, senescence-associated heterochromatic foci, cell cycle arrest in the G1 phase, and accumulation of DNA damage with the high level of ATM, p53, p16, and p21. Although cellular senescence is a beneficial process to suppress the accumulation of aberrant cells caused by stress in young organisms, it is detrimental in older organisms to induce agerelated phenotypes. In addition, senescent cells are known to be increased by aging (18). Although still under debate and not fully defined, growing evidence have shown that Sirtuin is an essential factor in delaying cellular senescence and extending organismal lifespan. Especially, the role of Sirtuin on the protection from cellular senescence has mainly been investigated with mammalian SIRT1 and SIRT6. The levels of Sirtuins, including SIRT1 and SIRT6 but not SIRT2, are reported to decrease in senescent cells of mouse embryonic fibroblasts, lung epithelial cells, human endothelial cells and macrophages exposed to oxidants. In addition, the reduction of SIRT1 and SIRT6 using pharmacological inhibitors, siRNA or miRNA, promotes premature senescence-like phenotypes in endothelial cells. Conversely, the overexpression of SIRT1 and SIRT6 suppresses the cellular senescence in angiotensin II-treated human coronary artery endothelial cells, primary porcine aortic endothelial cells, and stress-exposed lung cells. Taken together, these results support that Sirtuins have a role in cellular senescence. The Sirtuin-related suppression of cellular senescence is mainly mediated through the prevention of telomere attrition and the promotion of DNA damage repair. Sirtuins play vital roles in sustaining genome integrity, by contributing in maintaining the normal chromatin condensation state, and responding to DNA damage and repair. Especially, the nuclear form of Sirtuins, such as SIRT1, SIRT6 and SIRT7, act as transcriptional regulators to suppress gene expression by stabilizing the chromatin structure. SIRT1 deacetylates histones H3, H4 and H1 and more than 50 non-histone proteins, including DNMT1, transcription factors and DNA repair proteins. Similar to mammalian Sirtuins, Drosophila dSir2 is also involved in the epigenetic inheritance of silent chromatin states, and the mutation of dSir2 was reported to suppress the heterochromatin-mediated silencing phenomenon known as position effect variegation. SIRT1 and SIRT6 are known to regulate the expression of telomere reverse transcriptase required for ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 3 telomere elongation, and to deacetylate histone 3 lysine 9 (H3K9) and H3K56 resulting in maintaining the telomeric integrity. In addition, SIRT1 and SIRT6 were shown to be recruited to the damaged sites and promote DNA repair through deacetylating the repair proteins such as poly (ADP-ribose) polymerase (PARP)-1, Ku70, NBS, and Werner (WRN) helicase. SIRT4 also plays a role in DNA damage by regulating the mitochondrial glutamine metabolism. Furthermore, Sirtuins modulate cellular senescence through the deacetylation of a variety of signaling molecules such as FOXO, NFκB, and p53. SIRT1 deacetylates FOXO3 and FOXO4, potentiating the FOXO-induced cell cycle arrest, and deacetylates all the major acetylation site of p53, thereby suppressing the oncogene- or stress-induced cellular. Furthermore, SIRT6 regulates the RelA subunit of NFκB by modifying the cellular senescence-related gene expression. In addition to the suppression of senescence of mitotic cells, Sirtuin also modulates the senescence of stem cells, and is required for the maintenance of stem cell self-renewal. Theexpression level of SIRT1 is reported to be higher in embryonic stem cells, but decreases in differentiated cells through the miRNA-mediated post-transcriptional regulations. Reduction of SIRT1 resulted in increased DNA damage, and induced aging phenotypes in hematopoietic stem cells and endothelial progenitor cells, whereas an overexpression of SIRT1 delayed the senescence of bone marrow-derived mesenchymal stem cells. In addition to SIRT1, SIRT3 (a mitochondrial type of Sirtuin) is also highly expressed in hematopoietic stem cells, suggesting that SIRT3 might also function in stem cells. SIRTUINS IN LIFESPAN In addition to the roles in cellular senescence, it is well established that Sirtuin regulates the organismal lifespan in several animal models. Increased expression levels of Sirtuin, especially yeast SIR2 and its homologues, extends the lifespan of budding yeast S. cereviseasae, worms C. elegans, fruit flies D. melanogaster, and mice. The first investigation for the prolongevity effect of SIR2 was established using the yeast model system almost 20 years ago, in which the complex of SIR2/3/4 extended the replicative lifespan of S. cerevisiae by silencing the HM loci and preventing a/α co-expression; SIR2 alone also extended the lifespan by repressing the recombination and generation of toxic rDNA circles. The prolongevity effect of SIR2 has been confirmed in higher organisms, while there are different mechanisms of exerting prolongevity effects in yeast, including changes in mitochondrial function and biogenesis, suppression of inflammation, and regulation of genomic stability. A 7-fold overexpression of sir-2.1 extended the mean lifespan of worms by 14.8–50.5%, whereas a low-copy overexpression of sir2.1 extended the lifespan by 26.2%. In addition, sir-2.1 mutation resulted in decreased lifespan of C. elegans. In Drosophila, overexpression of dSir2 using a P-element mediated insertion of the UAS sequence upstream of dSir2 extended the lifespan, whereas dSir2 null mutants showed a shortened lifespan. Of note, the overexpression of dSir2 in the pan-neuronal cells or fat body extended the lifespan up to 52% and 32.2%, respectively, but the dSir2 induction in motoneuron or muscles had no effect on the lifespan. These results indicate that the prolongevity effect of Sirtuin is tissue-specific. Similarly, mice overexpressing SIRT1 specifically in the hypothalamus had increased median lifespan by 16% in females and 9% in males. ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 4 Sirtuins other than SIRT1 are also reported to exert a prolongevity effect. The transgenic male mice overexpressing SIRT6 showed a significantly longer lifespan than wild-type mice by 16% whereas the SIRT6- and SIRT7-deficient mice lived shorter than controls. In addition, a polymorphism in SIRT3 has been reported in European centenarians. In Drosophila, the overexpression of dSirt4 (which has a mitochondria-targeting sequence) in the whole body or fat body, was reported to extend the lifespan and increase the resistance to starvation. The expression of dSirt4 was induced by starvation in the fat body, and a deficiency of dSirt4 resulted in decreased fertility, locomotion activity, and lifespan. The molecular targets of this longevity effect of Sirtuins have been actively investigated. Sirtuins are found to especially interact with all the major conserved longevity pathways, such as AMPactivated protein kinase (AMPK), insulin/IGF-1 signaling (IIS), target of rapamycin (TOR), and forkhead box O (FOXO). Of these, FOXO transcription factor is the most fascinating target of Sirtuin. In C. elegans, the extension of lifespan by elevation of sir-2.1 was shown to be dependent on daf-16, the homologue of FOXO in worms. Loss of daf-16 using mutants or RNAi treatment abolished the lifespan extension of sir-2.1 overexpression. Daf-16 was reported to physically interact with Sir-2.1 under heat stress, and sir-2.1 reduction completely prevented the subsequent activation of Daf-16 target genes, although reduction or overexpression of sir2.1 had no effect on the nuclear translocation of Daf-16. Conversely, dSir2 is also shown to be important for dFOXO-dependent lifespan extension in Drosophila. Lifespan extension by the overexpression of constitutively active dFOXO in the adult fat body was abrogated when dSir2 was knocked down using dSir2 RNAi. Considering that FOXO is a major component in the IIS cascade to promote lifespan extension and stress resistance, several pieces of evidence have reported the association of the IIS pathway with the prolongevity effect of Sirtuin. In C. elegans, Sir-2.1 does not interact physically with Daf-16 when the expression of insulin-like receptor (daf-2) was decreased via daf-2 RNAi, and the deletion of sir-2.1 had no effect on the lifespan of a long-living daf-2 mutant. In addition, deletion of daf-2 did not result in further extending the lifespan of worms overexpressing sir-2.1 and the reduction of dSir2 by mutation or RNAi expression showed a decrease in starvation survival and systemic insulin signaling in Drosophila. These results indicate that the lifespan extension by IIS reduction is associated with SIR2. In mammals, the relationship of IIS and Sirtuin has also been well investigated. SIRT1 is reported to play a crucial role in metabolic homeostasis and IIS. In addition, SIRT6 transgenic mice express lower serum levels of IGF1, higher levels of IGF-binding protein 1, and altered phosphorylation levels of major components of IGF1 signaling. AMPK signaling belongs to the protein kinase family and restores cellular energy levels. Increased AMPK activity is known to extend the lifespan of some model organisms. The mutation of AMPK (aak-2) in C. elegans abrogated the lifespan extension by sir-2.1 expression, indicating that AMPK also contributes to the Sirtuin-induced lifespan extension. SIRT1 activates AMPK through the direct deacetylation of LKB1, a regulator of AMPK, and AMPK is known to activate SIRT1 through the elevation of NAD+ levels. In addition, AMPK contributes to the ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 5 prolongevity effect of IIS, suggesting that these longevity pathways intricately cross-talk with each other. Apart from these, several other molecules are also reported to mediate lifespan extension by Sirtuin overexpression, including 14-3-3, kat-1, hcf-1, and cts-1 in C. elegans. The 14-3-3 protein is a small acidic protein that alters the subcellular localization of its target. The mutation of par5 and ftt-2, encoding the two proteins of a conserved 14-3-3 family in worms, abolished the lifespan extension by sir-2.1 overexpression. In addition, in vivo GST-pull down assay and immunoprecipitation assay revealed that Sir-2.1 directly interacts with PAR-5 and FTT-2, and both of which are necessary for the SIR-2.1-mediated transcription activation of the DAF-16 target genes sod-3 and hsp-16.2. These results support the concept that the lifespan-extension effect of SIR2 is mediated by the 14-3-3 protein. In addition, a study of mutant screening reported that loss-of-function mutations of ketoacyl thiolase (kat-1) resulted in premature aging and fully suppressed the lifespan extension exerted by overexpression of sir-2.1. Also, host cell factor-1 (hcf-1), a nuclear co-repressor of FOXO, was shown to act downstream of sir-2.1 to modulate the lifespan in C. elegant. Furthermore, mitochondrial regulators such as cts1 and fzo-1, and the mitochondrial unfolded protein response (UPRmt) gene hsp-6, were reported to increase by sir-2.1 overexpression, and the knock-down of UPRmt regulator ubl5 using RNAi almost completely suppressed the lifespan extension by sir-2.1 overexpression, thereby indicating that the effects of sir-2.1 are dependent on UPRmt . POSSIBLE ROLE OF SIRTUINS IN LIFESPAN EXTENSION Although numerous evidences indicate that overexpression of Sirtuin delays the cellular senescence and extends the lifespan of organisms, several reports have challenged this theory. The outcrossing abrogated the lifespan extension phenotype of geIn3 worm strains, used for overexpressing Sirtuin 10–30 fold, indicating that the longevity effect of this Sirtuin overexpressing strain is due to a lack of genetic background standardization. In addition, geIn3 strain also has an unlinked dyf mutation which attributes to the longevity effect of the strain, indicating that the lifespan extension of geIn3 is also due to incorrectly matched controls. The report also showed that overexpression of dSir2 using outcrossed dSir2EP2300 and the two newly constructed lines containing inducible UAS-dSir2 under ubiquitously expressing Tubulin-Gal4 driver did not extend the lifespan compared to the flies expressing the Gal4 driver only. Independently, a reduction in the expression of dSir2 using Sir2 RNAi in the fat body did not affect the lifespan of flies. Later, this argument was also refuted. Viswanathan and Guarente showed that geIn3 worms still have long lifespan after outcrossing compared to outcrossed control lines. These contradictory results concerning the effect of Sirtuin overexpression on lifespan might be explained by the extent of overexpression of Sirtuin. Whitaker et al. showed that highly expressed (45-fold increase) dSir2 in the whole fly shortened the lifespan, but modest levels (2– 11-fold increase) of dSir2 resulted in extended lifespan. Thus, they asserted that dSir2 expression in previous reports might not be relevant each other, since the extent of overexpression varied depending on the controls that were used for comparison. In the previous ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 6 reports showing the lifespan extension by dSir2, the dSir2 was overexpressed 3–4-fold. The dose-dependent effect of Sirtuin was also presented in a mice model. Alcendor et al. showed that 2.5–7.5-fold mild increase of SIRT1 in mouse heart prevented age-related cardiac hypertrophy by eliciting an increase in the level of antioxidant enzymes, but a 12.5-fold increase of SIRT1 increased the oxidative stress and promoted cardiac hypertrophy. CALORIC RESTRICTION Calorie restriction (CR), also known as dietary restriction, is a proven intervention to extend lifespan in almost all animal models including non-human primate, which experimentally means a reduction in calorie intake by 10–50% compared to the ad libitum intake without malnutrition. Although still controversial, it is believed that the beneficial effects of CR on lifespan extension and prevention of age-related diseases is mediated by the induction of Sirtuins. In many animal models, the expression and activity of Sirtuin was reported to be increased by CR and nutritional deprivation, and the activation of Sirtuin by CR was mediated by the upregulation of AMPK and increase of NAD+ levels. In C. elegans, the expression levels of sir-2.1 tagged with mCherry fluorescence increased due to starvation in the intestine and muscle cells, but not in nerve cells. In D. melanogaster, dSir2 expression increased in the flies fed low-calorie food. In mammals, the levels of Sirtuins (except SIRT4) were also reported to increase after CR, which is known to be tissue specific. Increased levels of SIRT1 by CR were observed in white adipose tissue, skeletal muscle, kidney, brain and intestine,. However, in liver, the response of SIRT1 expression to CR is debatable; one paper showed induction of SIRT1 in the liver of CR-experienced mice, but another report showed a reduction in levels. Furthermore, the latter report also showed that knock-out of SIRT1 in liver is dispensable in this tissue. In addition, SIRT6 is indirectly activated by CR by upregulating the SIRT1, FOXO3a, and nuclear respiratory factor 1 (NRF). SIRT3 are induced by CR in diverse tissues, including muscle, white adipose tissue, and liver; SIRT3 is suggested to be essential for cochlea neurons against oxidative damage. These studies indicate that the expression of Sirtuin is regulated by CR with tissue-specificity, suggesting that a more elaborate investigation is required to understand Sirtuin regulation by CR. Numerous researches have also reported the requirement of Sirtuins in the lifespan-extension effect of CR in various organisms. In budding yeast, CR does not extend the lifespan of SIR2 mutant strain, and the lifespan extension by CR was reported to require the nicotinamidase PNC1, an enzyme that recycles NAD+, which is critical for Sirtuin-dependent functions. This indicates that SIR2 is indispensable for mediating the positive effects of CR in yeast. In C. elegans, the requirement of Sirtuin is dependent with respect to the strain and genetic background. The strain of eat-2 such as ad465, ad113, ad116 that have defective pumping and are considered as the CR model of worms, lived longer than the wild-type N2 stain . The sir-2.1 mutation suppressed this longevity effect of eat-2 in ad465 and ad113 strain but not in the ad116 strain, in which CR is more extreme. In Drosophila, the lifespan extending effects of CR were partially mediated by dSir2, and mammalian Sirtuins are well known to mediate the beneficial effect of CR. SIRT1 knockout mice did not normally display the metabolic response triggered by CR, and the increased physical activity by CR mice was not seen ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 7 in SIRT1 knockout mice. Knockout of SIRT3 increased the oxidative stress and damage by CR . In addition to these CR-related outputs, SIRT1 knockout mice failed to show the lifespan extension in response to CR (91), indicating that SIRT1 mediates the lifespan-extension by CR. The role of Sirtuins in lifespan extension by CR has long been challenged. Several reports asserted that Sirtuins are not required for lifespan extension by CR in yeast, C. elegans, and fruit flies. In yeast, the SIR2 mutation suppresses the replicative lifespan extension in the CR model strain gpa2Δ and hsk2Δ, but not in fob1Δ . In addition, SIR2 overexpression increases the replicative lifespan of yeast in low glucose. Furthermore, SIR2 mutation did not suppress the chronological lifespan extension by CR, and deletion of all Sirtuin family in yeast did not prevent the effect of CR. In C. elegans, the role of Sirtuins in the lifespan extension of CR is shown as dependent with the protocols of CR. Although the lifespan extension in the pumping defective eat-2 worms requires Sir-2.1, the lifespan extension by CR through food deprivation or bacteria dilution does not require Sir-2.1. In D. melanogaster, the homozygotic mutant dSir24.5 and dSir217 respond normally to CR, and the CR increased the lifespan of these mutant flies. In addition, the dSirt4 knockout flies also responded normally to CR with the expected increase in lifespan. These controversial results are suggested to be due to differences in strain background, CR protocols, or Sirtuin gene redundancy. In addition, it is suggested that there may be both Sirtuin-dependent and -independent pathways that play a role in extending the lifespan by CR. SIRTUIN ACTIVATORS Since Sirtuin is commonly believed to mediate the beneficial effects of CR, the activators of Sirtuin are considered to mimic these beneficial effects and are hence attractive therapeutics for age-related diseases. Subsequently, high-throughput screening has identified over 14,000 Sirtuin-activating compounds (STACs). STACS In 2003, a screen for activators of the mammalian SIRT1 identified 15 small molecules including quercetin, butein, fisetin, and piceatannol. This study further revealed the most potent activator of SIRT1 to be resveratrol (3,5,4′-trihydroxystilbene), a polyphenol found in red wine, which extended the replicative lifespan of budding yeast by 70% at 10 μM. In addition, the lifespanextension effect of resveratrol was abrogated by the SIR2 mutation, indicating that resveratrol extends the lifespan of yeast through the activation of Sirtuin. Interestingly, higher concentrations of resveratrol did not further increase the lifespan, and resveratrol failed to extend the chronological lifespan. Another study in the following year showed that fisetin, butein, and resveratrol also activated levels of Sir-2.1 of C. elegans up to 2.5-fold, and dSir2 of D. melanogaster up to 2.4-fold. In addition, a single amino acid of SIRT1 (E230) was proved to be critical for binding to STACs and inducing activation. Supplementation of resveratrol at 100 μM extended the lifespan of C. elegans and D. melanogaster by 14% and 29%, respectively, but was ineffective on the Sir2 mutant, suggesting that resveratrol extends the lifespan in a Sir2- dependent manner. However, the lifespan of transgenic worms overexpressing sir-2.1 were ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 8 extended by 39% following resveratrol treatment, thereby suggesting that sir-2.1 exerts its effect of extending the lifespan independent of resveratrol. In addition, the study also revealed that resveratrol-induced lifespan extension is mediated by the ER stress gene, abu-11, whose expression is regulated by sir-2.1. The lifespan extending effect of resveratrol and other related STACs was also established in the short lived fish Nothobranchius furzeri and in the honeybee Apis mellifera. Other than resveratrol, natural compounds including cilostazol, paeonol, statins, hydrogen sulfide, Icariin, persimmon, melatonin, and curcumin are also reported as potent STACs. Some of these STACs have exhibited a prolongevity effect on model animals. For example, pretreatment with curcumin or alkylresorcinols enhanced the SIRT1 activity, and extended the lifespan of Drosophila. Natural STACs are hydrophobic in nature with low solubility and low bioavailability. To overcome these weaknesses, synthetic STACs were developed by using drug design approaches. To date, more than 14,000 STACs have been synthesized up to the 5th generation, and dozens of these have been tested in animal disease models. Several STACs are also currently undergoing clinical trials. These synthetic STACs are reported to be beneficial for several age-related diseases, and have demonstrated protection from cancer, neurodegeneration, cardiovascular disease and diabetes, with some compounds exerting an extended lifespan. Of these, SRT1720, SRT2104, SRT1460, SRT2183, STAC-5, STAC-9, and STAC-10 have attracted attention due to their increased potency, solubility, and bioavailability as compared to resveratrol. Especially, SRT1720, a synthetic STACs structurally unrelated to resveratrol, has been reported to improve insulin sensitivity and mitochondrial capacity in obese rodents, and has extended the lifespan of mice fed a high-calorie diet, to a similar extent as resveratrol. In addition, SRT2104 mimics aspects of CR and extends the lifespan of male mice fed a standard diet. In addition to these STACs, several oxazolo(4,5-β)pyridine and imidazo(1,2-β)thiazole derivatives have also been identified as activators of SIRT1. Especially, 1,4-dihydropyridine derivatives activate several Sirtuins (SIRT1- SIRT3) in a dose-dependent manner, and synthetic iso-nicotinamide (iNAM) also acts as a Sirtuin-activator. The requirement of SIR2 in the lifespan extension by these STACs is controversial, and several increasing evidences show that the lifespan extending effects of STACs are Sirtuin-independent. Predominantly, it is contentiously debated whether or not resveratrol and synthetic STACs directly activate SIRT1. The original report reveals the activation of SIRT1 by resveratrol using a fluorescence-conjugated peptide substrate, Fluor-de-Lys. However, resveratrol and STACs showed no activation of Sirtuin using in vitro full-length endogenous substrates (such as p53 and PGC1α) or short, fluorescence-unconjugated peptide substrates. In addition, it was suggested that the effect of resveratrol on SIRT1 was due to the off-target effects on other enzymes such as phosphodiesterase. Recently, a newly developed assay for detection of Sirtuin activity, named as CycLex, revealed that SRT1720 does not activate SIRT1. In addition, several reports show that resveratrol treatment does not extend the lifespan of S. cerevisiae and D. melanogaster, and only slightly extends the lifespan of C. elegans independent with sir-2.1. ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 9 Additionally, resveratrol and STACs show no beneficial effects when administrated to the obese mouse model. More precisely controlled protocols to detect the Sirtuin activity, and more discreetly managed experiments to investigate the role of STACs on longevity are required. THE NAD+ BOOSTER An alternative approach to activating Sirtuins is regulating NAD+ levels by activating enzymes involved in biosynthesis of NAD. NAD is an essential cofactor for electron transfer and for regulating metabolic homeostasis, and its levels decrease with aging in the liver and muscle, as could partially be explained due to the decreased activity of Sirtuin upon aging. The supplementation of NAD extended the lifespan of C. elegans, Drosophila, and the premature mice model, which is mediated by Sirtuin activation. During deacetylation reaction by Sirtuin, NAD+ is converted to nicotinamide (NAM), and NAM is recycled into NAD or other nicotinic acid (NA) derivatives through the NAD salvage pathway. Thus, the administration of NAM increases the level of NAD with subsequent SIRT1 activation. NAM is also methylated by nicotinamide N-methyltransferase (NNMT) to 1-methylnicotinamide (MNA). The treatment of NAM, NA, or MNA, and the overexpression of anmt-1, the C. elegans NNMT, results in extending the lifespan of worms. Supplementation of NA extends the lifespan similar to the extent with sir-2.1 overexpression. In the mutation of anmt-1, NA and NAM are unable to extend the lifespan, but MNA is still capable of extending the lifespan of worms. In addition, the overexpression of nicotinamide phosphoribosyltransferase (NAMPT), the enzyme converts NAM into nicotinamide mononucleotide (NMN), is also reported to increase the SIRT1 activity. Overexpression of the NAMPT orthologue dNAAM is shown to increase the Drosophila lifespan in a dSir2-dependent manner. Furthermore, the inhibitor of NAD+ consuming enzyme PARPs also increases the C. elegans lifespan in a Sirtuin-dependent manner, and the inhibition of CD38 results in NAD+ accumulation and subsequent SIRT1 activation in mice, rendering a protective effect against high-fat-diet-induced obesity. An increase of NAD+ levels is also observed in energy deficient conditions, such as fasting, CR or low glucose feeding. Compounds that raise NAD levels, such as nicotinamide riboside and NMN, are potential candidates as CR mimetics that are shown to extend lifespan. Under conditions of starvation, AMPK is activated and alters the intracellular metabolism, resulting in an increase in NAD levels with a concomitant increase in SIRT1 activity. In yeast, the longevity effect of CR is reported to require PNC-1, a homologue of NAMPT. However, contrarily, several reports have revealed that the level of NAD does not increase in yeast exposed to CR, and NAD supplementation further extended the lifespan of eat-2 mutant worms, indicating that lifespan extension by NAD worked along a different pathway. ©ESSENTIALS SPA 2020 DAY ZERO THERAPEUTICS® IS A REGISTERED TRADEMAR OF ESSENTIALS SPA 10 CONCLUSTIONS For over 20 years, the Sirtuin family has been actively investigated for its function in delaying cellular senescence and extending longevity. In addition, based on the role of Sirtuin on the beneficial effect of CR, therapeutic trials using activators of Sirtuin have actively proceeded to protect age-related diseases. Growing evidences have principally supported that Sirtuin is an attractive anti-aging molecule involved in improving health through the target molecules participating in diverse biological processes; however, the role of Sirtuin on longevity, and the longevity effect of CR, are still controversial. In addition, numerous questions remain unresolved, such as the role of other Sirtuins in addition to SIRT1 and SIRT6 on aging, the redundancy of the Sirtuin family members to regulate lifespan, whether other enzymatic activities (apart from deacetylation activity) participate in the process of aging, and whether STACs could be promoted as drugs to treat aging or age-related diseases in humans. These questions will be answered soon, and Sirtuin may provide the effective approach to extend lifespan and improve our quality of life
A nicotinic hypothesis for Covid-19 with preventive and therapeutic implications
121 jean-pierre CHANGEUX, Zahir Amoura , Felix Rey , Makoto Miyara
SARS-CoV-2 epidemics raises a considerable issue of public health at the planetary scale. There is a pressing urgency to find treatments based upon currently available scientific knowledge. Therefore, we tentatively propose a hypothesis which hopefully might ultimately help saving lives. Based on the current scientific literature and on new epidemiological data which reveal that current smoking status appears to be a protective factor against the infection by SARS-CoV-2 , we hypothesize that the nicotinic acetylcholine receptor (nAChR) plays a key role in the pathophysiology of Covid-19 infection and might represent a target for the prevention and control of Covid- 19 infection.
Symptomatic Covid-19 disease (as caused by SARS-CoV-2 virus) is observed in 2.5 percent of infected individuals  indicating an individual variability in the clinical presentation. Among the epidemiological and clinical features of Covid-19, the following features are of special interest for understanding the patho-physiolology, namely: (1) in outpatients with favorable outcome : neurological/psychiatric disorders, especially loss of sense of smell which is specific of the disease and (2) in hospitalized older patients with a poor prognosis : systemic hyperinflammatory syndrome with increased levels of circulating cytokines and atypical acute respiratory distress syndrome with loss of
neurological control of lung perfusion regulation and hypoxic vasoconstriction . This raises the issue of the basis of inter-individual variability for the susceptibility to
The nAChR appears as a hypothetical clue for the main clinical manifestations of Covid- 19. It is accepted that the angiotensin converting enzyme 2 (ACE2), represents the principal receptor molecule for SARS-CoV-2 [4-6]. ACE2 is expressed at the transcriptomic level in the lung, the small intestine and colon, in the kidney, in the testis, in the heart muscle and in the brain, yet the protein is not detected in the lung . In the brain, ACE2 is expressed in both neurons and glia and particularly present in the brain stem and in the regions responsible for the regulation of cardiovascular functions, including the subfornical organ, paraventricular nucleus, nucleus of the tractus solitarius, and rostral ventrolateral medulla . Additional receptors or co-receptors are, however, not excluded. The relationship between nicotine and ACE2 has been explored in the framework of cardiovascular and pulmonary diseases . Accordingly, in the ACE/ANG II/AT1R arm, nicotine increases the expression and/or activity of renin, ACE and AT1R, whereas in the compensatory ACE2/ANG-(1–7)/MasR arm, nicotine down regulates the expression and/or activity of ACE2 and AT2R, thus suggesting a possible contribution of acetylcholine receptors in ACE2 regulation. This possibility has not yet been explored in the framework of viral neuroinfections.
There is strong evidence for a neurotropic action of SARS-CoV-2 infection. It has been demonstrated that β-coronaviruses to which the SARS- CoV-2 belongs, do not limit their presence to the respiratory tract and have been shown to frequently invade the CNS  . This propensity has been convincingly documented for the SARS-CoV-1, MERS- CoV and the coronavirus responsible for porcine hemagglutinating encephalomyelitis (HEV 67N). In light of the high similarity between SARS-CoV-1 and SARS-CoV-2, it is quite likely that SARS-CoV-2 also possesses a similar potential. Neuroinfection has
been proposed to potentially contribute to the pathophysiology and clinical manifestations of Covid-19  with the neuroinvasive potential of SARS-CoV-2 suggested to play a role in the respiratory failure of Covid‐19 patients [11, 12]. Our nicotinic hypothesis proposes that the virus could enter the body through neurons of the olfactory system and/or through the lung leading to different clinical features with different outcome, and contrasts with the currently accepted view that ACE2 is the principal receptor of SARS-CoV-2 for its entry into cells.
As mentioned, loss of sense of smell frequently occurs in Covid‐19 patients . Furthermore, several studies have reported that some patients infected with SARS‐ CoV‐2 show neurologic signs such as headache (about 8%), nausea and vomiting (1%) . More recently, a study of 214 Covid‐19 patients  further found that about 88% (78/88) of the severe patients displayed neurologic manifestations including acute cerebrovascular diseases and impaired consciousness. Based on an epidemiological survey on Covid‐19, the median time from the first symptom to dyspnea was 5.0 days, to hospital admission was 7.0 days, and to the intensive care was 8.0 days . Therefore, the latency period may be adequate for the virus to enter the nervous system, invade the brain stem and affect the medullary neurons of the respiratory centers. However, variability of the neurological signs was observed with patients having anosmia, showing in general a mild evolution without pulmonary attack, in contrast with those without anosmia suggesting a diversity in the mode of proliferation and /or progression of the virus.
More than 20 years ago, Mohammed, Norrby & Kristensson , in a pioneering
study, showed with a broad diversity of viruses (Poliovirus, Herpes simplex virus, West Nile virus , Vesicular Stomatitis Virus, influenza H1N1 virus ), that viruses enter the olfactory epithelium and progress first through the olfactory pathway in an anterograde direction and then in a retrograde manner to the reticular neurons projecting to the olfactory bulbs, the median raphe neurons (serotoninergic) and the ventral and
horizontal diagonal band (cholinergic) [16, 18]. This olfactory infection route scheme  has been recently extended to Covid-19 infection [2, 11]. To further investigate the molecular aspects of Covid-19 propagation in the brain and its pharmacology, we have been aided by abundant studies on rabies virus (RABV) a negative polarity, single- strand RNA virus that is distinct from the coronaviruses [18-20]. nAChRs were shown to be the first receptors for RABV . Structural studies further revealed that a short region in the ectodomain of the rabies virus glycoprotein shows sequence similarity to some snake toxins [20, 22] that were initially used to isolate the nAChR from fish electric organs ). These snake toxins  are known to bind with high affinity and exquisite selectivity to the peripheral muscle receptor, while also to some brain receptors [25, 26]. The neurotoxin-like region of the rabies virus glycoprotein inhibited acetylcholine responses of α4β2 nAChRs in vitro, as did the full length ectodomain of the rabies virus glycoprotein . The same peptides significantly altered a nAChR elicited behaviour in C. elegans and increased locomotor activity levels when injected into the CNS of mice . The nAChR thus plays a critical role in the host-pathogen interaction in the case of the RABV. Furthermore, a broad variety of nAChR oligomers are distributed throughout the brain, including the reticular core neurons and the spinal cord, with the 𝛼𝛼4𝛽𝛽2 and 𝛼𝛼7 nAChR oligomers being the most frequent . The hypothesis we wish to explore is to what implications these data may hold for SARS- CoV-2 infection and we suggest a strong role of nAChR in the disorder.
The nAChR pathway is hypothesized to be engaged in the Covid-19 inflammatory syndrome. The nervous system, through the vagus nerve, can significantly and rapidly inhibit the release of macrophage TNF, and attenuate systemic inflammatory responses ). This physiological mechanism, termed the ‘cholinergic anti-inflammatory pathway’ has major implications in immunology and in therapeutics. The cytokine production of macrophages – one of the main cell types found in the bronchoalveolar fluid – is under the physiological control of auto/paracrine acetylcholine through their nAChRs .
Following dysregulation of macrophage nAChRs, the profile of cytokines massively secreted include Il1, Il6, TNF et Il18. This cytokine profile shows striking analogies with the cytokine storm syndrome, leading to the hyperinflammatory syndrome described in a subgroup of Covid-19 patients ; Systemic coagulopathy with venous and arterial thrombosis is one of the critical aspects of the morbidity and mortality of Covid-19. In line with our hypothesis, one should note that hematopoietic 𝛼𝛼7-nAChR deficiency increases platelet reactive status, which could explain the thrombogenic presentation of Covid-19 .
Although selective cytokine blockers (eg, IL1-receptor antagonist anakinra or anti-IL6 tocilizumab) have been proposed for the control of Covid-19 cytokine storm, their efficacy is still to be explored. Interestingly, 𝛼𝛼7 agonists, including nicotine, have proven to be effective in reducing macrophage cytokine production and inflammation in animal models of pancreatitis  and peritonitis . In this setting, a nicotinic treatment that might possibly antagonize the blocking action of SARS-CoV-2 on the AChR through a possible modulation of the ACE2 – nAChR interaction, would act earlier than anti- cytokine therapies. nAChR modulation by Covid-19 might tentatively account for the hyperinflammatory features observed in a subgroup of Covid-19 patients, mimicking bona fide the macrophage activation syndrome.
Of note, our hypothesis could explain the high prevalence of obesity and diabetes mellitus observed in severe forms of Covid19. The diminished vagus nerve activity previously described in these two illnesses could be potentiated by the Covid-19 elicited nicotinic receptor dysregulation, leading to a hyperinflammatory state often reported in obese patients .
nAChRs are present in the lung epithelium. The non-neuronal cholinergic system contributes to the regulation of cell functions such as cell-cell interaction, apoptosis, and proliferation and it is well established that human bronchial epithelial cells contain nAChRs. The airway epithelium expresses 𝛼𝛼3, 𝛼𝛼4, 𝛼𝛼5, 𝛼𝛼7, 𝛼𝛼9, 𝛽𝛽2, and 𝛽𝛽4-nAChRs
subunits [34-37] and their contribution has been discussed in the framework of airway epithelial basal cell proliferation-differentiation and their alteration in lung cancers . These nAChRs are mentioned here as possible targets of Covid-19 infection of the lung, which would take place concomitantly with, and/or as a consequence of, the neuro- infection. Additionally, nAChRs are involved in lung perfusion regulation, which seems to be disrupted in the atypical acute respiratory distress syndrome reported in Covid-19 patients .
A potential protective effect of smoking and of nicotine on SARS-CoV-2 infection has been noted. Until recently , no firm conclusions could be drawn from studies evaluating the rates of current smokers in Covid-19. All these studies [40-48], although reporting low rates of current smokers, ranging from 1.4% to 12.5%, did not take into account the main potential confounders of smoking including age and sex. In the study that two of us are reporting , the rates of current smoking remain below 5 % even when main confounders for tobacco consumption, i.e. age and sex, in- or outpatient status, were considered. Compared to the French general population, the Covid-19 population exhibited a significantly weaker current daily smoker rate by 80.3 % for outpatients and by 75.4 % for inpatients. Thus, current smoking status appears to be a protective factor against the infection by SARS-CoV-2. Although the chemistry of tobacco smoke is complex, these data are consistent with the hypothesis that its protective role takes place through direct action on various types of nAChRs expressed in neurons, immune cells (including macrophages), cardiac tissue, lungs, and blood vessels.
Mechanisms engaged in Covid-19 as nAChR disease might be tentatively suggested. There is structural evidence supporting the hypothesis that SARS-CoV-2
virus is a nicotinic agent. The recently reported X-ray structure of the RABV glycoprotein (G) ectodomain  shows that the region corresponding to the neurotoxin-like peptide
is exposed at the G surface, in agreement with the fact that this region is part of the major antigenic region II of RABV . The recently published cryo-EM structure of the trimeric SARS-CoV-2 spike (S) protein [51, 52] revealed an insertion with respect to that of SARS-CoV-1, in a loop that is disordered in the reported structure, and which has a polybasic sequence that corresponds to a furin site. Importantly, this exposed loop of the SARS-CoV-2 S protein also contains a motif that is homologous to that of snake neurotoxins and to the RABV neurotoxin-like region (Figure). This observation supports the hypothesis that SARS-CoV-2 virus itself is a nAChR blocker.
Figure: The neurotoxin motifs. Amino acid sequence alignment of the motifs found in toxins from snakes of the Ophiophagus (cobra) and Bungarus genera, in G from three RABV strains and in S from SARS-CoV-2
Nicotine may be suggested as a potential preventive agent against Covid-19 infection. Both the epidemiological/clinical evidence and the in silico findings may suggest that Covid-19 infection is a nAChR disease that could be prevented and may be controlled by nicotine. Nicotine would then sterically or allosterically compete with the SARS-CoV- 2 binding to the nAChR. This legitimates the use of nicotine as a protective agent against SARS-CoV-2 infection and the subsequent deficits it causes in the CNS. Thus,
in order to prevent the infection and the retro-propagation of the virus through the CNS, we plan a therapeutic assay against Covid-19 with nicotine (and other nicotinic agents) patches or other delivery methods (like sniffing/chewing) in hospitalized patients and in the general population
In conclusion, we propose, and try to justify, the hypothesis that nAChRs play a critical role in the pathophysiology of SARS-CoV-2 infection and as a consequence propose nicotine and nicotinic orthosteric and/or allosteric agents as a possible therapy for SARS-CoV-2 infection. Interestingly, ivermectin, which has been recently shown to inhibit the replication of SARS-CoV-2 in cells in vitro , is a positive allosteric modulator of a7 nAChR . The nicotinic hypothesis might be further challenged by additional clinical studies and by experimental observations determining whether SARS- CoV-2 physically interacts with the nAChR in vitro, for instance by electrophysiological recordings, high resolution EM and by animal model studies. Further work should also specify the still enigmatic relationships between ACE2 and nAChRs in the nervous system.
One should not forget that nicotine is a drug of abuse (53) responsible for smoking addiction. Smoking has severe pathological consequences and remains a serious danger for health. Yet under controlled settings, Nicotinic agents could provide an efficient treatment for an acute infection such as Covid-19.